G2Cdb::Gene report

Gene id
G00002004
Gene symbol
MYH9 (HGNC)
Species
Homo sapiens
Description
myosin, heavy chain 9, non-muscle
Orthologue
G00000755 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000030429 (Vega human gene)
Gene
ENSG00000100345 (Ensembl human gene)
4627 (Entrez Gene)
171 (G2Cdb plasticity & disease)
MYH9 (GeneCards)
Literature
160775 (OMIM)
Marker Symbol
HGNC:7579 (HGNC)
Protein Sequence
P35579 (UniProt)

Synonyms (5)

  • EPSTS
  • FTNS
  • MHA
  • NMHC-II-A
  • NMMHCA

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000133: May-Hegglin anomaly N Y (16978745) Unknown (?) Y
D00000134: MYH9-related disease N Y (16818291) Microinsertion (MI) Y
D00000134: MYH9-related disease N Y (16806139) Single nucleotide polymorphism (SNP) Y
D00000133: May-Hegglin anomaly N Y (16642488) Unknown (?) Y
D00000134: MYH9-related disease N Y (16162639) Nonsense (No) Y
D00000134: MYH9-related disease N Y (16098078) Microinsertion (MI) Y
D00000134: MYH9-related disease N Y (16077952) Polymorphism (P) N
D00000134: MYH9-related disease N Y (15667538) Frameshift mutation (FS) Y
D00000134: MYH9-related disease N Y (15613099) Deletion (D) Y
D00000134: MYH9-related disease N Y (15339844) Polymorphism (P) Y

References

  • Identification of a novel MYH9 mutation in a patient with May-Hegglin anomaly.

    Otsubo K, Kanegane H, Nomura K, Ogawa J, Miyawaki T and Kunishima S

    Pediatric blood & cancer 2006;47;7;968-9

  • Analysis of clinical manifestations, mutant gene and encoded protein in two Chinese MYH9-related disease families.

    Yi Y, Sen Zhang G, Xu M, San Ling Z, Ru Shao X, Zeng Li J and Ma J

    Division of Hematology/Institute of Molecular Hematology, the Second Xiang-ya Hospital, Central South University, Changsha, Hunan 410011, P.R. China.

    Background: MYH9-related disease is a rare autosomal dominant disorder characterized by the triad of giant platelet, thrombocytopenia and inclusion bodies in neutrophil. In recent years, much progress has been made in the investigation of its clinical feature and pathogenesis.

    Methods: Clinical manifestations were analyzed in two Chinese MYH9-related disease families. Polymerase chain reaction (PCR), DNA sequencing and CpoI restrictive endonuclease map analysis were used to identify spot mutation in nonmuscle myosin heavy chain 9 (MYH9) gene. Indirect immunofluence combined propidium iodine (PI) nuclei count-staining technology was applied to probe nonmuscle myosin heavy chain IIA (NMMHC-A) in MYH9-related disease neutrophils and platelets. Western blot was undergone to examine the expression of NMMHC-A in MYH9-related disease patients.

    Results: All of the patients manifested with the typical triad, mild to moderate bleeding tendency were their common clinical feature, some patients were accompanied by renal lesion. G5521A mutation in MYH9 gene was identified in both families. Spindle-like inclusions with yellow fluorescence in MYH9-related disease neutrophils were clearly revealed by indirect immunofluence combined PI nuclei count-staining technology, which matched very well with the inclusions, detected by Wright-Giemsa's stain. An upregulation of NMMHC-A in MYH9-related disease neutrophils was observed by Western blotting analysis.

    Conclusion: Mutation of MYH9 gene exists in cases of Chinese MYH9-related disease. In the two families, the point mutation was located in exon 38(G5521A), and the transference rule of the MYH9 gene mutation is corresponding with clinical phenotype distribution. Indirect immunofluorescence combining with PI nuclei staining technology is sensitive and more specific than Wright-Giemsa's staining in detecting MYH9-related disease inclusions, with which we might easily distinguish MYH9-related disease inclusions from infection-associated inclusions. The expression of the NMMHC-A in MYH9-related disease neutrophils was upregulated than normal control.

    Clinica chimica acta; international journal of clinical chemistry 2006;373;1-2;49-54

  • [The MYH9 syndrome: report of a new case with a new mutation of the MYH9 gene].

    Schleinitz N, Favier R, Mazodier K, Difeo A, Ebbo M, Veit V, Berda-Haddad Y, Bernit E, Heudier P, Kaplanski G, Camoin L, Bardet V and Harle JR

    Service de médecine interne, CHU la Conception, APHM, boulevard Baille, 13385 Marseille cedex 05, France. nicolas.schleinitz@ap-hm.fr

    Introduction: Familial macrothrombocytopenias are a group of rare autosomal dominant platelet disorders including many syndromes in particular the May-Hegglin anomaly. They are characterized by thrombocytopenia with giant platelets and in some cases neutrophilic inclusions in peripheral blood granulocytes. Recently these different clinical entities have been demonstrated to be linked to mutations in the same gene, MYH9.

    We report in a young African woman presenting as a May-Hegglin anomaly a new mutation of the MYH9 gene. In regard of this case we present a brief review of the MYH9 syndrome.

    Conclusion: The MYH9 syndrome includes now several clinical entities who share some common clinical and biological characteristics such as a thrombocytopenia with giant platelets, presence or absence of other manifestations including Dohle like bodies, nephritis, sensorineural hearing loss, cataract. We report a new case in which a new mutation of the MYH9 gene was evidenced.

    La Revue de medecine interne 2006;27;10;783-6

  • Hematologic and genetic characterization of an MYH9-related disorder in a Chinese family.

    Ma ES, Wong CL, Shek TW and Hui SP

    We describe a Chinese family with an MYH9-related disorder in which a novel mutation V1516L at exon 31 of the MYH9 gene was identified. To the best of our knowledge, this is the first reported Chinese family with MYH9 mutation and supports the pan-ethnic nature of the disorder.

    Haematologica 2006;91;7;1002-3

  • Pathogenetic mechanisms of hematological abnormalities of patients with MYH9 mutations.

    Pecci A, Canobbio I, Balduini A, Stefanini L, Cisterna B, Marseglia C, Noris P, Savoia A, Balduini CL and Torti M

    Department of Internal Medicine, University of Pavia, Italy. alessandro.pecci@unipv.it

    Mutations of MYH9, the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA), cause a complex clinical phenotype characterized by macrothrombocytopenia and granulocyte inclusion bodies, often associated with deafness, cataracts and/or glomerulonephritis. The pathogenetic mechanisms of these defects are either completely unknown or controversial. In particular, it is a matter of debate whether haploinsufficiency or a dominant-negative effect of mutant allele is responsible for hematological abnormalities. We investigated 11 patients from six pedigrees with different MYH9 mutations. We evaluated NMMHC-IIA levels in platelets and granulocytes isolated from peripheral blood and in megakaryocytes (Mks) cultured from circulating progenitors. NMMHC-IIA distribution in Mks and granulocytes was also assessed. We demonstrated that all the investigated patients had a 50% reduction of NMMHC-IIA expression in platelets and that a similar defect was present also in Mks. In subjects with R1933X and E1945X mutations, the whole NMMHC-IIA of platelets and Mks was wild-type. No NMMHC-IIA inclusions were observed at any time of Mk maturation. In granulocytes, the extent of NMMHC-IIA reduction in patients with respect to control cells was significantly greater than that measured in platelets and Mks, and we found that wild-type protein was sequestered within most of the NMMHC-IIA inclusions. Altogether these results indicate that haploinsufficiency of NMMHC-IIA in megakaryocytic lineage is the mechanism of macrothrombocytopenia consequent to MYH9 mutations, whereas in granulocytes a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies. The finding that the same mutations act through different mechanisms in different cells is surprising and requires further investigation.

    Funded by: Telethon: GP0019Y01, TGM06S01

    Human molecular genetics 2005;14;21;3169-78

  • Dissecting clinical findings: platelet defects segregate independently of deafness and cataract in a family affected by an apparent syndromic form of macrothrombocytopenia.

    Gangarossa S, Seri M, Pecci A, Di Bari F, Cusano R, Balduini C, Gasparini P and Savoia A

    ASL 7 Ragusa, Università di Bologna, Italy.

    We studied a family with a suspected diagnosis of MYH9-related disease, which is one of the most common forms of autosomal dominant macrothrombocytopenias associated with hearing impairment, cataracts and nephritis. No mutation of the MYH9 gene was identified. Moreover, the A156V variant of the GPIbalpha gene, responsible for 30% of macrothrombocytopenias in Italy, was not detected in the family. Therefore, we hypothesized that the clinical symptoms were caused by mutations in different genes. The screening of the candidate genes for deafness and/or cataract allowed us to identify two variants, M34T and S19T, of the GJB2 gene in family members with hearing impairment. Because of the relatively common occurrence of inherited hearing loss and, at least in the Mediterranean area, of platelet macrocytosis, the two traits occurred by chance in the same family and mimicked the MYH9-related disease.

    Funded by: Telethon: F04001

    International journal of molecular medicine 2005;16;3;437-41

  • Genotype-phenotype correlation in MYH9-related thrombocytopenia.

    Dong F, Li S, Pujol-Moix N, Luban NL, Shin SW, Seo JH, Ruiz-Saez A, Demeter J, Langdon S and Kelley MJ

    Department of Medicine, Duke University Medical Center and Hematology/Oncology, Durham Veterans Affairs Hospital, Durham, NC 27705, USA.

    Mutation of the non-muscle myosin heavy chain type II-A results in MYH9-related hereditary macrothrombocytopenia (HMTC), including four autosomal dominant platelet disorders: May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FS) and Epstein (EPS) syndrome. Denaturing high-performance liquid chromatography (DHPLC) was optimised for rapid screening of the seven exons harbouring all but one of the previously reported mutations of MYH9. Individuals from 13 families with phenotypes suggestive of MYH9-related HMTC were screened for mutations by DHPLC followed by direct sequencing of samples with aberrant column retention time. Mutations were identified in all 13 families. Six distinct missense heterozygous mutations were found in 10 families, including six families with MHA or SBS (E1841K, D1424N), three families with FS (R702H, R1165C, and D1424Y), and one family with EPS (S96L). A truncating mutation (R1933X) was found in three MHA families. A review of all published mutations suggests that mutation in the C-terminal coiled coil region or truncation of the tailpiece is associated with haematological-only phenotype, while mutation of the head ATPase domain frequently is associated with nephropathy and/or hearing loss. Mutations of other regions have intermediate expression of non-haematological characteristics. Further study is required to confirm these associations and understand the molecular basis for this genotype-phenotype relationship.

    Funded by: NCI NIH HHS: 1U01-CA-96123-01, 5R21 CA91565-02; NHLBI NIH HHS: 1R01HL66192-02, R01 HL066192

    British journal of haematology 2005;130;4;620-7

  • First description of somatic mosaicism in MYH9 disorders.

    Kunishima S, Matsushita T, Yoshihara T, Nakase Y, Yokoi K, Hamaguchi M and Saito H

    Department of Haemostasis and Thrombosis, Clinical Research Centre, National Hospital Organization Nagoya Medical Centre, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Dohle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, which encodes non-muscle myosin heavy chain-A (NMMHCA). These disorders are known to be transmitted in an autosomal dominant manner, although about 20% of cases are considered to be sporadic. We report here the first case of a MYH9 disorder because of somatic mosaicism. The patient was the father of a male with typical May-Hegglin anomaly. The father had normal platelet counts, however, both normal-sized and giant platelets were observed on his peripheral blood smears. In addition, 14% of neutrophils contained inclusion bodies and the rest showed a normal morphology. Quantitative fluorescent polymerase chain reaction analysis showed that only 6% of DNA from peripheral blood leucocytes harboured the mutation. The mosaicism was demonstrated at a similar rate in different tissues, buccal mucosa cells and hair bulb cells, implying that the mutation had occurred before gastrulation. Mosaicism might account for some de novo mutations in MYH9 disorders.

    British journal of haematology 2005;128;3;360-5

  • Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly.

    Franke JD, Dong F, Rickoll WL, Kelley MJ and Kiehart DP

    Department of Biology, Developmental Cell and Molecular Biology Group, Duke University Medical Center, Durham, NC 27708-1000, USA.

    MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the alpha-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.

    Funded by: NHLBI NIH HHS: HL 66192, R01 HL066192; NIGMS NIH HHS: GM 33830

    Blood 2005;105;1;161-9

  • Detection of unique neutrophil non-muscle myosin heavy chain-A localization by immunofluorescence analysis in MYH9 disorder presented with macrothrombocytopenia without leukocyte inclusions and deafness.

    Kunishima S, Matsushita T, Shiratsuchi M, Ikuta T, Nishimura J, Hamaguchi M, Naoe T and Saito H

    Department of Hemostasis and Thrombosis, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders are autosomal-dominant macrothrombocytopenias with leukocyte inclusions caused by mutations in the MYH9 gene, which encodes the non-muscle myosin heavy chain-A (NMMHCA). We report a patient with an MYH9 disorder who presented with macrothrombocytopenia without leukocyte inclusions and severe bilateral sensory deafness. Conventional May-Grunwald-Giemsa staining failed to detect granulocyte cytoplasmic inclusions, whereas immunofluorescence analysis clearly demonstrated abnormal neutrophil NMMHCA localization. Genetic analyses revealed a novel heterozygous 18 base deletion in MYH9, leading to a six-amino acid in-frame deletion (N76_S81del) in NMMHCA. These results further support the usefulness of immunofluorescence analysis in differential diagnosis of MYH9 disorders.

    European journal of haematology 2005;74;1;1-5

Literature (135)

Pubmed - human_disease

  • Identification of a novel MYH9 mutation in a patient with May-Hegglin anomaly.

    Otsubo K, Kanegane H, Nomura K, Ogawa J, Miyawaki T and Kunishima S

    Pediatric blood & cancer 2006;47;7;968-9

  • [The MYH9 syndrome: report of a new case with a new mutation of the MYH9 gene].

    Schleinitz N, Favier R, Mazodier K, Difeo A, Ebbo M, Veit V, Berda-Haddad Y, Bernit E, Heudier P, Kaplanski G, Camoin L, Bardet V and Harle JR

    Service de médecine interne, CHU la Conception, APHM, boulevard Baille, 13385 Marseille cedex 05, France. nicolas.schleinitz@ap-hm.fr

    Introduction: Familial macrothrombocytopenias are a group of rare autosomal dominant platelet disorders including many syndromes in particular the May-Hegglin anomaly. They are characterized by thrombocytopenia with giant platelets and in some cases neutrophilic inclusions in peripheral blood granulocytes. Recently these different clinical entities have been demonstrated to be linked to mutations in the same gene, MYH9.

    We report in a young African woman presenting as a May-Hegglin anomaly a new mutation of the MYH9 gene. In regard of this case we present a brief review of the MYH9 syndrome.

    Conclusion: The MYH9 syndrome includes now several clinical entities who share some common clinical and biological characteristics such as a thrombocytopenia with giant platelets, presence or absence of other manifestations including Dohle like bodies, nephritis, sensorineural hearing loss, cataract. We report a new case in which a new mutation of the MYH9 gene was evidenced.

    La Revue de medecine interne 2006;27;10;783-6

  • Hematologic and genetic characterization of an MYH9-related disorder in a Chinese family.

    Ma ES, Wong CL, Shek TW and Hui SP

    We describe a Chinese family with an MYH9-related disorder in which a novel mutation V1516L at exon 31 of the MYH9 gene was identified. To the best of our knowledge, this is the first reported Chinese family with MYH9 mutation and supports the pan-ethnic nature of the disorder.

    Haematologica 2006;91;7;1002-3

  • Dissecting clinical findings: platelet defects segregate independently of deafness and cataract in a family affected by an apparent syndromic form of macrothrombocytopenia.

    Gangarossa S, Seri M, Pecci A, Di Bari F, Cusano R, Balduini C, Gasparini P and Savoia A

    ASL 7 Ragusa, Università di Bologna, Italy.

    We studied a family with a suspected diagnosis of MYH9-related disease, which is one of the most common forms of autosomal dominant macrothrombocytopenias associated with hearing impairment, cataracts and nephritis. No mutation of the MYH9 gene was identified. Moreover, the A156V variant of the GPIbalpha gene, responsible for 30% of macrothrombocytopenias in Italy, was not detected in the family. Therefore, we hypothesized that the clinical symptoms were caused by mutations in different genes. The screening of the candidate genes for deafness and/or cataract allowed us to identify two variants, M34T and S19T, of the GJB2 gene in family members with hearing impairment. Because of the relatively common occurrence of inherited hearing loss and, at least in the Mediterranean area, of platelet macrocytosis, the two traits occurred by chance in the same family and mimicked the MYH9-related disease.

    Funded by: Telethon: F04001

    International journal of molecular medicine 2005;16;3;437-41

  • Genotype-phenotype correlation in MYH9-related thrombocytopenia.

    Dong F, Li S, Pujol-Moix N, Luban NL, Shin SW, Seo JH, Ruiz-Saez A, Demeter J, Langdon S and Kelley MJ

    Department of Medicine, Duke University Medical Center and Hematology/Oncology, Durham Veterans Affairs Hospital, Durham, NC 27705, USA.

    Mutation of the non-muscle myosin heavy chain type II-A results in MYH9-related hereditary macrothrombocytopenia (HMTC), including four autosomal dominant platelet disorders: May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FS) and Epstein (EPS) syndrome. Denaturing high-performance liquid chromatography (DHPLC) was optimised for rapid screening of the seven exons harbouring all but one of the previously reported mutations of MYH9. Individuals from 13 families with phenotypes suggestive of MYH9-related HMTC were screened for mutations by DHPLC followed by direct sequencing of samples with aberrant column retention time. Mutations were identified in all 13 families. Six distinct missense heterozygous mutations were found in 10 families, including six families with MHA or SBS (E1841K, D1424N), three families with FS (R702H, R1165C, and D1424Y), and one family with EPS (S96L). A truncating mutation (R1933X) was found in three MHA families. A review of all published mutations suggests that mutation in the C-terminal coiled coil region or truncation of the tailpiece is associated with haematological-only phenotype, while mutation of the head ATPase domain frequently is associated with nephropathy and/or hearing loss. Mutations of other regions have intermediate expression of non-haematological characteristics. Further study is required to confirm these associations and understand the molecular basis for this genotype-phenotype relationship.

    Funded by: NCI NIH HHS: 1U01-CA-96123-01, 5R21 CA91565-02; NHLBI NIH HHS: 1R01HL66192-02, R01 HL066192

    British journal of haematology 2005;130;4;620-7

  • First description of somatic mosaicism in MYH9 disorders.

    Kunishima S, Matsushita T, Yoshihara T, Nakase Y, Yokoi K, Hamaguchi M and Saito H

    Department of Haemostasis and Thrombosis, Clinical Research Centre, National Hospital Organization Nagoya Medical Centre, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Dohle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, which encodes non-muscle myosin heavy chain-A (NMMHCA). These disorders are known to be transmitted in an autosomal dominant manner, although about 20% of cases are considered to be sporadic. We report here the first case of a MYH9 disorder because of somatic mosaicism. The patient was the father of a male with typical May-Hegglin anomaly. The father had normal platelet counts, however, both normal-sized and giant platelets were observed on his peripheral blood smears. In addition, 14% of neutrophils contained inclusion bodies and the rest showed a normal morphology. Quantitative fluorescent polymerase chain reaction analysis showed that only 6% of DNA from peripheral blood leucocytes harboured the mutation. The mosaicism was demonstrated at a similar rate in different tissues, buccal mucosa cells and hair bulb cells, implying that the mutation had occurred before gastrulation. Mosaicism might account for some de novo mutations in MYH9 disorders.

    British journal of haematology 2005;128;3;360-5

  • Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly.

    Franke JD, Dong F, Rickoll WL, Kelley MJ and Kiehart DP

    Department of Biology, Developmental Cell and Molecular Biology Group, Duke University Medical Center, Durham, NC 27708-1000, USA.

    MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the alpha-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.

    Funded by: NHLBI NIH HHS: HL 66192, R01 HL066192; NIGMS NIH HHS: GM 33830

    Blood 2005;105;1;161-9

  • Detection of unique neutrophil non-muscle myosin heavy chain-A localization by immunofluorescence analysis in MYH9 disorder presented with macrothrombocytopenia without leukocyte inclusions and deafness.

    Kunishima S, Matsushita T, Shiratsuchi M, Ikuta T, Nishimura J, Hamaguchi M, Naoe T and Saito H

    Department of Hemostasis and Thrombosis, Clinical Research Center, Nagoya Medical Center, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders are autosomal-dominant macrothrombocytopenias with leukocyte inclusions caused by mutations in the MYH9 gene, which encodes the non-muscle myosin heavy chain-A (NMMHCA). We report a patient with an MYH9 disorder who presented with macrothrombocytopenia without leukocyte inclusions and severe bilateral sensory deafness. Conventional May-Grunwald-Giemsa staining failed to detect granulocyte cytoplasmic inclusions, whereas immunofluorescence analysis clearly demonstrated abnormal neutrophil NMMHCA localization. Genetic analyses revealed a novel heterozygous 18 base deletion in MYH9, leading to a six-amino acid in-frame deletion (N76_S81del) in NMMHCA. These results further support the usefulness of immunofluorescence analysis in differential diagnosis of MYH9 disorders.

    European journal of haematology 2005;74;1;1-5

Pubmed - other

  • The association of the MYH9 gene and kidney outcomes in American Indians: the Strong Heart Family Study.

