G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
dynactin 1
G00000763 (Mus musculus)

Databases (7)

ENSG00000204843 (Ensembl human gene)
1639 (Entrez Gene)
1200 (G2Cdb plasticity & disease)
DCTN1 (GeneCards)
601143 (OMIM)
Marker Symbol
HGNC:2711 (HGNC)
Protein Sequence
Q14203 (UniProt)

Literature (86)

Pubmed - other

  • The retromer component SNX6 interacts with dynactin p150(Glued) and mediates endosome-to-TGN transport.

    Hong Z, Yang Y, Zhang C, Niu Y, Li K, Zhao X and Liu JJ

    Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.

    The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network (TGN). It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins (SNXs). Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2's dimerization partners, SNX6, interacts with the p150(Glued) subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-p150(Glued) interaction. Disruption of the SNX6-p150(Glued) interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.

    Cell research 2009;19;12;1334-49

  • Dynactin subunit p150Glued isoforms notable for differential interaction with microtubules.

    Zhapparova ON, Bryantseva SA, Dergunova LV, Raevskaya NM, Burakov AV, Bantysh OB, Shanina NA and Nadezhdina ES

    Institute of Protein Research of Russian Academy of Sciences, 34 Vavilova Str., Moscow 117334, Russia.

    Dynactin is a multiprotein complex that enhances dynein activity. The largest dynactin subunit, p150Glued, interacts with microtubules through its N-terminal region that contains a globular cytoskeleton-associated protein (CAP)-Gly domain and basic microtubule-binding domain of unknown structure. The p150Glued gene has a complicated intron-exon structure, and many splice isoforms of p150Glued protein have been predicted. Here we describe novel natural 150 kDa isoforms: the p150Glued-1A isoform, whose basic domain is composed of 41 amino acids, and p150Glued-1B with a basic domain of 21 aa because of the lack of exons 5-7 in the corresponding messenger RNA (mRNA). According to reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot data, p150Glued-1A is expressed in nerve tissues, in cultured cells and in embryonic tissues, while 1B is expressed ubiquitously. Overexpression of GFP-p150Glued-1A and -1B fusion proteins and immunostaining of cultured cells with 1A-specific antibodies show that the p150Glued-1A isoform is distributed along microtubules, whereas 1B is associated with microtubule plus-ends. The higher affinity of the p150Glued-1A isoform for microtubules is confirmed by a co-pelleting assay. In fibroblast-like cells, the interaction of p150Glued-1A with microtubules is less dependent on EB1/EB3 and CLIP170 proteins, compared with p150Glued-1B. In polarized cells, p150Glued-1A decorates microtubules that face the leading edge of the cell. The pattern of p150Glued-1A and p150Glued-1B interaction with microtubules and their tissue-specific expression patterns suggest that these isoforms might be involved in cell differentiation and proliferation.

    Traffic (Copenhagen, Denmark) 2009;10;11;1635-46

  • Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

    Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W and Neefjes J

    Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands.

    Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

    The Journal of cell biology 2009;185;7;1209-25

  • Characterization of DCTN1 genetic variability in neurodegeneration.

    Vilariño-Güell C, Wider C, Soto-Ortolaza AI, Cobb SA, Kachergus JM, Keeling BH, Dachsel JC, Hulihan MM, Dickson DW, Wszolek ZK, Uitti RJ, Graff-Radford NR, Boeve BF, Josephs KA, Miller B, Boylan KB, Gwinn K, Adler CH, Aasly JO, Hentati F, Destée A, Krygowska-Wajs A, Chartier-Harlin MC, Ross OA, Rademakers R and Farrer MJ

    Molecular Genetics Laboratory and Core, Morris K. Udall Parkinson's Disease Research Center of Excellence, Mayo Clinic, Department of Neuroscience, 4500 San Pablo Road, Jacksonville, FL 32224, USA. VilarinoGuell.Carles@mayo.edu

    Objective: Recently, mutations in DCTN1 were found to cause Perry syndrome, a parkinsonian disorder with TDP-43-positive pathology. Previously, mutations in DCTN1 were identified in a family with lower motor neuron disease, in amyotrophic lateral sclerosis (ALS), and in a family with ALS/frontotemporal dementia (FTD), suggesting a central role for DCTN1 in neurodegeneration.

    Methods: In this study we sequenced all DCTN1 exons and exon-intron boundaries in 286 samples diagnosed with Parkinson disease (PD), frontotemporal lobar degeneration (FTLD), or ALS.

    Results: This analysis revealed 36 novel variants (9 missense, 5 silent, and 22 noncoding). Segregation analysis in families and association studies in PD, FTLD, and ALS case-control series did not identify any variants segregating with disease or associated with increased disease risk.

    Conclusions: This study suggests that pathogenic mutations in DCTN1 are rare and do not play a common role in the development of Parkinson disease, frontotemporal lobar degeneration, or amyotrophic lateral sclerosis.

    Funded by: NIA NIH HHS: P50 AG16574; NINDS NIH HHS: P50 NS40256

    Neurology 2009;72;23;2024-8

  • Mitotic control of kinetochore-associated dynein and spindle orientation by human Spindly.

    Chan YW, Fava LL, Uldschmid A, Schmitz MH, Gerlich DW, Nigg EA and Santamaria A

    Department of Cell Biology, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany.

    Mitotic spindle formation and chromosome segregation depend critically on kinetochore-microtubule (KT-MT) interactions. A new protein, termed Spindly in Drosophila and SPDL-1 in C. elegans, was recently shown to regulate KT localization of dynein, but depletion phenotypes revealed striking differences, suggesting evolutionarily diverse roles of mitotic dynein. By characterizing the function of Spindly in human cells, we identify specific functions for KT dynein. We show that localization of human Spindly (hSpindly) to KTs is controlled by the Rod/Zw10/Zwilch (RZZ) complex and Aurora B. hSpindly depletion results in reduced inter-KT tension, unstable KT fibers, an extensive prometaphase delay, and severe chromosome misalignment. Moreover, depletion of hSpindly induces a striking spindle rotation, which can be rescued by co-depletion of dynein. However, in contrast to Drosophila, hSpindly depletion does not abolish the removal of MAD2 and ZW10 from KTs. Collectively, our data reveal hSpindly-mediated dynein functions and highlight a critical role of KT dynein in spindle orientation.

    The Journal of cell biology 2009;185;5;859-74

  • Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration.

    Moore JK, Sept D and Cooper JA

    Department of Cell Biology and Physiology, Washington University, Saint Louis, MO 63110, USA.

    Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1-4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150(glued) subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5-8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement.

    Funded by: NCI NIH HHS: T-32-CA113275, T32 CA113275; NIGMS NIH HHS: GM47337, R01 GM047337, R01 GM067246

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;13;5147-52

  • DCTN1 mutations in Perry syndrome.

    Farrer MJ, Hulihan MM, Kachergus JM, Dächsel JC, Stoessl AJ, Grantier LL, Calne S, Calne DB, Lechevalier B, Chapon F, Tsuboi Y, Yamada T, Gutmann L, Elibol B, Bhatia KP, Wider C, Vilariño-Güell C, Ross OA, Brown LA, Castanedes-Casey M, Dickson DW and Wszolek ZK

    Division of Neurogenetics, Department of Neuroscience, Mayo Clinic Florida, Jacksonville, Florida 32224, USA. farrer.matthew@mayo.edu

    Perry syndrome consists of early-onset parkinsonism, depression, severe weight loss and hypoventilation, with brain pathology characterized by TDP-43 immunostaining. We carried out genome-wide linkage analysis and identified five disease-segregating mutations affecting the CAP-Gly domain of dynactin (encoded by DCTN1) in eight families with Perry syndrome; these mutations diminish microtubule binding and lead to intracytoplasmic inclusions. Our findings show that DCTN1 mutations, previously associated with motor neuron disease, can underlie the selective vulnerability of other neuronal populations in disti 119c nct neurodegenerative disorders.

    Funded by: NINDS NIH HHS: P01 NS040256, P01 NS040256-019001, P01 NS040256-01S19001, P01 NS040256-029001, P01 NS040256-04, P01 NS040256-05, P50 NS040256, P50 NS40256

    Nature genetics 2009;41;2;163-5

  • Huntingtin regulates RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) nuclear trafficking indirectly through a complex with REST/NRSF-interacting LIM domain protein (RILP) and dynactin p150 Glued.

