Myosin Va

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Hanshermann Gerdes - One of the best experts on this subject based on the ideXlab platform.

  • Versatile roles for Myosin Va in dense core vesicle biogenesis and function. Biochem Soc Trans 2010;38:199–204
    2020
    Co-Authors: Tanja Kögel, Rudiger Rudolf, Claudia Margarethe Bittins, Hanshermann Gerdes
    Abstract:

    Abstract The motor protein Myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of Myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems

  • Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules
    Cellular and Molecular Neurobiology, 2010
    Co-Authors: Tanja Kögel, Hanshermann Gerdes
    Abstract:

    Neuroendocrine secretory granules (SGs) are formed at the trans -Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and remoVal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of Myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of Myosin Va or Rab3D blocked the remoVal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant Myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of Myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks.

  • Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles
    Cellular and Molecular Neurobiology, 2010
    Co-Authors: Claudia Margarethe Bittins, John A. Hammer, Tilo Wolf Eichler, Hanshermann Gerdes
    Abstract:

    Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein Myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of Myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where Myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

  • versatile roles for Myosin Va in dense core vesicle biogenesis and function
    Biochemical Society Transactions, 2010
    Co-Authors: Tanja Kögel, Rudiger Rudolf, Claudia Margarethe Bittins, Hanshermann Gerdes
    Abstract:

    The motor protein Myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of Myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems.

  • expression of the dominant negative tail of Myosin Va enhances exocytosis of large dense core vesicles in neurons
    Cellular and Molecular Neurobiology, 2009
    Co-Authors: Claudia Margarethe Bittins, Tilo Wolf Eichler, Hanshermann Gerdes
    Abstract:

    Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein Myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of Myosin Va in this process, neurons were transfected with the dominant-negative tail domain of Myosin Va (MyosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This Value was doubled to 1.80% of the vesicles when MyosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and MyosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by MyosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium-calmodulin-dependent kinase II. We suggest that MyosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons.

John A. Hammer - One of the best experts on this subject based on the ideXlab platform.

  • creation of a Myosin Va tap tagged mouse and identification of potential Myosin Va interacting proteins in the cerebellum
    Cytoskeleton, 2018
    Co-Authors: Christopher J Alexander, Nancy A Jenkins, Neal G Copeland, Wolfgang Wagner, John A. Hammer
    Abstract:

    The actin-based motor Myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this Myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleaVage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of Myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known Myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates Myosin Va, and Gnao1's localization to PN dendritic spines depends on Myosin Va. The mouse model created here should facilitate the identification of novel Myosin Va-binding partners, which in turn should adVance our understanding of the roles played by this important Myosin in vivo.

  • rab10 and Myosin Va mediate insulin stimulated glut4 storage vesicle translocation in adipocytes
    Journal of Cell Biology, 2012
    Co-Authors: Yu Chen, John A. Hammer, Yan Wang, Jinzhong Zhang, Yongqiang Deng, Li Jiang, Eli Song, Jennifer Lippincottschwartz
    Abstract:

    Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with Myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

  • Myosin Va transports the endoplasmic reticulum into the dendritic spines of purkinje neurons
    Nature Cell Biology, 2011
    Co-Authors: W Wagner, Stephan D Brenowitz, John A. Hammer
    Abstract:

    The role of actin-based motors and the mode of endoplasmic reticulum (ER) transport into spines had remained unclear. Myosin-Va is now shown to act as a point-to-point ER transporter into dendritic spines.

  • Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles
    Cellular and Molecular Neurobiology, 2010
    Co-Authors: Claudia Margarethe Bittins, John A. Hammer, Tilo Wolf Eichler, Hanshermann Gerdes
    Abstract:

    Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein Myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of Myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where Myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.

  • role of Myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo
    PLOS ONE, 2008
    Co-Authors: Ira V Roder, John A. Hammer, Yvonne Petersen, Kyeong Rok Choi, Veit Witzemann, Rudiger Rudolf
    Abstract:

    Background Myosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of Myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ). Methodology/Principal Findings Dilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of Myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous Myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length Myosin Va as well as a cargo-binding fragment of Myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with Myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins. Conclusions/Significance In summary, our data show a crucial role of Myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.

Wanxi Yang - One of the best experts on this subject based on the ideXlab platform.

