G2Cdb::Gene report

Gene id
Gene symbol
Actn2 (MGI)
Mus musculus
actinin alpha 2
G00001792 (Homo sapiens)

Databases (9)

Curated Gene
OTTMUSG00000017028 (Vega mouse gene)
ENSMUSG00000052374 (Ensembl mouse gene)
11472 (Entrez Gene)
4 (G2Cdb plasticity & disease)
Gene Expression
NM_033268 (Allen Brain Atlas)
11472 (Genepaint)
102573 (OMIM)
Marker Symbol
MGI:109192 (MGI)
Protein Sequence
Q9JI91 (UniProt)

Literature (43)

Pubmed - other

  • DOT1L regulates dystrophin expression and is critical for cardiac function.

    Nguyen AT, Xiao B, Neppl RL, Kallin EM, Li J, Chen T, Wang DZ, Xiao X and Zhang Y

    Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, NC 27599, USA.

    Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin-glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.

    Funded by: Howard Hughes Medical Institute; NCI NIH HHS: CA119133, R01 CA119133

    Genes & development 2011;25;3;263-74

  • Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology.

    Gokhin DS, Lewis RA, McKeown CR, Nowak RB, Kim NE, Littlefield RS, Lieber RL and Fowler VM

    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.

    Funded by: NEI NIH HHS: P30 EY012598, P30-EY12598; NHLBI NIH HHS: HL007195, HL083464, R01 HL083464, T32 HL007089, T32 HL007195; NIAMS NIH HHS: AR40050, F32 AR055870, R01 AR040050

    The Journal of cell biology 2010;189;1;95-109

  • G-CSF promotes the proliferation of developing cardiomyocytes in vivo and in derivation from ESCs and iPSCs.

    Shimoji K, Yuasa S, Onizuka T, Hattori F, Tanaka T, Hara M, Ohno Y, Chen H, Egasgira T, Seki T, Yae K, Koshimizu U, Ogawa S and Fukuda K

    Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo 160-8582, Japan.

    During a screen for humoral factors that promote cardiomyocyte differentiation from embryonic stem cells (ESCs), we found marked elevation of granulocyte colony-stimulating factor receptor (G-CSFR) mRNA in developing cardiomyocytes. We confirmed that both G-CSFR and G-CSF were specifically expressed in embryonic mouse heart at the midgestational stage, and expression levels were maintained throughout embryogenesis. Intrauterine G-CSF administration induced embryonic cardiomyocyte proliferation and caused hyperplasia. In contrast, approximately 50% of csf3r(-/-) mice died during late embryogenesis because of the thinning of atrioventricular walls. ESC-derived developing cardiomyocytes also strongly expressed G-CSFR. When extrinsic G-CSF was administered to the ESC- and human iPSC-derived cardiomyocytes, it markedly augmented their proliferation. Moreover, G-CSF-neutralizing antibody inhibited their proliferation. These findings indicated that G-CSF is critically involved in cardiomyocyte proliferation during development, and may be used to boost the yield of cardiomyocytes from ESCs for their potential application to regenerative medicine.

    Cell stem cell 2010;6;3;227-37

  • BPAG1 isoform-b: complex distribution pattern in striated and heart muscle and association with plectin and alpha-actinin.

    Steiner-Champliaud MF, Schneider Y, Favre B, Paulhe F, Praetzel-Wunder S, Faulkner G, Konieczny P, Raith M, Wiche G, Adebola A, Liem RK, Langbein L, Sonnenberg A, Fontao L and Borradori L

    Clinic of Dermatology, Geneva University Hospital, Rue Micheli-du-Crest 24, 1211 Geneva, Switzerland.

    BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

    Funded by: Austrian Science Fund FWF: P 17862, P 20744; NINDS NIH HHS: NS047711; Telethon: GGP04088

    Experimental cell research 2010;316;3;297-313

  • Alpha-actinin2 cytoskeletal protein is required for the functional membrane localization of a Ca2+-activated K+ channel (SK2 channel).

    Lu L, Timofeyev V, Li N, Rafizadeh S, Singapuri A, Harris TR and Chiamvimonvat N

    Division of Cardiovascular Medicine, Department of Medicine, University of California, Davis, CA 95616, USA. linglu@njnu.edu.cn.

    The importance of proper ion channel trafficking is underpinned by a number of channel-linked genetic diseases whose defect is associated with failure to reach the cell surface. Conceptually, it is reasonable to suggest that the function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane, which is determined jointly by the secretory and endocytic pathways. Yet the precise mechanisms of the entire ion channel trafficking pathway remain unknown. Here, we directly demonstrate that proper membrane localization of a small-conductance Ca(2+)-activated K(+) channel (SK2 or K(Ca)2.2) is dependent on its interacting protein, alpha-actinin2, a major F-actin crosslinking protein. SK2 channel localization on the cell-surface membrane is dynamically regulated, and one of the critical steps includes the process of cytoskeletal anchoring of SK2 channel by its interacting protein, alpha-actinin2, as well as endocytic recycling via early endosome back to the cell membrane. Consequently, alteration of these components of SK2 channel recycling results in profound changes in channel surface expression. The importance of our findings may transcend the area of K(+) channels, given that similar cytoskeletal interaction and anchoring may be critical for the membrane localization of other ion channels in neurons and other excitable cells.

    Funded by: BLRD VA: I01 BX000576; NHLBI NIH HHS: HL85727, HL85844, R01 HL075274, R01 HL085727, R01 HL085844, T32 HL086350

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;43;18402-7

  • Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.

    Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM and Srivastava D

    Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, 94158, USA.

    Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

    Funded by: NCRR NIH HHS: C06 RR018928; NHLBI NIH HHS: P01 HL046345, P01 HL089707, P01 HL089707-01A1, R01 HL057181, R01 HL057181-13, R01 HL057181-14, R01 HL080592, R01 HL080592-05, R01 HL080592-06, R01 HL088390, R01 HL094414

    Developmental cell 2009;16;2;233-44

  • Prox1 maintains muscle structure and growth in the developing heart.

    Risebro CA, Searles RG, Melville AA, Ehler E, Jina N, Shah S, Pallas J, Hubank M, Dillard M, Harvey NL, Schwartz RJ, Chien KR, Oliver G and Riley PR

    Molecular Medicine Unit, UCL Institute of Child Health, London WC1N 1EH, UK.

    Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the murine homeobox transcription factor Prox1 results in the disruption of expression and localisation of sarcomeric proteins, gross myofibril disarray and growth-retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of the genes encoding the structural proteins alpha-actinin, N-RAP and zyxin, which collectively function to maintain an actin-alpha-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and the manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study reveals an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling foetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.

    Funded by: British Heart Foundation; Medical Research Council: G0400153; NHLBI NIH HHS: R01 HL073402, R01 HL073402-06, R01-HL073402

    Development (Cambridge, England) 2009;136;3;495-505

  • alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering.

    Dobbins GC, Luo S, Yang Z, Xiong WC and Mei L

    Institute of Molecular Medicine and Genetics, Department of Neurobiology, Medical College of Georgia, Augusta, Georgia 30912, USA. gclemd@uab.edu

    AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that α-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. α-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the rapsyn-α-actinin interaction is increased by agrin, a factor known to stimulate AChR clustering. Downregulation of α-actinin expression inhibits agrin-mediated AChR clustering. Furthermore, the rapsyn-α-actinin interaction can be disrupted by inhibiting Abl and by cholinergic stimulation. Together these results indicate a role for α-actinin in AChR clustering.

    Funded by: NINDS NIH HHS: R01 NS040480

    Molecular brain 2008;1;18

  • Essential role of developmentally activated hypoxia-inducible factor 1alpha for cardiac morphogenesis and function.

    Krishnan J, Ahuja P, Bodenmann S, Knapik D, Perriard E, Krek W and Perriard JC

    Institute of Cell Biology, Eidgenössische Technische Hochschule, Zürich, Switzerland.

    Development of the mammalian heart is governed by precisely orchestrated interactions between signaling pathways integrating environmental cues and a core cardiac transcriptional network that directs differentiation, growth and morphogenesis. Here we report that in mice, at about embryonic day (E)8.5 to E10.0, cardiac development proceeds in an environment that is hypoxic and characterized by high levels of hypoxia-inducible factor (HIF)1alpha protein. Mice lacking HIF1alpha in ventricular cardiomyocytes exhibit aborted development at looping morphogenesis and embryonic lethality between E11.0 to E12.0. Intriguingly, HIF1alpha-deficient hearts display reduced expression of the core cardiac transcription factors Mef2C and Tbx5 and of titin, a giant protein that serves as a template for the assembly and organization of the sarcomere. Chromatin immunoprecipitation experiments revealed that Mef2C, Tbx5, and titin are direct target genes of HIF1alpha in vivo. Thus, hypoxia signaling controls cardiac development through HIF1alpha-mediated transcriptional regulation of key components of myofibrillogenesis and the cardiac transcription factor network, thereby providing a mechanistic basis of how heart development, morphogenesis, and function is coupled to low oxygen tension during early embryogenesis.

    Circulation research 2008;103;10;1139-46

  • Deficiency of Myo18B in mice results in embryonic lethality with cardiac myofibrillar aberrations.

    Ajima R, Akazawa H, Kodama M, Takeshita F, Otsuka A, Kohno T, Komuro I, Ochiya T and Yokota J

    Biology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan.

    Myo18B is an unconventional myosin family protein expressed predominantly in muscle cells. Although conventional myosins are known to be localized on the A-bands and function as a molecular motor for muscle contraction, Myo18B protein was localized on the Z-lines of myofibrils in striated muscles. Like Myo18A, another 18th class of myosin, the N-terminal unique domain of the protein and not the motor domain and the coiled-coil tail is critical for its localization to F-actin in myocytes. Myo18B expression was induced by myogenic differentiation through the binding of myocyte-specific enhancer factor-2 to its promoter. Deficiency of Myo18B caused an embryonic lethality in mice accompanied by disruption of myofibrillar structures in cardiac myocytes at embryonic day 10.5. Thus, Myo18B is a unique unconventional myosin that is predominantly expressed in myocytes and whose expression is essential for the development and/or maintenance of myofibrillar structure.

    Genes to cells : devoted to molecular & cellular mechanisms 2008;13;10;987-99

  • Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion.

    Nygren JM, Liuba K, Breitbach M, Stott S, Thorén L, Roell W, Geisen C, Sasse P, Kirik D, Björklund A, Nerlov C, Fleischmann BK, Jovinge S and Jacobsen SE

    Hematopoietic Stem Cell Laboratory, Lund University, BMC B10, Klinikgatan 26, 221 84 Lund, Sweden.

    Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation.

    Funded by: Medical Research Council: G0501838

    Nature cell biology 2008;10;5;584-92

  • Identification of transcripts with enriched expression in the developing and adult pancreas.

    Hoffman BG, Zavaglia B, Witzsche J, Ruiz de Algara T, Beach M, Hoodless PA, Jones SJ, Marra MA and Helgason CD

    Department of Cancer Endocrinology, BC Cancer Research Center, West 10th Ave, Vancouver, BC V5Z 1L3, Canada. bhoffman@bccrc.ca

    Background: Despite recent advances, the transcriptional hierarchy driving pancreas organogenesis remains largely unknown, in part due to the paucity of comprehensive analyses. To address this deficit we generated ten SAGE libraries from the developing murine pancreas spanning Theiler stages 17-26, making use of available Pdx1 enhanced green fluorescent protein (EGFP) and Neurog3 EGFP reporter strains, as well as tissue from adult islets and ducts.

    Results: We used a specificity metric to identify 2,536 tags with pancreas-enriched expression compared to 195 other mouse SAGE libraries. We subsequently grouped co-expressed transcripts with differential expression during pancreas development using K-means clustering. We validated the clusters first using quantitative real time PCR and then by analyzing the Theiler stage 22 pancreas in situ hybridization staining patterns of over 600 of the identified genes using the GenePaint database. These were then categorized into one of the five expression domains within the developing pancreas. Based on these results we identified a cascade of transcriptional regulators expressed in the endocrine pancreas lineage and, from this, we developed a predictive regulatory network describing beta-cell development.

    Conclusion: Taken together, this work provides evidence that the SAGE libraries generated here are a valuable resource for continuing to elucidate the molecular mechanisms regulating pancreas development. Furthermore, our studies provide a comprehensive analysis of pancreas development, and insights into the regulatory networks driving this process are revealed.

    Genome biology 2008;9;6;R99

  • Target gene selectivity of the myogenic basic helix-loop-helix transcription factor myogenin in embryonic muscle.