    Shimojo M

    Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0509, USA. mshim1@uky.edu

    Huntingtin has been reported to regulate the nuclear translocation of the transcriptional repressor RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). The REST/NRSF-interacting LIM domain protein (RILP) has also been shown to regulate REST/NRSF nuclear translocation. Therefore, we were prompted to address the question of how two distinct proteins could have the same function. We initially used a yeast two-hybrid screen to look for an interaction between huntingtin and RILP. This screen identified dynactin p150(Glued) as an interacting protein. Coimmunoprecipitation of proteins in vitro expressed in a reticulocyte lysate system showed an interaction between REST/NRSF and RILP as well as between RILP and dynactin p150(Glued). Coimmunoprecipitation analysis further showed a complex containing RILP, dynactin p150(Glued), and huntingtin. Huntingtin did not interact directly with either REST/NRSF or RILP, but did interact with dynactin p150(Glued). The N-terminal fragment of wild-type huntingtin did not affect the interaction between dynactin p150(Glued) and RILP; however, mutant huntingtin weakened this interaction. We further show that HAP1 (huntingtin-associated protein-1) prevents this complex from translocating REST/NRSF to the nucleus. Thus, this study suggests that REST/NRSF, dynactin p150(Glued), huntingtin, HAP1, and RILP form a complex involved in the translocation of REST/NRSF into the nucleus and that HAP1 controls REST/NRSF cellular localization in neurons.

    Funded by: NCRR NIH HHS: P20RR020171; NIMH NIH HHS: K01MH067123

    The Journal of biological chemistry 2008;283;50;34880-6

  • Regulation of dynactin through the differential expression of p150Glued isoforms.

    Dixit R, Levy JR, Tokito M, Ligon LA and Holzbaur EL

    Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. We have identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, we used RNAi to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; here we investigate the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell.

    Funded by: NIA NIH HHS: T32 AG00255; NIGMS NIH HHS: GM068591, GM48661

    The Journal of biological chemistry 2008;283;48;33611-9

  • Lysosomal proliferation and distal degeneration in motor neurons expressing the G59S mutation in the p150Glued subunit of dynactin.

    Chevalier-Larsen ES, Wallace KE, Pennise CR and Holzbaur EL

    Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA.

    An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.

    Funded by: NIGMS NIH HHS: GM48661, R01 GM048661, R01 GM048661-15

    Human molecular genetics 2008;17;13;1946-55

  • Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype-phenotype correlation study.

    Dierick I, Baets J, Irobi J, Jacobs A, De Vriendt E, Deconinck T, Merlini L, Van den Bergh P, Rasic VM, Robberecht W, Fischer D, Morales RJ, Mitrovic Z, Seeman P, Mazanec R, Kochanski A, Jordanova A, Auer-Grumbach M, Helderman-van den Enden AT, Wokke JH, Nelis E, De Jonghe P and Timmerman V

    Peripheral Neuropathy Group, VIB Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium.

    Distal hereditary motor neuropathy (HMN) is a clinically and genetically heterogeneous group of disorders affecting spinal alpha-motor neurons. Since 2001, mutations in six different genes have been identified for autosomal dominant distal HMN; glycyl-tRNA synthetase (GARS), dynactin 1 (DCTN1), small heat shock 27 kDa protein 1 (HSPB1), small heat shock 22 kDa protein 8 (HSPB8), Berardinelli-Seip congenital lipodystrophy (BSCL2) and senataxin (SETX). In addition a mutation in the (VAMP)-associated protein B and C (VAPB) was found in several Brazilian families with complex and atypical forms of autosomal dominantly inherited motor neuron disease. We have investigated the distribution of mutations in these seven genes in a cohort of 112 familial and isolated patients with a diagnosis of distal motor neuropathy and found nine different disease-causing mutations in HSPB8, HSPB1, BSCL2 and SETX in 17 patients of whom 10 have been previously reported. No mutations were found in GARS, DCTN1 and VAPB. The phenotypic features of patients with mutations in HSPB8, HSPB1, BSCL2 and SETX fit within the distal HMN classification, with only one exception; a C-terminal HSPB1-mutation was associated with upper motor neuron signs. Furthermore, we provide evidence for a genetic mosaicism in transmitting an HSPB1 mutation. This study, performed in a large cohort of familial and isolated distal HMN patients, clearly confirms the genetic and phenotypic heterogeneity of distal HMN and provides a basis for the development of algorithms for diagnostic mutation screening in this group of disorders.

    Brain : a journal of neurology 2008;131;Pt 5;1217-27

  • Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking.

    Laird FM, Farah MH, Ackerley S, Hoke A, Maragakis N, Rothstein JD, Griffin J, Price DL, Martin LJ and Wong PC

    Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, approximately 5-10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150(Glued) subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150(Glued) causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150(Glued). Neuronal expression of mutant, but not wild type, dynactin p150(Glued) causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150(Glued) mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease.

    Funded by: NINDS NIH HHS: R01 NS040014, R01 NS40014

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;9;1997-2005

  • Novel interaction partners of Bardet-Biedl syndrome proteins.

    Oeffner F, Moch C, Neundorf A, Hofmann J, Koch M and Grzeschik KH

    Center of Human Genetics, University of Marburg, 35037 Marburg, Germany. oeffner@staff.uni-marburg.de

    Bardet-Biedl syndrome (BBS) is a rare, developmental disorder characterized by six major symptoms: rod-cone dystrophy, obesity, polydactyly, renal abnormalities, learning difficulties, and hypogonadism. Secondary features include cardiac and hepatic anomalies, metabolic disturbancies, and hearing loss. BBS is genetically heterogeneous with 12 disease genes (BBS1-BBS12) described thus far. Current data suggest a functional disturbance in ciliary function and intraflagellar transport being associated with the phenotype. However, the precise functions of the BBS proteins have yet to be elucidated. This study focuses on the detection of protein factors interacting with BBS proteins. Applying yeast two-hybrid (Y2H) technology we found a series of novel, functionally potentially plausible binding partners of BBS1, BBS2, BBS4, and BBS7. Protein interactions were supported by coimmunoprecipitation analyses (ALDOB, EPAS1) and substantiated by colocalization studies at the subcellular level (ALDOB, EXOC7, FLOT1, KRT18, PAX2). Our work provides new insights into the understanding of BBS interactions and thus their biological function.

    Cell motility and the cytoskeleton 2008;65;2;143-55

  • Suppression of microtubule dynamic instability by the +TIP protein EB1 and its modulation by the CAP-Gly domain of p150glued.

    Manna T, Honnappa S, Steinmetz MO and Wilson L

    Department of Molecular, Cellular, and Developmental Biology and the Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California 93106, USA.

    The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.

    Funded by: NINDS NIH HHS: NS13560

    Biochemistry 2008;47;2;779-86

  • The G59S mutation in p150(glued) causes dysfunction of dynactin in mice.

    Lai C, Lin X, Chandran J, Shim H, Yang WJ and Cai H

    Unit of Transgenesis, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The G59S missense mutation at the conserved microtubule-binding domain of p150(glued), a major component of dynein/dynactin complex, has been linked to an autosomal dominant form of motor neuron disease (MND). To study how this mutation affects the function of the dynein/dynactin complex and contributes to motor neuron degeneration, we generated p150(glued) G59S knock-in mice. We found that the G59S mutation destabilizes p150(glued) and disrupts the function of dynein/dynactin complex, resulting in early embryonic lethality of homozygous knock-in mice. Heterozygous knock-in mice, which developed normally, displayed MND-like phenotypes after 10 months of age, including excessive accumulation of cytoskeletal and synaptic vesicle proteins at neuromuscular junctions, loss of spinal motor neurons, increase of reactive astrogliosis, and shortening of gait compared with wild-type littermates and age-matched p150(glued) heterozygous knock-out mice. Our findings indicate that the G59S mutation in p150(glued) abrogates the normal function of p150(glued) and accelerates motor neuron degeneration.

    Funded by: Intramural NIH HHS: Z01 AG000959-04, Z99 AG999999

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;51;13982-90

  • Interaction of tau protein with the dynactin complex.