    Franceschini N, Voruganti VS, Haack K, Almasy L, Laston S, Goring HH, Umans JG, Lee ET, Best LG, Fabsitz RR, MacCluer JW, Howard BV, North KE and Cole SA

    Department of Epidemiology, University of North Carolina, Chapel Hill, NC 27514, USA. noraf@unc.edu

    Chronic kidney disease (CKD) is an important public health problem in American Indian populations. Recent research has identified associations of polymorphisms in the myosin heavy chain type II isoform A (MYH9) gene with hypertensive CKD in African-Americans. Whether these associations are also present among American Indian individuals is unknown. To evaluate the role of genetic polymorphisms in the MYH9 gene on kidney disease in American Indians, we genotyped 25 SNPs in the MYH9 gene region in 1,119 comparatively unrelated individuals. Four SNPs failed, and one SNP was monomorphic We inferred haplotypes using seven SNPs within the region of the previously described E haplotype using Phase v2.1. We studied the association between 20 MYH9 SNPs with kidney function (estimated glomerular filtration rate, eGFR) and CKD (eGFR\60 ml/min/1.73 m(2) or renal replacement therapy or kidney transplant) using age-, sex- and center-adjusted models and measured genotyped within the variance component models. MYH9 SNPs were not significantly associated with kidney traits in additive or recessive genetic adjusted models. MYH9 haplotypes were also not significantly associated with kidney outcomes. In conclusion, common variants in MYH9 polymorphisms may not confer an increased risk of CKD in American Indian populations. Identification of the actual functional genetic variation responsible for the associations seen in African-Americans will likely help to clarify the lack of replication of this gene in our population of American Indians.

    Funded by: NHLBI NIH HHS: R01 HL089651, R01 HL089651-01A1, U01 HL041642, U01 HL041652, U01 HL041654, U01 HL065520, U01 HL065521, U01-HL41642, U01-HL41652, U01-HL41654, U01-HL65520, U01-HL65521; NIMH NIH HHS: MH59490, R01 MH059490, R37 MH059490

    Human genetics 2010;127;3;295-301

  • Assessment of a polymorphism of SDK1 with hypertension in Japanese Individuals.

    Oguri M, Kato K, Yokoi K, Yoshida T, Watanabe S, Metoki N, Yoshida H, Satoh K, Aoyagi Y, Nozawa Y and Yamada Y

    Department of Cardiology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan.

    Background: Hypertension is a major risk factor for cardiovascular disease. Although genetic studies have suggested that several genetic variants increase the risk for hypertension, the genes that underlie genetic susceptibility to this condition remain to be identified definitively. The purpose of the present study was to identify genetic variants that confer susceptibility to hypertension in Japanese individuals.

    Methods: A total of 5,734 Japanese individuals from two independent populations were examined: subject panel A comprised 2,066 hypertensive individuals and 824 controls; and subject panel B comprised 834 hypertensive individuals and 2,010 controls. The 150 polymorphisms examined in the present study were selected by genome-wide association studies of myocardial infarction and ischemic stroke with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix).

    Results: The chi(2)-test revealed that 10 polymorphisms were significantly (P < 0.05) related to the prevalence of hypertension in subject panel A. To validate the relations, these polymorphisms were examined in subject panel B. The A-->G polymorphism (rs645106) of SDK1 and the C-->G polymorphism (rs12078839) of RABGAP1L were significantly associated with hypertension in subject panel B. Multivariable logistic regression analysis with adjustment for covariates, as well as a stepwise forward selection procedure revealed that the A-->G polymorphism of SDK1 was significantly associated with hypertension in both subject panels A and B, with the G allele protecting against this condition.

    Conclusions: SDK1 may be a susceptibility gene for hypertension in Japanese individuals, although the functional relevance of the identified polymorphism was not determined.

    American journal of hypertension 2010;23;1;70-7

  • Association among polymorphisms at MYH9, environmental factors, and nonsyndromic orofacial clefts in western China.

    Jia ZL, Li Y, Chen CH, Li S, Wang Y, Zheng Q and Shi B

    State Key Laboratory of Oral Disease and Department of Cleft Lip and Palate Surgery, West China College of Stomatology, Sichuan University, Chengdu, People's Republic of China.

    Myosin heavy chain 9, nonmuscle (MYH9) and environmental factors have been shown to be associated with nonsyndromic cleft lip with or without cleft palate in several populations. Our study aimed to confirm the contribution of MYH9 and environmental factors to nonsyndromic orofacial cleft risk in western Han Chinese. Four single-nucleotide polymorphisms were investigated in 180 case trios and 224 normal peers in western China using transmission disequilibrium test, family-based association test analysis, and logistic regression models. Strong evidence of linkage disequilibrium was found between these markers and the disease by both single-nucleotide polymorphism analysis (G allele at rs2269529 and T allele at rs16996652) and haplotype analysis (G-T [for rs2269529 and rs16996652] and G-A-T [for rs2269529, rs3752462, and rs16996652] among others). Mothers' illness, medication, and passive smoking during the first trimester may increase the risk of nonsyndromic orofacial clefts, but mothers' vitamin (including folic acid) supplementation during the first trimester is a protective factor. Interactions between mothers' passive smoking during the first trimester and T/T genotype of rs16996652 had statistical significance. Risk factors identified in our study may provide a better understanding of the etiological role of MYH9 and influence of environmental factors in nonsyndromic orofacial cleft incidence.

    DNA and cell biology 2010;29;1;25-32

  • Gene-gene and gene-environment interactions in HIV-associated nephropathy: A focus on the MYH9 nephropathy susceptibility gene.

    Núñez M, Saran AM and Freedman BI

    Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1053, USA.

    HIV-associated nephropathy (HIVAN) is a leading cause of ESRD in African Americans. The HIV-1 virus infects podocytes, cells integral to formation of the glomerular filtration barrier, often leading to focal segmental glomerulosclerosis. HIVAN is typically a complication of late-stage HIV infection, associated with low CD4 cell counts and elevated serum HIV RNA levels. Highly active antiretroviral therapy is partially protective and has altered the natural history of HIV-associated kidney disease. Nonetheless, HIVAN remains an important public health concern among HIV-infected African Americans. Although polymorphisms in the MYH9 gene on chromosome 22 are strongly associated with HIVAN, as well as with idiopathic focal segmental glomerulosclerosis and global glomerulosclerosis (historically labeled "hypertensive nephrosclerosis"), the majority of HIV-infected patients who are genetically at risk from MYH9 do not appear to develop severe kidney disease. Therefore, we postulate that additional environmental exposures and/or inherited factors are necessary to initiate human HIVAN. Gene-environment interactions have also been proposed as necessary for the initiation of HIVAN in murine models. It is important that these novel risk factors be identified because prevention of environmental exposures and targeting of additional gene products may reduce the risk for HIVAN, even among those harboring 2 risk alleles in MYH9.

    Funded by: NIDDK NIH HHS: R01 DK 070941, R01 DK070941, R01 DK070941-04, R01 DK084149, R01 DK084149-01, R01DK084149

    Advances in chronic kidney disease 2010;17;1;44-51

  • Non-muscle myosin heavy chain 9 gene MYH9 associations in African Americans with clinically diagnosed type 2 diabetes mellitus-associated ESRD.

    Freedman BI, Hicks PJ, Bostrom MA, Comeau ME, Divers J, Bleyer AJ, Kopp JB, Winkler CA, Nelson GW, Langefeld CD and Bowden DW

    1Internal Medicine/Nephrology, 2Biochemistry, 3Biostatistical Sciences, Wake Forest University School of Medicine, Winston-Salem, NC, USA. bfreedma@wfubmc.edu

    Background: Although MYH9 is strongly associated with biopsy-proven idiopathic and HIV-associated focal segmental glomerulosclerosis (FSGS) and clinically diagnosed 'hypertension-associated' end-stage renal disease (ESRD) in African Americans, its role in type 2 diabetes mellitus (T2DM)-associated ESRD is unclear.

    Methods: To assess whether MYH9 was associated with T2DM-ESRD, 751 African Americans with T2DM-ESRD, 227 with T2DM lacking nephropathy and 925 non-diabetic non-nephropathy controls were genotyped for 14 MYH9 SNPs. Association analyses used SNPGWA and Dandelion.

    Results: Comparing T2DM-ESRD cases with non-diabetic controls, single SNP associations were detected with 8 of 14 SNPs, gender- and admixture-adjusted P-values 0.047-0.005 [recessive model, odds ratio (OR) range 1.30-1.55]. The previously associated MYH9 E1 and L1 haplotypes were associated with T2DM-ESRD (E1: OR 1.27, 95% CI 1.04-1.56, P = 0.021 recessive and L1: OR 1.43, 95% CI 1.09-1.87, P = 0.009 dominant). Contrasting the 751 T2DM-ESRD cases with 227 T2DM non-nephropathy controls revealed that E1 haplotype SNPs rs4821480, rs2032487 and rs4821481 were associated with kidney failure (OR 1.38-1.40 recessive, all P < 0.048). Among E1 and L1 risk homozygotes, respectively, mean (SD) diabetes duration prior to renal replacement therapy was 16.6 (9.7) and 16.4 (10.0) years, and 65% had diabetic retinopathy.

    Conclusions: Genetic dissection of T2DM-associated ESRD reveals that MYH9 underlies a portion of this clinically diagnosed disorder in African Americans. It is likely that a subset of African Americans with T2DM and coincident nephropathy have primary MYH9-related kidney disease (e.g. FSGS or global glomerulosclerosis), although renal biopsy studies need to be performed.

    Funded by: CCR NIH HHS: HHSN261200800001C; Intramural NIH HHS; NCI NIH HHS: HHSN261200800001E, N01 CO012400, N01-CO-12400, N01CO12400; NHLBI NIH HHS: R01 HL055673, R01 HL055673-10; NIDDK NIH HHS: R01 DK 070941, R01 DK053591, R01 DK053591-09, R01 DK070941, R01 DK53591; PHS HHS: HHSN261200800001E

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2009;24;11;3366-71

  • Mutations in MYH9 exons 1, 16, 26, and 30 are infrequently found in Japanese patients with nonsyndromic deafness.

    Kunishima S, Matsunaga T, Ito Y and Saito H

    Mutations in MYH9 result in the autosomal dominant giant platelet disorders with leukocyte inclusion bodies with varying degrees of Alport manifestations, including nephritis, deafness, and cataracts. A specific MYH9 mutation in exon 16, R705H, causes nonsyndromic deafness DFNA17. We searched for mutations in MYH9 exons 1, 16, 26, and 30 in a total of 157 Japanese patients with nonsyndromic deafness without known cause of hearing loss, but no mutations were found. We conclude that mutations in MYH9 are infrequently found in patients with nonsyndromic deafness and suggest that MYH9 mutations infrequently cause isolated sensorineural hearing loss. Thus, MYH9 may not currently be a good candidate gene for efficient screening of genetic causes in nonsyndromic deafness.

    Genetic testing and molecular biomarkers 2009;13;5;705-7

  • Identification of three in-frame deletion mutations in MYH9 disorders suggesting an important hot spot for small rearrangements in MYH9 exon 24.

    Miyazaki K, Kunishima S, Fujii W and Higashihara M

    Department of Hematology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, 228-8555 Kanagawa, Japan. miyazakk@med.kitasato-u.ac.jp

    MYH9 disorders include hereditary macrothrombocytopenias with leukocyte inclusion bodies. Among more than 200 genetically confirmed families, the vast majority of cases exhibit single point mutations including substitutions and deletions of the COOH-terminus in the protein-coding sequence of MYH9. Only four in-frame deletions have been reported to date. In the current study, we describe three in-frame deletions including p.E1084del, p.E1066_A1072del and p.G1055_Q1068del, all of which are localized to exon 24. Interestingly, these three deletions were found to induce the diverse clinical manifestations on the non-hematological symptoms, while they equally demonstrated type I staining of inclusion bodies. As a result of these findings, we suggest that exon 24 represents a potential 'hot spot' for unequal homologous recombination, which may generate in-frame deletions in the coiled-coil rod of non-muscle myosin heavy chain-IIA. The exact length and position of these deletions may also determine the severity of the non-hematological manifestations, however does not appear to affect the morphology of the leukocyte inclusion bodies. These findings further our current understanding of the molecular pathogenesis underlying MYH9 disorders.

    European journal of haematology 2009;83;3;230-4

  • Clinical manifestation and molecular genetic characterization of MYH9 disorders.

    Provaznikova D, Geierova V, Kumstyrova T, Kotlin R, Mikulenkova D, Zurkova K, Matoska V, Hrachovinova I and Rittich S

    Institute of Haematology and Blood Transfusion, Prague, Czech Republic. dana.provaznikova@uhkt.cz

    Currently, the May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndrome are considered to be distinct clinical manifestations of a single disease caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). Manifestations of these disorders include giant platelets, thrombocytopenia and combinations of the presence of granulocyte inclusions, deafness, cataracts and renal failure. We examined 15 patients from 10 unrelated families on whom we performed immunostaining of NMMHC-IIA in blood samples. Polymerase chain reaction (PCR) analysis of selected exons of the MYH9 gene revealed mutations in nine samples with one novel mutation. Results of fluorescence and mutational analysis were compared with clinical manifestations of the MYH9 disorder. We also determined the number of glycoprotein sites on the surface of platelets. Most patients had an increased number of glycoproteins, which could be due to platelet size.

    Platelets 2009;20;5;289-96

  • Genome-wide linkage analysis of serum creatinine in three isolated European populations.

    Pattaro C, Aulchenko YS, Isaacs A, Vitart V, Franklin CS, Polasek O, Kolcic I, Biloglav Z, Campbell S, Hastie N, Lauc G, Meitinger T, Oostra BA, Gyllensten U, Wilson JF, Pichler I, Hicks AA, Campbell H, Wright AF, Rudan I, van Duijn CM, Riegler P, Marroni F, Pramstaller PP and EUROSPAN Consortium

    Institute of Genetic Medicine, European Academy Bozen/Bolzano (EURAC), Bolzano, Italy.

    There is increasing evidence for a role of genetic predisposition in the etiology of kidney disease, but linkage scans have been poorly replicated. Here we performed a genome-wide linkage analysis of serum creatinine on 2859 individuals from isolated villages in South Tyrol (Italy), Rucphen (The Netherlands) and Vis Island (Croatia), populations that have been stable and permanently resident in their region. Linkage of serum creatinine levels to loci on chromosomes 7p14, 9p21, 11p15, 15q15-21, 16p13, and 18p11 was successfully replicated in at least one discovery population or in the pooled analysis. A novel locus was found on chromosome 10p11. Linkage to chromosome 22q13, independent of diabetes and hypertension, was detected over a region containing the non-muscle myosin heavy chain type II isoform A (MYH9) gene (LOD score=3.52). In non-diabetic individuals, serum creatinine was associated with this gene in two of the three populations and in meta-analysis (SNP rs11089788, P-value=0.0089). In populations sharing a homogeneous environment and genetic background, heritability of serum creatinine was higher than in outbred populations, with consequent detection of a larger number of loci than reported before. Our finding of a replicated association of serum creatinine with the MYH9 gene, recently linked to pathological renal conditions in African Americans, suggests that this gene may also influence kidney function in healthy Europeans.

    Funded by: Chief Scientist Office: CZB/4/710; Medical Research Council: MC_U127561128

    Kidney international 2009;76;3;297-306

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Identification of the first duplication in MYH9-related disease: a hot spot for unequal crossing-over within exon 24 of the MYH9 gene.

    De Rocco D, Pujol-Moix N, Pecci A, Faletra F, Bozzi V, Balduini CL and Savoia A

    Medical Genetics, Department of Reproductive and Developmental Sciences, Institute for Maternal and Child Health-IRCCS Burlo Garofolo, University of Trieste, Via dell'Istria 65/1, 34137 Trieste, Italy.

    MYH9-related disease (MYH9RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. The spectrum of mutations so far identified is peculiar, consisting of mostly missense mutations. Others are nonsense and frameshift mutations, all localized in the COOH terminus of the protein, or in-frame deletions. We report a family with three affected members carrying a novel mutation, the first duplication (p.E1066_A1072dup), of MYH9. The mutation was localized within exon 24, where the presence of a 16 nucleotide repeat was likely to be responsible for unequal crossing-over. Of note, a deletion of the same amino acids 1066_1072 was also identified in another MHY9RD family. Since two of the four patients with the duplication or the deletion in exon 24 were affected with bilateral neonatal cataracts, we speculate that these mutations might correlate with the ocular defect, which is reported only in 16% of MYH9RD patients.

    European journal of medical genetics 2009;52;4;191-4

  • Megakaryocytes of patients with MYH9-related thrombocytopenia present an altered proplatelet formation.

    Pecci A, Malara A, Badalucco S, Bozzi V, Torti M, Balduini CL and Balduini A

    Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation, University of Pavia, Pavia, Italy. alessandro.pecci@unipv.it

    MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations of MYH9, the gene for the heavy chain of myosin-IIA. Pathogenesis of thrombocytopenia of MYH9-RD is unknown. Recent studies in mice demonstrated that myosin-IIA is an inhibitor of proplatelet formation (PPF), and suggested that it could be involved in the suppression of PPF exerted by megakaryocyte adhesion to type I collagen, which regulates the timing of platelet release within bone marrow. However, the consequences on PPF of the heterozygous mutations causative of the MYH9-RD have never been investigated. We studied the in-vitro PPF by megakaryocytes obtained from four patients carrying the p.D1424N or the p.R1933X mutations. We demonstrated that MYH9-RD megakaryocytes completely lose the physiologic suppression of proplatelet extension exerted by interaction with type I collagen, thus supporting the hypothesis that a premature platelet release within bone marrow contributes to pathogenesis of MYH9-related thrombocytopenia. Moreover, proplatelets extended by MYH9-RD megakaryocytes presented a significant defect in branching in secondary processes (p=0.001) and formed a significantly lower number of proplatelet tips (p=0.005). Since platelets are assembled at the level of proplatelet tips, this defect could further contribute to pathogenesis of thrombocytopenia of MYH9-RD patients.

    Thrombosis and haemostasis 2009;102;1;90-6

  • Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse.

    Sanborn KB, Rak GD, Maru SY, Demers K, Difeo A, Martignetti JA, Betts MR, Favier R, Banerjee PP and Orange JS

    Immunology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    NK cell cytotoxicity requires the formation of an actin-rich immunological synapse (IS) with a target cell and the polarization of perforin-containing lytic granules toward the IS. Following the polarization of lytic granules, they traverse through the actin-rich IS to join the NK cell membrane in order for directed secretion of their contents to occur. We examined the role of myosin IIA as a candidate for facilitating this prefinal step in lytic NK cell IS function. Lytic granules in and derived from a human NK cell line, or ex vivo human NK cells, were constitutively associated with myosin IIA. When isolated using density gradients, myosin IIA-associated NK cell lytic granules directly bound to F-actin and the interaction was sensitive to the presence of ATP under conditions of flow. In NK cells from patients with a truncation mutation in myosin IIA, NK cell cytotoxicity, lytic granule penetration into F-actin at the IS, and interaction of isolated granules with F-actin were all decreased. Similarly, inhibition of myosin function also diminished the penetration of lytic granules into F-actin at the IS, as well as the final approach of lytic granules to and their dynamics at the IS. Thus, NK cell lytic granule-associated myosin IIA enables their interaction with actin and final transit through the actin-rich IS to the synaptic membrane, and can be defective in the context of naturally occurring human myosin IIA mutation.

    Funded by: NIAID NIH HHS: R01 AI067946, R01 AI067946-02, R01-AI067946; NIGMS NIH HHS: T32 GM007229, T32-GM07229

    Journal of immunology (Baltimore, Md. : 1950) 2009;182;11;6969-84

  • The collagen receptor DDR1 regulates cell spreading and motility by associating with myosin IIA.

    Huang Y, Arora P, McCulloch CA and Vogel WF

    Department of Laboratory Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

    The spreading and migration of cells on adhesive substrates is regulated by the counterbalance of contractile and protrusive forces. Non-muscle myosin IIA, an ubiquitously expressed contractile protein and enzyme, is implicated in the regulation of cell spreading and directional migration in response to various stimuli. Here we show that discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor activated by type I collagen, associates with the non-muscle myosin IIA heavy chain (NMHC-IIA) upon ligand stimulation. An association was also indicated by coimmunoprecipitation of NMHC-IIA with full-length DDR1, but not with the truncated DDR1d-isoform lacking the kinase domain. DDR1 was important for assembly of NMHC-IIA into filaments on cells plated on collagen. DDR1 expression inhibited cell spreading over collagen but promoted cell migration. By contrast, blockade of non-muscle myosin II activity by blebbistatin enhanced cell spreading but inhibited migration over collagen. We propose that myosin and DDR1 impact cell spreading and migration by regulating adhesive contacts with collagen.

    Journal of cell science 2009;122;Pt 10;1637-46

  • Further evidence for the involvement of MYH9 in the etiology of non-syndromic cleft lip with or without cleft palate.

    Birnbaum S, Reutter H, Mende M, de Assis NA, Diaz-Lacava A, Herms S, Scheer M, Lauster C, Braumann B, Schmidt G, Martini M, Hemprich A, Pötzsch S, Knapp M, Nöthen MM, Kramer FJ and Mangold E

    Institute of Human Genetics, University of Bonn, Wilhelmstrasse 31, Bonn, Germany. birnbaum@uni-bonn.de

    Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects and has a multifactorial etiology that includes both genetic and environmental components. MYH9, the gene coding for the heavy chain of non-muscle myosin II, has been considered as a good candidate gene in NSCL/P on the basis of its expression profile during craniofacial morphogenesis. Reports in an Italian sample, as well as in an ethnically mixed North American sample, of a positive association between single-nucleotide polymorphisms in the MYH9 gene and NSCL/P have provided further support for the role of MYH9 in the development of NSCL/P. In the present study, we aimed to replicate these findings by conducting a family-based association study with seven single nucleotide polymorphisms in MYH9 using a sample of 248 NSCL/P patients and their parents. Single marker analysis resulted in a highly significant association for rs7078. In haplotype analysis, the most significant result was obtained for the SNP combination (rs7078; rs2071731; rs739097; rs5995288). Our results thus confirm the potential involvement of MYH9 in the etiology of NSCL/P in our patients of Central European origin, although further studies are warranted to determine its exact pathogenetic role.

    European journal of oral sciences 2009;117;2;200-3

  • Identification of the first in cis mutations in MYH9 disorder.