    Davie JK, Cho JH, Meadows E, Flynn JM, Knapp JR and Klein WH

    Department of Biochemistry and Molecular Biology, Unit 1000, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

    The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin(+/+) and myogenin(-/-) mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin(-/-) mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences.

    Funded by: NCI NIH HHS: CA16672

    Developmental biology 2007;311;2;650-64

  • Extraocular muscle morphogenesis and gene expression are regulated by Pitx2 gene dose.

    Diehl AG, Zareparsi S, Qian M, Khanna R, Angeles R and Gage PJ

    Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor 48105, USA.

    Purpose: PITX2 gene dose plays a central role in Axenfeld-Rieger syndrome. The purpose of this study was to test the hypothesis that the effects of Pitx2 gene dose on eye development can be molecularly dissected in available Pitx2 mutant mice.

    Methods: A panel of mice with Pitx2 gene dose ranging from wild-type (+/+) to none (-/-) was generated. Eye morphogenesis was assessed in animals with each Pitx2 gene dose. We also compared global gene expression in eye primordia taken from e12.5 Pitx2+/+, Pitx2+/-, Pitx2-/- embryos using gene microarrays. The validity of microarray results was confirmed by qRT-PCR.

    Results: Morphogenesis of all extraocular muscle bundles correlated highly with Pitx2 gene dose, but there were some differences in sensitivity among muscle groups. Superior and inferior oblique muscles were most sensitive and disappeared before the four rectus muscles. Expression of muscle-specific genes was globally sensitive to Pitx2 gene dose, including the muscle-specific transcription factor genes Myf5, Myog, Myod1, Smyd1, Msc, and Csrp3.

    Conclusions: Pitx2 gene dose regulates both morphogenesis and gene expression in developing extraocular muscles. The expression of key muscle-specific transcription factor genes is regulated by Pitx2 gene dose, suggesting that sufficient levels of PITX2 protein are essential for early initiation of the myogenic regulatory cascade in extraocular muscles. These results document the first ocular tissue affected by Pitx2 gene dose in a model organism, where the underlying mechanisms can be analyzed, and provide a paradigm for future experiments designed to elucidate additional effects of Pitx2 gene dose during eye development.

    Funded by: NEI NIH HHS: EY014126, EY07003, P30 EY007003, R01 EY014126

    Investigative ophthalmology & visual science 2006;47;5;1785-93

  • Role of p90 ribosomal S6 kinase-mediated prorenin-converting enzyme in ischemic and diabetic myocardium.

    Itoh S, Ding B, Shishido T, Lerner-Marmarosh N, Wang N, Maekawa N, Berk BC, Takeishi Y, Yan C, Blaxall BC and Abe J

    Cardiovascular Research Institute, University of Rochester, Rochester, NY, USA.

    Background: Epidemiological data strongly indicate that diabetes increases the incidence of heart failure. Although the benefit of angiotensin-converting enzyme inhibitor (ACE-I) treatment during and after myocardial infarction has been found to be greater in diabetics than nondiabetics and activation of the renin-angiotensin system (RAS) has been implicated, the molecular basis of these actions remains unclear.

    We generated transgenic mice with cardiac-specific overexpression of wild-type p90 ribosomal S6 kinase (WT-p90RSK-Tg) and a dominant-negative form of p90RSK (DN-p90RSK-Tg). Recovery of cardiac function after ischemia/reperfusion in WT-p90RSK-Tg isolated mouse hearts was significantly impaired. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed specific induction of prorenin-converting enzyme (PRECE) in WT-p90RSK-Tg mice. mRNA induction of PRECE was confirmed with serial angiotensinogen protein reduction after perfusion in WT-p90RSK-Tg mice, suggesting an increase of angiotensinogen cleavage and subsequent RAS activation in WT-p90RSK-Tg mice. We investigated the role of the RAS in WT-p90RSK-Tg animals after ischemia/reperfusion with the use of an ACE-I (captopril) and an angiotensin II type 1 receptor blocker (olmesartan). We did not observe any effect of these inhibitors in non-Tg littermate controls, thus corroborating other reports in rodents. In contrast, both captopril and olmesartan significantly improved cardiac function and reduced infarct size in WT-p90RSK-Tg mice. At 8 months of age, WT-p90RSK-Tg mice developed cardiac dysfunction. p90RSK activity and PRECE mRNA were both increased by streptozotocin-induced hyperglycemia in non-Tg littermate controls, whereas DN-p90RSK-Tg animals exposed to streptozotocin did not have PRECE induction.

    Conclusions: This study demonstrates the critical role of p90RSK in hyperglycemia-mediated myocardial PRECE induction, which may explain the augmentation of the RAS in diabetic hearts and provide an alternative therapeutic approach to treat diabetic cardiomyopathy.

    Funded by: NHLBI NIH HHS: HL-66919, HL44721; NIGMS NIH HHS: GM-071485-01A1

    Circulation 2006;113;14;1787-98

  • Myospryn is a direct transcriptional target for MEF2A that encodes a striated muscle, alpha-actinin-interacting, costamere-localized protein.

    Durham JT, Brand OM, Arnold M, Reynolds JG, Muthukumar L, Weiler H, Richardson JA and Naya FJ

    Department of Biology, Program in Cell and Molecular Biology, Boston University, Boston, Massachusetts 02215, USA.

    The full repertoire of proteins that comprise the striated muscle Z-disc and peripheral structures, such as the costamere, have yet to be discovered. Recent studies suggest that this elaborate protein network, which acts as a structural and signaling center for striated muscle, harbors factors that function as mechanosensors to ensure coordinated contractile activity. Mutations in genes whose products reside in this region often result in skeletal and cardio myopathies, demonstrating the importance of this macromolecular complex in muscle structure and function. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the costamere in striated muscle. This gene, called myospryn, was identified by microarray analysis as a transcript down-regulated in MEF2A knock-out mice. MEF2A knock-out mice develop cardiac failure during the perinatal period with mutant hearts exhibiting several cardiac abnormalities including myofibrillar disarray. Myospryn is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy-associated gene 5 (CMYA5). Myospryn is expressed as a single, large transcript of approximately 12 kilobases in adult heart and skeletal muscle with an open reading frame of 3739 amino acids. This protein, belonging to the tripartite motif superfamily of proteins, contains a B-box coiled-coil (BBC), two fibronectin type III (FN3) repeats, and SPRY domains and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that myospryn functions directly downstream of MEF2A at the costamere in striated muscle potentially playing a role in myofibrillogenesis.