    Magnani E, Fan J, Gasparini L, Golding M, Williams M, Schiavo G, Goedert M, Amos LA and Spillantini MG

    Department of Clinical Neurosciences, Brain Repair Centre, University of Cambridge, Cambridge, UK.

    Tau is an axonal microtubule-associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and tau aggregates are present in Alzheimer's disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated. The dynein-activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N-terminal projection domain of tau binds to the C-terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N-terminus of tau, found in patients with FTDP-17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule-binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.

    Funded by: Medical Research Council: G0301152, MC_U105184291, MC_U105184313, U.1051.04.002(78842)

    The EMBO journal 2007;26;21;4546-54

  • The p150 subunit of dynactin (DCTN1) gene in multiple sclerosis.

    Münch C, Meyer R, Linke P, Meyer T, Ludolph AC, Haas J and Hemmer B

    Department of Neurology, Jewish Hospital, Berlin, Germany. muench@jkb-online.de

    Objectives: Mutations in the p150 subunit of the axonal transport protein dynactin (DCTN1) have been reported in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the common features of neurodegeneration in multiple sclerosis (MS), FTD and ALS, sequence variants of the DCTN1 gene may be a contributory factor to neurodegeneration in MS.

    Methods: We investigated a total of 200 MS patients and 200 controls. A total of 100 patients had a relapsing-remitting form of MS, 100 cases were primary progressive. Sequence alterations were screened for in the coding region of DCTN1 using heteroduplex and sequence analyses.

    Results: Two heterozygous missense mutations (T1249I, I196V) were found in two healthy control subjects. No mutations were identified in 200 MS patients. The frequency of a known single nucleotide polymorphism (R495Q) was not significantly different between patients and controls.

    Conclusion: The results indicate that the DCTN1 gene is probably not influencing susceptibility to neurodegeneration in MS.

    Acta neurologica Scandinavica 2007;116;4;231-4

  • Structure-function relationship of CAP-Gly domains.

    Weisbrich A, Honnappa S, Jaussi R, Okhrimenko O, Frey D, Jelesarov I, Akhmanova A and Steinmetz MO

    Biomolecular Research, Structural Biology, Paul Scherrer Insititut, CH-5232 Villigen PSI, Switzerland.

    In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions. We demonstrate that the conserved GKNDG motif of CAP-Gly domains is responsible for targeting to the C-terminal EEY/F sequence motifs of CLIP170, EB proteins and microtubules. The CAP-Gly-EEY/F interaction is essential for the recruitment of the dynactin complex by CLIP170 and for activation of CLIP170. Our findings define the molecular basis of CAP-Gly domain function, including the tubulin detyrosination-tyrosination cycle. They further establish fundamental roles for the interaction between CAP-Gly proteins and C-terminal EEY/F sequence motifs in regulating complex and dynamic cellular processes.

    Nature structural & molecular biology 2007;14;10;959-67

  • FBXL5 interacts with p150Glued and regulates its ubiquitination.

    Zhang N, Liu J, Ding X, Aikhionbare F, Jin C and Yao X

    Division of Cellular Dynamics, Hefei National Laboratory for Physical Sciences and Chinese University of Science & Technology, Hefei 230027, China.

    The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150(Glued) is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao, Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394-402]. Here we show that F-box protein FBXL5 interacts with p150(Glued) and orchestrates its turnover via ubiquitination. FBXL5 binds to p150(Glued)in vitro and in vivo. FBXL5 and p150(Glued) co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150(Glued) and protein turnover of p150(Glued). Our findings provide a potential mechanism by which p150(Glued) protein function is regulated by SCFs.

    Funded by: NCI NIH HHS: CA92080; NIDDK NIH HHS: DK56292

    Biochemical and biophysical research communications 2007;359;1;34-9

  • Requirement of dynactin p150(Glued) subunit for the functional integrity of the keratinocyte microparasol.

    Byers HR, Dykstra SG and Boissel SJ

    Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. hrbyers@bu.edu

    The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.

    Funded by: NCI NIH HHS: CA-45587

    The Journal of investigative dermatology 2007;127;7;1736-44

  • Dynactin is required for coordinated bidirectional motility, but not for dynein membrane attachment.

    Haghnia M, Cavalli V, Shah SB, Schimmelpfeng K, Brusch R, Yang G, Herrera C, Pilling A and Goldstein LS

    Howard Hughes Medical Institute and Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0683, USA.

    Transport of cellular and neuronal vesicles, organelles, and other particles along microtubules requires the molecular motor protein dynein (Mallik and Gross, 2004). Critical to dynein function is dynactin, a multiprotein complex commonly thought to be required for dynein attachment to membrane compartments (Karki and Holzbaur, 1999). Recent work also has found that mutations in dynactin can cause the human motor neuron disease amyotrophic lateral sclerosis (Puls et al., 2003). Thus, it is essential to understand the in vivo function of dynactin. To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors (King and Schroer, 2000).

    Funded by: NIGMS NIH HHS: GM46295, R01 GM046295

    Molecular biology of the cell 2007;18;6;2081-9

  • 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

  • GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome.

    Fumoto K, Hoogenraad CC and Kikuchi A

    Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.

    Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.

    The EMBO journal 2006;25;24;5670-82

  • Microtubule plus-end loading of p150(Glued) is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells.

    Watson P and Stephens DJ

    Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK.

    Microtubule dynamics and function are regulated, at least in part, by a family of proteins that localize to microtubule plus-ends, and include EB1, CLIP-170 and the dynactin component p150(Glued). Plus-end pools of these proteins, notably dynactin, have been invoked in a number of ;search-and-capture' mechanisms, including the attachment of microtubules to kinetochores during mitosis and to endomembranes prior to the initiation of intracellular transport. Here we show that, in mammalian cells, EB1 is required for the plus-end localization of CLIP-170, and that this is in turn required to localize p150(Glued) to plus-ends. Specific depletion of CLIP-170 results in defects in microtubule dynamics, cell polarization in response to scratch wounding and a loss of p150(Glued) from plus ends. By contrast, removal of p150(Glued) from plus-ends by depletion of either EB1 or CLIP-170 caused no defects in the localization of intracellular organelles, the dynamics of ER-to-Golgi transport, the efficiency of transferrin uptake or the motility of early endosomes or lysosomes. In addition to labelling microtubule plus-ends, we show that GFP-p150(Glued) becomes incorporated into the dynactin complex and labels small, highly dynamic, punctate structures that move along microtubules. A subset of these structures colocalizes with ER-Golgi transport intermediates. Together, these data show that the function of CLIP-170 and p150(Glued) in membrane trafficking is not associated with their plus-end localization.

    Funded by: Medical Research Council: G117/554, G117/554(71630); Wellcome Trust: 071233

    Journal of cell science 2006;119;Pt 13;2758-67

  • The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

    Petretti C, Savoian M, Montembault E, Glover DM, Prigent C and Giet R

    CNRS UMR 6061 Université de Rennes I, Equipe Labellisée Ligue Nationale Contre le Cancer, IFR140 GFAS, Faculté de Médecine, France.

    The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.

    EMBO reports 2006;7;4;418-24

  • A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

    Levy JR, Sumner CJ, Caviston JP, Tokito MK, Ranganathan S, Ligon LA, Wallace KE, LaMonte BH, Harmison GG, Puls I, Fischbeck KH and Holzbaur EL

    Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

    The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

    Funded by: Intramural NIH HHS; NIA NIH HHS: T32 AG000255, T32 AG00255; NIGMS NIH HHS: GM48661, R01 GM048661; NINDS NIH HHS: K22 NS048199, K22-NS0048199-01

    The Journal of cell biology 2006;172;5;733-45

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD.

    Münch C, Rosenbohm A, Sperfeld AD, Uttner I, Reske S, Krause BJ, Sedlmeier R, Meyer T, Hanemann CO, Stumm G and Ludolph AC

    Department of Neurology, Jewish Hospital, Berlin.

    A heterozygous R1101K mutation of the p150 subunit of dynactin (DCTN1) is reported in a family with amyotrophic lateral sclerosis (ALS) and co-occurrence of frontotemporal dementia (FTD). Two members of our kindred were affected with motor neuron disease and two with dementia in an autosomal dominant pattern of inheritance. We excluded the involvement of the ALS and FTD-linked genes for copper/zinc superoxide dismutase (SOD1) and tau. The R1101K sequence alteration of the DCTN1 gene may predispose subjects to ALS and FTD.

