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// 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.

Weibel–Palade body (WPB)–actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A–MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP–actin interactions can also occur. To evaluate the specific contribution of MyRIP–actin and MyRIP–MyoVa binding in WPB trafficking and Ca2+-driven exocytosis, we used EGFP–MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca2+-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa., Summary: The role of MyRIP in restricting the movement and exocytosis of Weibel–Palade bodies in endothelial cells is mediated primarily through its actin- rather than myosin-Va-binding properties.

The alpha1 (α1) subunit of the sodium/potassium ATPase (i.e., Na+/K+-ATPase α1), the prototypical sodium pump, is expressed in each eukaryotic cell. They pump out three sodium ions in exchange for two extracellular potassium ions to establish a cellular electrochemical gradient important for firing of neuronal and cardiac action potentials. We hypothesized that myosin (myo or myh) motor proteins might interact with Na+/K+-ATPase α1 subunits in order for them to play an important role in the transport and trafficking of sodium pump. To this end immunoassays were performed to determine whether class II non-muscle myosins (i.e., NMHC-IIA/myh9, NMHC-IIB/myh10 or NMHC-IIC/myh14), myosin Va (myoVa) and myosin VI (myoVI) would interact with Na+/K+-ATPase α1 subunits. Immunoprecipitation of myh9, myh10, myh14, myoVa and myoVI from rat brain tissues led to the co-immunoprecipitation of Na+/K+-ATPase α1 subunits expressed there. Heterologous expression studies using HEK293 cells indicated that recombinant myh9, myh10, myh14 and myoVI interact with Na+/K+-ATPase α1 subunits expressed in HEK293 cells. Additional results indicated that loss of tail regions in recombinant myh9, myh10, myh14 and myoVI did not affect their interaction with Na+/K+-ATPase α1 subunits. However, recombinant myh9, myh10 and myh14 mutants having reduced or no actin binding ability, as a result of loss of their actin binding sites, displayed greatly reduced or null interaction with Na+/K+-ATPase α1 subunits. These results suggested the involvement of the actin binding site, but not tail regions, of NMHC-IIs in their interaction with Na+/K+-ATPase α1 subunits. Overall these results suggest a role for these diverse myosins in the trafficking and transport of sodium pump in neuronal and non-neuronal tissues. Electronic supplementary material The online version of this article (10.1186/s13041-018-0388-1) contains supplementary material, which is available to authorized users.

Myosin Va is an actin-based molecular motor that is involved in organelle transport and membrane trafficking. Here, we explored the role of myosin Va in the formation of synaptic circuitry by examining climbing fiber (CF) innervation of Purkinje cells (PCs) in the cerebella of dilute-neurological (d-n) mice and dilute-opisthotonus (dop) rats that have mutations in dilute–encoded myosin Va. Anterograde labeling of CFs with biotinylated dextran amine (BDA) revealed that they arborized poorly and that their tips extended only half way through the thickness of the molecular layer (ML) in adult d-n mice. Using immunohistochemistry specific for vesicular glutamate transporter 2 (VGluT2) to visualize CF synaptic terminals, we found that during development and in adulthood, these terminals did not ascend as far along the proximal shaft dendrites of PCs in d-n mice and dop rats as they did in normal animals. An irregular distribution of BDA-labeled bulbous varicosities and VGluT2 spots along CF branches were also noted in these animals. Finally, VGluT2-positive CF terminals were occasionally localized on the PC somata of adult d-n cerebella. These phenotypes are consistent with our electrophysiological findings that CF-mediated excitatory postsynaptic currents (EPSCs) were significantly smaller in amplitude and faster in decay in adult d-n mice, and that the regression of multiple CFs was slightly delayed in developing d-n mice. Taken together, our results suggest that myosin Va is essential for terminal CF extension and for the establishment of CF synapses within the proper dendritic territories of PCs., Running title: Climbing fibers in myosin Va mutants

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.

The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes.