    Funded by: NHLBI NIH HHS: HL73304

    The Journal of biological chemistry 2006;281;10;6841-9

  • Establishment of cardiac cytoarchitecture in the developing mouse heart.

    Hirschy A, Schatzmann F, Ehler E and Perriard JC

    Institute of Cell Biology, ETH Zurich-Hönggerberg, CH-8093 Zurich, Switzerland.

    Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.

    Funded by: Medical Research Council: G0400153

    Developmental biology 2006;289;2;430-41

  • Mammalian E4 is required for cardiac development and maintenance of the nervous system.

    Kaneko-Oshikawa C, Nakagawa T, Yamada M, Yoshikawa H, Matsumoto M, Yada M, Hatakeyama S, Nakayama K and Nakayama KI

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan.

    Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a(-/-) mouse embryonic fibroblasts. Furthermore, Ufd2a(+/-) mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.

    Molecular and cellular biology 2005;25;24;10953-64

  • N-RAP expression during mouse heart development.

    Lu S, Borst DE and Horowits R

    Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.

    N-RAP gene expression and N-RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. N-RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5-10.5 heart tissue, N-RAP protein was primarily associated with developing premyofibril structures containing alpha-actinin, as well as with the Z-lines and M-lines of more-mature myofibrils. In contrast, N-cadherin was concentrated in patches at the periphery of the cardiomyocytes. N-RAP labeling markedly increased between E10.5 and E16.5; almost all of the up-regulated N-RAP was associated with intercalated disk structures, and the proportion of mature sarcomeres containing N-RAP decreased. In adult hearts, specific N-RAP staining was only observed at the intercalated disks and was not found in the sarcomeres. The results are consistent with N-RAP functioning as a catalytic scaffolding molecule, with low levels of the scaffold being sufficient to repetitively catalyze key steps in myofibril assembly.

    Funded by: NEI NIH HHS: EY11726

    Developmental dynamics : an official publication of the American Association of Anatomists 2005;233;1;201-12

  • Synaptopodin regulates the actin-bundling activity of alpha-actinin in an isoform-specific manner.

    Asanuma K, Kim K, Oh J, Giardino L, Chabanis S, Faul C, Reiser J and Mundel P

    Department of Medicine, Albert Einstein College of Medicine, New York, New York, USA.

    Synaptopodin is the founding member of a novel class of proline-rich actin-associated proteins highly expressed in telencephalic dendrites and renal podocytes. Synaptopodin-deficient (synpo(-/-)) mice lack the dendritic spine apparatus and display impaired activity-dependent long-term synaptic plasticity. In contrast, the ultrastructure of podocytes in synpo(-/-) mice is normal. Here we show that synpo(-/-) mice display impaired recovery from protamine sulfate-induced podocyte foot process (FP) effacement and LPS-induced nephrotic syndrome. Similarly, synpo(-/-) podocytes show impaired actin filament reformation in vitro. We further demonstrate that synaptopodin exists in 3 isoforms, neuronal Synpo-short (685 AA), renal Synpo-long (903 AA), and Synpo-T (181 AA). The C terminus of Synpo-long is identical to that of Synpo-T. All 3 isoforms specifically interact with alpha-actinin and elongate alpha-actinin-induced actin filaments. synpo(-/-) mice lack Synpo-short and Synpo-long expression but show an upregulation of Synpo-T protein expression in podocytes, though not in the brain. Gene silencing of Synpo-T abrogates stress-fiber formation in synpo(-/-) podocytes, demonstrating that Synpo-T serves as a backup for Synpo-long in synpo(-/-) podocytes. In concert, synaptopodin regulates the actin-bundling activity of alpha-actinin in highly dynamic cell compartments, such as podocyte FPs and the dendritic spine apparatus.

    Funded by: NIDA NIH HHS: DA18886, R01 DA018886; NIDDK NIH HHS: DK062472, DK064236, DK57683, P50 DK064236, R01 DK057683, R01 DK062472

    The Journal of clinical investigation 2005;115;5;1188-98

  • Temporal and spatial expression pattern of beta1 sodium channel subunit during heart development.

    Domínguez JN, Navarro F, Franco D, Thompson RP and Aránega AE

    Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain.

    Objectives: The aim of this study is to analyze Scn1b mRNA expression levels and protein distribution of Scn1b, a putative modulator of the pore-forming Na(+) channel subunit in the heart, during mouse cardiac development.

    Methods: Scn1b mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E18.5 as well as in postnatal and adult heart. Scn1b protein distribution and subcellular localization during cardiogenesis were analyzed by immunohistochemistry and confocal microscopy.

    Results: Scn1b mRNA showed a dynamic expression pattern, peaking at stage E12.5 and decreasing at E15.5. Scn1b mRNA increased at later embryonic and neonatal stages, being maximal in the adult heart. Immunohistochemistry experiments revealed comparable distribution of Scn1b protein between the different cardiac chambers at early embryonic stages. With further development, Scn1b protein showed an enhanced expression in the trabeculated myocardium and the bundle branches. At the subcellular level in later embryonic and postnatal mouse cardiomyocytes, Scn1b was present in T-tubules as identified by immunostaining of alpha-actinin, and in the intercalated disks as identified by immunostaining of connexin 43.

    Conclusion: These results demonstrate that Scn1b is expressed during mouse heart development, suggesting it can play an important role in the action potential configuration of the cardiomyocytes during heart morphogenesis.

    Cardiovascular research 2005;65;4;842-50

  • Attenuation of cardiac remodeling after myocardial infarction by muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc.

    Heineke J, Ruetten H, Willenbockel C, Gross SC, Naguib M, Schaefer A, Kempf T, Hilfiker-Kleiner D, Caroni P, Kraft T, Kaiser RA, Molkentin JD, Drexler H and Wollert KC

    Department of Cardiology, Hanover Medical School, 30625 Hanover, Germany.