  • kifc1 and Myosin Va two motors for acrosomal biogenesis and nuclear shaping during spermiogenesis of portunus trituberculatus
    Cell and Tissue Research, 2017
    Co-Authors: Mengying Pan, Fuqing Tan, Congcong Hou, Wanxi Yang
    Abstract:

    To investigate the molecular mechanisms underlying the spermiogenesis of the swimming crab Portunus trituberculatus, full lengths of motor proteins KIFC1 and Myosin Va were cloned by rapid-amplification of cDNA ends from P. trituberculatus testes cDNA, and their respective probes and specific antibodies were used to track their localization during sperm maturation. Antisense probes were designed from the gene sequences and used to detect the mRNA levels of each gene. According to the results of fluorescence in situ hybridization (FISH), the transcription of kifc1 and Myosin Va began at the mid-stage of spermatids, with the kifc1 mRNA being most active at the location where the acrosome cap was formed and the Myosin Va was more concentrated in the acrosome complex. Immunofluorescence results showed that KIFC1 and Myosin Va were highly expressed in each stage of spermigenesis. In the early spermatids, they were randomly dispersed in the cytoplasm together with cytoskeletons. At the mid-stage, the motors were gathered above one side of the nucleus where the acrosome would later form. In the late spermatids and mature sperm, the KIFC1 was closely distributed in the perinuclear region, indicating its role in nucleus deformation. Myosin Va was distributed in the acrosome complex until sperm maturity. This suggests Myosin Va's potential role in material transportation during acrosome formation and maturation. The above results provide a preliminary illustration of the essential roles of KIFC1 and Myosin Va in the spermiogenesis of the swimming crab P. trituberculatus.

  • Myosin Va plays essential roles in maintaining normal mitosis enhancing tumor cell motility and viability
    Oncotarget, 2017
    Co-Authors: Ai Zhong, Han Dong, Fuqing Tan, Wanxi Yang
    Abstract:

    // Yan-Ruide Li 1 , Ai Zhong 1 , Han Dong 1 , Lu-Han Ni 1 , Fu-Qing Tan 2 and Wan-Xi Yang 1 1 The Sperm Laboratory, College of Life Sciences, Zhejiang University, Hangzhou, China 2 The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China Correspondence to: Wan-Xi Yang, email: wxyang@spermlab.org Keywords: Myosin Va, testicular cancer, prostate cancer, tumorigenesis, actin Received: April 15, 2017      Accepted: May 03, 2017      Published: May 17, 2017 ABSTRACT Myosin Va, a member of Class V Myosin, functions in organelle motility, spindle formation, nuclear morphogenesis and cell motility. The purpose of this study is to explore the expression and localization of Myosin Va in testicular cancer and prostate cancer, and its specific roles in tumor progression including cell division, migration and proliferation. We detected Myosin Va in testicular and prostate tumor tissues using sqRT-PCR, western blot, and immunofluorescence. Tumor samples showed an increased expression of Myosin Va, abnormal actin and Myosin Va distribution. Immunofluorescence images during the cell cycle showed that Myosin Va tended to gather at cytoplasm during anaphase but co-localized with nucleus during other phases, suggesting the roles of Myosin Va in disassembly of spindle microtubule, movement of chromosomes and normal cytokinesis. In addition, multi-nucleation and aberrant nuclear morphology were observed in Myosin Va-knockdown cells. Wounding assay and CCK-8-based cell counting were conducted to explore Myosin Va roles in cell migration, viability and proliferation. Our results suggest that Myosin Va plays essential roles in maintaining normal mitosis, enhancing tumor cell motility and viability, and these properties are the hallmark of tumor progression and metastasis development. Therefore, an increased understanding of Myosin Va expression and function will assist in the development of future oncodiagnosis and -therapy.