    Annals of neurology 2005;58;5;777-80

  • 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

  • RPGR-ORF15, which is mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule transport proteins.

    Khanna H, Hurd TW, Lillo C, Shu X, Parapuram SK, He S, Akimoto M, Wright AF, Margolis B, Williams DS and Swaroop A

    Department of Ophthalmology & Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105, USA.

    Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene account for almost 20% of patients with retinitis pigmentosa. Most mutations are detected in alternatively spliced RPGR-ORF15 isoform(s), which are primarily but not exclusively expressed in the retina. We show that, in addition to the axoneme, the RPGR-ORF15 protein is localized to the basal bodies of photoreceptor connecting cilium and to the tip and axoneme of sperm flagella. Mass spectrometric analysis of proteins that were immunoprecipitated from the retinal axoneme-enriched fraction using an anti-ORF15 antibody identified two chromosome-associated proteins, structural maintenance of chromosomes (SMC) 1 and SMC3. Using pulldown assays, we demonstrate that the interaction of RPGR with SMC1 and SMC3 is mediated, at least in part, by the RCC1-like domain of RPGR. This interaction was not observed with phosphorylation-deficient mutants of SMC1. Both SMC1 and SMC3 localized to the cilia of retinal photoreceptors and Madin-Darby canine kidney cells, suggesting a broader physiological relevance of this interaction. Additional immunoprecipitation studies revealed the association of RPGR-ORF15 isoform(s) with the intraflagellar transport polypeptide IFT88 as well as microtubule motor proteins, including KIF3A, p150Glued, and p50-dynamitin. Inhibition of dynein function by overexpressing p50 abrogated the localization of RPGR-ORF15 to basal bodies. Taken together, these results provide novel evidence for the possible involvement of RPGR-ORF15 in microtubule organization and regulation of transport in primary cilia.

    Funded by: NEI NIH HHS: EY07003, EY07961, EY12598, EY13408, F31 EY007003, P30 EY007003, P30 EY012598, R01 EY007042, R01 EY007961, R01 EY013408; NIDDK NIH HHS: DK069605, R01 DK069605

    The Journal of biological chemistry 2005;280;39;33580-7

  • 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

  • Coupling of ER exit to microtubules through direct interaction of COPII with dynactin.

    Watson P, Forster R, Palmer KJ, Pepperkok R and Stephens DJ

    Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK.

    Transport of proteins from the endoplasmic reticulum (ER) to the Golgi is mediated by the sequential action of two coat complexes: COPII concentrates cargo for secretion at ER export sites, then COPI is subsequently recruited to nascent carriers and retrieves recycling proteins back to the ER. These carriers then move towards the Golgi along microtubules, driven by the dynein/dynactin complexes. Here we show that the Sec23p component of the COPII complex directly interacts with the dynactin complex through the carboxy-terminal cargo-binding domain of p150(Glued). Functional assays, including measurements of the rate of recycling of COPII on the ER membrane and quantitative analyses of secretion, indicate that this interaction underlies functional coupling of ER export to microtubules. Together, our data suggest a mechanism by which membranes of the early secretory pathway can be linked to motors and microtubules for subsequent organization and movement to the Golgi apparatus.

    Funded by: Wellcome Trust: 071233

    Nature cell biology 2005;7;1;48-55

  • Assay and properties of rab6 interaction with dynein-dynactin complexes.

    Fuchs E, Short B and Barr FA

    RAB GTPases help to maintain the fidelity of membrane trafficking events by recruiting cytosolic tethering and motility factors to vesicle and organelle membranes. In the case of Rab6, it recruits the dynein-dynaction complex to Golgi-associated vesicles via an adaptor protein of the Bicaudal-D family. Here we describe methods for the identification of Rab6-binding partners in cell extracts. We then focus on the biochemical analysis of interactions with the dynein-dynactin complex and the adaptor proteins Bicaudal-D1 and -D2. Standard protocols for yeast two-hybrid analysis, and biochemical assays for the analysis of the interactions between Rab6, Bicaudal-D, and the subunits of the dynein-dynactin complex are outlined.

    Methods in enzymology 2005;403;607-18

  • p150(Glued), Dynein, and microtubules are specifically required for activation of MKK3/6 and p38 MAPKs.

    Cheung PY, Zhang Y, Long J, Lin S, Zhang M, Jiang Y and Wu Z

    Department of Biochemistry, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    To look for regulators of the mitogen-activated protein kinase (MAPK) kinase 6 (MKK6), a yeast two-hybrid screen was initiated using MKK6 as bait. p150(Glued) dynactin, a key component of the cytoplasmic dynein-dynactin motor complex, was found to specifically interact with MKK6 and its close homologue MKK3. Silencing of p150(Glued) expression by small interference RNA reduced the stimulus-induced phosphorylation of MKK3/6 and p38 MAPKs. The similar adverse effect was also seen when the cytoplasmic dynein motor was disrupted by other means. Like p150(Glued), MKK3/6 directly associate with microtubules. Disruption of microtubules prior to cell stimulation specifically inhibits the stimulus-induced phosphorylation of both MKK3/6 and p38 MAPKs. Our unexpected findings reveal a specific requirement for p150(Glued)/dynein/functional microtubules in activation of MKK3/6 and p38 MAPKs in vivo.

    The Journal of biological chemistry 2004;279;44;45308-11

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS.

    Münch C, Sedlmeier R, Meyer T, Homberg V, Sperfeld AD, Kurt A, Prudlo J, Peraus G, Hanemann CO, Stumm G and Ludolph AC

    Department of Neurology, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. christoph.muench@medizin.uni-ulm.de

    The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

    Neurology 2004;63;4;724-6

  • The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression.

    Kim JC, Badano JL, Sibold S, Esmail MA, Hill J, Hoskins BE, Leitch CC, Venner K, Ansley SJ, Ross AJ, Leroux MR, Katsanis N and Beales PL

    Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr., Burnaby BC, V5A 1S6, Canada.

    BBS4 is one of several proteins that cause Bardet-Biedl syndrome (BBS), a multisystemic disorder of genetic and clinical complexity. Here we show that BBS4 localizes to the centriolar satellites of centrosomes and basal bodies of primary cilia, where it functions as an adaptor of the p150(glued) subunit of the dynein transport machinery to recruit PCM1 (pericentriolar material 1 protein) and its associated cargo to the satellites. Silencing of BBS4 induces PCM1 mislocalization and concomitant deanchoring of centrosomal microtubules, arrest in cell division and apoptotic cell death. Expression of two truncated forms of BBS4 that are similar to those found in some individuals with BBS had a similar effect on PCM1 and microtubules. Our findings indicate that defective targeting or anchoring of pericentriolar proteins and microtubule disorganization contribute to the BBS phenotype and provide new insights into possible causes of familial obesity, diabetes and retinal degeneration.

    Nature genetics 2004;36;5;462-70

  • Dynactin affects extension and assembly of adherens junctions in Drosophila photoreceptor development.

    Fan SS

    Department of Biology and Life Science Research Center, Tunghai University, Taichung, Taiwan, ROC. sfan@mail.thu.edu.tw

    Drosophila eye development is a progressive process including cell fate determination, pattern formation, and rhabdomere morphogenesis. During eye development, a dramatic change in cell shape, which involves turning and extension of the photoreceptor apical surface, occurs in the early pupal stages. It is known that assembly and extension of adherens junctions (AJs) play an important role in this process. In the present study, I show that mutation of the largest subunit of dynactin complexes encoded by Glued (Gl) affects the extension and assembly of Ajs in developing photoreceptors. In Gl(1)/(+) mutants and transgenic flies expressing the dominant negative form of Glued, the AJs failed to properly assemble and extend. In addition, the morphogenesis of rhabdomeres was also affected in these flies. Taken together, these results suggest that the extension and assembly of AJs as well as determination of the rhabdomere domain in photoreceptor development are Gl dependent.

    Journal of biomedical science 2004;11;3;362-9

  • TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly.

    Cassimeris L and Morabito J

    Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, USA. lc07@lehigh.edu

    The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336-341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by approximately 20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by approximately 20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.