    Adverse left ventricular (LV) remodeling after myocardial infarction (MI) is a major cause for heart failure. Molecular modifiers of the remodeling process remain poorly defined. Patients with heart failure after MI have reduced LV expression levels of muscle LIM protein (MLP), a component of the sarcomeric Z-disk that is involved in the integration of stress signals in cardiomyocytes. By using heterozygous MLP mutant (MLP+/-) mice, we explored the role of MLP in post-MI remodeling. LV dimensions and function were similar in sham-operated WT and MLP+/- mice. After MI, however, MLP+/- mice displayed more pronounced LV dilatation and systolic dysfunction and decreased survival compared with WT mice, indicating that reduced MLP levels predispose to adverse LV remodeling. LV dilatation in MLP+/- mice was associated with reduced thickening but enhanced elongation of cardiomyocytes. Activation of the stress-responsive, prohypertrophic calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway was reduced in MLP+/- mice after MI, as shown by a blunted transcriptional activation of NFAT in cardiomyocytes isolated from MLP+/-/NFAT-luciferase reporter gene transgenic mice. Calcineurin was colocalized with MLP at the Z-disk in WT mice but was displaced from the Z-disk in MLP+/- mice, indicating that MLP is essential for calcineurin anchorage to the Z-disk. In vitro assays in cardiomyocytes with down-regulated MLP confirmed that MLP is required for stress-induced calcineurin-NFAT activation. Our study reveals a link between the stress sensor MLP and the calcineurin-NFAT pathway at the sarcomeric Z-disk in cardiomyocytes and indicates that reduced MLP-calcineurin signaling predisposes to adverse remodeling after MI.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;5;1655-60

  • Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase.

    Keicher C, Gambaryan S, Schulze E, Marcus K, Meyer HE and Butt E

    Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany.

    LIM and SH3 domain protein (LASP-1) is a specific focal adhesion protein involved in cell migration. Overlay studies demonstrate that LASP-1 directly binds to the proline-rich domains of zyxin, lipoma preferred partner (LPP), and vasodilator-stimulated phosphoprotein (VASP), with zyxin being the most prominent interacting partner. Despite the LIM/zinc-finger domain, hypothesized to be involved in homodimerization, LASP-1 exists as a monomer. In vitro phosphorylation of recombinant mouse LASP-1 by cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) occurs at serine 61, serine 99, and threonine 156 whereas in intact cells mouse LASP-1 is phosphorylated only at threonine 156. This site is different from the known in vivo phosphorylation sites in human (serine 146) and rabbit (serine 99 and serine 146). Nevertheless, immunofluorescence of LASP-1 in human and mouse mesangial cells revealed no difference in subcellular distribution. Exposure of the cells to forskolin induced a translocation of both, human and mouse LASP-1, from the focal contacts to the cell interior without affecting F-actin structure. Immunoblotting of LASP-1 in various mouse and human tissues detected a similar prominent expression in non-muscle tissue. Altogether, our data suggest so far no functional differences between human and mouse LASP-1.

    Biochemical and biophysical research communications 2004;324;1;308-16

  • The enhancement of nuclear receptor transcriptional activation by a mouse actin-binding protein, alpha actinin 2.

    Huang SM, Huang CJ, Wang WM, Kang JC and Hsu WC

    Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan 114, Republic of China.

    The p160 coactivators, steroid receptor coactivator 1, glucocorticoid receptor interacting protein 1 (GRIP1) and the activator of thyroid and retinoic acid receptor, have two activation domains, AD1 and AD2, which transmit the activation signal from the DNA-bound nuclear receptor to the chromatin and/or transcription machinery. In screening for mammalian proteins that bind the AD2 of GRIP1, we identified a mouse actin-binding protein, alpha actinin 2 (mACTN2). mACTN2 was expressed in the heart, skeletal muscle, lung, brain and testis, but there was no expression in the spleen, liver or kidney. Interestingly, the expression level of mACTN2 in the developing embryo depended on the embryonic stage. We further demonstrated that mACTN2 could enhance two transactivation activities of GRIP1, which in turn could enhance the homodimerization of mACTN2. Importantly, mACTN2 not only served as a primary coactivator for androgen receptor, estrogen receptor and thyroid receptor activities, but also acted synergistically with GRIP1 to enhance these nuclear receptor (NR) functions. However, the NR binding motif, LXXLL, conserved in mACTN2 and other actinin family proteins, might be a dispensable domain for its coactivator roles in NRs. These findings suggested that mACTN2 might play an important role in GRIP1-induced NR coactivator functions.

    Journal of molecular endocrinology 2004;32;2;481-96

  • GenePaint.org: an atlas of gene expression patterns in the mouse embryo.

    Visel A, Thaller C and Eichele G

    Max Planck Institute of Experimental Endocrinology, Feodor-Lynen-Strasse 7, D-30625 Hannover, Germany.

    High-throughput instruments were recently developed to determine gene expression patterns on tissue sections by RNA in situ hybridization. The resulting images of gene expression patterns, chiefly of E14.5 mouse embryos, are accessible to the public at http://www.genepaint.org. This relational database is searchable for gene identifiers and RNA probe sequences. Moreover, patterns and intensity of expression in approximately 100 different embryonic tissues are annotated and can be searched using a standardized catalog of anatomical structures. A virtual microscope tool, the Zoom Image Server, was implemented in GenePaint.org and permits interactive zooming and panning across approximately 15,000 high-resolution images.

    Nucleic acids research 2004;32;Database issue;D552-6

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Mice deficient in alpha-actinin-4 have severe glomerular disease.

    Kos CH, Le TC, Sinha S, Henderson JM, Kim SH, Sugimoto H, Kalluri R, Gerszten RE and Pollak MR

    Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    Dominantly inherited mutations in ACTN4, which encodes alpha-actinin-4, cause a form of human focal and segmental glomerulosclerosis (FSGS). By homologous recombination in ES cells, we developed a mouse model deficient in Actn4. Mice homozygous for the targeted allele have no detectable alpha-actinin-4 protein expression. The number of homozygous mice observed was lower than expected under mendelian inheritance. Surviving mice homozygous for the targeted allele show progressive proteinuria, glomerular disease, and typically death by several months of age. Light microscopic analysis shows extensive glomerular disease and proteinaceous casts. Electron microscopic examination shows focal areas of podocyte foot-process effacement in young mice, and diffuse effacement and globally disrupted podocyte morphology in older mice. Despite the widespread distribution of alpha-actinin-4, histologic examination of mice showed abnormalities only in the kidneys. In contrast to the dominantly inherited human form of ACTN4-associated FSGS, here we show that the absence of alpha-actinin-4 causes a recessive form of disease in mice. Cell motility, as measured by lymphocyte chemotaxis assays, was increased in the absence of alpha-actinin-4. We conclude that alpha-actinin-4 is required for normal glomerular function. We further conclude that the nonsarcomeric forms of alpha-actinin (alpha-actinin-1 and alpha-actinin-4) are not functionally redundant. In addition, these genetic studies demonstrate that the nonsarcomeric alpha-actinin-4 is involved in the regulation of cell movement.