    Miyajima Y and Kunishima S

    Department of Pediatrics, Anjo Kosei Hospital, Anjo, Aichi, Japan. miyajima@kosei.anjo.aichi.jp

    Here, we report the first in cis mutations in exon 1 of the MYH9 gene in a patient with MYH9 disorder. The patient was a 5-yr-old girl with macrothrombocytopenia and conspicuous cytoplasmic inclusion bodies in neutrophils. Immunofluorescence analysis of neutrophil non-muscle myosin heavy chain-II A (NMMHC-IIA) indicated several cytoplasmic spots of NMMHC-IIA aggregates that were circular to oval in shape (type II pattern). Mutational analysis showed two mutations, c.99G > T and c.103C > G, which would result in p.W33C and p.P35A, respectively, in exon 1 of the MYH9 gene. In addition, concurrent mutations were present on the same chromosome. Inclusion bodies are usually faint or mostly invisible in MYH9 disorders with a mutation in exon 1. In this case, double mutations might have caused the large myosin protein aggregation and accumulation. Although not observed in this patient, the development of Alport manifestations should be monitored by careful follow-up.

    European journal of haematology 2009;82;4;288-91

  • Polymorphisms in the non-muscle myosin heavy chain 9 gene (MYH9) are strongly associated with end-stage renal disease historically attributed to hypertension in African Americans.

    Freedman BI, Hicks PJ, Bostrom MA, Cunningham ME, Liu Y, Divers J, Kopp JB, Winkler CA, Nelson GW, Langefeld CD and Bowden DW

    Internal Medicine/Nephrology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1053, USA. bfreedma@wfubmc.edu

    African Americans have high incidence rates of end-stage renal disease (ESRD) labeled as due to hypertension. As recent studies showed strong association with idiopathic and HIV-related focal segmental glomerulosclerosis and non-muscle myosin heavy chain 9 (MYH9) gene polymorphisms in this ethnic group, we tested for MYH9 associations in a variety of kidney diseases. Fifteen MYH9 single-nucleotide polymorphisms were evaluated in 175 African Americans with chronic glomerulonephritis-associated ESRD, 696 African Americans reportedly with hypertension-associated ESRD, and 948 control subjects without kidney disease. Significant associations were detected with 14 of the 15 polymorphisms in all 871 non-diabetic patients with ESRD. In hypertension-associated ESRD cases alone, significant associations were found with 13 MYH9 polymorphisms and the previously reported E1 haplotype. Thus, hypertension-associated ESRD in African Americans is substantially related to MYH9 gene polymorphisms and this may explain the poor response to blood pressure control in those diagnosed with hypertensive nephrosclerosis. It is possible that many African Americans classified as having hypertension-associated ESRD have occult MYH9-associated segmental or global glomerulosclerosis. Our study shows that gene-environment and/or gene-gene interactions may initiate kidney disease in genetically susceptible individuals, because African Americans homozygous for MYH9 risk alleles do not universally develop kidney disease.

    Funded by: CCR NIH HHS: HHSN261200800001C; Intramural NIH HHS; NCI NIH HHS: HHSN261200800001E, N01 CO012400, N01-CO-12400, N01CO12400; NIDDK NIH HHS: R01 DK 070942, R01 DK053591, R01 DK053591-02, R01 DK053591-03, R01 DK053591-03S1, R01 DK053591-04, R01 DK053591-05A1, R01 DK053591-06, R01 DK053591-07, R01 DK053591-08, R01 DK070941, R01 DK070941-01A1, R01 DK070941-02, R01 DK070941-03, R01 DK53591; PHS HHS: HHSN261200800001E

    Kidney international 2009;75;7;736-45

  • Genomic screening identifies novel linkages and provides further evidence for a role of MYH9 in nonsyndromic cleft lip and palate.

    Chiquet BT, Hashmi SS, Henry R, Burt A, Mulliken JB, Stal S, Bray M, Blanton SH and Hecht JT

    Department of Pediatrics, University of Texas Medical School at Houston, Houston, TX 77225, USA.

    Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth anomaly that requires prolonged multidisciplinary rehabilitation. Although variation in several genes has been identified as contributing to NSCLP, most of the genetic susceptibility loci have yet to be defined. To identify additional contributory genes, a high-throughput genomic scan was performed using the Illumina Linkage IVb Panel platform. We genotyped 6008 SNPs in nine non-Hispanic white NSCLP multiplex families and a single large African-American NSCLP multiplex family. Fourteen chromosomal regions were identified with LOD>1.5, including six regions not previously reported. Analysis of the data from the African-American and non-Hispanic white families revealed two likely chromosomal regions: 8q21.3-24.12 and 22q12.2-12.3 with LOD scores of 2.98 and 2.66, respectively. On the basis of biological function, syndecan 2 (SDC2) and growth differentiation factor 6 (GDF6) in 8q21.3-24.12 and myosin heavy-chain 9, non-muscle (MYH9) in 22q12.2-12.3 were selected as candidate genes. Association analyses from these genes yielded marginally significant P-values for SNPs in SDC2 and GDF6 (0.01<or=P<0.05). Evidence for an altered transmission was found for four MYH9 SNPs (P<0.01). SNP rs1002246 exhibited altered transmission by all analytic methods. However, analysis of two SNP MYH9 haplotypes did not identify a single high-risk haplotype. Our results confirm a previous report that 8q21.3-24.12 may harbor a clefting gene and identify 22q12.2-12.3 as a new candidate region that contains MYH9. Most importantly, we confirm the previous report of an association with MYH9.

    Funded by: NIDCR NIH HHS: F30 DE019343, F30 DE019343-01, R01 DE011931, R01 DE011931-08, R01-DE011931, T32 DE015355, T32-DE015355-05

    European journal of human genetics : EJHG 2009;17;2;195-204

  • Non-muscle myosin IIA differentially regulates intestinal epithelial cell restitution and matrix invasion.

    Babbin BA, Koch S, Bachar M, Conti MA, Parkos CA, Adelstein RS, Nusrat A and Ivanov AI

    Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia, USA.

    Epithelial cell motility is critical for self-rejuvenation of normal intestinal mucosa, wound repair, and cancer metastasis. This process is regulated by the reorganization of the F-actin cytoskeleton, which is driven by a myosin II motor. However, the role of e26 myosin II in regulating epithelial cell migration remains poorly understood. This study addressed the role of non-muscle myosin (NM) IIA in two different modes of epithelial cell migration: two-dimensional (2-D) migration that occurs during wound closure and three-dimensional (3-D) migration through a Matrigel matrix that occurs during cancer metastasis. Pharmacological inhibition or siRNA-mediated knockdown of NM IIA in SK-CO15 human colonic epithelial cells resulted in decreased 2-D migration and increased 3-D invasion. The attenuated 2-D migration was associated with increased cell adhesiveness to collagen and laminin and enhanced expression of beta1-integrin and paxillin. On the 2-D surface, NM IIA-deficient SK-CO15 cells failed to assemble focal adhesions and F-actin stress fibers. In contrast, the enhanced invasion of NM IIA-depleted cells was dependent on Raf-ERK1/2 signaling pathway activation, enhanced calpain activity, and increased calpain-2 expression. Our findings suggest that NM IIA promotes 2-D epithelial cell migration but antagonizes 3-D invasion. These observations indicate multiple functions for NM IIA, which, along with the regulation of the F-actin cytoskeleton and cell-matrix adhesions, involve previously unrecognized control of intracellular signaling and protein expression.

    Funded by: NIDDK NIH HHS: DK 064399, DK 55679, DK 59888, DK 61379, DK 72564, K08 DK074706, K08 DK074706-01, R01 DK055679, R01 DK059888, R01 DK061379, R01 DK072564, R24 DK064399, R29 DK055679

    The American journal of pathology 2009;174;2;436-48

  • [May-Hegglin anomaly: past and present--novel diagnostic test and new concept of the disease].

    Kunishima S

    Laboratory of Molecular Diagnosis, Department of Advanced Diagnosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya 460-0001, Japan. kunishis@nnh.hosp.go.jp

    May-Hegglin anomaly(MHA) is the prototype of the autosomal dominant macrothrombocytopenia with leukocyte inclusion bodies/MYH9 disorders that result from mutations in MYH9, the gene for nonmuscle myosin heavy chain-IIA(NMMHC-IIA). Others include Sebastian(SBS), Fechtner(FTNS), and Epstein (EPS) syndromes. Clear phenotype-genotype relationships have not been found; however, patients with an MYH9 head domain mutation tend to develop Alport manifestation more frequently than those with a rod mutation. The hallmark of MYH9 disorders is the presence of granulocyte inclusion bodies. We revealed that mutant NMMHC-IIA was present and sequestered only in inclusion bodies within neutrophils, diffusely distributed throughout lymphocyte cytoplasm, sparsely localized on a diffuse cytoplasmic background in monocytes, and uniformly distributed at diminished levels only in large platelets. We showed the differential expression of mutant NMMHC-IIA, and postulated that cell-specific regulation mechanisms function in MYH9 disorders.

    Rinsho byori. The Japanese journal of clinical pathology 2009;57;1;54-9

  • Multiple regulatory steps control mammalian nonmuscle myosin II assembly in live cells.

    Breckenridge MT, Dulyaninova NG and Egelhoff TT

    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

    To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (DeltaIQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Deltatailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Deltatailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.

    Funded by: NIDDK NIH HHS: DK007678, T32 DK007678; NIGMS NIH HHS: GM 077224, R01 GM077224

    Molecular biology of the cell 2009;20;1;338-47

  • Polymorphisms in the nonmuscle myosin heavy chain 9 gene (MYH9) are associated with albuminuria in hypertensive African Americans: the HyperGEN study.

    Freedman BI, Kopp JB, Winkler CA, Nelson GW, Rao DC, Eckfeldt JH, Leppert MF, Hicks PJ, Divers J, Langefeld CD and Hunt SC

    Section on Nephrology, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1053, USA. bfreedma@wfubmc.edu

    Background: MYH9 is a podocyte-expressed gene encoding nonmuscle myosin IIA that is associated with idiopathic and human immunodeficiency virus-associated focal segmental glomerulosclerosis (FSGS) and hypertensive end-stage renal disease in African Americans.

    Methods: Four single nucleotide polymorphisms comprising the major MYH9 E1 risk haplotype were tested for association with estimated glomerular filtration rate (eGFR) and urine albumin:creatinine ratio (ACR) in 2,903 HyperGEN participants (1,458 African Americans (AA) in 895 families and 1,445 European Americans (EA) in 859 families) to determine the role of MYH9 in subclinical nephropathy. Association analyses employed general linear models in unrelated probands and generalized estimating equations in families. Adjustment was performed for age, sex, diabetes, BMI, medications, and mean arterial pressure separately in each race.

    Results: Mean (SD) eGFR and ACR were 74.3 (16.0) ml/min/1.73 m(2) and 20.3 (119.9) mg/g in EA, and 88.6 (20.9) ml/min/1.73 m(2) and 76.8 (394.5) mg/g in AA (both p < 0.0001 across ethnicities). Urine ACR was associated with rs3752462 (p = 0.01) and rs4821481 (p = 0.05) in unrelated AA and with rs4821481 (p = 0.03), 1f40 rs2032487 (p = 0.04) and the E1 3224 haplotype (p = 0.013) in AA families. Single nucleotide polymorphisms and the haplotype were not associated with ACR in EA or with eGFR in either ethnic group.

    Conclusions: MYH9 variants are associated with albuminuria in hypertensive AA. The strength of the association was weaker than that in FSGS and hypertensive end-stage renal disease. MYH9 risk variants appear to be associated with primary FSGS with secondary hypertension, although nephrosclerosis may develop in response to hypertension in subjects homozygous for the MYH9 E1 risk haplotype.

    Funded by: CCR NIH HHS: HHSN261200800001C; NCI NIH HHS: HHSN261200800001E, N01 CO012400, N01CO12400; NHLBI NIH HHS: HL54471, HL54472, HL54473, HL54495, HL54496, HL54497, HL54509, HL54515, R01 HL055673, R01 HL055673-04, U01 HL054471, U01 HL054471-10, U01 HL054472, U01 HL054472-06, U01 HL054473, U01 HL054473-13, U01 HL054495, U01 HL054495-10, U01 HL054496, U01 HL054496-10, U01 HL054509, U01 HL054509-10, U10 HL054471, U10 HL054472, U10 HL054473, U10 HL054495, U10 HL054496, U10 HL054509, U10 HL054515-05; NIDDK NIH HHS: R01 DK070941, R01 DK070941-01A1, R01 DK070941-02, R01 DK070941-03

    American journal of nephrology 2009;29;6;626-32

  • Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA.

    Chu CC, Catera R, Hatzi K, Zhang L, Wang XB, Fales HM, Allen SL, Kolitz JE, Rai KR and Chiorazzi N

    The Feinstein Institute for Medical Research, North Shore-Long Island Jewish (LIJ) Health System, Manhasset, NY, USA.

    Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

    Funded by: Intramural NIH HHS; NCI NIH HHS: CA81554, CA87956, R01 CA081554, R01 CA087956; NCRR NIH HHS: M01 RR018535, RR018535

    Blood 2008;112;13;5122-9

  • The C-terminal tail region of nonmuscle myosin II directs isoform-specific distribution in migrating cells.

    Sandquist JC and Means AR

    Department of Pharmacology and Cancer Biology, Duke University Medical Center; Durham, NC 27710, USA.

    Nonmuscle myosin II isoforms A and B (hereafter, IIA and IIB) perform unique roles in cell migration, even though both isoforms share the same basic molecular functions. That IIA and IIB assume distinct subcellular distribution in migrating cells suggests that discrete spatiotemporal regulation of each isoform's activity may provide a basis for its unique migratory functions. Here, we make the surprising finding that swapping a small C-terminal portion of the tail between IIA and IIB inverts the distinct distribution of these isoforms in migrating cells. Moreover, swapping this region between isoforms also inverts their specific turnover properties, as assessed by fluorescence recovery after photobleaching and Triton solubility. These data, acquired through the use of chimeras of IIA and IIB, suggest that the C-terminal region of the myosin heavy chain supersedes the distinct motor properties of the two isoforms as the predominant factor directing isoform-specific distribution. Furthermore, our results reveal a correlation between isoform solubility and distribution, leading to the proposal that the C-terminal region regulates isoform distribution by tightly controlling the amount of each isoform that is soluble and therefore available for redistribution into new protrusions.

    Funded by: Howard Hughes Medical Institute; NCI NIH HHS: CA-082845, R01 CA082845

    Molecular biology of the cell 2008;19;12;5156-67

  • Cellular nonmuscle myosins NMHC-IIA and NMHC-IIB and vertebrate heart looping.

    Lu W, Seeholzer SH, Han M, Arnold AS, Serrano M, Garita B, Philp NJ, Farthing C, Steele P, Chen J and Linask KK

    Department of Pediatrics, USF/ACH-Children's Research Institute, St. Petersburg, Florida 33701, USA.

    Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the Nodal(Delta 600) homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping.

    Developmental dynamics : an official publication of the American Association of Anatomists 2008;237;12;3577-90

  • RASD2, MYH9, and CACNG2 genes at chromosome 22q12 associated with the subgroup of schizophrenia with non-deficit in sustained attention and executive function.

    Liu YL, Fann CS, Liu CM, Chen WJ, Wu JY, Hung SI, Chen CH, Jou YS, Liu SK, Hwang TJ, Hsieh MH, Chang CC, Yang WC, Lin JJ, Chou FH, Faraone SV, Tsuang MT and Hwu HG

    Division of Mental Health and Substance Abuse Research, National Health Research Institutes, Taipei, Taiwan.

    Background: In a previous linkage study of schizophrenia that included Taiwanese samples, the marker D22S278 (22q12.3) was significantly linked to schizophrenia (p = .001).

    Methods: We conducted fine mapping of the implicated genomic region, with 47 validated single nucleotide polymorphism (SNP) markers around 1 Mb of D22S278, in a Taiwanese sample of 218 pedigrees with at least 2 siblings affected with schizophrenia. We examined the association of these SNPs and their haplotypes with schizophrenia and with subgroups defined by the presence and absence of deficits in sustained attention as assessed by undegraded and degraded continuous performance tests (CPTs). We also examined subgroups defined by deficits in categories achieved in the Wisconsin Card Sort Test (WCST).

    Results: Three of five candidate vulnerability genes (RASD2, APOL5, MYH9, EIF3S7, and CACNG2), which had marginally significant associations with schizophrenia, had significant associations with schizophrenic patients who did not have deficits in sustained attention on the undegraded CPT (RASD2 gene SNP rs736212; p = .0008 with single locus analysis) and the degraded CPT (MYH9 gene haplotype 1-1-1-1 of SNP rs3752463 - rs1557540 - rs713839 - rs739097; p = .0059 with haplotype analysis). We also found a significant association for patients who showed no deficits in executive function as measured by categories achieved in the WCST (CACNG2 gene haplotype 2-1-1-1 of SNP rs2267360 - rs140526 - rs1883987 - rs916269; p = .0163 with haplotype analysis).

    Conclusions: The genes RASD2, MYH9, and CACNG2 might be vulnerability genes for neuropsychologically defined subgroups of schizophrenic patients.

    Funded by: NIMH NIH HHS: 1R01 MH59624-01

    Biological psychiatry 2008;64;9;789-96

  • Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.

    Balduini A, Pallotta I, Malara A, Lova P, Pecci A, Viarengo G, Balduini CL and Torti M

    Department of Biochemistry, University of Pavia, Pavia, Italy.

    Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called prop 1f40 latelets. In humans, the regulation of this process is still poorly characterized.

    Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors.

    Methods: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34(+) cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy.

    Results: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin alphaIIbbeta3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets.

    Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.

    Funded by: Telethon: GGP06177

    Journal of thrombosis and haemostasis : JTH 2008;6;11;1900-7

  • MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis.

    Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, Bowden DW, Oleksyk T, McKenzie LM, Kajiyama H, Ahuja TS, Berns JS, Briggs W, Cho ME, Dart RA, Kimmel PL, Korbet SM, Michel DM, Mokrzycki MH, Schelling JR, Simon E, Trachtman H, Vlahov D and Winkler CA

    Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The increased burden of chronic kidney and end-stage kidney diseases (ESKD) in populations of African ancestry has been largely unexplained. To identify genetic variants predisposing to idiopathic and HIV-1-associated focal segmental glomerulosclerosis (FSGS), we carried out an admixture-mapping linkage-disequilibrium genome scan on 190 African American individuals with FSGS and 222 controls. We identified a chromosome 22 region with a genome-wide logarithm of the odds (lod) score of 9.2 and a peak lod of 12.4 centered on MYH9, a functional candidate gene expressed in kidney podocytes. Multiple MYH9 SNPs and haplotypes were recessively associated with FSGS, most strongly a haplotype spanning exons 14 through 23 (OR = 5.0, 95% CI = 3.5-7.1; P = 4 x 10(-23), n = 852). This association extended to hypertensive ESKD (OR = 2.2, 95% CI = 1.5-3.4; n = 433), but not type 2 diabetic ESKD (n = 476). Genetic variation at the MYH9 locus substantially explains the increased burden of FSGS and hypertensive ESKD among African Americans.

    Funded by: Intramural NIH HHS: Z01 DK043308-12; NCI NIH HHS: N01-CO-12400, N01CO12400; NIDDK NIH HHS: R01 DK059997, R01 DK059997-04, R01 DK067528, R01 DK067528-05, R01 DK070941, R01 DK072348, R01 DK072348-04, Z01 DK043308, ZO-1 DK043308

    Nature genetics 2008;40;10;1175-84

  • MYH9 is associated with nondiabetic end-stage renal disease in African Americans.

    Kao WH, Klag MJ, Meoni LA, Reich D, Berthier-Schaad Y, Li M, Coresh J, Patterson N, Tandon A, Powe NR, Fink NE, Sadler JH, Weir MR, Abboud HE, Adler SG, Divers J, Iyengar SK, Freedman BI, Kimmel PL, Knowler WC, Kohn OF, Kramp K, Leehey DJ, Nicholas SB, Pahl MV, Schelling JR, Sedor JR, Thornley-Brown D, Winkler CA, Smith MW, Parekh RS and Family Investigation of Nephropathy and Diabetes Research Group

    Department of Epidemiology, School of Medicine and Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21287, USA. wkao@jhsph.edu

    As end-stage renal disease (ESRD) has a four times higher incidence in African Americans compared to European Americans, we hypothesized that susceptibility alleles for ESRD have a higher frequency in the West African than the European gene pool. We carried out a genome-wide admixture scan in 1,372 ESRD cases and 806 controls and found a highly significant association between excess African ancestry and nondiabetic ESRD (lod score = 5.70) but not diabetic ESRD (lod = 0.47) on chromosome 22q12. Each copy of the European ancestral allele conferred a relative risk of 0.50 (95% CI = 0.39-0.63) compared to African ancestry. Multiple common SNPs (allele frequencies ranging from 0.2 to 0.6) in the gene encoding nonmuscle myosin heavy chain type II isoform A (MYH9) were associated with two to four times greater risk of nondiabetic ESRD and accounted for a large proportion of the excess risk of ESRD observed in African compared to European Americans.

    Funded by: AHRQ HHS: HS08365, R01 HS008365; Intramural NIH HHS; NCI NIH HHS: N01-CO-12400, N01CO12400; NCRR NIH HHS: M01 RR000080, M01 RR000425, M01 RR000827, M01 RR001346, M01 RR007122, M01-RR-000080, M01-RR-00425, M01-RR-00827-29, M01-RR-01346, M01-RR-07122; NHLBI NIH HHS: HL62985, R01 HL062985; NIDDK NIH HHS: DK07024, K01 DK067207, K01DK067207, R01 DK059997, R01 DK059997-04, R01 DK067528, R01 DK067528-05, R01 DK072348, R01 DK072348-04, U01 DK057292, U01 DK057304, U01 DK070657, U01 DK070657-01, U01 DK070657-02, U01 DK070657-03, U01DK070657, U01DK57292, U01DK57304

    Nature genetics 2008;40;10;1185-92

  • High-resolution melting analysis for detection of MYH9 mutations.

    Provaznikova D, Kumstyrova T, Kotlin R, Salaj P, Matoska V, Hrachovinova I and Rittich S

    Institute of Haematology and Blood Transfusion, Coagulation Laboratory, Prague, Czech Republic. dana.provaznikova@uhkt.cz

    May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As t 1f40 he MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

    Platelets 2008;19;6;471-5

  • Identification and characterization of the first large deletion of the MYH9 gene associated with MYH9 disorders.