    Funded by: NIGMS NIH HHS: GM58025, R01 GM058025

    Molecular biology of the cell 2004;15;4;1580-90

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Characterization of functional domains of human EB1 family proteins.

    Bu W and Su LK

    Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

    EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.

    Funded by: NCI NIH HHS: CA 6672, CA 70371

    The Journal of biological chemistry 2003;278;50;49721-31

  • Preferentially localized dynein and perinuclear dynactin associate with nuclear pore complex proteins to mediate genomic union during mammalian fertilization.

    Payne C, Rawe V, Ramalho-Santos J, Simerly C and Schatten G

    Program in Molecular and Cellular Biosciences, Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97201, USA.

    Fertilization is complete once the parental genomes unite, and requires the migration of the egg nucleus to the sperm nucleus (female and male pronuclei, respectively) on microtubules within the inseminated egg. Neither the molecular mechanism of pronucleus binding to microtubules nor the role of motor proteins in regulating pronuclear motility has been fully characterized, and the failure of zygotic development in some patients suggests that they contribute to human infertility. Based on the minus-end direction of female pronuclear migration, we propose a role for cytoplasmic dynein and dynactin in associating with the pronuclear envelope and mediating genomic union. Our results show that dynein intermediate and heavy chains preferentially concentrate around the female pronucleus, whereas dynactin subunits p150Glued, p50 and p62 localize to the surfaces of both pronuclei. Transfection of antibodies against dynein and dynactin block female pronuclear migration in zygotes. Both parthenogenetic activation in oocytes and microtubule depolymerization in zygotes significantly reduce the localization of dynein to the female pronucleus but do not inhibit the pronuclear association of dynactin. When immunoprecipitated from zygotes, p150Glued associates with nuclear pore complex proteins, as well as the intermediate filament vimentin and dynein. Antibodies against nucleoporins and vimentin inhibit pronuclear apposition when transfected into zygotes. We conclude that preferentially localized dynein and perinuclear dynactin associate with the nuclear pore complex and vimentin and are required to mediate genomic union. These data suggest a model in which dynein accumulates and binds to the female pronucleus on sperm aster microtubules, where it interacts with dynactin, nucleoporins and vimentin.

    Funded by: NICHD NIH HHS: R00 HD055330

    Journal of cell science 2003;116;Pt 23;4727-38

  • LIS1 association with dynactin is required for nuclear motility and genomic union in the fertilized mammalian oocyte.

    Payne C, St John JC, Ramalho-Santos J and Schatten G

    Program in Molecular and Cellular Biosciences, Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, USA.

    Mutations in the human LIS1 gene cause the devastating brain disorder lissencephaly. LIS1 also regulates microtubule dynamics; it interacts with the molecular motor cytoplasmic dynein and its cofactor dynactin, and is necessary for neuronal migration. Recently, LIS1 has been suggested to mediate pronuclear migration during fertilization. Here we use rhesus monkey and bovine oocytes, as well as pronucleate-stage bovine zygotes, to determine: Lis1 RNA expression using reverse transcription-polymerase chain reaction; LIS1 protein association with dynactin using immunoprecipitation, Western blot analysis, and immunocytochemistry; and LIS1 function in mediating genomic union using antibody transfection. We find that Lis1 RNA expression increases during fertilization, that LIS1 and dynactin subunit p150/(Glued) co-immunoprecipitate and co-localize to pronuclear surfaces, and that anti-LIS1 antibodies transfected into zygotes dramatically inhibit pronuclear migration and apposition. LIS1 is, therefore, essential to mediate genomic union in a process that involves the dynein-dynactin complex. These results shed light on an additional role for LIS1 and raise implications for human reproduction.

    Cell motility and the cytoskeleton 2003;56;4;245-51

  • BPAG1n4 is essential for retrograde axonal transport in sensory neurons.

    Liu JJ, Ding J, Kowal AS, Nardine T, Allen E, Delcroix JD, Wu C, Mobley W, Fuchs E and Yang Y

    Department of Neurology, Stanford University School of Medicine, Stanford, CA 94305-5489, USA. yanmin.yang@stanford.edu

    Disruption of the BPAG1 (bullous pemphigoid antigen 1) gene results in progressive deterioration in motor function and devastating sensory neurodegeneration in the null mice. We have previously demonstrated that BPAG1n1 and BPAG1n3 play important roles in organizing cytoskeletal networks in vivo. Here, we characterize functions of a novel BPAG1 neuronal isoform, BPAG1n4. Results obtained from yeast two-hybrid screening, blot overlay binding assays, and coimmunoprecipitations demonstrate that BPAG1n4 interacts directly with dynactin p150Glued through its unique ezrin/radixin/moesin domain. Studies using double immunofluorescent microscopy and ultrastructural analysis reveal physiological colocalization of BPAG1n4 with dynactin/dynein. Disruption of the interaction between BPAG1n4 and dynactin results in severe defects in retrograde axonal transport. We conclude that BPAG1n4 plays an essential role in retrograde axonal transport in sensory neurons. These findings might advance our understanding of pathogenesis of axonal degeneration and neuronal death.

    Funded by: NIA NIH HHS: AG16999, R01 AG016999; NIAMS NIH HHS: AR27883, R01 AR027883, R37 AR027883; NINDS NIH HHS: K02 NS043281, NS24054, NS38869, NS42791, NS43281, R01 NS024054, R01 NS038869, R01 NS042791

    The Journal of cell biology 2003;163;2;223-9

  • A role for Plk1 phosphorylation of NudC in cytokinesis.

    Zhou T, Aumais JP, Liu X, Yu-Lee LY and Erikson RL

    Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA. tzhou@mcb.harvard.edu

    Polo-like kinase 1 (Plk1) plays essential roles at multiple events during cell division, yet little is known about its physiological substrates. In a cDNA phage display screen using Plk1 C-terminal affinity columns, we identified NudC (nuclear distribution gene C) as a Plk1 binding protein. Here, we characterize the interaction between Plk1 and NudC, show that Plk1 phosphorylates NudC at conserved S274 and S326 residues in vitro, and present evidence that NudC is also a substrate for Plk1 in vivo. Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, which are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites. These results suggest that Plk1 phosphorylation of NudC may influence cytokinesis.

    Funded by: NIGMS NIH HHS: GM59172

    Developmental cell 2003;5;1;127-38

  • CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms.

    Goodson HV, Skube SB, Stalder R, Valetti C, Kreis TE, Morrison EE and Schroer TA

    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA. hgoodson@nd.edu

    CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.

    Funded by: NIGMS NIH HHS: GM44589

    Cell motility and the cytoskeleton 2003;55;3;156-73

  • Interactions between the evolutionarily conserved, actin-related protein, Arp11, actin, and Arp1.

    Eckley DM and Schroer TA

    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.

    The dynein activator dynactin is a multiprotein complex with distinct microtubule- and cargo-binding domains. The cargo-binding domain contains a short, actin-like filament of the actin-related protein Arp1, a second actin-related protein, Arp11, and conventional actin. The length of this filament is invariant in dynactin isolated from multiple species and tissues, suggesting that activities that regulate Arp1 polymerization are important for dynactin assembly. Arp11 is present in a protein complex localized at the pointed end of the Arp1 minifilament, whereas actin capping protein (CapZ) is present at the barbed end. Either might cooperate with conventional actin to cap Arp1. We tested the ability of Arp11 to interact with conventional actin and found it could coassemble. Like Arp1, cytosolic Arp11 is found only in dynactin, suggesting that Arp11 and free cytosolic actin do not interact significantly. Recombinant Arp11 and Arp1 were demonstrated to interact by coprecipitation. We developed an in vivo assay for Arp11-Arp1 interaction based on previous observations that Arp1 forms filamentous assemblies when overexpressed in cultured cells. Arp11 significantly decreases the formation of these organized Arp1 assemblies. Finally, this assay was used to confirm the identity of a putative Arp11 homolog in Drosophila melanogaster.

    Funded by: NIGMS NIH HHS: GM-44589, R01 GM044589, R56 GM044589

    Molecular biology of the cell 2003;14;7;2645-54

  • Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Casenghi M, Meraldi P, Weinhart U, Duncan PI, Körner R and Nigg EA

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152, Martinsried, Germany.