    Funded by: NHLBI NIH HHS: HL-07208, HL-65584, R01 HL065584, T32 HL007208; NIDDK NIH HHS: DK-51711, DK-55001, DK-59588, R01 DK055001, R01 DK059588, R37 DK059588

    The Journal of clinical investigation 2003;111;11;1683-90

  • An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles.

    Pun S, Sigrist M, Santos AF, Ruegg MA, Sanes JR, Jessell TM, Arber S and Caroni P

    Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.

    We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed "Fast Synapsing" (FaSyn) and "Delayed Synapsing" (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.

    Neuron 2002;34;3;357-70

  • Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins.

    Frey N and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.

    The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.

    The Journal of biological chemistry 2002;277;16;13998-4004

  • Gene defect in ectodermal dysplasia implicates a death domain adapter in development.

    Headon DJ, Emmal SA, Ferguson BM, Tucker AS, Justice MJ, Sharpe PT, Zonana J and Overbeek PA

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, 77030, USA.

    Members of the tumour-necrosis factor receptor (TNFR) family that contain an intracellular death domain initiate signalling by recruiting cytoplasmic death domain adapter proteins. Edar is a death domain protein of the TNFR family that is required for the development of hair, teeth and other ectodermal derivatives. Mutations in Edar-or its ligand, Eda-cause hypohidrotic ectodermal dysplasia in humans and mice. This disorder is characterized by sparse hair, a lack of sweat glands and malformation of teeth. Here we report the identification of a death domain adapter encoded by the mouse crinkled locus. The crinkled mutant has an hypohidrotic ectodermal dysplasia phenotype identical to that of the edar (downless) and eda (Tabby) mutants. This adapter, which we have called Edaradd (for Edar-associated death domain), interacts with the death domain of Edar and links the receptor to downstream signalling pathways. We also identify a missense mutation in its human orthologue, EDARADD, that is present in a family affected with hypohidrotic ectodermal dysplasia. Our findings show that the death receptor/adapter signalling mechanism is conserved in developmental, as well as apoptotic, signalling.

    Funded by: Medical Research Council: G9800001

    Nature 2001;414;6866;913-6

  • Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

    Hüttelmaier S, Illenberger S, Grosheva I, Rüdiger M, Singer RH and Jockusch BM

    Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.

    By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

    The Journal of cell biology 2001;155;5;775-86

  • Differential expression of the actin-binding proteins, alpha-actinin-2 and -3, in different species: implications for the evolution of functional redundancy.

    Mills M, Yang N, Weinberger R, Vander Woude DL, Beggs AH, Easteal S and North K

    Neurogenetics Research Unit, Children's Hospital at Westmead, Westmead, Sydney, NSW 2145, Australia.

    The alpha-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of alpha-actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that alpha-actinin-3 is absent in approximately 18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true alpha-actinin-3 deficiency. Absence of alpha-actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that alpha-actinin-2 is able to compensate for alpha-actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, alpha-actinin-2 expression does not completely overlap alpha-actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken alpha-actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models.

    Funded by: NIAMS NIH HHS: K02 AR02026, R01 AR44345

    Human molecular genetics 2001;10;13;1335-46

  • The small muscle-specific protein Csl modifies cell shape and promotes myocyte fusion in an insulin-like growth factor 1-dependent manner.

    Palmer S, Groves N, Schindeler A, Yeoh T, Biben C, Wang CC, Sparrow DB, Barnett L, Jenkins NA, Copeland NG, Koentgen F, Mohun T and Harvey RP

    Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia.

    We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer-binding factor (MEF)2, were also enhanced in an IGF-1 signaling-dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.

    Funded by: NIA NIH HHS: R03 AG14811; Wellcome Trust

    The Journal of cell biology 2001;153;5;985-98

  • Calsarcins, a novel family of sarcomeric calcineurin-binding proteins.

    Frey N, Richardson JA and Olson EN

    Departments of Molecular Biology and Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    The calcium- and calmodulin-dependent protein phosphatase calcineurin has been implicated in the transduction of signals that control the hypertrophy of cardiac muscle and slow fiber gene expression in skeletal muscle. To identify proteins that mediate the effects of calcineurin on striated muscles, we used the calcineurin catalytic subunit in a two-hybrid screen for cardiac calcineurin-interacting proteins. From this screen, we discovered a member of a novel family of calcineurin-interacting proteins, termed calsarcins, which tether calcineurin to alpha-actinin at the z-line of the sarcomere of cardiac and skeletal muscle cells. Calsarcin-1 and calsarcin-2 are expressed in developing cardiac and skeletal muscle during embryogenesis, but calsarcin-1 is expressed specifically in adult cardiac and slow-twitch skeletal muscle, whereas calsarcin-2 is restricted to fast skeletal muscle. Calsarcins represent a novel family of sarcomeric proteins that link calcineurin with the contractile apparatus, thereby potentially coupling muscle activity to calcineurin activation.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;26;14632-7

  • Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

    Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG and Ko MS

    Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.

    cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9127-32

  • Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion.

    Galliano MF, Huet C, Frygelius J, Polgren A, Wewer UM and Engvall E

    Burnham Institute, La Jolla Cancer Research Center, La Jolla, California 92037, USA.

    ADAM12 belongs to the transmembrane metalloprotease ADAM ("a disintegrin and metalloprotease") family. ADAM12 has been implicated in muscle cell differentiation and fusion, but its precise function remains unknown. Here, we show that ADAM12 is dramatically up-regulated in regenerated, newly formed fibers in vivo. In C2C12 cells, ADAM12 is expressed at low levels in undifferentiated myoblasts and is transiently up-regulated at the onset of differentiation when myoblasts fuse into multinucleated myotubes, whereas other ADAMs, such as ADAMs 9, 10, 15, 17, and 19, are expressed at all stages of differentiation. Using the yeast two-hybrid screen, we found that the muscle-specific alpha-actinin-2 strongly binds to the cytoplasmic tail of ADAM12. In vitro binding assays with GST fusion proteins confirmed the specific interaction. The major binding site for alpha-actinin-2 was mapped to a short sequence in the membrane-proximal region of ADAM12 cytoplasmic tail; a second binding site was identified in the membrane-distal region. Co-immunoprecipitation experiments confirm the in vivo association of ADAM12 cytoplasmic domain with alpha-actinin-2. Overexpression of the entire cytosolic ADAM12 tail acted in a dominant negative fashion by inhibiting fusion of C2C12 cells, whereas expression of a cytosolic ADAM12 lacking the major alpha-actinin-2 binding site had no effect on cell fusion. Our results suggest that interaction of ADAM12 with alpha-actinin-2 is important for ADAM12 function.