    Kunishima S, Matsushita T, Hamaguchi M and Saito H

    Department of Hemostasis and Thrombosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders are autosomal dominant macrothrombocytopenias with leukocyte inclusion bodies. Single point mutations in the protein-coding sequence of the MYH9 gene are the most common cause. So far no large gene deletion/insertion and splicing defects have been reported. Conventional DNA sequencing of each MYH9-coding exon showed no abnormalities in a patient. Reverse transcription- polymerase chain reaction (PCR) amplification and sequencing of neutrophil mRNA identified an inframe deletion of exon 25. Further long-range PCR amplification of genomic DNA revealed a deletion of 1220 nucleotides including entire exon 25. Immunoblot analysis showed a small, abnormal protein in neutrophils but not in platelets. This is the first report of a large deletion of the MYH9 gene leading to the development of MYH9 disorders.

    European journal of haematology 2008;80;6;540-4

  • TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation.

    Clark K, Middelbeek J, Lasonder E, Dulyaninova NG, Morrice NA, Ryazanov AG, Bresnick AR, Figdor CG and van Leeuwen FN

    Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

    Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian alpha-kinase TRPM7 inhibits myosin II-based contractility in a Ca(2+)- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains--the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the alpha-helical tail of the myosin IIA heavy chain.

    Funded by: NIGMS NIH HHS: R01 GM069945

    Journal of molecular biology 2008;378;4;790-803

  • MYH9-siRNA and MYH9 mutant alleles: expression in cultured cell lines and their effects upon cell structure and function.

    Li Y, Friedmann DR, Mhatre AN and Lalwani AK

    Laboratory of Molecular Otology, Department of Otolaryngology, New York University School of Medicine, New York, New York 10016, USA.

    MYH9 encodes a class II nonmuscle myosin heavy chain-A (NMHC-IIA), a widely expressed 1960 amino acid polypeptide, with translated molecular weight of 220 kDa. From studies of type II myosin in invertebrates and analogy with the skeletal and smooth muscle myosin II, NMHC-IIA is considered to be involved in diverse cellular functions, including cell shape, motility and division. The current study assessed the consequences of two separate, naturally occurring MYH9 dominant mutant alleles, MYH9(R702C) and MYH9(R705H) linked to syndromic and nonsyndromic hearing loss, respectively, upon diverse NMHC-IIA related functions in two separate cultured cell lines. MYH9-siRNA-induced inhibition of NMHC-IIA in HeLa cells or HEK293 cells resulted in alterations in their shape, actin cytoskeleton and adhesion properties. However, HeLa or HEK293 cells transfected with naturally occurring MYH9 mutant alleles, MYH9(R702C) or MYH9(R705H), as well as in vitro generated deletion derivatives, MYH9(DeltaN592) or MYH9(DeltaC570), were unaffected. The effects of MYH9-siRNA-induced suppression underline the critical role of NMHC-IIA in maintenance of cell shape and adhesion. However, the results also indicate that the NMHC-IIA mutants, R702C and R705H do not inactivate or suppress the endogenous wild type NMHC-IIA within the HeLa or HEK293 cell assay system.

    Funded by: NIDCD NIH HHS: DC005199

    Cell motility and the cytoskeleton 2008;65;5;393-405

  • Differential expression of wild-type and mutant NMMHC-IIA polypeptides in blood cells suggests cell-specific regulation mechanisms in MYH9 disorders.

    Kunishima S, Hamaguchi M and Saito H

    Department of Hemostasis and Thrombosis, Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA, and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils, diffusely distributed throughout lymphocyte cytoplasm, sparsely localized on a diffuse cytoplasmic background in monocytes, and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34(+) cells of patients, but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC-IIA and postulate that cell-specific regulation mechanisms function in MYH9 disorders.

    Blood 2008;111;6;3015-23

  • Inhibition of "self" engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.

    Tsai RK and Discher DE

    Biophysical Engineering Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA.

    Phagocytosis of foreign cells or particles by macrophages is a rapid process that is inefficient when faced with "self" cells that display CD47-although signaling mechanisms in self-recognition have remained largely unknown. With human macrophages, we show the phagocytic synapse at cell contacts involves a basal level of actin-driven phagocytosis that, in the absence of species-specific CD47 signaling, is made more efficient by phospho-activated myosin. We use "foreign" sheep red blood cells (RBCs) together with CD47-blocked, antibody-opsonized human RBCs in order to visualize synaptic accumulation of phosphotyrosine, paxillin, F-actin, and the major motor isoform, nonmuscle myosin-IIA. When CD47 is functional, the macrophage counter-receptor and phosphatase-activator SIRPalpha localizes to the synapse, suppressing accumulation of phosphotyrosine and myosin without affecting F-actin. On both RBCs and microbeads, human CD47 potently inhibits phagocytosis as does direct inhibition of myosin. CD47-SIRPalpha interaction initiates a dephosphorylation cascade directed in part at phosphotyrosine in myosin. A point mutation turns off this motor's contribution to phagocytosis, suggesting that self-recognition inhibits contractile engulfment.

    The Journal of cell biology 2008;180;5;989-1003

  • Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease.

    Pecci A, Panza E, Pujol-Moix N, Klersy C, Di Bari F, Bozzi V, Gresele P, Lethagen S, Fabris F, Dufour C, Granata A, Doubek M, Pecoraro C, Koivisto PA, Heller PG, Iolascon A, Alvisi P, Schwabe D, De Candia E, Rocca B, Russo U, Ramenghi U, Noris P, Seri M, Balduini CL and Savoia A

    Department of Internal Medicine, University of Pavia and Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Policlinico San Matteo Foundation, Pavia, Italy. alessandro.pecci@unipv.it

    MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafn 8b1 ess, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease.

    Funded by: Telethon: GGP06177

    Human mutation 2008;29;3;409-17

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.

    Morin NA, Oakes PW, Hyun YM, Lee D, Chin YE, Chin EY, King MR, Springer TA, Shimaoka M, Tang JX, Reichner JS and Kim M

    Department of Surgery, Rhode Island Hospital and Brown Medical School, Providence, RI 02903, USA.

    Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function-associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

    Funded by: NCI NIH HHS: R37 CA031798, R37 CA031798-29; NHLBI NIH HHS: HL087088, R01 HL087088, R01 HL087088-01A1; NIGMS NIH HHS: GM066194, R01 GM066194, R56 GM066194

    The Journal of experimental medicine 2008;205;1;195-205

  • An MYH9 human disease model in flies: site-directed mutagenesis of the Drosophila non-muscle myosin II results in hypomorphic alleles with dominant character.

    Franke JD, Montague RA, Rickoll WL and Kiehart DP

    Department of Biology, DCMB Group, Duke University, Durham, NC 27708-0338, USA.

    We investigated whether or not human disease-causing, amino acid substitutions in MYH9 could cause dominant phenotypes when introduced into the sole non-muscle myosin II heavy chain in Drosophila melanogaster (zip/MyoII). We characterized in vivo the effects of four MYH9-like mutations in the myosin rod-R1171C, D1430N, D1847K and R1939X-which occur at highly conserved residues. These engineered mutant heavy chains resulted in D. melanogaster non-muscle myosin II with partial wild-type function. In a wild-type genetic background, mutant heavy chains were overtly recessive and hypomorphic: each was able to substitute partially for endogenous non-muscle myosin II heavy chain in animals lacking zygotically produced heavy chain (but the penetrance of rescue was below Mendelian expectation). Moreover, each of the four mutant heavy chains exhibits dominant characteristics when expressed in a sensitized genetic background (flies heterozygous for RhoA mutations). Thus, these zip/MyoII(MYH9) alleles function, like certain other hypomorphic alleles, as excellent bait in screens for genetic interactors. Our conjecture is that these mutations in D. melanogaster behave comparably to their parent mutations in humans. We further characterized these zip/MyoII(MYH9) alleles, and found that all were capable of correct spatial and temporal localization in animals lacking zygotic expression of wild-type zip/MyoII. In vitro, we demonstrate that mutant heavy chains can dimerize with endogenous, wild-type heavy chains, fold into coiled-coil structures and assemble into higher-order structures. Our work further supports D. melanogaster as a model system for investigating the basis of human disease.

    Funded by: NIGMS NIH HHS: GM07184, GM33830

    Human molecular genetics 2007;16;24;3160-73

  • Myosin IIA is required for cytolytic granule exocytosis in human NK cells.

    Andzelm MM, Chen X, Krzewski K, Orange JS and Strominger JL

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

    Natural killer (NK) cell cytotoxicity involves the formation of an activating immunological synapse (IS) between the effector and target cell through which granzymes and perforin contained in lytic granules are delivered to the target cell via exocytosis. Inhibition of nonmuscle myosin II in human NK cells with blebbistatin or ML-9 impaired neither effector-target cell conjugation nor formation of a mature activating NK cell IS (NKIS; formation of an actin ring and polarization of the microtubule-organizing center and cytolytic granules to the center of the ring). However, membrane fusion of lytic granules, granzyme secretion, and NK cell cytotoxicity were all effectively blocked. Specific knockdown of the myosin IIA heavy chain by RNA interference impaired cytotoxicity, membrane fusion of lytic granules, and granzyme secretion. Thus, myosin IIA is required for a critical step between NKIS formation and granule exocytosis.

    Funded by: NIAID NIH HHS: AI-50207, AI-55602, K08 AI055602, K08 AI055602-01, K08 AI055602-02, K08 AI055602-03, K08 AI055602-04, K08 AI055602-05, R01 AI050207

    The Journal of experimental medicine 2007;204;10;2285-91

  • MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

    Calaminus SD, Auger JM, McCarty OJ, Wakelam MJ, Machesky LM and Watson SP

    Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK. sdc379@bham.ac.uk

    Background: MyosinIIs are adenosine triphosphate-driven molecular motors that form part of a cell's contractile machinery. They are activated by phosphorylation of their light chains, by either activation of myosin light chain (MLC) kinase or inhibition of MLC phosphatase via Rho kinase (ROCK). MyosinIIa phosphorylation underlies platelet rounding and stress fiber formation.

    Objective: To identify the functional significance of myosinIIa in platelet spreading and thrombus formation on collagen using inhibitors of ROCK (Y27632) and myosinII (blebbistatin).

    Results: Stress fiber formation on collagen is inhibited by both Y27632 and blebbistatin. A substantial proportion of spread platelets generate internal holes or splits on collagen, presumably because of a reduction in contractile strength. Platelet integrity, however, is maintained. In an in vitro model, thrombus embolization on collagen is increased in the presence of Y27632 and blebbistatin at intermediate shear, leading to a reduction in platelet aggregate growth. Moreover, Y27632 causes a marked reduction in thrombus formation in an in vivo laser-injury model.

    Conclusions: MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability.

    Funded by: Medical Research Council: G0300102, G117/569; Wellcome Trust

    Journal of thrombosis and haemostasis : JTH 2007;5;10;2136-45

  • Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells.

    Dulyaninova NG, House RP, Betapudi V and Bresnick AR

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.

    Funded by: NIGMS NIH HHS: GM069945, R01 GM069945

    Molecular biology of the cell 2007;18;8;3144-55

  • The critical role of myosin IIA in platelet internal contraction.

    Johnson GJ, Leis LA, Krumwiede MD and White JG

    Hematology/Oncology, Veterans Affairs Medical Center, Minneapolis, MN 55417, USA. mailto:johns337@umn.edu

    Background: Shape change and centralization of granules surrounded by a microtubular coil (internal contraction) are among the earliest morphologic changes observed following platelet activation. Myosin IIA contributes to initiation of platelet shape change, but its role in internal contraction has not been defined.

    Objective: To define the contribution of myosin IIA to platelet internal contraction.

    Methods: Aspirin-treated platelets suspended in calcium-free buffer were activated with a low concentration (25 nm) of the thromboxane A(2) analog U46619 which initiated shape change and internal contraction via a Rho kinase pathway. Shape change and internal contraction were assessed by aggregometry and transmission electron microscopy (TEM), and Rho activation and myosin regulatory light chain (MRLC) phosphorylation were studied concurrently.

    Low-concentration blebbistatin (10 microm) inhibited internal contraction in the majority of platelets with minimal inhibition of shape change without significant suppression of MRLC phosphorylation. Higher blebbistatin concentrations (25-100 microm) produced concentration-dependent inhibition of aggregation, shape change, Rho activation, and MRLC phosphorylation. These data demonstrate: (i) direct platelet myosin IIA participation in internal contraction; and (ii) inhibition of Rho activation and MRLC phosphorylation by >10 microm blebbistatin.

    Journal of thrombosis and haemostasis : JTH 2007;5;7;1516-29

  • Cleft lip with or without cleft palate: implication of the heavy chain of non-muscle myosin IIA.

    Martinelli M, Di Stazio M, Scapoli L, Marchesini J, Di Bari F, Pezzetti F, Carinci F, Palmieri A, Carinci P and Savoia A

    Non-syndromic cleft lip with or without palate (CL/P) is one of the most common malformations among live births, but most of the genetic components and environmental factors involved remain to be identified. Among the different causes, MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA, was considered a potential candidate, because it was found to be abundantly and specifically expressed in epithelial cells of palatal shelves before fusion. After fusion, its expression level was shown to decrease and to become limited to epithelial triangles before disappearing, as fusion is completed. To determine whether MYH9 plays a role in CL/P aetiology, a family-based association analysis was performed in 218 case/parent triads using single-nucleotide polymorphism (SNP) markers. Pairwise and multilocus haplotype analyses identified linkage disequilibrium between polymorphism alleles at the MYH9 locus and the disease. The strongest deviation from a null hypothesis of random sharing was obtained with two adjacent SNPs, rs3752462 and rs2009930 (global p value = 0.001), indicating that MYH9 might be a predisposing factor for CL/P, although its pathogenetic role needs to be investigated more accurately.

    Funded by: Telethon: GGP05147

    Journal of medical genetics 2007;44;6;387-92

  • Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1alpha.

    Rey M, Valenzuela-Fernández A, Urzainqui A, Yáñez-Mó M, Pérez-Martínez M, Penela P, Mayor F and Sánchez-Madrid F

    Servicio de Inmunología, Hospital Universitario de la Princesa, Diego de León, 62, 28006 Madrid, Spain.

    Endocytosis of chemokine receptors regulates signal transduction initiated by chemokines, but the molecular mechanisms underlying this process are not fully defined. In this work, we assessed the involvement of the motor protein nonmuscle myosin heavy chain IIA (MIIA) in the endocytosis of CXCR4 induced by SDF-1alpha (also known as CXCL12) in T lymphocytes. Overexpression of the C-terminal half of MIIA inhibited the ligand-induced endocytosis of CXCR4, but not that of transferrin receptor. Targeting MIIA either by silencing its expression with small interfering RNA (siRNA) or by blebbistatin treatment also inhibited endocytosis of CXCR4. Inhibition of endocytosis of CXCR4 by targeting endogenous MIIA resulted in an increased migration of T cells induced by SDF-1alpha, and in the inhibition of the HIV-1-Env antifusogenic activity of this chemokine. Coimmunoprecipitation and protein-protein binding studies demonstrated that MIIA interacts with both the cytoplasmic tail of CXCR4 and beta-arrestin. Moreover, SDF-1alpha promotes a rapid MIIA-beta-arrestin dissociation. Our data reveal a novel role for MIIA in CXCR4 endocytosis, which involves its dynamic association with beta-arrestin and highlights the role of endogenous MIIA as a regulator of CXCR4 internalization and, therefore, the onset of SDF-1alpha signaling.

    Journal of cell science 2007;120;Pt 6;1126-33

  • Haematological characteristics of MYH9 disorders due to MYH9 R702 mutations.

    Kunishima S, Yoshinari M, Nishio H, Ida K, Miura T, Matsushita T, Hamaguchi M and Saito H

    Department of Haemostasis and Thrombosis, Clinical Research Centre, National Hospital Organization Nagoya Medical Centre, Nagoya, Japan. kunishis@nnh.hosp.go.jp

    Objective: MYH9 disorders are characterised by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9, the gene for non-muscle myosin heavy chain-IIA (NMMHC-IIA). MYH9 R702 mutations are highly associated with Alport manifestations and result in Epstein syndrome. The aim of our study was to determine the haematological characteristics of MYH9 disorders as a result of R702 mutations to aid in making a proper diagnosis.

    Platelet size of patients with MYH9 disorders was determined as platelet diameter by microscopic observation of 200 platelets on stained peripheral blood smears. Double in situ hybridisation using a biotinylated oligo(dT) probe and immunofluorescence analysis of neutrophil NMMHC-IIA was performed on peripheral blood smears.

    Results: Patients carrying R702 mutations had significantly larger platelets than those with other MYH9 mutations. Although granulocyte inclusion bodies were mostly invisible on stained blood smears, immunofluorescence analysis for NMMHC-IIA showed an abnormal type II localisation in all neutrophils. We first showed that poly(A)+ RNA coincided with accumulated NMMHC-IIA at inclusion bodies in patients with MYH9 disorders. However, no condensation of poly(A)+ RNA at inclusion bodies was observed in patients with R702 mutations.

    Conclusion: Our study shows that R702 mutations result in especially large platelets and inclusion bodies being faint and mostly invisible on conventionally stained blood smears. We further demonstrated that poly(A)+ RNA content but not NMMHC-IIA accumulation is responsible for the morphological appearance/stainability of inclusion bodies on stained blood smears and the amount of poly(A)+ RNA is decreased in those with R702 mutations.

    European journal of haematology 2007;78;3;220-6

  • Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.

    Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS and Yamada KM

    Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health, Bethesda, MD 20892, USA.

    Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.

    Funded by: Intramural NIH HHS

    Nature cell biology 2007;9;3;299-309

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration.

    Sandquist JC, Swenson KI, Demali KA, Burridge K and Means AR

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.

    Funded by: NCI NIH HHS: CA 082845; NIGMS NIH HHS: GM 29860

    The Journal of biological chemistry 2006;281;47;35873-83

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Analysis of clinical manifestations, mutant gene and encoded protein in two Chinese MYH9-related disease families.

    Yi Y, Sen Zhang G, Xu M, San Ling Z, Ru Shao X, Zeng Li J and Ma J

    Division of Hematology/Institute of Molecular Hematology, the Second Xiang-ya Hospital, Central South University, Changsha, Hunan 410011, P.R. China.

    Background: MYH9-related disease is a rare autosomal dominant disorder characterized by the triad of giant platelet, thrombocytopenia and inclusion bodies in neutrophil. In recent years, much progress has been made in the investigation of its clinical feature and pathogenesis.

    Methods: Clinical manifestations were analyzed in two Chinese MYH9-related disease families. Polymerase chain reaction (PCR), DNA sequencing and CpoI restrictive endonuclease map analysis were used to identify spot mutation in nonmuscle myosin heavy chain 9 (MYH9) gene. Indirect immunofluence combined propidium iodine (PI) nuclei count-staining technology was applied to probe nonmuscle myosin heavy chain IIA (NMMHC-A) in MYH9-related disease neutrophils and platelets. Western blot was undergone to examine the expression of NMMHC-A in MYH9-related disease patients.

    Results: All of the patients manifested with the typical triad, mild to moderate bleeding tendency were their common clinical feature, some patients were accompanied by renal lesion. G5521A mutation in MYH9 gene was identified in both families. Spindle-like inclusions with yellow fluorescence in MYH9-related disease neutrophils were clearly revealed by indirect immunofluence combined PI nuclei count-staining technology, which matched very well with the inclusions, detected by Wright-Giemsa's stain. An upregulation of NMMHC-A in MYH9-related disease neutrophils was observed by Western blotting analysis.

    Conclusion: Mutation of MYH9 gene exists in cases of Chinese MYH9-related disease. In the two families, the point mutation was located in exon 38(G5521A), and the transference rule of the MYH9 gene mutation is corresponding with clinical phenotype distribution. Indirect immunofluorescence combining with PI nuclei staining technology is sensitive and more specific than Wright-Giemsa's staining in detecting MYH9-related disease inclusions, with which we might easily distinguish MYH9-related disease inclusions from infection-associated inclusions. The expression of the NMMHC-A in MYH9-related disease neutrophils was upregulated than normal control.

    Clinica chimica acta; international journal of clinical chemistry 2006;373;1-2;49-54

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin.

    Huang Y, Shi H, Zhou H, Song X, Yuan S and Luo Y

    Laboratory of Protein Chemistry, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China.

    Nucleolin, originally described as a nuclear protein, was recently found to be expressed on the surface of endothelial cells during angiogenic. However, the functions of cell-surface nucleolin in angiogenic remain mysterious. Here we report that upon endothelial cells adhering to extracellular matrix components, vascular endothelial growth factor (VEGF) mobilizes nucleolin from nucleus to cell surface. Functional blockage or down-regulation of the expression of cell-surface nucleolin in endothelial cells significantly inhibits the migration of endothelial cells and prevents capillary-tubule formation. Moreover, nonmuscle myosin heavy chain 9 (MyH9), an actin-based motor protein, is identified as a nucleolin-binding protein. Subsequent studies reveal that MyH9 serves as a physical linker between nucleolin and cytoskeleton, thus modulating the translocation of nucleolin. Knocking down endogenous MyH9, specifically inhibiting myosin activity, or overexpressing functional deficient MyH9 disrupts the organization of cell-surface nucleolin and inhibits its angiogenic function. These studies indicate that VEGF, extracellular matrix, and intracellular motor protein MyH9 are all essential for the novel function of nucleolin in angiogenic.

    Blood 2006;107;9;3564-71

  • Non-muscle myosin heavy chain IIA and IIB interact and co-localize in living cells: relevance for MYH9-related disease.

    Marini M, Bruschi M, Pecci A, Romagnoli R, Musante L, Candiano G, Ghiggeri GM, Balduini C, Seri M and Ravazzolo R

    Laboratory of Molecular Genetics, G. Gaslini Institute, 16147 Genova, Italy.