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

    Developmental cell 2003;5;1;113-25

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Mutant dynactin in motor neuron disease.

    Puls I, Jonnakuty C, LaMonte BH, Holzbaur EL, Tokito M, Mann E, Floeter MK, Bidus K, Drayna D, Oh SJ, Brown RH, Ludlow CL and Fischbeck KH

    Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA. pulsi@ninds.nih.gov

    Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

    Nature genetics 2003;33;4;455-6

  • Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex.

    Matanis T, Akhmanova A, Wulf P, Del Nery E, Weide T, Stepanova T, Galjart N, Grosveld F, Goud B, De Zeeuw CI, Barnekow A and Hoogenraad CC

    Department of Experimental Tumorbiology, University of Muenster, Badestrasse 9, D-48149 Muenster, Germany.

    The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport.

    Nature cell biology 2002;4;12;986-92

  • Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes.

    Quintyne NJ and Schroer TA

    Department of Biology, Johns Hopkins University, Charles & 34th Streets, Baltimore, MD 21218, USA.

    Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.

    Funded by: NIDDK NIH HHS: DK 44375; NIGMS NIH HHS: GM 44589, R01 GM044589, R56 GM044589

    The Journal of cell biology 2002;159;2;245-54

  • The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes.

    Short B, Preisinger C, Schaletzky J, Kopajtich R and Barr FA

    Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried, Germany.

    Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes.

    Current biology : CB 2002;12;20;1792-5

  • Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome.

    Askham JM, Vaughan KT, Goodson HV and Morrison EE

    Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds LS9 7TF, United Kingdom. rmrjma@leeds.ac.uk

    EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

    Molecular biology of the cell 2002;13;10;3627-45

  • Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

    Takahashi M, Yamagiwa A, Nishimura T, Mukai H and Ono Y

    Biosignal Research Center, Kobe University, Japan.

    Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

    Molecular biology of the cell 2002;13;9;3235-45

  • Pim-1 associates with protein complexes necessary for mitosis.

    Bhattacharya N, Wang Z, Davitt C, McKenzie IF, Xing PX and Magnuson NS

    Cancer Prevention and Research Center, WSU, Pullman, WA 99164-4234, USA.

    The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.

    Chromosoma 2002;111;2;80-95

  • Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.

    Tai CY, Dujardin DL, Faulkner NE and Vallee RB

    University of Massachusetts Medical School, Department of Cell Biology, Worcester, MA 01605, USA.

    Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.

    Funded by: NICHD NIH HHS: HD61982; NIGMS NIH HHS: GM47434, R01 GM047434, R37 GM047434

    The Journal of cell biology 2002;156;6;959-68

  • beta III spectrin binds to the Arp1 subunit of dynactin.

    Holleran EA, Ligon LA, Tokito M, Stankewich MC, Morrow JS and Holzbaur EL

    Department of Cell and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.

    Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

    Funded by: NIGMS NIH HHS: GM48661

    The Journal of biological chemistry 2001;276;39;36598-605

  • Mammalian Golgi-associated Bicaudal-D2 functions in the dynein-dynactin pathway by interacting with these complexes.

    Hoogenraad CC, Akhmanova A, Howell SA, Dortland BR, De Zeeuw CI, Willemsen R, Visser P, Grosveld F and Galjart N

    MGC Department of Cell Biology, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands.

    Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

    The EMBO journal 2001;20;15;4041-54

  • Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

    Lane JD, Vergnolle MA, Woodman PG and Allan VJ

    School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

    Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

    The Journal of cell biology 2001;153;7;1415-26

  • Genomic organization of the DCTN1-SLC4A5 locus encoding both NBC4 and p150(Glued).

    Pushkin A, Abuladze N, Newman D, Tatishchev S and Kurtz I

    Division of Nephrology, UCLA School of Medicine, Los Angeles, CA 90095, USA. Apushkin@mednet.ucla.edu

    In eukaryotes, it is rare for a single gene to encode two functionally unrelated proteins. p150(Glued) is a component of the dynactin heteromultimeric complex of proteins which is required for dynein-mediated vesicle and organelle transport by microtubules. NBC4 is an electrogenic sodium bicarbonate cotransporter, which regulates intracellular pH. Here we report that NBC4 and p150(Glued) are encoded by the same locus, DCTN1-SLC4A5. We have characterized the genomic organization of the human DCTN1-SLC4A5 locus which spans approximately 230 kilobases on chromosome 2p13 and contains 66 exons. This information should allow the study of potential genomic alterations of DCTN1-SLC4A5 in patients with diseases mapping to this genomic region.

    Funded by: NIDDK NIH HHS: DK07789, DK58563

    Cytogenetics and cell genetics 2001;95;3-4;163-8

  • Colocalization of dynactin subunits P150Glued and P50 with melanosomes in normal human melanocytes.

    Vancoillie G, Lambert J, Haeghen YV, Westbroek W, Mulder A, Koerten HK, Mommaas AM, Van Oostveldt P and Naeyaert JM

    Department of Dermatology, University Hospital, Ghent, Belgium.

    Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.

    Pigment cell research 2000;13;6;449-57

  • The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

    Mayor T, Stierhof YD, Tanaka K, Fry AM and Nigg EA

    Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

    The Journal of cell biology 2000;151;4;837-46

  • A dynactin subunit with a highly conserved cysteine-rich motif interacts directly with Arp1.

    Karki S, Tokito MK and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Dynactin is a multisubunit complex and a required cofactor for most, or all, of the cellular processes powered by the microtubule-based motor cytoplasmic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequences were used to clone human cDNAs encoding p62. Sequence analysis of the predicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn(2+)-binding RING domain. We have characterized p62 as an integral component of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that p62 binds directly to the Arp1 subunit of dynactin. Immunocytochemistry with antibodies to p62 demonstrates that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin. In transfected cells, overexpression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the protein, suggesting that this polypeptide may be targeted to the nucleus at very high expression levels.

    Funded by: NIGMS NIH HHS: GM48661

    The Journal of biological chemistry 2000;275;7;4834-9

  • Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the arp1 minifilament pointed end.

    Eckley DM, Gill SR, Melkonian KA, Bingham JB, Goodson HV, Heuser JE and Schroer TA

    Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, USA.

    The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

    Funded by: NIGMS NIH HHS: R0 GM44598

    The Journal of cell biology 1999;147;2;307-20

  • Specific isoforms of actin-binding proteins on distinct populations of Golgi-derived vesicles.

    Heimann K, Percival JM, Weinberger R, Gunning P and Stow JL

    Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia.

    Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with beta-COP, gamma-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. beta-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with beta-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

    The Journal of biological chemistry 1999;274;16;10743-50

  • A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein.

    Mattagajasingh SN, Huang SC, Hartenstein JS, Snyder M, Marchesi VT and Benz EJ

    Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.

    Funded by: NHLBI NIH HHS: HL44985, HL61295, R01 HL044985, R01 HL061295, R29 HL061295

    The Journal of cell biology 1999;145;1;29-43

  • Self-regulated polymerization of the actin-related protein Arp1.

    Bingham JB and Schroer TA

    Department of Biology The Johns Hopkins University Baltimore Maryland 21218 USA.

    The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery [1] [2] [3]. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin [4] [5] [6] and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S [7] suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament [8]. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit (D.M. Eckley, S.R. Gill, J.B.B., J.E. Heuser, T.A.S., unpublished observations) at the other [8]. In the present study, we analyzed the behavior of highly purified, native Arp1. Arp1 was found to polymerize rapidly into short filaments that were similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments.

    Current biology : CB 1999;9;4;223-6

  • The genomic structure of DCTN1, a candidate gene for limb-girdle muscular dystrophy (LGMD2B).

    Tokito MK and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 143 Rosenthal Building, 3800 Spruce Street, Philadelphia, PA 19104-6046, USA.

    Dynactin is a required activator for the molecular motor cytoplasmic dynein, and is likely to be essential for normal neuronal development. Previously we mapped the human gene encoding the p150Glued subunit of dynactin to 2p13, in the vicinity of the locus linked to limb-girdle muscular dystrophy (LGMB2B). We now report the genomic organization of DCTN1. We have identified 32 exons in the gene which spans approximately 25 kb. Alternative splicing of several of the exons generates functionally distinct isoforms of the p150Glued polypeptide.