    The Journal of biological chemistry 2000;275;18;13933-9

  • Melusin is a new muscle-specific interactor for beta(1) integrin cytoplasmic domain.

    Brancaccio M, Guazzone S, Menini N, Sibona E, Hirsch E, De Andrea M, Rocchi M, Altruda F, Tarone G and Silengo L

    Department of Genetics, University of Torino, Torino 10126, Italy.

    Here we describe the isolation and partial characterization of a new muscle-specific protein (Melusin) which interacts with the integrin cytoplasmic domain. The cDNA encoding Melusin was isolated in a two-hybrid screening of a rat neonatal heart library using beta(1)A and beta(1)D integrin cytoplasmic regions as baits. Melusin is a cysteine-rich cytoplasmic protein of 38 kDa, with a stretch of acidic amino acid residues at the extreme carboxyl-terminal end. In addition, putative binding sites for SH3 and SH2 domains are present in the amino-terminal half of the molecule. Chromosomic analysis showed that melusin gene maps at Xq12.1/13 in man and in the synthenic region X band D in mouse. Melusin is expressed in skeletal and cardiac muscles but not in smooth muscles or other tissues. Immunofluorescence analysis showed that Melusin is present in a costamere-like pattern consisting of two rows flanking alpha-actinin at Z line. Its expression is up-regulated during in vitro differentiation of the C2C12 murine myogenic cell line, and it is regulated during in vivo skeletal muscle development. A fragment corresponding to the tail region of Melusin interacted strongly and specifically with beta(1) integrin cytoplasmic domain in a two-hybrid test, but the full-length protein did not. Because the tail region of Melusin contains an acidic amino acid stretch resembling high capacity and low affinity calcium binding domains, we tested the possibility that Ca(2+) regulates Melusin-integrin association. In vitro binding experiments demonstrated that interaction of full-length Melusin with detergent-solubilized integrin heterodimers occurred only in absence of cations, suggesting that it can be regulated by intracellular signals affecting Ca(2+) concentration.

    Funded by: Telethon: E.0672

    The Journal of biological chemistry 1999;274;41;29282-8

  • Cypher, a striated muscle-restricted PDZ and LIM domain-containing protein, binds to alpha-actinin-2 and protein kinase C.

    Zhou Q, Ruiz-Lozano P, Martone ME and Chen J

    Department of Medicine, UCSD-Salk Program in Molecular Medicine, University of California at San Diego, School of Medicine, La Jolla, California 92093-0613, USA.

    We have cloned and characterized a novel striated muscle-restricted protein (Cypher) that has two mRNA splice variants, designated Cypher1 and Cypher2. Both proteins contain an amino-terminal PDZ domain. Cypher1, but not Cypher2, contains three carboxyl-terminal LIM domains and an amino acid repeat sequence that exhibits homology to a repeat sequence found in the largest subunit of RNA polymerase II. cypher1 and cypher2 mRNAs exhibited identical expression patterns. Both are exclusively expressed in cardiac and striated muscle in embryonic and adult stages. By biochemical assays, we have demonstrated that Cypher1 and Cypher2 bind to alpha-actinin-2 via their PDZ domains. This interaction has been further confirmed by immunohistochemical studies that demonstrated co-localization of Cypher and alpha-actinin at the Z-lines of cardiac muscle. We have also found that Cypher1 binds to protein kinase C through its LIM domains. Phosphorylation of Cypher by protein kinase C has demonstrated the functional significance of this interaction. Together, our data suggest that Cypher1 may function as an adaptor in striated muscle to couple protein kinase C-mediated signaling, via its LIM domains, to the cytoskeleton (alpha-actinin-2) through its PDZ domain.

    Funded by: NCRR NIH HHS: RR04050

    The Journal of biological chemistry 1999;274;28;19807-13

  • p27Kip1 is expressed transiently in developing myotomes and enhances myogenesis.

    Zabludoff SD, Csete M, Wagner R, Yu X and Wold BJ

    Division of Biology, California Institute of Technology, Pasadena 91125, USA.

    Vertebrate skeletal muscle development is characterized by tight coupling of muscle differentiation with cell cycle arrest in G1/G0. Key regulators of G1 progression are the G1 cyclin-dependent kinases, their positive regulators, the G1 cyclins, and their negative regulators, the cyclin-dependent kinase inhibitors (CDIs). Here we show that p27Kip1 protein, a G1 CDI, is expressed in a prominent but transient wave in the developing myotomes of the mouse embryo. We relate its expression to expression of MyoD and myogenin proteins, which are determination and differentiation class myogenic regulatory factors, respectively. Functional assays showed that ectopic p27 expression can powerfully enhance the efficiency of MyoD-initiated muscle differentiation in cell culture. When considered together with the myotomal expression patterns of p18, p21, and p57, these results suggest a model in which p27 acts as a "trigger" CDI while myoblasts are exiting the cell cycle and initiating differentiation. At later times, when p27 protein has been down-regulated, it is proposed that accumulation of p18, p21, and p57 maintain the differentiated myocytes in a postmitotic state.

    Funded by: NIAMS NIH HHS: AR40708, AR42671

    Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 1998;9;1;1-11

  • Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.

    Yoon JK, Olson EN, Arnold HH and Wold BJ

    Division of Biology, 156-29, California Institute of Technology, Pasadena, California, 91125, USA.