    Myosins of class II constitute part of a superfamily of several classes of proteins expressed in almost all eukaryotic cell types. Differences in the heavy chains produce three isoforms of class II non-muscle myosins (A, B and C), which are widely distributed in most tissues and thought to be components of the cell motor systems, although specific functional roles are largely unknown. In particular, it is still a matter of debate whether they interact and have overlapping or distinct functions. This argument is relevant not only to cell physiology, but also to human pathology since mutations of the MYH9 gene encoding non-muscle myosin heavy chain II A (NMMHC-A) cause MYH9-related disease (MYH9-RD), an autosomal dominant disorder characterized by platelet macrocytosis, thrombocytopenia and leukocyte inclusions, variably associated with sensorineural hearing loss, cataracts and/or glomerulonephritis. In this study, we report the results of yeast two-hybrid screening showing that the C-terminals of NMMHC-A and -B interact. This interaction was confirmed by immunoprecipitation in transfected COS-7 cells and in skin fibroblasts naturally expressing both isoforms. Moreover, our immunomorphological study revealed that isoforms A and B co-localize in fibroblasts, erythroblasts and kidney cells. These results suggest that isoforms A and B are strictly related molecules and support the hypothesis that their interrelationship could be involved both in the variability of clinical phenotype and selectivity of tissue damage of MYH9-RD.

    International journal of molecular medicine 2006;17;5;729-36

  • Identification and characterization of oviductal glycoprotein-binding protein partner on gametes: epitopic similarity to non-muscle myosin IIA, MYH 9.

    Kadam KM, D'Souza SJ, Bandivdekar AH and Natraj U

    National Institute for Research in Reproductive Health, Indian Council for Medical Research, Mumbai, Maharashtra.

    The mammalian estrogen induced oviductal glycoprotein (OGP) has been known to associate with capacitated sperm, oocytes and developing embryos. This study aimed to identify the putative binding partner of OGP on gametes using N-terminal peptide of bonnet monkey (Macaca radiata) OGP, Nmon, as bait. A protein(s) of molecular size approximately 54 kDa was detected by far-western blot analysis of detergent solubilized human sperm proteins. MALDI-TOF mass spectra analysis of approximately 54 kDa tryptic peptides gave a significant hit to non-muscle myosin heavy chain. Biochemical characterization of approximately 54 kDa was done with antibodies specific to non-muscle myosin IIA, MYH9. The approximately 54 kDa protein, possible breakdown product of MYH9, immunoreacted with MYH9 antibody in western blot analysis. OGP binding to approximately 54 kDa could also be demonstrated in far-western blot analysis of detergent solubilized human sperm proteins and nuclear matrix intermediate filament (NM-IF) preparations from human sperm and mouse oocytes. Far-western blot analysis of MYH9 enriched by immunoprecipitation identified the native approximately 220 kDa protein as OGP-binding partner. The identical and characteristic immunogold localization pattern of Nmon and MYH9 on sperm NM-IF preparation substantiated these findings. The results suggest that OGP binds to both gametes through its interaction with MYH9 through the non-glycosylated N-terminal conserved region of OGP, spanning the residues 11-137.

    Molecular human reproduction 2006;12;4;275-82

  • Signaling via the angiotensin-converting enzyme results in the phosphorylation of the nonmuscle myosin heavy chain IIA.

    Kohlstedt K, Kellner R, Busse R and Fleming I

    Vascular Signaling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.

    The phosphorylation of the short C-terminal cytoplasmic domain of the somatic angiotensin-converting enzyme (ACE) is involved in the regulation of enzyme shedding. We determined whether the phosphorylation of the cytoplasmic domain of ACE (ACEct) on Ser1270 regulates the cleavage/secretion of the enzyme by affecting its association with other proteins. ACE was associated with beta-actin and the nonmuscle myosin heavy chain IIA (MYH9) in endothelial cells, as determined by coimmunoprecipitation experiments as well as an ACEct affinity column. The ACE-associated MYH9 immunoprecipitated from (32)P-labeled endothelial cells was basally phosphorylated and cell stimulation with ACE inhibitors, or with bradykinin, increased the phosphorylation of MYH9. Casein kinase 2 (CK2) but not protein kinase C phosphorylated MYH9 in vitro, CK2 coprecipitated with MYH9 from endothelial cells and the phosphorylation of MYH9 in intact cells paralleled the phosphorylation of ACE on Ser1270 by CK2. The CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole attenuated the phosphorylation of ACE and MYH9, disrupted their association, and enhanced the cleavage/secretion of ACE from the plasma membrane. Cytochalasin D decreased the interaction between ACE and MYH9 and stimulated ACE shedding. Although MYH9 was still able to associate with residual amounts of a nonphosphorylatable S1270A ACE mutant, no ACE inhibitor-induced increase in MYH9 phosphorylation could be detected in S1270A-expressing cells. These data indicate that the interaction of ACE with MYH9 determines ACE shedding and is modulated by phosphorylation processes. Furthermore, because ACE inhibitors affect the phosphorylation of MYH9, the phosphorylation of this class II myosin might contribute to the phenomenon of ACE signaling in endothelial cells.

    Molecular pharmacology 2006;69;1;19-26

  • Pathogenetic mechanisms of hematological abnormalities of patients with MYH9 mutations.

    Pecci A, Canobbio I, Balduini A, Stefanini L, Cisterna B, Marseglia C, Noris P, Savoia A, Balduini CL and Torti M

    Department of Internal Medicine, University of Pavia, Italy. alessandro.pecci@unipv.it

    Mutations of MYH9, the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA), cause a complex clinical phenotype characterized by macrothrombocytopenia and granulocyte inclusion bodies, often associated with deafness, cataracts and/or glomerulonephritis. The pathogenetic mechanisms of these defects are either completely unknown or controversial. In particular, it is a matter of debate whether haploinsufficiency or a dominant-negative effect of mutant allele is responsible for hematological abnormalities. We investigated 11 patients from six pedigrees with different MYH9 mutations. We evaluated NMMHC-IIA levels in platelets and granulocytes isolated from peripheral blood and in megakaryocytes (Mks) cultured from circulating progenitors. NMMHC-IIA distribution in Mks and granulocytes was also assessed. We demonstrated that all the investigated patients had a 50% reduction of NMMHC-IIA expression in platelets and that a similar defect was present also in Mks. In subjects with R1933X and E1945X mutations, the whole NMMHC-IIA of platelets and Mks was wild-type. No NMMHC-IIA inclusions were observed at any time of Mk maturation. In granulocytes, the extent of NMMHC-IIA reduction in patients with respect to control cells was significantly greater than that measured in platelets and Mks, and we found that wild-type protein was sequestered within most of the NMMHC-IIA inclusions. Altogether these results indicate that haploinsufficiency of NMMHC-IIA in megakaryocytic lineage is the mechanism of macrothrombocytopenia consequent to MYH9 mutations, whereas in granulocytes a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies. The finding that the same mutations act through different mechanisms in different cells is surprising and requires further investigation.

    Funded by: Telethon: GP0019Y01, TGM06S01

    Human molecular genetics 2005;14;21;3169-78

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.

    Tao WA, Wollscheid B, O'Brien R, Eng JK, Li XJ, Bodenmiller B, Watts JD, Hood L and Aebersold R

    The Bindley Bioscience Center and Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.

    We present a robust and general method for the identification and relative quantification of phosphorylation sites in complex protein mixtures. It is based on a new chemical derivatization strategy using a dendrimer as a soluble polymer support and tandem mass spectrometry (MS/MS). In a single step, phosphorylated peptides are covalently conjugated to a dendrimer in a reaction catalyzed by carbodiimide and imidazole. Modified phosphopeptides are released from the dendrimer via acid hydrolysis and analyzed by MS/MS. When coupled with an initial antiphosphotyrosine protein immunoprecipitation step and stable-isotope labeling, in a single experiment, we identified all known tyrosine phosphorylation sites within the immunoreceptor tyrosine-based activation motifs (ITAM) of the T-cell receptor (TCR) CD3 chains, and previously unknown phosphorylation sites on total 97 tyrosine phosphoproteins and their interacting partners in human T cells. The dynamic changes in phosphorylation were quantified in these proteins.

    Funded by: NHLBI NIH HHS: N01-HV-28179

    Nature methods 2005;2;8;591-8

  • Global phosphoproteome of HT-29 human colon adenocarcinoma cells.

    Kim JE, Tannenbaum SR and White FM

    Biological Engineering Division, Massachusetts Institute of Technology, 77 Massassachusetts Avenue, Cambridge, MA 02139, USA.

    Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.

    Funded by: NIEHS NIH HHS: P30 ES 002109, P30 ES002109; NIGMS NIH HHS: 1P50 GM 68762-01

    Journal of proteome research 2005;4;4;1339-46

  • Disease-associated mutations and alternative splicing alter the enzymatic and motile activity of nonmuscle myosins II-B and II-C.

    Kim KY, Kovács M, Kawamoto S, Sellers JR and Adelstein RS

    Laboratory of Molecular Cardiology and Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Human families with single amino acid mutations in nonmuscle myosin heavy chain (NMHC) II-A (MYH9) and II-C (MYH14) have been described as have mice generated with a point mutation in NMHC II-B (MYH10). These mutations (R702C and N93K in human NMHC II-A, R709C in murine NMHC II-B, and R726S in human NMHC II-C) result in phenotypes affecting kidneys, platelets, and leukocytes (II-A), heart and brain (II-B), and the inner ear (II-C). To better understand the mechanisms underlying these defects, we characterized the in vitro activity of mutated and wild-type baculovirus-expressed heavy meromyosin (HMM) II-B and II-C. We also expressed two alternatively spliced isoforms of NMHC II-C which differ by inclusion/exclusion of eight amino acids in loop 1, with and without mutations. Comparison of the actin-activated MgATPase activity and in vitro motility shows that mutation of residues Asn-97 and Arg-709 in HMM II-B and the homologous residue Arg-722 (Arg-730 in the alternatively spliced isoform) in HMM II-C decreases both parameters but affects in vitro motility more severely. Analysis of the transient kinetics of the HMM II-B R709C mutant shows an extremely tight affinity of HMM for ADP and a very slow release of ADP from acto-HMM. Although mutations generally decreased HMM activity, the R730S mutation in HMM II-C, unlike the R730C mutation, had no effect on actin-activated MgATPase activity but decreased the rate of in vitro motility by 75% compared with wild type. Insertion of eight amino acids into the HMM II-C heavy chain increases both actin-activated MgATPase activity and in vitro motility.

    The Journal of biological chemistry 2005;280;24;22769-75

  • Vertebrate nonmuscle myosin II isoforms rescue small interfering RNA-induced defects in COS-7 cell cytokinesis.

    Bao J, Jana SS and Adelstein RS

    Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.

    RNA interference (RNAi) treatment of monkey COS-7 cells, a cell line that lacks nonmuscle myosin heavy chain II-A (NMHC II-A) but contains NMHC II-B and II-C, was used to investigate the participation of NMHC isoforms in cytokinesis. We specifically suppressed the expression of NMHC II-B or II-C using 21 nucleotide small interfering RNA (siRNA) duplexes. Down-regulation of NMHC II-B protein expression to 10.2 +/- 0.7% inhibited COS-7 cell proliferation by 50% in the RNAi-treated cells compared with control cells. Moreover, whereas 8.7 +/- 1.0% of control cells were multinucleated, 62.4 +/- 8.8% of the NMHC II-B RNAi-treated cells were multinucleated 72 h after transfection. The RNAi-treated cells had increased surface areas and, unlike control cells, lacked actin stress fibers. Treatment of the COS-7 cells with NMHC II-C siRNA decreased NMHC II-C expression to 5.2 +/- 0.1% compared with the endogenous content of II-C; however, down-regulation of NMHC II-C did not cause increased multinucleation. Immunoblot analysis using a pan-myosin antibody showed that the content of NMHC II-C was less than one-twentieth the amount of NMHC II-B, thereby explaining the lack of response to II-C siRNA. Introducing green fluorescent protein (GFP)-tagged NMHC II isoforms into II-B siRNA-treated cells resulted in reduction of multinucleation from 62.4 +/- 8.8% to 17.8 +/- 2.2% using GFP-NMHC II-B, to 29.8 +/- 7.4% using GFP-NMHC II-A, and to 34.1 +/- 8.6% using NMHC II-C-GFP. These studies have shown that expression of endogenous NMHC II-C in COS-7 cells is insufficient for normal cytokinesis and that exogenous NMHC II-A and NMHC II-C can, at least partially, rescue the defect in cytokinesis due to the loss of NMHC II-B.

    The Journal of biological chemistry 2005;280;20;19594-9

  • Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation.

    Dulyaninova NG, Malashkevich VN, Almo SC and Bresnick AR

    Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

    Previous studies suggested that heavy chain phosphorylation regulates non-muscle myosin-II assembly in an isoform-specific manner, affecting the assembly of myosin-IIB, but not myosin-IIA. We re-examined the effects of heavy chain phosphorylation on myosin-IIA filament formation and also examined mts1 binding. We demonstrated that heavy chain phosphorylation by either protein kinase C (PKC) or casein kinase 2 (CK2) inhibits the assembly of myosin-IIA into filaments. PKC phosphorylation had no affect on mts1 binding, but CK2 phosphorylation decreased the affinity of mts1 for the myosin-IIA rod by approximately 6.5-fold. Mts1 destabilized PKC-phosphorylated myosin-IIA filaments and inhibited the assembly of myosin-IIA monomers with maximal inhibition of assembly and promotion of disassembly occurring at a molar ratio of one mts1 dimer per myosin-IIA rod. At this molar ratio, mts1 only weakly disassembled CK2-phosphorylated myosin-IIA filaments and weakly inhibited the assembly of CK2-phosphorylated myosin-IIA monomers. These observations demonstrate that CK2 phosphorylation of the myosin-IIA heavy chain protects against mts1-induced filament disassembly and inhibition of assembly, and suggest that heavy chain phosphorylation provides an additional level of regulation for the mts1-myosin-IIA interaction.

    Funded by: NIGMS NIH HHS: GM069945

    Biochemistry 2005;44;18;6867-76

  • Altered cytoskeleton organization in platelets from patients with MYH9-related disease.

    Canobbio I, Noris P, Pecci A, Balduini A, Balduini CL and Torti M

    Centre of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, Pavia, Italy.

    MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

    Funded by: Telethon: GP0019Y01

    Journal of thrombosis and haemostasis : JTH 2005;3;5;1026-35

  • Vector-capping: a simple method for preparing a high-quality full-length cDNA library.

    Kato S, Ohtoko K, Ohtake H and Kimura T

    Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for Persons with Disabilities, 4-1 Namiki, Tokorozawa, Saitama 359-8555, Japan. seishi@rehab.go.jp

    Full-length cDNAs play an essential role in identifying genes and determining their promoter regions. Here we describe a simple method for constructing a full-length cDNA library, which has the following advantages: (i) it consists of only three steps including direct ligation between a vector and a cDNA strand using T4 RNA ligase, (ii) it contains neither a PCR process generating mutations nor restriction enzyme treatment causing truncation of cDNA, (iii) the intactness of cDNA is assured due to the presence of an additional dGMP at its 5' end, (iv) approximately 95% of cDNA clones are full-length when cultured cells or fresh tissues are used, (v) several micrograms of total RNA without mRNA purification is sufficient for preparation of a library containing >10(5) independent clones, and (vi) a long-sized full-length cDNA up to 9.5 kbp can be cloned. This method will accelerate comprehensive gene analysis in a variety of eukaryotes.

    DNA research : an international journal for rapid publication of reports on genes and genomes 2005;12;1;53-62

  • Cleavage of nonmuscle myosin heavy chain-A during apoptosis in human Jurkat T cells.

    Kato M, Fukuda H, Nonaka T and Imajoh-Ohmi S

    Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639.

    We have previously reported that calpastatin, an endogenous inhibitory protein of calpain, is cleaved by a caspase-3-like protease during apoptosis in human Jurkat T cells [Kato, M. et al. (2000) J. Biochem. 127, 297-305]. In this study, we found that nonmuscle myosin heavy chain-A (NMHC-A) is cleaved during apoptosis in Jurkat cells by using a cleavage-site-directed antibody for calpastatin. The cleavage-site-directed antibody was raised against the amino-terminal fragment of calpastatin, and this antibody detected the in vitro cleaved calpastatin fragment. Although cleaved calpastatin was not detected, a 95-kDa polypeptide (p95) was detected in apoptotic cells by this antibody. This p95 was identified as the carboxyl-terminal fragment of NMHC-A based on the results of peptide mass spectrometry fingerprinting and amino-terminal sequencing. Furthermore, two cleavage sites on NMHC-A, Asp-1153 and Asp-1948, were determined, and three cleaved fragments of NMHC-A, one cleaved at Asp-1153 and the other two cleaved at Asp-1948, were detected by cleavage-site-directed antibodies against each cleavage site. The results of confocal immunofluorescence microscopic analysis show that the cleavage at Asp-1948 occurs faster than that at Asp-1153 during apoptosis. In addition, the Asp-1153 cleaved fragment was distributed diffusely in the cytoplasm of apoptotic cells, whereas the Asp-1948 cleaved fragments were detected as condensed dots. In conclusion, our findings can be summarized as follows: (i) NMHC-A is cleaved at two sites during apoptosis, (ii) the timing of cleavage is different between these two cleavage sites, and (iii) the distribution of cleaved fragments is different in apoptotic cells.

    Journal of biochemistry 2005;137;2;157-66

  • Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy.

    Zhang S, Wang G, Fernig DG, Rudland PS, Webb SE, Barraclough R and Martin-Fernandez M

    Cancer and Polio Research Fund Laboratories, School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, UK.

    Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.

    European biophysics journal : EBJ 2005;34;1;19-27

  • Nucleolar proteome dynamics.

    Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI and Mann M

    Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.

    The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • Targeted disruption of mouse ortholog of the human MYH9 responsible for macrothrombocytopenia with different organ involvement: hematological, nephrological, and otological studies of heterozygous KO mice.

    Matsushita T, Hayashi H, Kunishima S, Hayashi M, Ikejiri M, Takeshita K, Yuzawa Y, Adachi T, Hirashima K, Sone M, Yamamoto K, Takagi A, Katsumi A, Kawai K, Nezu T, Takahashi M, Nakashima T, Naoe T, Kojima T and Saito H

    Department of Hematology, Nagoya University Graduate School of Medicine, Japan. tmatsu@med.nagoya-u.ac.jp

    Among three different isoforms of non-muscle myosin heavy chains (NMMHCs), only NMMHCA is associated with inherited human disease, called MYH9 disorders, characterized by macrothrombocytopenia and characteristic granulocyte inclusions. Here targeted gene disruption was performed to understand fundamental as well as pathological role of the gene for NMMHCA, MYH9. Heterozygous intercrosses yielded no homozygous animals among 552 births, suggesting that MYH9 expression is required for embryonic development. In contrast, MYH9+/- mice were viable and fertile without gross anatomical, hematological, and nephrological abnormalities. Immunofluorescence analysis also showed the normal cytoplasmic distribution of NMMHCA. We further measured the auditory brainstem response and found two of six MYH9+/- mice had hearing losses, whereas the remaining four were comparable to wild-type mice. Such observation may parallel the diverse expression of Alport's manifestations of human individuals with MYH9 disorders and suggest the limited requirement of the gene for maintenance and function of specific organs.

    Biochemical and biophysical research communications 2004;325;4;1163-71

  • Application of a diagnostic algorithm for inherited thrombocytopenias to 46 consecutive patients.

    Noris P, Pecci A, Di Bari F, Di Stazio MT, Di Pumpo M, Ceresa IF, Arezzi N, Ambaglio C, Savoia A and Balduini CL

    Department of Internal Medicine, IRCCS Policlinico San Matteo-University of Pavia, Italy.

    The Italian Gruppo di Studio delle Piastrine recently developed a diagnostic algorithm to assist clinicians in the diagnosis of inherited thrombocytopenias. This algorithm is based on the simplest possible diagnostic investigations and can also be used in centers that are not highly specialized. The aim of the present study was to validate this diagnostic algorithm by applying it to a case series of genetic thrombocytopenias.

    The diagnostic algorithm was applied retrospectively to 46 consecutive patients observed during the last five years at a single institution. Twenty-eight were affected by defined illnesses or their variants, while 18 had a disorder that did not fit the criteria for any known genetic thrombocytopenia. The study was based on the evaluation of clinical records and laboratory tests.

    Results: The diagnostic algorithm recognized: 4 homozygous and 4 heterozygous Bernard-Soulier syndromes, 11 MYH9-related diseases, one von WillebrandOs disease type 2B, one gray platelet syndrome and one X-linked thrombocytopenia with thalassemia. Moreover, it identified 4 patients with the clinical and laboratory features of heterozygous Bernard-Soulier syndrome not caused by mutations in the coding region of the GPIbalpha, GPIbbeta, GPIX or GPV genes, and two patients with the clinical phenotype of MYH9-related disease but without MYH9 mutations. Since the diagnostic flow chart did not allow prompt recognition of two subjects with MYH9-related disease, we introduced a small change to the previously proposed flow chart to obviate this defect.

    The diagnostic algorithm correctly diagnosed 26 of 28 patients with known disorders or phenotypic variants of known disorders. By a simple modification of the investigation sequence, its sensitivity reached 100%. The algorithm also identified 18 patients with new, as yet uncharacterized forms of genetic thrombocytopenia.

    Funded by: Telethon: GP0019Y01

    Haematologica 2004;89;10;1219-25

  • Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A.

    Timmermann M, Buck F, Sorg C and Högger P

    Institut für Pharmazie und Lebensmittelchemie, Bayerische Julius-Maximilians-Universität, Würzburg, Germany.

    CD163 is a monocyte/macrophage-specific scavenger receptor that undergoes ectodomain shedding upon an inflammatory stimulus. Soluble CD163 (sCD163) actively inhibits lymphocyte proliferation, but to date exactly how it interacts with these cells has remained elusive. We screened T lymphocytes and endothelial cells for proteins binding to sCD163. In both cell types a high affinity binding protein was detected. Partial sequencing of the protein revealed sequence identity to a non-muscle myosin heavy chain type A. Employing labelled sCD163 we found little specific binding of sCD163 to the extracellular domains of T lymphocytes and human umbilical vein endothelial cells (HUVEC). In activated T lymphocytes we demonstrated specific binding of sCD163 to intracellular structures as well as the presence of the native protein within the cell after co-incubation with purified sCD163. Furthermore, we developed a novel ELISA for highly specific detection of sCD163-myosin complexes. These complexes were present in activated T lymphocytes after incubation with shed sCD163. Co-localization of sCD163 and cellular myosin in T lymphocytes was further confirmed by fluorescence microscopy. Our results suggest that sCD163 associates with cellular myosin, thereby possibly modulating the cells' response to an inflammatory stimulus.