  • gene expression pattern of Myosin Va during spermatogenesis of chinese mitten crab eriocheir sinensis
    Gene, 2012
    Co-Authors: Xiao Sun, Haitao Mao, Wanxi Yang
    Abstract:

    Abstract Myosin Va is an F-actin dependent molecular motor with multiple functions that are essential for acrosome formation in mouse spermiogenesis. The spermatozoon of the crab has a complicated acrosome surrounded by a cup-shaped nucleus. In the present study, the Myosin Va cDNA was cloned from the testis of the Chinese mitten crab Eriocheir sinensis using degenerate PCR and rapid amplification of cDNA ends (RACE). The Myosin Va cDNA consists of a 125 bp 5′-untranslated region (5′ UTR), a 5331 bp open reading frame (ORF) and a 590 bp 3′ UTR. The putative Myosin Va protein contains the head domain, neck domain and tail domain. Multiple alignment and phylogenetic tree showed that E. sinensis Myosin Va is more closely related to the vertebrate Myosin Va than to the invertebrate Myosin V. E. sinensis Myosin Va was expressed in Various tissues. In situ hybridization demonstrated that Myosin Va mRNA is located in the entire process of spermatogenesis. Quantitative real-time PCR indicated that the expression level at the mitotic and meiotic phases is higher than the spermiogenesis phase. Taken together, our work suggests that Myosin Va may function in E. sinensis spermatogenesis.

  • Myosin Va participates in acrosomal formation and nuclear morphogenesis during spermatogenesis of chinese mitten crab eriocheir sinensis
    PLOS ONE, 2010
    Co-Authors: Xiao Sun, Lin Hou, Wanxi Yang
    Abstract:

    BACKGROUND The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is aVailable in crustacean species. Myosin Va, a member of Class V Myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of Myosin Va during spermatogenesis in E. sinensis. METHODOLOGY/PRINCIPAL FINDINGS Western blot demonstrated that Myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that Myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, Myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, Myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, Myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, Myosin Va predominates in acrosomal tubule (AT) and nucleus. CONCLUSIONS/SIGNIFICANCE Our study demonstrates that Myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of Myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of Myosin Va in crustacean spermatogenesis.

Rudiger Rudolf - One of the best experts on this subject based on the ideXlab platform.

  • Versatile roles for Myosin Va in dense core vesicle biogenesis and function. Biochem Soc Trans 2010;38:199–204
    2020
    Co-Authors: Tanja Kögel, Rudiger Rudolf, Claudia Margarethe Bittins, Hanshermann Gerdes
    Abstract:

    Abstract The motor protein Myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of Myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems

  • participation of Myosin Va and pka type i in the regeneration of neuromuscular junctions
    PLOS ONE, 2012
    Co-Authors: Ira V Roder, Rudiger Rudolf, Siegfried Strack, Markus Reischl, Oliver Dahley, Muzamil Majid Khan, Olivier Kassel, Manuela Zaccolo
    Abstract:

    Background: The unconventional motor protein, Myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and Myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of Myosin Va and PKA in NMJ recovery during muscle regeneration. Methodology/Principal Findings: To address a putative role of Myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems Myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate Myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology. Conclusions/Significance: Our data show a correlation between the subsynaptic accumulation of Myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of Myosin Va and PKA type I for the maturation of NMJs in regenerating muscle.

  • distinct roles of Myosin Va in membrane remodeling and exocytosis of secretory granules
    Traffic, 2010
    Co-Authors: Tanja Kögel, Rudiger Rudolf, Andrea Hellwig, Sergei A. Kuznetsov, Erlend Hodneland, Florian Seiler, Thomas H Sollner
    Abstract:

    Hormone- and neuropeptide-containing secretory granules (SGs) of neuroendocrine PC12 cells are formed at the trans- Golgi network as immature SGs. These intermediates are converted to mature SGs in a complex maturation process, including matrix condensation, processing of cargo proteins and remoVal of proteins and membrane in clathrin-coated vesicles. The resulting mature SGs undergo Ca2+-dependent exocytosis upon an appropriate stimulus. We here show that the motor protein Myosin Va is implicated in a maturation step of SGs, their binding to F-actin and their stimulated exocytosis. Interference with Myosin Va function blocked the remoVal of the transmembrane protein furin from maturing SGs without affecting condensation and processing of proteins of the SG lumen. Furthermore, the ATP-inhibited binding of SGs to F-actin decreased with progressive maturation and upon interference with Myosin Va function. Moreover, the expression of a dominant-negative Myosin Va-tail or shRNA-based downregulation of Myosin Va interfered with stimulated exocytosis of SGs. In summary, our data suggest an essential function of Myosin Va in the membrane remodeling of SGs during maturation and a role in their exocytosis.