Transcytosis of proteins and hydrodynamic flow of cytoplasm is a major mechanism to sustain physiology in all cells, observable from gametes (1) to mature adult cells and tissues (2, 3). Mammalian cells involved in secretion discretely need to respond to the environment and move components within the cells and position them at appropriate locations for secretion (4, 5). This process involves force generation using Gibbs free energy of hydrolysis of adenosine triphosphate (ATP). The ATPase is most often myosin, a naturally occurring cellular ATPase known for its wide role in generation of cellular force (6). The nanomechanics of transport involve the necessary target cargoes, in association with myosin and track on actin filaments, which are ubiquitous cellular cytoskeletal scaffolds of metazoan cells (7). Cellular secretion encompasses multiple physiological systems operating on wide range of time scales including the processes of exocrine and endocrine glandular secretions and neuronal secretion in response to discrete electrical field stimulation, commonly referred to as neurotransmission (8, 9).

To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va–associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein–myosin Va tail construct. Myosin Va–associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va–associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Submitted by Boris Flegr (boris@uerj.br) on 2021-01-07T15:13:52Z No. of bitstreams: 1 Elisama Azevedo Cardoso Tese completa.pdf: 2063255 bytes, checksum: 704e01090c87796880fcc8b4c1ffd7df (MD5) Made available in DSpace on 2021-01-07T15:13:52Z (GMT). No. of bitstreams: 1 Elisama Azevedo Cardoso Tese completa.pdf: 2063255 bytes, checksum: 704e01090c87796880fcc8b4c1ffd7df (MD5) Previous issue date: 2012-05-30 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The members of the genus Leishmania are responsible for a group of parasitic diseases that vary from self-limited lesions to severe tissue injury. These parasites are obligatory intracellular protozoa that use human macrophages as hosts. Their entry into macrophages occurs through phagocytosis that involves the cytoskeleton, comprised of actin and myosin, to form the phagosomes. Actin and myosin are also involved in other cellular processes including cytokinesis, intracellular trafficking of organelles and vesicles, which may interfere with parasite entry. Previous studies have analyzed the expression, localization and role of both actin and myosin in Leishmania. Their participation in various processes vital to the biology of the parasite suggests new targets for therapeutic intervention. Since myosin is required for intracellular transport, attempts have been made to examine the intracellular localization and expression of Leishmania myosin. Studies have shown the presence of kinase activity of CK2 in various trypanosomes linked to cell growth, morphology and infectivity of macrophages by promastigotes. Therefore, the objective of this thesis was to investigate the role of myosin, actin and CK2 for infectivity of Leishmania brasiliensis. In addition, the influence of these proteins in cytokine production and microbiocidal activity in human macrophages was investigated. Drug-based inhibition using lipoxin, latrunculin, nocodozole and TBB caused at least a 50% decrease in growth of L. brasiliensis. The CK2 secreted by the parasite was purified from the supernatant of cultures by HPLC and fraction 44 was determined to correspond to this protein. Lipoxin and TBB were shown to inhibit CK2 activity, contrary to latrunculin that increased its activity. Pretreatment of parasites or macrophages with either lipoxin, latrunculin, nocodozole or TBB lead to a ~50% decrease in the association index between L. brasiliensis and macrphages, with or without preactivation with LPS and INF-γ. Latrunculin and TBB increased the NO in non-activated macrophages and parasites did not infect activated macrophages treated with latrunculin. The other drugs all decreased the production of NO. The release of IL-10 was decreased after treatment with all drugs in non-activated macrophages in the presence of parasites and in activated macrophages in the presence of parasites. For the production of TNF-α, there was a significant decrease in activated macrophages by latrunculin, nocodozole and TBB. When macrophages were activated and infected, treatment with lipoxin showed an increase in the production of this cytokine, opposite to the reduction observed with TBB. When evaluating the integrity