    Funded by: NIGMS NIH HHS: GM48661

    Biochimica et biophysica acta 1998;1442;2-3;432-6

  • Human DCTN1: genomic structure and evaluation as a candidate for Alström syndrome.

    Collin GB, Nishina PM, Marshall JD and Naggert JK

    The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine, 04609-1500, USA.

    The human dynactin 1 gene (DCTN1) is positioned on chromosome 2p13, the candidate region for various diseases including Alström syndrome, limb-girdle muscle dystrophy, and Miyoshi myopathy. Here, we report the exon-intron structure of DCTN1 along with characterization of the 5' upstream sequence and alternative splice variants previously identified by Tokito et al. (1996), Mol. Biol. Cell 7: 1167-1180). Knowledge of the genomic structure of DCTN1allowed us to design intronic primers necessary for analyzing mutations in families segregating for diseases linked to this gene. These primers were tested on a French Acadian kindred segregating for Alström syndrome. No mutations were observed within the coding region of DCTN1 in this family. However, the intronic primers should allow for the rapid amplification of the coding region for mutational analysis of additional Alström families and other diseases tightly linked to the DCTN1locus on chromosome 2p13.

    Funded by: NCI NIH HHS: CA-34196; NICHD NIH HHS: R01 HD 36878; NIDDK NIH HHS: DK46977

    Genomics 1998;53;3;359-64

  • Characterization of the p22 subunit of dynactin reveals the localization of cytoplasmic dynein and dynactin to the midbody of dividing cells.

    Karki S, LaMonte B and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA.

    Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. We have characterized p22 as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein was also localized to these structures. We therefore propose that dynein/dynactin complexes may have a novel function during cytokinesis.

    Funded by: NIGMS NIH HHS: GM48661, R01 GM048661

    The Journal of cell biology 1998;142;4;1023-34

  • Interaction of huntingtin-associated protein with dynactin P150Glued.

    Li SH, Gutekunst CA, Hersch SM and Li XJ

    Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    Huntingtin is the protein product of the gene for Huntington's disease (HD) and carries a polyglutamine repeat that is expanded in HD (>36 units). Huntingtin-associated protein (HAP1) is a neuronal protein and binds to huntingtin in association with the polyglutamine repeat. Like huntingtin, HAP1 has been found to be a cytoplasmic protein associated with membranous organelles, suggesting the existence of a protein complex including HAP1, huntingtin, and other proteins. Using the yeast two-hybrid system, we found that HAP1 also binds to dynactin P150(Glued) (P150), an accessory protein for cytoplasmic dynein that participates in microtubule-dependent retrograde transport of membranous organelles. An in vitro binding assay showed that both huntingtin and P150 selectively bound to a glutathione transferase (GST)-HAP1 fusion protein. An immunoprecipitation assay demonstrated that P150 and huntingtin coprecipitated with HAP1 from rat brain cytosol. Western blot analysis revealed that HAP1 was enriched in rat brain microtubules and comigrated with P150 and huntingtin in sucrose gradients. Immunofluorescence showed that transfected HAP1 colocalized with P150 and huntingtin in human embryonic kidney (HEK) 293 cells. We propose that HAP1, P150, and huntingtin are present in a protein complex that may participate in dynein-dynactin-associated intracellular transport.

    Funded by: NINDS NIH HHS: NS01624

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;4;1261-9

  • Huntingtin-associated protein 1 (HAP1) interacts with the p150Glued subunit of dynactin.

    Engelender S, Sharp AH, Colomer V, Tokito MK, Lanahan A, Worley P, Holzbaur EL and Ross CA

    Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Huntington's disease (HD) is an inherited neurodegenerative disease caused by expansion of a polyglutamine repeat in the HD protein huntingtin. Huntingtin's localization within the cell includes an association with cytoskeletal elements and vesicles. We previously identified a protein (HAP1) which binds to huntingtin in a glutamine repeat length-dependent manner. We now report that HAP1 interacts with cytoskeletal proteins, namely the p150 Glued subunit of dynactin and the pericentriolar protein PCM-1. Structural predictions indicate that both HAP1 and the interacting proteins have a high probability of forming coiled coils. We examined the interaction of HAP1 with p150 Glued . Binding of HAP1 to p150 Glued (amino acids 879-1150) was confirmed in vitro by binding of p150 Glued to a HAP1-GST fusion protein immobilized on glutathione-Sepharose beads. Also, HAP1 co-immunoprecipitated with p150 Glued from brain extracts, indicating that the interaction occurs in vivo . Like HAP1, p150 Glued is highly expressed in neurons in brain and both proteins are enriched in a nerve terminal vesicle-rich fraction. Double label immunofluorescence experiments in NGF-treated PC12 cells using confocal microscopy revealed that HAP1 and p150 Glued partially co-localize. These results suggest that HAP1 might function as an adaptor protein using coiled coils to mediate interactions among cytoskeletal, vesicular and motor proteins. Thus, HAP1 and huntingtin may play a role in vesicle trafficking within the cell and disruption of this function could contribute to the neuronal dysfunction and death seen in HD.

    Funded by: NIGMS NIH HHS: GM48661; NIMH NIH HHS: MH01152; NINDS NIH HHS: NS16375

    Human molecular genetics 1997;6;13;2205-12

  • The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport.

    Waterman-Storer CM, Karki SB, Kuznetsov SA, Tabb JS, Weiss DG, Langford GM and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. waterman@email.unc.edu

    Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell bod 1f40 y. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150(Glued) subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150(Glued) disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150(Glued) antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.

    Funded by: NIGMS NIH HHS: GM48661, R01 GM048661

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;22;12180-5

  • Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.

    Blangy A, Arnaud L and Nigg EA

    Swiss Institute for Experimental Cancer Research (ISREC), 155, Chemin des Boveresses, CH-1066 Epalinges, Switzerland.

    The kinesin-related motor HsEg5 is essential for centrosome separation, and its association with centrosomes appears to be regulated by phosphorylation of tail residue threonine 927 by the p34(cdc2) protein kinase. To identify proteins able to interact with the tail of HsEg5, we performed a yeast two-hybrid screen with a HsEg5 stalk-tail construct as bait. We isolated a cDNA coding for the central, alpha-helical region of human p150(Glued), a prominent component of the dynactin complex. The interaction between HsEg5 and p150(Glued) was enhanced upon activation of p34(CDC28), the budding yeast homolog of p34(cdc2), provided that HsEg5 had a phosphorylatable residue at position 927. Phosphorylation also enhanced the specific binding of p150(Glued) to the tail domain of HsEg5 in vitro, indicating that the two proteins are able to interact directly. Immunofluorescence microscopy revealed co-localization of HsEg5 and p150(Glued) during mitosis but not during interphase, consistent with a cell cycle-dependent association between the two proteins. Taken together, these results suggest that HsEg5 and p150(Glued) may interact in mammalian cells in vivo and that p34(cdc2) may regulate this interaction. Furthermore, they imply that the dynactin complex may functionally interact not only with dynein but also with kinesin-related motors.

    The Journal of biological chemistry 1997;272;31;19418-24

  • Integrated radiation hybrid map of human chromosome 2p13: possible involvement of dynactin in neuromuscular diseases.

    Korthaus D, Wedemeyer N, Lengeling A, Ronsiek M, Jockusch H and Schmitt-John T

    Developmental Biology, University of Bielefeld, Germany.

    The genes for the human neuromuscular diseases limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy are located on chromosome 2p13-p14, and two neuromuscular mutations of the mouse have been mapped to regions homologous to human chromosome 2p13 by conserved synteny, wobbler (wr) on proximal Chr 11 and motor neuron degeneration 2 (mnd2) on Chr 6. Neither one is a mouse homologue of LGMD2B. Recently the gene DCTN1, coding for the large subunit of the cytoskeletal protein dynactin, was shown by FISH to be located in this region and therefore should be considered a candidate for all these disease genes. Here we present mapping data based on radiation hybrid and physical mapping that more precisely define the location of nine genetic markers in the critical region and the homology relationship of human chromosome 2p with mouse proximal Chr 11 and Chr 6. The human dynactin gene was mapped between markers TGFA and D2S1394, implying that the mouse dynactin gene Dctn1 is located on Chr 6, distal to mnd2. Thus DCTN1/Dctn1 is a candidate for LGMD2B but not for mnd2 or wr.