    Immunology and cell biology 2004;82;5;479-87

  • Heterodimeric interaction and interfaces of S100A1 and S100P.

    Wang G, Zhang S, Fernig DG, Spiller D, Martin-Fernandez M, Zhang, Ding Y, Rao Z, Rudland PS and Barraclough R

    Cancer and Polio Research Fund Laboratories Molecular Medicine Group, School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK. wangg@liv.ac.uk

    With the widespread use of yeast two-hybrid systems, many heterodimeric forms of S100 proteins have been found, although their biological significance is unknown. In the present study, S100A1 was found to interact with another S100 protein, S100P, by using the yeast two-hybrid system. The binding parameters of the interaction were obtained using an optical biosensor and show that S100P has a slightly higher affinity for S100A1 (K(d)=10-20 nM) when compared with that for self-association (K(d)=40-120 nM). The physical interaction of S100A1 and S100P was also demonstrated in living mammalian cells using a fluorescence resonance energy transfer technique. Preincubation of recombinant S100P with S100A1, before the biosensor assay, reduced by up to 50% the binding of S100P to a recombinant C-terminal fragment of non-muscle myosin A, one of its target molecules. Site-specific mutations of S100P and S100A1, combined with homology modelling of an S100P/S100A1 heterodimer using known S100P and S100A1 structures, allowed the hydrophobic interactions at the dimeric interface of the heterodimer to be defined and provide an explanation for the heterodimerization of S100P and S100A1 at the molecular level. These results have revealed the similarities and the differences between the S100P homodimer and the S100A1/S100P heterodimer.

    The Biochemical journal 2004;382;Pt 1;375-83

  • Circular rapid amplification of cDNA ends for high-throughput extension cloning of partial genes.

    Fu GK, Wang JT, Yang J, Au-Young J and Stuve LL

    Incyte Corporation, Palo Alto, CA 94304, USA. gfu@incyte.com

    The rapid amplification of cDNA ends (RACE) procedure is a widely used PCR-based method to clone the cDNA ends of mRNA transcripts. Current RACE methods often produce a high background of nonspecific PCR products, which can exclude the identification of the target cDNA of interest. We describe here an improved RACE procedure using circular cDNA templates and demonstrate the successful extension cloning of 4406 cDNAs.

    Genomics 2004;84;1;205-10

  • A single class II myosin modulates T cell motility and stopping, but not synapse formation.

    Jacobelli J, Chmura SA, Buxton DB, Davis MM and Krummel MF

    Department of Pathology, University of California at San Francisco, 513 Parnassus Ave., San Francisco, California 93143, USA.

    Upon encountering an antigen, motile T cells stop crawling, change morphology and ultimately form an 'immunological synapse'. Although myosin motors are thought to mediate various aspects of this process, the molecules involved and their exact roles are not defined. Here we show that nonmuscle myosin heavy chain IIA, or MyH9, is the only class II myosin expressed in T cells and is associated with the uropod during crawling. MyH9 function is required for maintenance of the uropod and for T cell motility but is dispensable for synapse formation. Phosphorylation of MyH9 in its multimerization domain by T cell receptor-generated signals indicates that inactivation of this motor may be a key step in the 'stop' response during antigen recognition.

    Funded by: NIAID NIH HHS: R01 AI052116, R01-AI52116-01

    Nature immunology 2004;5;5;531-8

  • Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin.

    Kovács M, Tóth J, Nyitray L and Sellers JR

    Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.

    The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.

    Biochemistry 2004;43;14;4219-26

  • MARK4 is a novel microtubule-associated proteins/microtubule affinity-regulating kinase that binds to the cellular microtubule network and to centrosomes.

    Trinczek B, Brajenovic M, Ebneth A and Drewes G

    Department of Medicinal Chemistry, University of Kansas, Malott Hall, Lawrence, Kansas 66045, USA.

    The MARK protein kinases were originally identified by their ability to phosphorylate a serine motif in the microtubule-binding domain of tau that is critical for microtubule binding. Here, we report the cloning and expression of a novel human paralog, MARK4, which shares 75% overall homology with MARK1-3 and is predominantly expressed in brain. Homology is most pronounced in the catalytic domain (90%), and MARK4 readily phosphorylates tau and the related microtubule-associated protein 2 (MAP2) and MAP4. In contrast to the three paralogs that all exhibit uniform cytoplasmic localization, MARK4 colocalizes with the centrosome and with microtubules in cultured cells. Overexpression of MARK4 causes thinning out of the microtubule network, concomitant with a reorganization of microtubules into bundles. In line with these findings, we show that a tandem affinity-purified MARK4 protein complex contains alpha-, beta-, and gamma-tubulin. In differentiated neuroblastoma cells, MARK4 is localized prominently at the tips of neurite-like processes. We suggest that although the four MARK/PAR-1 kinases might play multiple cellular roles in concert with different targets, MARK4 is likely to be directly involved in microtubule organization in neuronal cells and may contribute to the pathological phosphorylation of tau in Alzheimer's disease.

    The Journal of biological chemistry 2004;279;7;5915-23

  • A genome annotation-driven approach to cloning the human ORFeome.

    Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, Huckle EJ, Beare DM and Dunham I

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.

    Genome biology 2004;5;10;R84

  • Mts1 regulates the assembly of nonmuscle myosin-IIA.

    Li ZH, Spektor A, Varlamova O and Bresnick AR

    Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

    The formation of myosin-II filaments is fundamental to contractile and motile processes in nonmuscle cells, and elucidating the mechanisms controlling filament assembly is essential for understanding how myosin-II rapidly responds to changing conditions within the cell. Several proteins including KRP and a novel 38 kDa protein (1, 2) have been shown to modulate filament assembly through the stabilization of myosin-II assemblies. In contrast, we demonstrate that mts1, a member of the Ca(2+)-regulated S100 family of proteins, may regulate the monomeric, unassembled state in an isoform-specific manner. Biochemical analyses demonstrate that mts1 has a 9-fold higher affinity for myosin-IIA filaments than for myosin-IIB filaments. At stoichiometric levels, mts1 inhibits the assembly of myosin-IIA monomers into filaments and promotes the disassembly of myosin-IIA filaments into monomers; however, mts1 has little effect on the assembly properties of myosin-IIB. Using a solution based-assay, we have demonstrated that mts1 binds to residues 1909-1924 of the myosin-IIA heavy chain, which is near the C-terminal tip of the alpha-helical coiled-coil. The observation that mts1 binds a linear sequence of approximately 16 amino acids is consistent with other S100 family members, which bind linear sequences of 13-22 residues in their protein targets. In addition, mts1 increases the critical monomer concentration for myosin-IIA filament assembly by approximately 11-fold. Kinetic assembly assays indicate that the elongation rate and the extent of polymerization depend on the initial myosin-IIA concentration; however, mts1 had only a small affect on the half-time for assembly and predominately affected the extent of myosin IIA polymerization. Altogether, these observations are consistent with mts1 regulating myosin IIA assembly by monomer sequestration and suggest that mts1 regulates cell shape and motility through the modulation of myosin-IIA function.

    Biochemistry 2003;42;48;14258-66

  • Menin, a tumor suppressor, associates with nonmuscle myosin II-A heavy chain.

    Obungu VH, Lee Burns A, Agarwal SK, Chandrasekharapa SC, Adelstein RS and Marx SJ

    Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Obunguvi@lilly.com

    Oncogene 2003;22;41;6347-58

  • Non-muscle myosin heavy chain (MYH9): a new partner fused to ALK in anaplastic large cell lymphoma.

    Lamant L, Gascoyne RD, Duplantier MM, Armstrong F, Raghab A, Chhanabhai M, Rajcan-Separovic E, Raghab J, Delsol G and Espinos E

    INSERM U-563, Department of Oncogenesis and Signaling in Hematopoietic Cells, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, France.

    In anaplastic large cell lymphoma, the ALK gene at 2p23 is known to be fused to NPM, TPM3, TPM4, TFG, ATIC, CLTC, MSN, and ALO17. All of these translocations result in the expression of chimeric ALK transcripts that are translated into fusion proteins with tyrosine kinase activity and oncogenic properties. We report a case showing a restricted cytoplasmic staining pattern of ALK and a novel chromosomal abnormality, t(2;22)(p23;q11.2), demonstrated by fluorescence in situ hybridization analysis. The result of 5' RACE analysis showed that the ALK gene was fused in-frame to a portion of the non-muscle myosin heavy chain gene, MYH9. Nucleotide sequence of the MYH9-ALK chimeric cDNA revealed that the ALK breakpoint was different from all those previously reported. It is localized in the same exonic sequence as MSN-ALK, but 6 bp downstream, resulting in an in-frame fusion of the two partner proteins. In contrast to the previously reported ALK fusion proteins, MYH9-ALK may lack a functional oligomerization domain. However, biochemical analysis showed that the new fusion protein is tyrosine phosphorylated in vivo but seems to lack tyrosine kinase activity in vitro. If further investigations confirm this latter result, the in vivo tyrosine phosphorylation of MYH9-ALK protein could involve mechanisms different from those described in the other ALK hybrid proteins.

    Funded by: NCI NIH HHS: 1-U01-CA84967-01

    Genes, chromosomes & cancer 2003;37;4;427-32

  • Asp1424Asn MYH9 mutation results in an unstable protein responsible for the phenotypes in May-Hegglin anomaly/Fechtner syndrome.

    Deutsch S, Rideau A, Bochaton-Piallat ML, Merla G, Geinoz A, Gabbiani G, Schwede T, Matthes T, Antonarakis SE and Beris P

    University of Geneva, Switzerland.

    May-Hegglin anomaly (MHA), Fechtner syndrome (FTNS), Sebastian syndrome (SBS), and Epstein syndrome (EPS) are a group of rare, autosomal dominant disorders characterized by thrombocytopenia, giant platelets, and Döhle-like inclusion bodies, together with variable manifestations of Alport-like symptoms that include high-tone sensorineural deafness, cataracts, and nephritis. These disorders result from mutations in the MYH9 gene, which encodes for the nonmuscle myosin heavy chain A protein (also known as NMMHC-A). To date 20 different mutations have been characterized for this gene, but no clear phenotype-genotype correlation has been established, and very little is known regarding the molecular pathogenesis of this group of diseases. Here, we describe 2 new families with MHA/FTNS phenotypes that have been characterized in terms of their mutations, protein localization in megakaryocytes, protein expression, and mRNA stability. Our findings suggest that, at least for the Asp1424Asn mutation in the MYH9 gene, the phenotypes result from a highly unstable protein. No abnormalities in protein localization or mRNA stability were observed. We hypothesize that haploinsufficiency of the MYH9 results in a failure to properly reorganize the cytoskeleton in megakaryocytes as required for efficient platelet production.

    Blood 2003;102;2;529-34

  • MYH9-related disease: May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but represent a variable expression of a single illness.

    Seri M, Pecci A, Di Bari F, Cusano R, Savino M, Panza E, Nigro A, Noris P, Gangarossa S, Rocca B, Gresele P, Bizzaro N, Malatesta P, Koivisto PA, Longo I, Musso R, Pecoraro C, Iolascon A, Magrini U, Rodriguez Soriano J, Renieri A, Ghiggeri GM, Ravazzolo R, Balduini CL and Savoia A

    Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genova, Italy.

    May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are autosomal dominant macrothrombocytopenias distinguished by different combinations of clinical and laboratory signs, such as sensorineural hearing loss, cataract, nephritis, and polymorphonuclear Döhle-like bodies. Mutations in the MYH9 gene encoding for the nonmuscle myosin heavy chain IIA (NMMHC-IIA) have been identified in all these syndromes. To understand the role of the MYH9 mutations, we report the molecular defects in 12 new cases, which together with our previous works represent a cohort of 19 families. Since no genotype-phenotype correlation was established, we performed an accurate clinical and biochemical re-evaluation of patients. In addition to macrothrombocytopenia, an abnormal distribution of NMMHC-IIA within leukocytes was observed in all individuals, including those without Döhle-like bodies. Selective, high-tone hearing deficiency and cataract was diagnosed in 83% and 23%, respectively, of patients initially referred as having May-Hegglin anomaly or Sebastian syndrome. Kidney abnormalities, such as hematuria and proteinuria, affected not only patients referred as Fechtner syndrome and Epstein syndrome but also those referred as May-Hegglin anomaly and Sebastian syndrome. These findings allowed us to conclude that May-Hegglin anomaly, Sebastian syndrome, Fechtner syndrome, and Epstein syndrome are not distinct entities but rather a single disorder with a continuous clinical spectrum varying from mild macrothrombocytopenia with leukocyte inclusions to a severe form complicated by hearing loss, cataracts, and renal failure. For this new nosologic entity, we propose the term "MHY9-related disease," which better interprets the recent knowledge in this field and identifies all patients at risk of developing renal, hearing, or visual defects.

    Funded by: Telethon: GP0019Y01

    Medicine 2003;82;3;203-15

  • Myosin is an in vivo substrate of the protein tyrosine phosphatase (SHP-1) after mIgM cross-linking.

    Baba T, Fusaki N, Shinya N, Iwamatsu A and Hozumi N

    Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, 278-0022, Chiba, Japan.

    SHP-1 plays an important role in negative signaling in many cell types. For example, after BCR stimulation in apoptotic B cells, SHP-1 has been shown to be recruited to phosphorylated ITIMs present in receptors such as CD72. However, the SHP-1 substrates in the chicken B cell line, DT40, have been poorly undefined. To identify SHP-1 substrates in DT40, we used a trapping mutant SHP-1 C/S (a catalytically inactive form). BCR stimulation induced hyper-phosphorylation of 230 kDa protein in C/S transfectants. MALDI-TOF/MS analysis revealed that this was myosin carrying ITIM. SHP-1 was shown to bind to this ITIM in synthetic peptide binding experiment. Thus, myosin is a direct SHP-1 substrate in B cells. The results suggest that SHP-1 plays a critical role in the reorganization of cytoskeletal architecture mediated via BCR stimulation.

    Biochemical and biophysical research communications 2003;304;1;67-72

  • A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

    Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.

    Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

    Nature biotechnology 2003;21;3;315-8

  • Macrothrombocytopenia and progressive deafness is due to a mutation in MYH9.

    Mhatre AN, Kim Y, Brodie HA and Lalwani AK

    Laboratory of Molecular Otology, Epstein Laboratories, Department of Otolaryngology-Head & Neck Surgery, University of California San Francisco, CA 94143, USA.

    Background: In 1992, a family with hereditary macrothrombocytopenia and progressive sensorineural hearing impairment without renal dysfunction was described. Recently, mutations in MYH9, a nonmuscle myosin heavy chain, have been found in several forms of hereditary macrothrombocytopenia.

    Hypothesis: The hereditary macrothrombocytopenia and hearing loss in the previously reported family is due to a mutation in MYH9 gene.

    Methods: Genomic DNA was extracted from the affected proband. Mutation screening of all MYH9 coding exons was carried out using denaturing high-performance liquid chromatography. Abnormal results were followed by direct sequencing of the exon and comparison of the sequence with the normal MYH9 sequence.

    Results: The results of denaturing high-performance liquid chromatography suggested a potential sequence alteration in exon 30 of MYH9. Direct sequence analysis of this exon in the affected individual identified a G to A single base pair transition at nucleotide 4270 altering codon 1424. This mutations leads to an amino acid change from aspartate (D) to asparagine (N) in the highly conserved coiled-coil domain.

    Conclusions: A single base pair transition in MYH9, resulting in an amino acid substitution D1424N, is responsible for macrothrombocytopenia and hearing loss in the kindred under study. The presence of hearing impairment and the absence of renal symptoms, as reported in other families with the same mutation MYH9, further highlights the role of genetic background in expression and modification of the affected phenotype.

    Otology & neurotology : official publication of the American Ot 1f40 ological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 2003;24;2;205-9

  • Genetics, clinical and pathological features of glomerulonephritis associated with mutations of nonmuscle myosin IIA (Fechtner syndrome).

    Ghiggeri GM, Caridi G, Magrini U, Sessa A, Savoia A, Seri M, Pecci A, Romagnoli R, Gangarossa S, Noris P, Sartore S, Necchi V, Ravazzolo R and Balduini CL

    Laboratory on Pathophysiology of Uremia and Laboratory of Molecular Genetics, Istituto di Ricerca e Cura a Carattere Scientifico G. Gaslini, Genova, Italy. labnefro@ospedale-gaslini.ge.it

    Background: Fechtner syndrome (FTNS), also known as Alport-like syndrome, is a rare inherited condition characterized by progressive nephritis, macrothrombocytopenia, Döhle-like leukocyte inclusions, deafness, and cataract. Although it recently was shown that FTNS derives from mutation of MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA), its pathophysiological characteristics remain unknown.

    Methods: We studied a large FTNS family in which 10 components carried a missense mutation of MYH9 determining the D1424H substitution.

    Results: All affected subjects presented with macrothrombocytopenia and leukocyte Döhle-like bodies consisting of macroaggregates of NMMHC-IIA, but only two subjects had major renal problems characterized by proteinuria and renal failure. Electron microscopy showed focal and segmental effacement of podocytes and loss of the interpodocyte slit diaphragm. Immunohistochemistry showed apical localization of NMMHC-IIA in tubular epithelia and less podocyte staining in the two patients, whereas it was diffuse in normal epithelia. Three patients presented with stable microhematuria, and another five patients had no renal lesions, although they carried the same mutation of MYH9. Therefore, MYH9 mutation per se was responsible for platelet and leukocyte abnormalities, whereas additional predisposing conditions and/or environmental factors are necessary for nephropathy, cataract, and deafness. Looking at podocyte components conferring permselectivity properties to the kidney, we characterized the haplotype of podocin and found cosegregation of one specific allele in the two patients with nephrotic syndrome, suggesting a relationship between podocin features and proteinuria.

    Conclusion: Our study indicates a major role for the NMMHC-IIA abnormality in the pathogenesis of leukocyte, platelet, and kidney defects in FTNS. The basic feature in all cases is aggregation and compartmentation of NMMHC-IIA. However, proteinuria and podocyte lesions are the hallmark of nephropathy in patients who develop renal failure, and podocin may have some function in this setting.

    Funded by: Telethon: E.1313, GP0019Y01, TGM06S01

    American journal of kidney diseases : the official journal of the National Kidney Foundation 2003;41;1;95-104

  • Immunofluorescence analysis of neutrophil nonmuscle myosin heavy chain-A in MYH9 disorders: association of subcellular localization with MYH9 mutations.

    Kunishima S, Matsushita T, Kojima T, Sako M, Kimura F, Jo EK, Inoue C, Kamiya T and Saito H

    Japanese Red Cross Aichi Blood Center, Seto, Japan.

    The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly, Sebastian syndrome, and Fechtner syndrome, are rare human disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. These disorders are caused by mutations in the same gene, the MYH9, which encodes the nonmuscle myosin heavy chain-A (NMMHCA). The term, MYH9 disorders, has been proposed, but the clinicopathologic basis of MYH9 mutations has been poorly investigated. In this study, a total of 24 cases with MYH9 disorders and suspected cases were subjected to immunofluorescence analysis by a polyclonal antibody against human platelet NMMHCA. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations. Comparison with May-Grünwald-Giemsa staining revealed that the NMMHCA always coexisted with the neutrophil inclusion bodies, suggesting that NMMHCA is associated with such bodies. In three cases, neutrophil inclusions were not detected on conventional May-Grünwald-Giemsa-stained blood smears but immunofluorescence analysis revealed the abnormal NMMHCA localization. In contrast, cases with Epstein syndrome and the isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. An antibody that recognizes the C-terminal 12 mer peptides showed similar immunoreactivity from the patients heterozygous for truncated mutations that abolished the C-terminal epitope, suggesting that normal NMMHCA dimerizes with abnormal NMMHCA to form inclusion bodies. We further propose that the localization pattern can be classified into three groups according to the number, size, and shape of the fluorescence-labeled NMMHCA granule. Immunofluorescence analysis of neutrophil NMMHCA is useful as a screening test for the clear hematopathologic classification of MYH9 disorders.

    Laboratory investigation; a journal of technical methods and pathology 2003;83;1;115-22

  • Expression of non-muscle type myosin heavy polypeptide 9 (MYH9) in mammalian cells.

    Takubo T, Wakui S, Daigo K, Kurokata K, Ohashi T, Katayama K and Hino M

    Clinical Hematology and Clinical Diagnostics, Graduate School of Medicine, Osaka City University, 1-4-3, Asahimachi, Abeno-ku, Osaka 545-8585, Japan. m3542254@med.osaka-cu.ac.jp

    Myosin is a functional protein associated with cellular movement, cell division, muscle contraction and other functions. Members of the myosin super-family are distinguished from the myosin heavy chains that play crucial roles in cellular processes. Although there are many studies of myosin heavy chains in this family, there are fewer on non-muscle myosin heavy chains than of muscle myosin heavy chains. Myosin is classified as type I (myosin I) or type II (myosin II). Myosin I, called unconventional myosin or mini-myosin, has one head, while myosin II, called conventional myosin, has two heads. We transfected myosin heavy polypeptide 9 (MYH9) into HeLa cells as a fusion protein with enhanced green fluorescent protein (EGFP) and analyzed the localization and distribution of MYH9 in parallel with those of actin and tubulin. The results indicate that MYH9 colocalizes with actin stress fibers. Since it has recently been shown by genetic analysis that autosomal dominant giant platelet syndromes are MYH9-related disorders, our development of transfected EGFP-MYH9 might be useful for predicting the associations between the function of actin polymerization, the MYH9 motor domain, and these disorders.

    European journal of histochemistry : EJH 2003;47;4;345-52

  • Mutations in human nonmuscle myosin IIA found in patients with May-Hegglin anomaly and Fechtner syndrome result in impaired enzymatic function.