of actin, all compounds were determined to influence the organization of actin leading to a decrease in the association index. The transfection of the tail of myosin Va fused to eGFP into macrophages was observed to decrease the association index by 94%. The data confirm the importance of CK2, actin and myosin Va in the process of interactions between parasites and macrophages. O gênero Leishmania é responsável por um grupo de parasitoses que podem variar desde lesões auto-limitadas até severa injúria de tecido. Estes protozoários são parasitos obrigatoriamente intracelulares, tendo o macrófago como célula hospedeira. Durante o processo de fagocitose os macrófagos utilizam a maquinaria presente em seu citoesqueleto, a qual compreende a participação de miosinas e actinas, para a formação do fagossoma. Estas proteínas estão envolvidas em processos como citocinese, tráfego intracelular de organelas e vesículas, podendo interferir com a penetração do parasito. Alguns trabalhos vêm sendo realizados visando analisar a expressão, localização e o papel de miosina e de actina em Leishamania. Estudos associados à participação destas proteínas motoras em processo vitais para a biologia do parasito podem auxiliar na compreensão de seu ciclo e permitir a geração de conhecimentos que apontem novos alvos para intervenções terapêuticas. Uma vez que a miosina é necessária no transporte intracelular, alguns estudos tentam analisar a expressão e a localização intracelular de miosinas na Leishmania. Estudos mostram a presença de atividades cinásicas do tipo CK2 em diversos tripanossomatídeos, ligadas ao crescimento celular, morfologia e infectividade de promastigotas para macrófagos. Desta maneira, como objetivo desta tese temos o estudo da participação das miosinas, actina e CK2 na infectividade da Leishmania braziliensis. Além disso, investigamos a influência destas proteínas na produção de citocinas pelos macrófagos e em sua atividade microbicida. Lipoxina, latrunculina, nocodazol e TBB promoveram uma inibição de, pelo menos, 50% no crescimento de L. braziliensis. A CK2 secretada pelo parasito foi purificada de seu sobrenadante através de coluna de HPLC e a fração 44 mostrou ser a fração correspondente a esta enzima. A lipoxina e o TBB promoveram a inibição da atividade desta enzima ao contrário da latrunculina que forneceu aumento dessa atividade. O pré-tratamento dos parasitos ou dos macrófagos com lipoxina, latrunculina, nocodazol e TBB promoveram uma inibição de cerca de 50% no índice de associação entre Leishmania e macrófagos não-ativados ou ativados por LPS e IFN-γ. Latrunculina e TBB aumentaram a produção de NO em macrófagos não ativados e não infectados enquanto que em macrófagos ativados à exceção do TBB, todas as drogas diminuíram a produção de NO. A liberação de IL-10 foi diminuída após tratamento com todas as drogas em macrófagos não ativados em ausência de promastigotas e ativados em presença do parasito. Para a produção de TNF-α há uma redução significativa em macrófagos ativados não infectados tratados com latrunculina, nocodazol e TBB. Quando ativados e infectados, os macrófagos tratados com lipoxina tiveram a produção dessa citocina aumentada, ao contrário do TBB em que houve redução. Quando avaliamos a integridade da actina verificamos que todos os compostos foram capazes de influenciar a distribuição dessa proteína, levando a uma redução no índice de associação. Ao transfectarmos a cauda da miosina Va fusionada a GFP nos macrófagos observamos que houve uma diminuição de 94% no índice de associação. Nossos dados confirmam a importância da CK2, actina e miosina Va no processo de interação parasito- macrófago.

Copyright © 2011 The Author(s). Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plusend binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect MlpMyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer. publishersversion published

Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVa(d)), ashen (Rab27a(ash)), and leaden (Mlph(ln)), act in the same or overlapping pathways. Here we have cloned and characterized a human melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimentation assays using recombinant proteins reveal that melanophilin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respectively. Moreover, we show that Rab27a, melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/granuphilin-like Rab effector family constitutes an alpha-helix-based coiled-coil structure.

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. publishedVersion