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Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization. Key words: actin cytoskeleton, myosin VI, neurons, PC12 cells, Rac GEF. La myosine VI (MVI), la seule myosine connue pour avancer vers l'extremite moins (-) des filaments d'actine, est impliquee dans plusieurs processus, notamment l'endocytose, la migration cellulaire et la cytocinese. Elle peut agir comme moteur de transport ou comme proteine engagee dans le remodelage du cytosquelette d'actine par l'intermediaire de ses partenaires de liaison qui interagissent avec sa queue globulaire C-terminale. A l'aide d'une technique de co- precipitation et par spectrometrie de masse, nous avons identifie DOCK7 (dedicator of cytokinesis 7) comme nouveau partenaire potentiel de la MVI chez les cellules neurosecretrices PC12. DOCK7, exprimee principalement chez les cellules neuronales, est un facteur d' echange de guanidine (GEF) pour les petites GTPases Rac1 et Cdc42 qui sont les regulateurs principaux du cytosquelette d' actine. L' interaction MVI-DOCK7 a ete de plus confirmee par la co-immunoprecipitation de la MIV endogene en complexe avec DOCK7. En outre, la MVI et DOCK7 etaient localisees au meme endroit dans les cellules en interphase et en division. Nous concluons que chez les cellules PC12, les complexes MVI-DOCK7 peuvent agir A differents endroits au cours du cycle cellulaire. Fait remarquable, la MVI et DOCK7 etaient aussi localisees au meme endroit dans les neurones d'hippocampe en culture primaire, notamment dans les excroissances. Nous emettons l'hypothese que cette interaction nouvellement identifiee entre la MVI et DOCK7 peut aider A comprendre le mecanisme de regulation de l'organisation du cytosquelette d' actine dependant de la MVI. Mots-cles : cytosquelette d'actine, myosine VI, neurones, cellules PC12, Rac GEF. [Traduit par la Redaction], Introduction Myosin VI (MVI) is an unconventional myosin implicated in a range of cellular processes including endocytosis, intracellular trafficking, cell migration, and cytokinesis (Chibalina et al. 2009; Sweeney and Houdusse [...]

Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.). Key words: myosin, actin cytoskeleton, pinocytosis, phagocytosis, motility. Nous avons trouve recemment chez l'amibe un analogue immunologique de la myosine VI de 130 kDa qui se lie a l'actine de facon dependante de l'ATP et qui est localise chez l'amibe en migration avec l'actine filamenteuse et les structures vesiculaires contenant la dynamine II. Afin de mieux caracteriser cette proteine, nous avons evalue son implication dans la pinocytose et la phagocytose chez l'amibe. La microscopie a fluorescence confocale et la microscopie electronique de cellules immunomarquees a l'or colloidal ont revele que chez les amibes en pinocytose ou en phagocytose, l'analogue immunologique de la myosine VI etait visible a travers toute la cellule, y compris dans les canaux et les vesicules pinocytaires ainsi que dans les phagosomes et dans les cupules phagocytaires emergentes. Le blocage de la proteine endogene avec un anticorps anti-myosine VI de porc (introduit dans les cellules par microinjection) a cause des defauts severes de la pinocytose et de la phagocytose. Comparativement aux cellules controles, les amibes traitees formaient approximativement 75 % moins de canaux pinocytaires et phagocytaient environ 65 % moins de Tetrahymena. Ces resultats indiquent que l'analogue immunologique de la myosine VI joue un role important dans la pinocytose et la phagocytose chez Amoeba proteus. Mots-cles : myosine, cytosquelette d'active, pinocytose, phagocytose, motilite. [Traduit par la Redaction], Introduction Myosins are actin-based molecular motors expressed as multiple isoforms in almost all eukaryotic cells and they are involved in various cellular functions such as cell motility, muscle contraction, and [...]

Abstract: A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. [...]