    Hu A, Wang F and Sellers JR

    Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.

    A family of autosomal-dominant diseases including May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome, Alport syndrome, and Epstein syndrome are commonly characterized by giant platelets and thrombocytopenia. In addition, there may be leukocyte inclusions, deafness, cataracts, and nephritis, depending on the syndrome. Mutations in the human nonmuscle myosin IIA heavy chain gene (MYH9) have been linked to these diseases. Two of the recently described mutations, N93K and R702C, are conserved in smooth and nonmuscle myosins from vertebrates and lie in the head domain of myosin. Interestingly, the two mutations lie within close proximity in the three-dimensional structure of myosin. These two mutations were engineered into a heavy meromyosin-like recombinant fragment of nonmuscle myosin IIA, which was expressed in baculovirus along with the appropriate light chains. The R702C mutant displays 25% of the maximal MgATPase activity of wild type heavy meromyosin and moves actin filaments at half the wild type rate. The effects of the N93K mutation are more dramatic. This heavy meromyosin has only 4% of the maximal MgATPase activity of wild type and does not translocate actin filaments in an in vitro motility assay. Biochemical characterization of the mutant is consistent with this mutant being unable to fully adopt the "on" conformation.

    The Journal of biological chemistry 2002;277;48;46512-7

  • Cutting edge: association of the motor protein nonmuscle myosin heavy chain-IIA with the C terminus of the chemokine receptor CXCR4 in T lymphocytes.

    Rey M, Vicente-Manzanares M, Viedma F, Yáñez-Mó M, Urzainqui A, Barreiro O, Vázquez J and Sánchez-Madrid F

    Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, c/Diego de León 62, E-28006 Madrid, Spain.

    The binding of chemokines to their receptors guides lymphocyte migration. However, the precise mechanism that links the chemotactic signals with the energy and traction force generated by the actomyosin complex of the cell has not been elucidated. Using biochemical approaches and mass spectrometry analysis, we found an association between the C-termini of CXCR4 and CCR5 and the motor protein nonmuscle myosin H chain-IIA. Immunoprecipitation experiments revealed that this association also occurs between the endogenous molecules in T lymphocytes. As expected, myosin L chain was also associated with CXCR4. Confocal microscopy analysis showed that CXCR4 and motor protein nonmuscle myosin H chain-IIA colocalize at the leading edge of migrating T lymphocytes, together with filamentous actin and myosin L chain. These results provide the first evidence of a biochemical association between chemokine receptors and motor proteins, a mechanosignaling mechanism that may have a key role in lymphocyte migration.

    Journal of immunology (Baltimore, Md. : 1950) 2002;169;10;5410-4

  • Defective expression of GPIb/IX/V complex in platelets from patients with May-Hegglin anomaly and Sebastian syndrome.

    Di Pumpo M, Noris P, Pecci A, Savoia A, Seri M, Ceresa IF and Balduini CL

    Department of Internal Medicine, IRCCS S. Matteo Universit di Pavia, Italy.

    May-Hegglin anomaly (MHA) and Sebastian syndrome (SBS) are inherited macrothrombocytopenias with D hle-like bodies in leukocytes. MHA-SBS are due to mutations of the gene (MYH9) for the heavy chain of non-muscle myosin IIA (NMMHC-IIA), the only myosin II expressed in platelets. The bleeding tendency is often more severe than expected on the basis of platelet count, but no abnormality of platelet function has been identified. To characterize platelet abnormalities deriving from MYH9 mutations better, we studied surface glycoproteins (GPs) in platelets from MHA-SBS patients.

    Eight patients from 4 unrelated families were studied. Platelet surface GPs were studied by flow cytometry in both the whole platelet population and subpopulations of platelets identified according to their size.

    Results: Flow cytometry identified a defect of the GPIb/IX/V complex in the whole platelet population in 7 of 8 patients. Moreover, in all patients the subpopulation of large platelets had defective expression of this complex.

    These findings indicate that MYH9 mutations may be responsible for reduced surface expression of GPIb/IX/V. This defect could contribute to the bleeding tendency of these patients. The identification of a GPIb/IX/V defect in MHA-SBS platelets raises the question of the differential diagnosis from heterozygous Bernard-Soulier syndrome.

    Funded by: Telethon: GP0019Y01, TGM06S01

    Haematologica 2002;87;9;943-7

  • PKC epsilon is associated with myosin IIA and actin in fibroblasts.

    England K, Ashford D, Kidd D and Rumsby M

    Department of Biology, University of York, York YO10 5DD, UK. k.england@ucc.ie

    Proteins coimmunoprecipitating with protein kinase C (PKC) epsilon in fibroblasts were identified through matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI TOF m/s). This method identified myosin IIA in PKC epsilon immunoprecipitates, as well as known PKC epsilon binding proteins, actin, beta'Cop and cytokeratin. Myosin is not a substrate for PKC epsilon. Immunofluorescence analysis showed that PKC epsilon is colocalised with actin and myosin in actomyosin stress fibers in fibroblasts. Inhibitors of PKC and myosin ATPase activity, as well as microfilament-disrupting drugs, all inhibited spreading of fibroblasts after passage, suggesting a role for a PKC epsilon-actin-myosin complex in cell spreading.

    Cellular signalling 2002;14;6;529-36

  • Immunocytochemistry for the heavy chain of the non-muscle myosin IIA as a diagnostic tool for MYH9-related disorders.

    Pecci A, Noris P, Invernizzi R, Savoia A, Seri M, Ghiggeri GM, Sartore S, Gangarossa S, Bizzaro N and Balduini CL

    Department of Internal Medicine, IRCCS San Matteo, University of Pavia, Italy.

    May-Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS) are autosomal-dominant macrothrombocytopenias with Döhle-like leucocyte inclusions. These diseases are due to mutations of the MHY9 gene, encoding the heavy chain of non-muscle myosin IIA (NMMHC-A). We investigated the NMMHC-A localization in blood cells from eight MHA, SBS or FTNS patients with known MYH9 mutations. All the patients showed an altered localization of NMMHC-A in granulocytes and platelets, suggesting that Döhle-like bodies are due to the aggregation of NMMHC-A in the cytoplasm. Therefore, immunocytochemistry for NMMHC-A is a simple and sensitive method to detect pathological phenotypes of granulocytes and platelets in the diagnosis of MYH9-related disorders.

    Funded by: Telethon: GP0019Y01, TGM06S01

    British journal of haematology 2002;117;1;164-7

  • Cloning of the murine non-muscle myosin heavy chain IIA gene ortholog of human MYH9 responsible for May-Hegglin, Sebastian, Fechtner, and Epstein syndromes.

    D'Apolito M, Guarnieri V, Boncristiano M, Zelante L and Savoia A

    Servizio di Genetica Medica, IRCCS-Ospedale CSS, I-71013 San Giovanni Rotondo, Foggia, Italy.

    Mutations in the non-muscle myosin heavy chain IIA gene (MYH9) are responsible for May-Hegglin anomaly, Sebastian, Fechtner and Epstein syndromes. These 'MYH9-related' diseases are inherited as an autosomal dominant trait and are characterized by a variable expressivity of clinical features, including macrothrombocytopenia, deafness, nephrites, cataract, and Döhle-like leukocyte inclusions. To gain information of the function of the non-muscle myosin heavy chain IIA protein (NMMHC-IIA), we have identified the murine orthologue Myh9 gene. The gene is localized in a region of chromosome 15 and encodes a predicted protein of 1960 amino acids. This protein shows a high homology to the human NMMHC-IIA with 98% identity. The Myh9 exon-intron junctions were deduced from a murine genomic clone that revealed a perfect conservation of the exon structure between the human and mouse gene. Myh9 is expressed in liver, kidney, lung, and spleen. A low level of transcripts was detected also in heart and brain while no expression was revealed in skeletal muscle and testis. In vertebrates, NMMHC-IIA shows a striking degree of homology to NMMHC-IIB, which is expressed at higher level in mouse brain and testis than in other tissues, confirming the hypothesis that the two non-muscle myosins have different functional roles within cells.

    Funded by: Telethon: GP0019Y01, TGM06S01

    Gene 2002;286;2;215-22

  • Epstein syndrome: another renal disorder with mutations in the nonmuscle myosin heavy chain 9 gene.

    Seri M, Savino M, Bordo D, Cusano R, Rocca B, Meloni I, Di Bari F, Koivisto PA, Bolognesi M, Ghiggeri GM, Landolfi R, Balduini CL, Zelante L, Ravazzolo R, Renieri A and Savoia A

    Laboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa, Italy.

    Epstein syndrome (EPTS) is an autosomal dominant disease characterized by nephritis, mild hearing loss, and thrombocytopenia with giant platelets. Renal and hearing abnormalities are indistinguishable from those observed in Fechtner syndrome (FTNS), an Alport-like variant. EPTS macrothrombocytopenia is similar to that described in FTNS, May-Hegglin anomaly (MHA), and Sebastian syndrome (SBS), three disorders caused by mutations in the nonmuscle heavy chain myosin IIA ( MYH9). Unlike FTNS, MHA, and SBS, EPTS does not show inclusion bodies in the leukocytes. The clinical features of EPTS and the chromosomal localization of the respective gene in the same region as MYH9 suggest that this disorder is allelic with the other giant platelet disorders. We identified a MYH9 missense mutation in two EPTS familial cases. In both families, an R702H substitution was found, probably inducing conformational changes to the myosin head. A different amino acid substitution at the same codon (R702C) has been previously identified in FTNS. On the basis of predictions from molecular modeling of the X-ray crystallographic structure of chick smooth muscle myosin, the mutated thiol reactive group of R702C may lead to intermolecular disulfide bridges, with the consequent formation of the inclusions typical of FTNS. On the contrary, the R702H mutation does not allow the protein to aggregate and thus to generate "Döhle-like" bodies, which are indeed absent in EPTS. In conclusion, our results extend the allelic heterogeneity of MYH9 mutations to another clinical syndrome and contribute to the clarification of the pathogenesis of the various inherited giant platelet disorders.

    Funded by: Telethon: GP0019Y01, TGM06S01

    Human genetics 2002;110;2;182-6

  • Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.

    Arrondel C, Vodovar N, Knebelmann B, Grünfeld JP, Gubler MC, Antignac C and Heidet L

    Inserm U 423, Université René Descartes, Hôpital Necker-Enfants Malades, Paris, France.

    Mutations in the MYH9 gene, which encodes the nonmuscle myosin heavy chain IIA, have been recently reported in three syndromes that share the association of macrothrombocytopenia (MTCP) and leukocyte inclusions: the May-Hegglin anomaly and Sebastian and Fechtner syndromes. Epstein syndrome, which associates inherited sensorineural deafness, glomerular nephritis, and MTCP without leukocyte inclusions, was shown to be genetically linked to the same locus at 22q12.3 to 13. The expression of MYH9 in the fetal and mature human kidney was studied, and the 40 coding exons of the gene were screened by single-strand conformation polymorphism in 12 families presenting with the association of MTCP and nephropathy. MYH9 is expressed in both fetal and mature kidney. During renal development, it is expressed in the late S-shaped body, mostly in its lower part, in the endothelial and the epithelial cell layers. Later, as well as in mature renal tissue, MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells. Four missense heterozygous mutations that are thought to be pathogenic were found in five families, including two families with Epstein syndrome. Three mutations were located in the coiled-coil rod domain of the protein, and one was in the motor domain. Two mutations (E1841K and D1424N) have been reported elsewhere in families with May-Hegglin anomaly. The two others (R1165L and S96L) are new mutations, although one of them affects a codon (R1165), found elsewhere to be mutated in Sebastian syndrome.

    Journal of the American Society of Nephrology : JASN 2002;13;1;65-74

  • Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes.

    Heath KE, Campos-Barros A, Toren A, Rozenfeld-Granot G, Carlsson LE, Savige J, Denison JC, Gregory MC, White JG, Barker DF, Greinacher A, Epstein CJ, Glucksman MJ and Martignetti JA

    Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA.

    May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.

    Funded by: NICHD NIH HHS: 5 P30 HD28822

    American journal of human genetics 2001;69;5;1033-45

  • Mutations in the NMMHC-A gene cause autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly/Sebastian syndrome).

    Kunishima S, Kojima T, Matsushita T, Tanaka T, Tsurusawa M, Furukawa Y, Nakamura Y, Okamura T, Amemiya N, Nakayama T, Kamiya T and Saito H

    First Department of Internal Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan.

    Macrothrombocytopenia with leukocyte inclusions is a rare autosomal dominant platelet disorder characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. A previous study mapped a locus for the disease on chromosome 22q12.3-q13.2 by genome-wide linkage analysis. In addition, the complete DNA sequence of human chromosome 22 allowed a positional candidate approach, and results here indicate that the gene encoding nonmuscle myosin heavy chain-A, NMMHC-A, is mutated in this disorder. Mutations were found in 6 of 7 Japanese families studied: 3 missense mutations, a nonsense mutation, and a one-base deletion resulting in a premature termination. Immunofluorescence studies revealed that NMMHC-A distribution in neutrophils appeared to mimic the inclusion bodies. These results provide evidence for the involvement of abnormal NMMHC-A in the formation of leukocyte inclusions and also in platelet morphogenesis.

    Blood 2001;97;4;1147-9

  • Identification of six novel MYH9 mutations and genotype-phenotype relationships in autosomal dominant macrothrombocytopenia with leukocyte inclusions.

    Kunishima S, Matsushita T, Kojima T, Amemiya N, Choi YM, Hosaka N, Inoue M, Jung, Mamiya S, Matsumoto K, Miyajima Y, Zhang G, Ruan C, Saito K, Song KS, Yoon HJ, Kamiya T and Saito H

    First Department of Internal Medicine, Nagoya University School of Medicine, Japan.

    The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly (MHA), Sebastian syndrome (SBS), and Fechtner syndrome (FTNS), are rare platelet disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. The locus for these disorders was previously mapped on chromosome 22q12.3-q13.2 and the disease gene was recently identified as MYH9, the gene encoding the nonmuscle myosin heavy chain-A. To elucidate the spectrum of MYH9 mutations responsible for the disorders and to investigate genotypephenotype correlation, we examined MYH9 mutations in an additional 11 families and 3 sporadic patients with the disorders from Japan. Korea, and China. All 14 patients had heterozygous MYH9 mutations, including three known mutations and six novel mutations (three missense and three deletion mutations). Two cases had Alport manifestations including deafness, nephritis, and cataracts and had R1165C and E1841K mutations, respectively. However, taken together with three previous reports, including ours, the data do not show clear phenotype-genotype relationships. Thus, MHA, SBS, and FTNS appear to represent a class of allelic disorders with variable phenotypic diversity.

    Journal of human genetics 2001;46;12;722-9

  • Autosomal-dominant giant platelet syndromes: a hint of the same genetic defect as in Fechtner syndrome owing to a similar genetic linkage to chromosome 22q11-13.

    Toren A, Rozenfeld-Granot G, Rocca B, Epstein CJ, Amariglio N, Laghi F, Landolfi R, Brok-Simoni F, Carlsson LE, Rechavi G and Greinacher A

    Pediatric Hemato-Oncology Department and the Institute of Hematology, the Chaim Sheba Medical Center, Tel-Hashomer, Tel-Aviv, Israel. amost@post.tau.ac.il

    Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations-deafness, cataracts, and nephritis-and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.

    Blood 2000;96;10;3447-51

  • Human nonsyndromic hereditary deafness DFNA17 is due to a mutation in nonmuscle myosin MYH9.

    Lalwani AK, Goldstein JA, Kelley MJ, Luxford W, Castelein CM and Mhatre AN

    Laboratory of Molecular Otology, Epstein Laboratories, Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco, San Francisco, CA 94143, USA. lalwani@itsa.ucsf.edu

    The authors had previously mapped a new locus-DFNA17, for nonsyndromic hereditary hearing impairment-to chromosome 22q12.2-q13. 3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle-myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase-PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G-->A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated.

    Funded by: NHLBI NIH HHS: R01 HL066192; NIDCD NIH HHS: F31 DC000112, K23DC00112

    American journal of human genetics 2000;67;5;1121-8

  • Localisation of the gene responsible for fechtner syndrome in a region <600 Kb on 22q11-q13.

    Cusano R, Gangarossa S, Forabosco P, Ghiggeri GM, Russo G, Iolascon A, Ravazzolo R and Seri M

    Laboratorio Genetica Molecolare, Istituto G Gaslini, Genova, Biologia e Genetica, Università di Genova, Italy.

    Fechtner syndrome is an autosomal dominant disorder which has been thought to be a variant of Alport syndrome. It is characterised by nephritis, sensorineural hearing loss and eye abnormalities, as well as by macrothrombocytopenia and polymorphonuclear inclusion bodies. Recently, the Fechtner syndrome has been mapped in a 5.5 Mb region on the long arm of chromosome 22 by linkage analysis in an extended Israeli family. We describe here the genetic refinement of the Fechtner critical interval to a region less than 600 Kb by linkage analysis performed in a large Italian pedigree. The presence of several recombination events allowed the disease gene to be localised between markers D22S278 and D22S426, in a region containing only two non-recombinant markers, D22S1173 and D22S283. This interval, spanning <600 Kb on genomic DNA, has been entirely sequenced and contains six known and three putative genes.

    European journal of human genetics : EJHG 2000;8;11;895-9

  • Conditional expression of a truncated fragment of nonmuscle myosin II-A alters cell shape but not cytokinesis in HeLa cells.

    Wei Q and Adelstein RS

    Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

    A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1-591, delta N592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and delta N592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and delta N592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed delta N592 did not bind to actin, suggesting that delta N592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of delta N592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of delta N592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not delta N592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.

    Molecular biology of the cell 2000;11;10;3617-27

  • Mutation of MYH9, encoding non-muscle myosin heavy chain A, in May-Hegglin anomaly.

    Kelley MJ, Jawien W, Ortel TL and Korczak JF

    Department of Medicine, Duke University, Durham, North Carolina, USA. kellyeym@duke.edu

    May-Hegglin anomaly (MHA) is an autosomal dominant macrothrombocytopenia of unclear pathogenesis characterized by thrombocytopenia, giant platelets and leukocyte inclusions. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. The disorder has been linked to chromosome 22q12-13. Here we sc 114b reen a candidate gene in this region, encoding non-muscle myosin heavy chain A (MYH9), for mutations in ten families. In each family, we identified one of three sequence variants within either the -helical coiled coil or the tailpiece domain that co-segregated with disease status. The E1841K mutation was found in 5 families and occurs at a conserved site in the rod domain. This mutation was not found in 40 normal individuals. Four families had a nonsense mutation that resulted in truncation of most of the tailpiece. One family had a T1155I mutation present in an affected mother and daughter, but not in the mother's parents, thus representing a new mutation. Among the 30 affected individuals, 21 unaffected individuals and 13 spouses in the 10 families, there was correlation of a variant of MYH9 with the presence of MHA. The identification of MYH9 as the disease gene for MHA establishes the pathogenesis of the disorder, should provide further insight into the processes of normal platelet formation and may facilitate identification of the genetic basis of related disorders.

    Funded by: NHLBI NIH HHS: R01 HL066192

    Nature genetics 2000;26;1;106-8

  • Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

    Seri M, Cusano R, Gangarossa S, Caridi G, Bordo D, Lo Nigro C, Ghiggeri GM, Ravazzolo R, Savino M, Del Vecchio, d'Apolito M, Iolascon A, Zelante LL, Savoia A, Balduini CL, Noris P, Magrini U, Belletti S, Heath KE, Babcock M, Glucksman MJ, Aliprandis E, Bizzaro N, Desnick RJ and Martignetti JA

    Laboratory of Molecular Genetics, Institute G. Gaslini, Genoa, Italy.

    The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

    Funded by: NCRR NIH HHS: 5 M01 RR00071; NICHD NIH HHS: 5 P30 HD28822; NIDDK NIH HHS: 5 R37 DK34045; Telethon: E.1313

    Nature genetics 2000;26;1;103-5

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Autosomal dominant macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is linked to chromosome 22q12-13.

    Kelley MJ, Jawien W, Lin A, Hoffmeister K, Pugh EW, Doheny KF and Korczak JF

    Department of Medicine, Duke University, Durham, NC, USA. kelleym@duke.edu

    Macrothrombocytopenia with leukocyte inclusions (May-Hegglin anomaly) is a rare autosomal dominant disorder characterized by thrombocytopenia, giant platelets, and Döhle body-like inclusions in leukocytes. To determine the genetic basis of this disorder, we performed a genome-wide screen for linkage in three families with May-Hegglin anomaly. For the pooled analysis of the three families, three markers on chromosome 22 had two-point logarithm-of-difference (lod) scores greater than 3, with a maximum lod score of 3.91 at a recombination fraction (theta) of 0.076 for marker D22S683. Within the largest family (MHA-1), the maximum lod score was 5.36 at theta=0 at marker D22S445. Fine mapping of recombination events using eight adjacent markers indicated that the minimal disease region of family MHA-1 alone is in the approximately 26 cM region from D22S683 to the telomere. The maximum lod score for the three families combined was 5.84 at theta=0 for marker IL2RB. With the assumption of locus homogeneity, haplotype analysis of family MHA-4 indicated the disease region is centromeric to marker D22S1045. These data best support a minimal disease region from D22S683 to D22S1045, a span of about 1 Mb of DNA that contains 17 known genes and 4 predicted genes. Further analysis of this region will identify the genetic basis of May-Hegglin anomaly, facilitating subsequent characterization of the biochemical role of the disease gene in platelet formation.

    Funded by: NHGRI NIH HHS: N01-HG-65403; NHLBI NIH HHS: R01 HL066192

    Human genetics 2000;106;5;557-64

  • Protein complexes involving alpha v beta 3 integrins, nonmuscle myosin heavy chain-A, and focal adhesion kinase from in thrombospondin-treated smooth muscle cells.