    Genomics 1997;43;2;242-4

  • Identification of a novel 135-kDa Grb2-binding protein in osteoclasts.

    Sahni M, Zhou XM, Bakiri L, Schlessinger J, Baron R and Levy JB

    Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    The tyrosine kinase receptor for macrophage colony-stimulating factor and the non-receptor tyrosine kinase c-Src play critical roles in osteoclast differentiation and function. Since the ubiquitously expressed adaptor protein Grb2 plays an important role in several tyrosine kinase signal transduction pathways, we used a filter binding assay to identify osteoclast proteins that bind to Grb2. In osteoclasts, there were three major Grb2-binding proteins, two of which, mSos and c-Cbl (p120), have been previously identified as Grb2-binding proteins in many cell types. The third protein, p135, had a restricted pattern of expression and was present at high levels in authentic osteoclasts and osteoclast-like cells formed in an in vitro co-culture system. In addition to binding Grb2 in the filter binding assay, p135 was isolated in complexes with endogenous Grb2 from osteoclast cell extracts. The association of p135 and Grb2 was dependent on an intact Src homology 3 domain and furthermore, was shown to preferentially interact with the N-terminal Src homology 3 domain of Grb2, which is similar to the interaction of mSos and Grb2 in other cell types. p135 was not recognized by antibodies against several known Grb2-binding proteins and thus may be a novel Grb2-binding protein.

    Funded by: NIAMS NIH HHS: 1F33AR08261-01, 5-R01-AR42927-01-02

    The Journal of biological chemistry 1996;271;51;33141-7

  • Centractin (ARP1) associates with spectrin revealing a potential mechanism to link dynactin to intracellular organelles.

    Holleran EA, Tokito MK, Karki S and Holzbaur EL

    Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia 19104, USA.

    Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our results indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network.

    Funded by: NIGMS NIH HHS: GM48661

    The Journal of cell biology 1996;135;6 Pt 2;1815-29

  • Functionally distinct isoforms of dynactin are expressed in human neurons.

    Tokito MK, Howland DS, Lee VM and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6046, USA.

    P150Glued is the largest subunit of dynactin, which binds to cytoplasmic dynein and activates vesicle transport along microtubules. We have isolated human cDNAs encoding p150Glued as well as a 135-kDa isoform; these isoforms are expressed in human brain by alternative mRNA splicing of the human DCTN1 gene. The p135 isoform lacks the consensus microtubule-binding motif shared by members of the p150Glued/Glued/CLIP-170/BIK1 family of microtubule-associated proteins and, therefore, is predicted not to bind directly to microtubules. We used transient transfection assays and in vitro microtubule-binding assays to demonstrate that the p150 isoform binds to microtubules, but the p135 isoform does not. However, both isoforms bind to cytoplasmic dynein, and both partition similarly into cytosolic and membrane cellular fractions. Sequential immunoprecipitations with an isoform-specific antibody for p150 followed by a pan-isoform antibody revealed that, in brain, these polypeptides assemble to form distinct complexes, each of which sediments at approximately 20 S. On the basis of these observations, we hypothesize that there is a conserved neuronal function for a distinct form of the dynactin complex that cannot bind directly to cellular microtubules.

    Funded by: NIGMS NIH HHS: GM-48661

    Molecular biology of the cell 1996;7;8;1167-80

  • Localization of the DCTN1 gene encoding p150Glued to human chromosome 2p13 by fluorescence in situ hybridization.

    Holzbaur EL and Tokito MK

    School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA. holzbaur/pobox.upenn.edu

    Genomics 1996;31;3;398-9

  • Cytoplasmic dynein binds dynactin through a direct interaction between the intermediate chains and p150Glued.

    Vaughan KT and Vallee RB

    Cell Biology Group, Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.

    Cytoplasmic dynein is a retrograde microtubule motor thought to participate in organelle transport and some aspects of minus end-directed chromosome movement. The mechanism of binding to organelles and kinetochores is unknown. Based on homology with the Chlamydomonas flagellar outer arm dynein intermediate chains (ICs), we proposed a role for the cytoplasmic dynein ICs in linking the motor protein to organelles and kinetochores. In this study two different IC isoforms were used in blot overlay and immunoprecipitation assays to identify IC-binding partners. In overlays of complex protein samples, the ICs bound specifically to polypeptides of 150 and 135 kD, identified as the p150Glued doublet of the dynactin complex. In reciprocal overlay assays, p150Glued specifically recognized the ICs. Immunoprecipitations from total Rat2 cell extracts, rat brain cytosol, and rat brain membranes further identified the dynactin complex as a specific target for IC binding. using truncation mutants, the sites of interaction were mapped to amino acids 1-123 of IC-1A and amino acids 200-811 of p150Glued. While cytoplasmic dynein and dynactin have been implicated in a common pathway by genetic analysis, our findings identify a direct interaction between two specific component polypeptides and support a role for dynactin as a dynein "receptor". Our data also suggest, however, that this interaction must be highly regulated.

    Funded by: NIGMS NIH HHS: GM15941, GM43474

    The Journal of cell biology 1995;131;6 Pt 1;1507-16

  • Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    Golsteyn RM, Mundt KE, Fry AM and Nigg EA

    Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

    The Journal of cell biology 1995;129;6;1617-28

  • The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1).

    Waterman-Storer CM, Karki S and Holzbaur EL

    Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6046.

    p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, we overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. We also tested for a direct interaction between p150Glued and centractin. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin.

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;5;1634-8

  • Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150GLUED and a novel actin.

    Paschal BM, Holzbaur EL, Pfister KK, Clark S, Meyer DI and Vallee RB

    Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.

    Earlier work identified a series of accessory polypeptides of 150, 74, 59, 57, 55, 53, 50, and 45 kDa copurifying with cytoplasmic dynein. In the present study immunoprecipitation of the 50-kDa polypeptide from bovine brain cytosol with a specific monoclonal antibody revealed coprecipitating components of 150, 135, 62, and 45 kDa, which were completely distinct from the polypeptides immunoprecipitated using an antibody to the well established 74-kDa cytoplasmic dynein subunit. The 150- and 135-kDa polypeptides reacted with an antibody to p150Glued, the mammalian homologue of the Drosophila Glued gene. N-terminal microsequencing of tryptic peptides of the major 45-kDa component of the complex revealed it to be the alpha-isoform of centractin, a novel form of actin. Immunoblotting of sucrose gradient-fractionated brain cytosol revealed p150Glued, p50, and centractin to cosediment exclusively at 20 S. Immunofluorescence microscopy using antibody to p150Glued revealed centrosomal staining, which was abolished by microtubule depolymerization. Together these results reveal the 50-kDa polypeptide to be part of a cytosolic complex distinct from cytoplasmic dynein. However, the immunolocalization data indicate an association with microtubule minus ends, suggesting a possible interaction with cytoplasmic dynein in the cell.

    Funded by: NIGMS NIH HHS: GM26701, GM47434

    The Journal of biological chemistry 1993;268;20;15318-23

  • Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued.

    Holzbaur EL, Hammarback JA, Paschal BM, Kravit NG, Pfister KK and Vallee RB

    Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts.

    Cytoplasmic dynein is a microtubule-activated ATPase which produces force towards the minus ends of microtubules. It is thought to be responsible for retrograde axonal transport and other aspects of organelle motility and may have a role in the poleward movement of mitotic chromosomes. Cytoplasmic dynein is an oligomeric complex of two catalytic heavy chains and a number of accessory subunits. We now report the cloning and sequencing of a complementary DNA for one of these species, a cytoplasmic dynein-associated polypeptide of relative molecular mass 150,000 (Mr 150K). A full-length cDNA was found to contain an open reading frame of 4.0 kilobases, which is predicted to encode a polypeptide of Mr 145K. It has extensive homology with the product of the Drosophila gene Glued, which encodes a polypeptide of Mr 148K. The Glued mutation is dominant, with pleiotropic developmental defects in heterozygotes and an embryonic lethal phenotype in homozygotes. As dominant mutations may involve disruption of normal protein-protein interactions, the Glued mutation should provide insight into the mode of action of cytoplasmic dynein in vivo.

    Nature 1991;351;6327;579-83

Gene lists (6)

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
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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