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression. [ABSTRACT FROM AUTHOR]

Ruk/CIN85 is an adaptor protein. Similar to many other proteins of this type, Ruk/CIN85 is known to take part in multiple cellular processes including signal transduction, vesicle-mediated transport, cytoskeleton remodelling, programmed cell death and viral infection. Recent studies have also revealed the potential importance of Ruk/CIN85 in cancer cell invasiveness. In this review we summarize the various roles of this protein as well as the potential contribution of Ruk/CIN85 to malignancy and the invasiveness of cancer cells. In the last section of the paper we also speculate on the utility of Ruk/CIN85 as a target for novel anti-cancer therapies. [ABSTRACT FROM AUTHOR]

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a "bait" identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression. [ABSTRACT FROM AUTHOR]

Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibrils dissolution and abnormal accumulation of degradation products. So far causative mutations have been identified in nine genes encoding Z-disk proteins, including αB-crystallin (CRYAB), a small heat shock protein (also called HSPB5). Here, we report a case study of a 63-year-old Polish female with a progressive lower limb weakness and muscle biopsy suggesting a myofibrillar myopathy, and extra-muscular multisystemic involvement, including cataract and cardiomiopathy. Five members of the proband's family presented similar symptoms. Whole exome sequencing followed by bioinformatic analysis revealed a novel D109A mutation in CRYAB associated with the disease. Molecular modeling in accordance with muscle biopsy microscopic analyses predicted that D109A mutation influence both structure and function of CRYAB due to decreased stability of oligomers leading to aggregate formation. In consequence disrupted sarcomere cytoskeleton organization might lead to muscle pathology. We also suggest that mutated RQDE sequence of CRYAB could impair CRYAB chaperone-like activity and promote aggregation of lens crystallins.

Muscle contraction and different forms of motility of nonmuscle cells depend on cyclic interactions between actin filaments and myosin motors. Calcium ions are the main intracellular signal, which induces activation of actin-myosin interactions. Depending on the cell type and the class of myosin, the molecular mechanisms of regulation are different and take place on two levels - actin filament and myosin. In striated muscle, the actin thin filament is regulated by the troponin-tropomyosin complex. In smooth muscle and nonmuscle cells, actin filaments are predominantly regulated by caldesmon. The control of myosin activity in these cells also depends on the myosin light chain phosphorylation and the phosphorylation of the heavy chain. Direct binding of calcium ions to the myosin light chains (which could be calmodulin molecules) was observed in myosin from molluscan muscle and in some unconventional myosins. Intensive world-wide studies allow us to understand details of the mechanisms of actin-myosin interactions. In this article, we present the contemporary view on these mechanisms.

Background: Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins., Results: In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division., Conclusion: Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division.

Amoeba proteus and smaller by an order of magnitude (and evolutionary younger) Acanthamoeba castellanii have been for many years model cells for studies of amoeboidal (crawling) type of movement, characteristic also for some of metazoan cells such as fibroblasts, granulocytes and macrophages. Amoeboidal migration is indispensable of organization and dynamics of actin-based cytoskeleton. While there is a number of data on molecular mechanisms of motility of A. castellanii, there is very little known about bases of migration of A. proteus. Noteworthy, a large A. proteus (length approximately 600 microm) have been from over a century an object for studies on biology and physiology of cellular migration. This review describes the current knowledge on molecular aspects of force generation required for migration of these two amoebae and attempts to compare the functioning and regulation of actin cytoskeleton in these free-living unicellular species.

Myosins, actin-dependent molecular motors are expressed in almost all eukaryotic cells where are engaged in a panoply of cellular processes such as muscle contraction, cell migration and adhesion, intracellular trafficking, cytokinesis, endocytosis and secretion. In recent years a number of reports has been published revealing nuclear localization of myosins as well as actin and actin-binding proteins. Namely, nuclear form of myosin IC (NMI), myosin VI, myosin XVIB and myosin XVIIIB were found in nucleus. NMI and myosin VI seem to be involved in gene transcription and possibly intranuclear transport. In this paper, current knowledge on the role of myosin motors in nucleus has been presented.

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.