    Three different null alleles of the myogenic bHLH gene MRF4/herculin/Myf-6 were created recently. The three alleles were similar in design but were surprisingly different in the intensity of their phenotypes, which ranged from complete viability of homozygotes to complete lethality. One possible explanation for these differences is that each mutation altered expression from the nearby Myf-5 gene to a different extent. This possibility was first raised by the observation that the most severe MRF4 knockout allele expresses no Myf-5 RNA and is a developmental phenocopy of the Myf-5 null mutation. Furthermore, initial studies of the two weaker alleles had shown that their differences in viability correlate with the intensity of rib skeletal defects, and the most extreme version of this rib defect is the hallmark phenotype of Myf-5 null animals. In the present study we tested this hypothesis for the two milder MRF4 alleles. By analyzing compound heterozygous animals carrying either the intermediate or the weakest MRF4 knockout allele on one chromosome 10 and a Myf-5 knockout allele on the other chromosome, we found that both of these MRF4 alleles apparently downregulate Myf-5 expression by a cis-acting mechanism. Compound heterozygotes showed increased mortality of the normally viable MRF4 allele, together with intensified rib defects for both MRF4 alleles and increased deficits in myotomal Myf-5 expression. The allele-specific gradation in phenotypes also suggested that rib morphogenesis is profoundly sensitive to quantitative differences in Myf-5 function if Myf-5 products drop below hemizygous levels. The mechanistic basis for cis interactions at the MRF4/Myf-5 locus was further examined by fusing a DNA segment containing the entire MRF4 structural gene, including all sequences deleted in the three MRF knockout alleles, with a basal promoter and a lacZ reporter. Transgenic embryos showed specific LacZ expression in myotomes in a pattern that closely resembles the expression of Myf-5 RNA. cis-acting interactions between Myf-5 and MRF4 may therefore play a significant role in regulating expression of these genes in the early myotomes of wildtype embryos.

    Funded by: NIAMS NIH HHS: AR40780, AR42671

    Developmental biology 1997;188;2;349-62

  • Comparative mapping in the beige-satin region of mouse chromosome 13.

    Perou CM, Perchellet A, Jago T, Pryor R, Kaplan J and Justice MJ

    Department of Pathology, University of Utah School of Medicine, Salt Lake City, 84132, USA.

    The proximal end of mouse chromosome (Chr) 13 contains regions conserved on human chromosomes 1q42-q44, 6p23-p21, and 7p22-p13. This region also contains mutations that may be models for human disease, including beige (human Chediak-Higashi syndrome). An interspecific backcross of SB/Le and Mus spretus mice was used to generate a molecular genetic linkage map of mouse chromosome 13 with an emphasis on the proximal region including beige (bg) and satin (sa). This map provides the gene order of the two phenotypic markers bg and sa relative to restriction fragment length polymorphisms and simple sequence length polymorphisms in 131 backcross animals. In parallel, we have created a physical map of the region using Nidogen (Nid) as a molecular starting point for cloning a YAC contig that was used to identify the beige gene. The physical map provides the fine-structure order of genes and anonymous DNA fragments that was not resolved by the genetic linkage mapping. The results show that the bg region of mouse Chr 13 is highly conserved on human Chr 1q42-q44 and provide a starting point for a complete functional analysis of the entire bg-sa interval.

    Funded by: NCI NIH HHS: 7R29CA63229-02; NHLBI NIH HHS: HL26922; NIGMS NIH HHS: T32GM07464

    Genomics 1997;39;2;136-46

  • Molecular diversity of myofibrillar proteins: gene regulation and functional significance.

    Schiaffino S and Reggiani C

    Department of Biomedical Sciences, University of Padova, Padua, Italy.

    Myofibrillar proteins exist as multiple isoforms that derive from multigene (isogene) families. Additional isoforms, including products of tropomyosin, myosin light chain 1 fast, troponin T, titin, and nebulin genes, can be generated from the same gene through alternative splicing or use of alternative promoters. Myofibrillar protein isogenes are differentially expressed in various muscle types and fiber types but can be coexpressed within the same fiber. Isogenes are regulated by transcriptional and posttranscriptional mechanisms; however, specific regulatory sequences and transcriptional factors have not yet been identified. The pattern of isogene expression varies during muscle development in relation to the different origin of myogenic cells and primary/secondary fiber generations and is affected by neural and hormonal influences. The variable expression of myofibrillar protein isoforms is a major determinant of the contractile properties of skeletal muscle fibers. The diversity among isomyosins is related to the differences in the parameters of chemomechanical transduction as ATP hydrolysis rate and shortening velocity. Troponin and tropomyosin isoforms determine the variable sensitivity to calcium, whereas titin isoforms dictate the elastic properties of muscle fibers at rest. Both myosin and troponin isoforms contribute to the differences in the resistance to fatigue of muscle fibers.

    Funded by: Telethon: 343, A.012

    Physiological reviews 1996;76;2;371-423

  • Disruption of the mouse MRF4 gene identifies multiple waves of myogenesis in the myotome.

    Patapoutian A, Yoon JK, Miner JH, Wang S, Stark K and Wold B

    Division of Biology 156-29, California Institute of Technology, Pasadena 91125, USA.

    MRF4 (herculin/Myf-6) is one of the four member MyoD family of transcription factors identified by their ability to enforce skeletal muscle differentiation upon a wide variety of nonmuscle cell types. In this study the mouse germline MRF4 gene was disrupted by targeted recombination. Animals homozygous for the MRF4bh1 allele, a deletion of the functionally essential bHLH domain, displayed defective axial myogenesis and rib pattern formation, and they died at birth. Differences in somitogenesis between homozygous MRF4bh1 embryos and their wild-type littermates provided evidence for three distinct myogenic regulatory programs (My1-My3) in the somite, which correlate temporally and spatially with three waves of cellular recruitment to the expanding myotome. The first program (My1), marked initially by Myf-5 expression and followed by myogenin, began on schedule in the MRF4bh1/bh1 embryos at day 8 post coitum (E8). A second program (My2) was highly deficient in homozygous mutant MRF4 embryos, and normal expansion of the myotome failed. Moreover, expression of downstream muscle-specific genes, including FGF-6, which is a candidate regulator of inductive interactions, did not occur normally. The onset of MyoD expression around E10.5 in wild-type embryos marks a third myotomal program (My3), the execution of which was somewhat delayed in MRF4 mutant embryos but ultimately led to extensive myogenesis in the trunk. By E15 it appeared to have largely compensated for the defective My2 program in MRF4 mutants. Homozygous MRF4bh1 animals also showed improper rib pattern formation perhaps due to the absence of signals from cells expressing the My2 program. Finally, a later and relatively mild phenotype was detected in intercostal muscles of newborn animals.

    Funded by: NIAMS NIH HHS: AR40780, AR42671

    Development (Cambridge, England) 1995;121;10;3347-58

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000019 G2C Mus musculus Pocklington M1 Cluster 1 (mouse) from Pocklington et al (2006) 21
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 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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