  • Myosin Va cooperates with pka riα to mediate maintenance of the endplate in vivo
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Ira V Roder, Markus Reischl, Manuela Zaccolo, Yvonne Petersen, Kyeongrock Choi, Markus E Diefenbacher, Tullio Pozzan, Rudiger Rudolf
    Abstract:

    Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that Myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, Myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denerVation. We hypothesize that neuronal ligands trigger local actiVation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, Myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.

  • versatile roles for Myosin Va in dense core vesicle biogenesis and function
    Biochemical Society Transactions, 2010
    Co-Authors: Tanja Kögel, Rudiger Rudolf, Claudia Margarethe Bittins, Hanshermann Gerdes
    Abstract:

    The motor protein Myosin Va is involved in multiple successive steps in the development of dense-core vesicles, such as in the membrane remodelling during their maturation, their transport along actin filaments and the regulation of their exocytosis. In the present paper, we summarize the current knowledge on the roles of Myosin Va in the different steps of dense-core vesicle biogenesis and exocytosis, and compare findings obtained from different cell types and experimental systems.

Mark S Mooseker - One of the best experts on this subject based on the ideXlab platform.

  • the tail domain of Myosin Va modulates actin binding to one head
    Journal of Biological Chemistry, 2006
    Co-Authors: Adrian O Olivares, Wakam Chang, Mark S Mooseker, David D Hackney, Enrique M De La Cruz
    Abstract:

    Calcium actiVates full-length Myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length Myosin Va purified from chick brains. Both heads of nucleotide-free Myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of Myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actoMyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-actiVated Pi release from Myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived Myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium actiVates Myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.

  • Myosin Va binds to and mechanochemically couples microtubules to actin filaments
    Molecular Biology of the Cell, 2003
    Co-Authors: Tracy T Cao, Wakam Chang, Sarah E Masters, Mark S Mooseker
    Abstract:

    Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native Myosin-Va purified from chick brain, as well as the expressed globular tail domain of this Myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of Myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound Myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of Myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that Myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.

  • high affinity binding of brain Myosin Va to f actin induced by calcium in the presence of atp
    Journal of Biological Chemistry, 2001
    Co-Authors: Mark S Mooseker, S B F Tauhata, Daniela Dos Santos, Edwin W Taylor, Roy E Larson
    Abstract:

    Abstract Brain Myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that Myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca2+ in a highly cooperative manner. We demonstrate here that Ca2+ also induces Myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca2+-induced cosedimentation. The Ca2+-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between Myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca2+ was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actoMyosin-Va under Ca2+-induced binding conditions showed tightly packed F-actin bundles cross-linked by Myosin-Va. Our data demonstrate that high affinity binding of Myosin-Va and F-actin in the presence of ATP or 5′-O-(thiotriphosphate) is induced by micromolar concentrations of Ca2+. Since Ca2+ regulates both the actin binding properties and actin-actiVated ATPase of Myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of Myosin Va.

  • the light chain composition of chicken brain Myosin Va calmodulin Myosin ii essential light chains and 8 kda dynein light chain pin
    Cytoskeleton, 2000
    Co-Authors: Foued Salmen Espindola, Tracy T Cao, Daniel M Suter, Leticia B E Partata, Joseph S Wolenski, Richard E Cheney, Stephen M King, Mark S Mooseker
    Abstract:

    Class V Myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on Myosin-Va from chick brain indicate that this Myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this Myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with Myosin-Va is established. The 23- and 17-kDa subunits are both members of the Myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of Myosin-Va.

  • molecular genetic dissection of mouse unconventional Myosin Va tail region mutations
    Genetics, 1998
    Co-Authors: Jiandong Huang, Neal G Copeland, Mark S Mooseker, Valerie Mermall, Marjorie C Strobel, Liane B Russell, Nancy A Jenkins
    Abstract:

    We used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles, a mouse-coat-color locus encoding unconventional Myosin-Va. Ten of the mutations mapped to the MyoVa tail and are reported here. These mutations represent the first extensive collection of tail mutations reported for any unconventional mammalian Myosin. They identify sequences important for tail function and identify domains potentially involved in cargo binding and/or proper folding of the MyoVa tail. Our results also provide support for the notion that different Myosin tail isoforms produced by alternative splicing encode important cell-type-specific functions.

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