    Sajid M, Hu Z, Lele M and Stouffer GA

    Sealy Center for Molecular Cardiology, University of Texas Medical Branch, Galveston, USA.

    alpha v beta 3 integrins have been implicated in regulating vascular healing in animal models of arterial injury. Because the specific cellular events mediated by alpha v beta 3 integrins are not completely understood, we examined alpha v beta 3 integrin-dependent cytoplasmic events in cultured human smooth muscle cells (SMC) following treatment with thrombospondin-1 (TSP), a glycoprotein concentrated at sites of blood vessel injury. TSP treatment elicited a time-dependent association of nonmuscle myosin heavy chain-A (NMHC-A) with alpha v beta 3 integrins. NMHC-A also associated with focal adhesion kinase (FAK) in TSP-treated SMC. FAK, a nonreceptor kinase implicated in integrin-mediated signaling, was phosphorylated on tyrosine in growth-arrested SMC, but levels of tyrosine phosphorylation increased following treatment with TSP. To test whether NMHC-A was regulated by vascular injury, we examined expression in baboon brachial arteries. In uninjured arteries, NMHC-A staining was present in the media. In arteries injured by balloon withdrawal, medial NMHC-A expression was increased with intense staining at specific sites. In summary, heteromeric protein complexes involving alpha v beta 3 integrins, NMHC-A, and FAK form following treatment of human SMC with TSP. These results suggest that the formation of protein signaling complexes is one mechanism whereby alpha v beta 3 integrins influence intracellular signaling pathways.

    Journal of investigative medicine : the official publication of the American Federation for Clinical Research 2000;48;3;190-7

  • The DNA sequence of human chromosome 22.

    Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Bruskiewich R, Beare DM, Clamp M, Smink LJ, Ainscough R, Almeida JP, Babbage A, Bagguley C, Bailey J, Barlow K, Bates KN, Beasley O, Bird CP, Blakey S, Bridgeman AM, Buck D, Burgess J, Burrill WD, O'Brien KP et al.

    Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. id1@sanger.ac.uk

    Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

    Nature 1999;402;6761;489-95

  • The interface between MyBP-C and myosin: site-directed mutagenesis of the CX myosin-binding domain of MyBP-C.

    Miyamoto CA, Fischman DA and Reinach FC

    Instituto de Química, Universidade de São Paulo, Brazil.

    Myosin-binding protein-C (MyBP-C or C-protein) is a ca. 130 kDa protein present in the thick filaments of all vertebrate striated muscle. The protein contains ten domains, each of ca. 90-100 amino acids; seven are members of the IgI family of proteins, three of the fibronectin type III family. The motifs are arranged in the following order (from N- to C-terminus): Ig-Ig-Ig-Ig-Ig-Fn-Fn-Ig-Fn-Ig. The C-terminal Ig motif (domain X or CX) contains its light meromyosin-binding site. A recombinant form of CX, beginning at Met-1027, exhibits saturable binding to myosin with an affinity comparable to the C-terminal 13 kDa chymotryptic fragment of native MyBP-C. To identify the surface in CX involved in its interaction with myosin, nine site-directed mutants (R37E, K43E, N49D, E52R, D56K, R73E, R74E, G80D and R103E) were constructed. Using a new assay for assessing the binding of CX with the light meromyosin (LMM) portion of myosin, we demonstrate that recombinant CX, just as the full-length protein, is able to facilitate LMM polymerization. Moreover, we show that residues Arg-37, Glu-52, Asp-56, Arg-73, and Arg-74 are involved in this interaction with the myosin rod. All of these amino acids interact with negatively charged residues of LMM, since the mutants R37E, R73E and R74E are unable to bind myosin, whereas E52R and D56K bind myosin with higher affinity than wild-type CX. Residues Lys-43 and Arg-103 show a small but significant influence on the binding reaction; residues Asn-49 and Gly-80 seem not to be involved in this interaction. Based on these data, a model is proposed for the interaction between MyBP-C CX and myosin filaments. In this model, CX interacts with four molecules of LMM at four different sites of the binding protein, thus explaining the effects of MyBP-C on the critical concentration of myosin polymerization.

    Funded by: NHLBI NIH HHS: HL45458; NIAMS NIH HHS: AR3214

    Journal of muscle research and cell motility 1999;20;7;703-15

  • Mapping of a myosin-binding domain and a regulatory phosphorylation site in M-protein, a structural protein of the sarcomeric M band.

    Obermann WM, van der Ven PF, Steiner F, Weber K and Fürst DO

    Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry, D-37077 Göttingen, Germany.

    The myofibrils of cross-striated muscle fibers contain in their M bands cytoskeletal proteins whose main function seems to be the stabilization of the three-dimensional arrangement of thick filaments. We identified two immunoglobin domains (Mp2-Mp3) of M-protein as a site binding to the central region of light meromyosin. This binding is regulated in vitro by phosphorylation of a single serine residue (Ser76) in the immediately adjacent amino-terminal domain Mp1. M-protein phosphorylation by cAMP-dependent kinase A inhibits binding to myosin LMM. Transient transfection studies of cultured cells revealed that the myosin-binding site seems involved in the targeting of M-protein to its location in the myofibril. Using the same method, a second myofibril-binding site was uncovered in domains Mp9-Mp13. These results support the view that specific phosphorylation events could be also important for the control of sarcomeric M band formation and remodeling.

    Molecular biology of the cell 1998;9;4;829-40

  • Effect of Mts1 on the structure and activity of nonmuscle myosin II.

    Ford HL, Silver DL, Kachar B, Sellers JR and Zain SB

    Department of Biochemistry and Cancer Center, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, USA.

    The mts1 gene codes for a 9 kDa protein belonging to the S100 subfamily of Ca2+-binding proteins and is known to play a role in metastasis. Its role in metastasis may be through cellular locomotion, as transfection of mts1 into mouse mammary adenocarcinoma cells increases cellular motility in modified Boyden chemotaxis chambers. The Mts1 protein interacts with nonmuscle myosin II in the presence of Ca2+ with an affinity of approximately 7.9 x 10(4) M-1 and an approximate stoichiometry of 3 mol of Mts1/mol of myosin heavy chain. No interaction was found with myosin I or myosin V. The binding site of Mts1 on myosin is in the rod region, particularly to the light meromyosin portion of the rod. To understand the mechanism by which Mts1 alters cellular motility, we examined its effect on myosin structure and activity. Cosedimentation analysis and electron microscopy suggest that Mts1 destabilizes myosin filaments. In the presence of Ca2+, Mts1 inhibits the actin-activated MgATPase activity of myosin in vitro. The data demonstrate an effect of Mts1 on both myosin structure and function, and suggest a route through which Mts1 affects motility as well as metastasis.

    Funded by: NCI NIH HHS: P30CA11198, R01CA62376

    Biochemistry 1997;36;51;16321-7

  • A five-generation family with late-onset progressive hereditary hearing impairment due to cochleosaccular degeneration.

    Lalwani AK, Linthicum FH, Wilcox ER, Moore JK, Walters FC, San Agustin TB, Mislinski J, Miller MR, Sinninger Y, Attaie A and Luxford WM

    Department of Otolaryngology, Head and Neck Surgery, University of California, San Francisco, USA. lalwani@itsa.ucsf.edu

    Cochleosaccular dysplasia or degeneration (Scheibe degeneration) is considered the most common cause of profound congenital hearing impairment, and accounts for approximately 70% of cases 2 with hereditary deafness. A five-generation family with hereditary hearing impairment associated with cochleosaccular degeneration has recently been identified. The diagnosis of classical Scheibe degeneration was based on histopathological findings in the temporal bones of the proband, a 61-year-old profoundly deaf male. Auditory structures in the brainstem of the proband were also studied. Twenty-two members of the family were contacted for surveys and blood samples. Of these, 6 males and 2 females have hearing impairment. Complete audiological evaluation was done on 12 family members, and prior audiologic records of the proband and affected family members were available for study. Affected family members suffer a mild bilateral high-frequency hearing loss during childhood and adolescence, and progress to moderate-to-profound deafness in the second and third decades of life. The family is suitable for linkage analysis and does not map to previously reported loci harboring autosomal dominant, nonsyndromic hereditary hearing impairment genes. The genetic study of this family will be helpful in identifying the genes which, when mutated, result in Scheibe degeneration.

    Audiology & neuro-otology 1997;2;3;139-54

  • Purification and bio 1f40 chemical characterization of myomesin, a myosin-binding and titin-binding protein, from bovine skeletal muscle.

    Obermann WM, Plessmann U, Weber K and Fürst DO

    Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Germany.

    We report a method for isolating homogeneous myomesin from mammalian skeletal muscle. The identity of the purified bovine protein was confirmed by its reactivity with myomesin-specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the amino-terminal and carboxy-terminal ends of the sequence predicted by the human myomesin cDNA. All partial sequences obtained from bovine myomesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods with a molecular length of about 50 nm. Circular dichroism spectra showed a high degree of beta structure as expected for a member of the immunoglobulin superfamily of proteins. Alignment of the sequences of the class I and II domains of myomesin with the sequences of domains of known three-dimensional structure provides a more detailed model of myomesin. In agreement with this view, the cleavage sites observed by limited proteolysis locate primarily between individual domains. In a solid-phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy-terminal 30-kDa fragment of LMM. The myosin-binding site seemed to be confined to the amino-terminal 240 residues of the molecule. The cross-reactivity of myomesin with the phosphorylation-dependent monoclonal neurofilament antibody NE14 [Shaw, G.E., Debus, E. & Weber, K. (1984) Eur. J. Cell Biol. 34, 130-136] was analyzed. NE14 reactivity of myomesin was abolished by alkaline phosphatase. Reactivity of the antibody on stable proteolytic fragments of myomesin showed that the phosphorylation site must reside within the carboxy-terminal 60 residues.

    European journal of biochemistry 1995;233;1;110-5

  • Differential localization of myosin-II isozymes in human cultured cells and blood cells.

    Maupin P, Phillips CL, Adelstein RS and Pollard TD

    Department of Cell Biology and Anatomy, Johns Hopkins Medical School, Baltimore, MD 21205-2196.

    We used purified polyclonal antibodies to human cytoplasmic myosin-IIA and myosin-IIB directly labeled with fluorescent dyes to localize these myosin-II isozymes in HeLa cells, melanoma cells and blood cells. Both antibodies react strongly with myosin-II isozymes in HeLa cells, melanoma cells and blood eosinophils, but only anti-myosin-IIA antibodies stain platelets, lymphocytes, neutrophils and monocytes in smears of human blood. Both antibodies stain small spots along the stress fibers of interphase HeLa cells and melanoma cells, but double staining revealed that the detailed distributions of myosin-IIA and myosin-IIB differ. A low concentration of diffuse myosin-IIB is present in the cortex, both in lamellar regions around the periphery of the cell and over the free surface. Myosin-IIB is also concentrated in spots along perinuclear stress fibers. Myosin-IIA is absent from the cortex but is concentrated in spots along stress fibers located near the basal surface of cultured cells. This population of peripheral stress fibers is highly enriched in myosin-IIA relative to myosin-IIB, but both are found together in centrally located stress fibers. In prophase and metaphase both isozymes are concentrated in the cortex in small spots less than 04.micron in size, similar to those in stress fibers. As the chromosomes begin the separate at anaphase, most of the myosin-II spots become concentrated in the outer 0.7 micron of the equatorial cortex in 100% of cells. This concentration of myosin-II isozymes in the cleavage furrow is maintained until the daughter cells separate. The superimposition of these small spots concentrated in the cleavage furrow produces the intense, uniform staining observed in conventional micrographs of whole cells.

    Funded by: NIGMS NIH HHS: GM-26132

    Journal of cell science 1994;107 ( Pt 11);3077-90

  • Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro.

    Eilertsen KJ, Kazmierski ST and Keller TC

    Molecular Biophysics Program, Florida State University, Tallahassee 32306-3050.

    We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.

    The Journal of cell biology 1994;126;5;1201-10

  • Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types.

    Bement WM, Hasson T, Wirth JA, Cheney RE and Mooseker MS

    Department of Biology, Yale University, New Haven, CT 06520-8103.

    Myosin diversity in the human epithelial cell line Caco-2BBe, the porcine epithelial cell line LLC-PK1 (CL-4), human peripheral blood leukocytes, and human liver was analyzed. PCR amplification yielded 8-11 putative myosins (depending on the cDNA source) representing six distinct myosin classes. Analysis of clones obtained by hybridization screening demonstrated that the original PCR products correspond to bona fide myosins, based on the presence of sequences highly conserved in other myosins. RNase protection analysis confirmed mRNA expression of 11 myosins in Caco-2BBe cells. Immunoblot analysis showed that at least 6 myosin immunogens are expressed in Caco-2BBe cells. The results reveal the existence of at least 11 unconventional human myosin genes, most of which are expressed in an overlapping fashion in different cell types. The abundance of myosins suggests that the myosin I vs. myosin II paradigm is inadequate to explain actin-based cellular motility.

    Funded by: NIDDK NIH HHS: DK 25387, DK 34989, DK 38979

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;14;6549-53

  • Cleavage of human and mouse cytoskeletal and sarcomeric proteins by human immunodeficiency virus type 1 protease. Actin, desmin, myosin, and tropomyosin.

    Shoeman RL, Sachse C, Höner B, Mothes E, Kaufmann M and Traub P

    Max-Planck-Institut für Zellbiologie, Ladenburg, Federal Republic of Germany.

    HeLa cell actin was cleaved by human immunodeficiency virus type 1 protease when in its soluble, globular form (G-actin). No cleavage of the polymerized, filamentous form of actin (F-actin) was observed when examined by denaturing gel electrophoresis; however, electron microscopy revealed a low level of cleavage of F-actin. Immunoblotting of mouse skeletal and human pectoral muscle myofibrils treated in vitro with human immunodeficiency virus type 1 protease showed that myosin heavy chain, desmin, tropomyosin, and a fraction of the actin were all cleaved. Electron microscopy of these myofibrils demonstrated changes consistent with cleavage of these proteins: Z-lines were rapidly lost, the length of the A bands was shortened, and the thick filaments (myosin filaments) were often laterally frayed such that the structures disintegrated. Nonmuscle myosin heavy chains were also cleaved by this enzyme in vitro. These data demonstrate that this protease can cause alterations in muscle cell ultrastructure in vitro that may be of clinical relevance in infected individuals.

    The American journal of pathology 1993;142;1;221-30

  • Large tidal volume ventilation improves pulmonary gas exchange during lower abdominal surgery in Trendelenburg's position.

    Tweed WA, Phua WT, Chong KY, Lim E and Lee TL

    Department of Anaesthesia, National University Hospital, National University of Singapore.

    Impaired pulmonary gas exchange is a common complication of general anaesthesia. Periodic hyperinflation of the lungs and large tidal volume ventilation were the first preventive measures to be widely embraced, but their effectiveness in clinical practice has never been clearly established by controlled clinical studies. To assess their effects in high-risk patients we studied 24 adults having lower abdominal gynaecological surgery in the Trendelenburg (head down) position. Pulmonary oxygen exchange was determined during four steady-states: awake control (AC), after 30 min of conventional tidal volume (CVT, 7.5 ml.kg-1) or high tidal volume (HVT, 12.7 ml.kg-1) ventilation, introduced in random order, and five minutes after manual hyperinflations (HI) of the lungs. The patients' lungs were ventilated with air/O2 by an Ohmeda volume-controlled ventilator via a circle system. The FIO2 was controlled at 0.5, and FETCO2 was controlled by adding dead space during HVT. Arterial blood gas analysis was used to calculate the oxygen tension-based indices of gas exchange. There was significant deterioration of (A-a)DO2 at 30 min in Group A, whose lungs were first ventilated with CVT (81.6 +/- 7.2 to 166.8 +/- 13.7 mmHg, P less than 0.001); but not in Group B, whose lungs were first ventilated with HVT (77.0 +/- 9.9 to 104.4 +/- 16.8 mmHg). When Group A and B data were pooled there was no difference between randomized CVT and HVT, but improvement occurred after HI. In this model of compromised O2 exchange large inflation volumes (HVT and HI) were of considerable clinical benefit, HVT prevented and HI reversed the gas exchange disorder.

    Canadian journal of anaesthesia = Journal canadien d'anesthesie 1991;38;8;989-95

  • Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation.

    Toothaker LE, Gonzalez DA, Tung N, Lemons RS, Le Beau MM, Arnaout MA, Clayton LK and Tenen DG

    Hematology/Oncology Division, Beth Israel Hospital, Boston, MA 02215.

    We have isolated 5' cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.

    Funded by: NCI NIH HHS: CA41456, CA42557

    Blood 1991;78;7;1826-33

  • Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes.

    Simons M, Wang M, McBride OW, Kawamoto S, Yamakawa K, Gdula D, Adelstein RS and Weir L

    Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

    We report the cloning of cDNAs encoding two different human nonmuscle myosin heavy chains designated NMMHC-A and NMMHC-B. The mRNAs encoding NMMHC-A and NMMHC-B are both 7.5 kb in size but are shown to be the products of different genes, which are localized to chromosome 22q11.2 and chromosome 17q13, respectively. In aggreement with previously reported results using avian tissues, we show that the mRNAs encoding the two myosin heavy chain isoforms are differentially expressed in rat nonmuscle and muscle tissues as well as in a number of human cell lines. The cDNA sequence encoding the 5' portion of the NMMHC-A isoform completes the previously published 3' cDNA sequence encoding a human myosin heavy chain, thus providing the cDNA sequence encoding the entire NMMHC-A amino acid sequence. Comparison of this sequence to cDNA clones encoding the amino-terminal one third of the NMMHC-B sequence (amino acids 58-718) shows them to be 89% identical at the amino acid level and 74% identical at the nucleotide level.

    Circulation research 1991;69;2;530-9

  • Human nonmuscle myosin heavy chain mRNA: generation of diversity through alternative polyadenylylation.

    Saez CG, Myers JC, Shows TB and Leinwand LA

    Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.

    Myosin is a ubiquitous eukaryotic contractile protein that generates the force responsible for such diverse cellular movements as muscle contraction and cytokinesis. Although there have been numerous studies of sarcomeric myosin heavy chain (MHC) genes, no molecular clones have been reported that encode mammalian nonmuscle MHC. This study presents the molecular genetic characterization of a human nonmuscle MHC that is expressed in fibroblasts, endothelial cells, and macrophages. Human nonmuscle MHC amino acids are weakly homologous (33%) to sarcomeric MHC but are approximately 72% identical to smooth muscle MHC. In contrast to vertebrate sarcomeric MHCs, which generate diversity through the expression of members of a multigene family, an alternative polyadenylylation site is used in the nonmuscle MHC gene to generate multiple transcripts that encode the same protein. We have mapped this gene to chromosome 22. It is thus unlinked to either of the sarcomeric MHC gene clusters on human chromosomes 14 and 17.

    Funded by: NHLBI NIH HHS: HL41882; NIGMS NIH HHS: GM20553, GM29090

    Proceedings of the National Academy of Sciences of the United States of America 1990;87;3;1164-8

  • Effect of C-protein on actomyosin ATPase.

    Moos C and Feng IN

    The effect of C-protein on the actin-activated ATPase of column-purified skeletal muscle myosin has been investigated at varied ionic strength. At ionic strengths below about 0.1, C-protein is a potent inhibitor. The inhibition is not reversed by increasing the actin concentration, showing that it is caused by C-protein bound to the myosin filaments. When the ionic strength is raised above about 0.12, on the other hand, the inhibition vanishes and C-protein becomes a mild activator of the actomyosin ATPase. Both effects appear rapidly upon addition of C-protein to pre-formed myosin filaments, so C-protein probably acts by binding to the surface of the filaments.

    Biochimica et biophysica acta 1980;632;2;141-9

  • The interaction of C-protein with heavy meromyosin and subfragment-2.

    Starr R and Offer G

    C-protein has previously been shown to bind to the light-meromyosin region of the myosin tail. Examination of mixtures of C-protein with heavy meromyosin or subfragment-2 or subfragment-1 in the analytical ultracentrifuge shows that there is also a binding site for C-protein in the subfragment-2 region of the tail.

    The Biochemical journal 1978;171;3;813-6

  • Enzymic and immunochemical properties of lysozyme. Accurate definition of the antigenic site around the disulphide bridge 30-115 (site 3) by 'surface-simulation' synthesis.

    Lee CL and Atassi MZ

    1. Previous reports from this laboratory have shown that both Lys-33 and Lys-116 are parts of an antigenic site in native lysozyme. Similar studies of tyrosine derivatives indicated that one or both of Tyr-20 and Tyr-23 are located in or very close to an antigenic site in lysozyme. The site, which was located around the disulphide bridge 30-115, was recently shown unequivocally to include the residues Tyr-20, Arg-21, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. This was confirmed by the ;surface-simulation' synthetic approach that we have recently developed, in which the foregoing eight surface residues were directly linked via peptide bonds, with intervening spacers where appropriate, into a single peptide. The peptide does not exist in native lysozyme, but simulates a surface region of it. 2. In the present work several surface-simulation peptides were synthesized representing various parts of the region, to determine the minimum structural feature that retains full antigenic reactivity and to investigate if the spatially constructed antigenic site has a preferred direction. 3. The peptide Lys-Asn-Arg-Gly-Phe-Lys exhibited a remarkable inhibitory activity towards the immune reaction of lysozyme and accounted entirely for the maximum expected reactivity of the site in the native protein (i.e. about one-third of the total lysozyme reactivity). An immunoadsorbent of the peptide bound about one-third of the total antibody to lysozyme. 4. The residues Tyr-20 and Arg-21 are not part of the site. The previously reported immunochemical effect observed on nitration of Tyr-20 was due to a deleterious ionic effect exerted by the modified tyrosine residue on the adjacent Lys-96, which is in an entirely different antigenic site of lysozyme. Thus the modification of Tyr-20 impairs the reactivity of an adjacent antigenic site, even though the residue itself is not part of a site. The conformational and immunochemical implications of this finding are discussed. 5. The antigenic site therefore comprises the five spatially adjacent residues Lys-116, Asn-113, Arg-114, Phe-34, Lys-33. The antigenic site exhibited a preferred direction (Lys-116 to Lys-33), since the reverse surface-simulation synthetic sequence was immunochemically inefficient. The site describes a line which circumscribes part [2.1nm in C((alpha))-C((alpha)) distance from Lys-116 to Lys-33] of the surface of the molecule.

    The Biochemical journal 1977;167;3;571-81

Gene lists (7)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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