Characterization of Native Myosin VI Isolated from Sea Urchin Eggs

Sakata, Souhei; Watanabe, Yuuko; Usukura, Jiro; Mabuchi, Issei

doi:10.1093/jb/mvm161

Cited by 6 publications

(9 citation statements)

References 48 publications

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“…To date, there are no reports on myosin VI immunoanalog in other than A. proteus protozoan cells so we cannot compare our observations with the analogous organism. Moreover, whereas myosin VI isoforms are known to be expressed in lower metazoans such as nematode (Caenorhabditis elegans; Kelleher et al 2000), sea urchin (Strongylocentrotus franciscanus; Sakata et al 2007), and fruit fly (Drosophila melanogaster Meigen, 1830;Kellerman and Miller 1992), most reports on this motor protein come from studies on mammalian cells (see Sweeney and Houdusse (2007) and Buss and Kendrick-Jones (2008)). Mammalian myosin VI is involved in a number of steps during clathrin-dependent and clathrin-independent endocytosis: from the transport and clustering of plasma membrane receptors to formation of endocytotic vesicle but probably also in the later transport of the vesicle.…”

Section: Discussionmentioning

confidence: 99%

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

Sobczak

Wasik

Kłopocka

et al. 2008

Biochem. Cell Biol.

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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.).

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Section: Discussionmentioning

confidence: 99%

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

Sobczak

Wasik

Kłopocka

et al. 2008

Biochem. Cell Biol.

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“…Finally, given that myosin VI is predominantly monomeric in mammalian cells (Lister et al, 2004), sea urchins (Sakata et al, 2007), and during spermatid individualization in Drosophila (this work), it is possible that myosin VI always functions as a monomer. Although several reports suggest the involvement of myosin VI in endocytosis as a processive cargo transporter, other mechanisms that do not require processivity, such as stabilizing actin that pushes vesicles or anchoring vesicles to filaments, are possible (Frank et al, 2004).…”

Section: Implications For Myosin VI Function In Other Systemsmentioning

confidence: 94%

“…However, when purified from native sources (Lister et al, 2004;Sakata et al, 2007) or expressed in the baculovirus heterologous expression system (Lister et al, 2004), myosin VI is a monomer. To examine whether myosin VI dimers were present in the testis, we performed cross-linking studies on testis extracts using the zero-length cross-linker EDC.…”

Section: The Majority Of Myosin VI In the Fly Is Monomericmentioning

confidence: 99%

“…Therefore, myosin VI's biochemical and biophysical properties have been defined in vitro by using altered versions that have been induced to dimerize by adding the well-characterized GCN4 leucine zipper sequence adjacent to the predicted coiled coil (De La Cruz et al, 2001;Rock et al, 2001;Altman et al, 2004;Yildiz et al, 2004;Balci et al, 2005). However, when purified from a heterologous expression system or from native tissue sources, myosin VI is monomeric (Lister et al, 2004;Sakata et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization inDrosophila melanogaster

Noguchi¹,

Frank

Isaji

et al. 2009

MBoC

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Myosin VI is a pointed-end-directed actin motor that is thought to function as both a transporter of cargoes and an anchor, capable of binding cellular components to actin for long periods. Dimerization via a predicted coiled coil was hypothesized to regulate activity and motor properties. However, the importance of the coiled-coil sequence has not been tested in vivo. We used myosin VI's well-defined role in actin stabilization during Drosophila spermatid individualization to test the importance in vivo of the predicted coiled coil. If myosin VI functions as a dimer, a forced dimer should fully rescue myosin VI loss of function defects, including actin stabilization, actin cone movement, and cytoplasmic exclusion by the cones. Conversely, a molecule lacking the coiled coil should not rescue at all. Surprisingly, neither prediction was correct, because each rescued partially and the molecule lacking the coiled coil functioned better than the forced dimer. In extracts, no cross-linking into higher molecular weight forms indicative of dimerization was observed. In addition, a sequence required for altering nucleotide kinetics to make myosin VI dimers processive is not required for myosin VI's actin stabilization function. We conclude that myosin VI does not need to dimerize via the predicted coiled coil to stabilize actin in vivo. INTRODUCTIONDue to the facts that myosin VI is unique in its ability to move toward the pointed or slow growing end of an actin filament (Wells et al., 1999) and that it is implicated in human disease, it has been the subject of intense study both in vivo and in vitro. Although mutations in myosin VI cause deafness and are associated with hypertrophic cardiomyopathy (Melchionda et al., 2001;Ahmed et al., 2003;Mohiddin et al., 2004), myosin VI expression is up-regulated in ovarian and prostate cancers, with its level correlating with metastatic potential (Yoshida et al., 2004;Dunn et al., 2006). Studies in Drosophila and vertebrates have implicated myosin VI in a number of cellular processes, including endocytosis, basolateral targeting and sorting, cell adhesion and epithelial integrity, cell migration, and actin structure stabilization (Kellerman and Miller, 1992;Mermall et al., 1994;Hicks et al., 1999;Buss et al., 2001;Geisbrecht and Montell, 2002;Aschenbrenner et al., 2003;Petritsch et al., 2003;Millo et al., 2004;Au et al., 2007;Maddugoda et al., 2007). In some processes, myosin VI is proposed to mediate translocation along actin, whereas in others it likely serves as a stable actin anchor. These dual roles are thought to be possible because of myosin VI's ability to move processively along an actin filament and stall in a tightly bound state when placed under load (Altman et al., 2004). How these properties are achieved is still under investigation, but require significant adaptations of the motor, compared with barbed/plus-end-directed motors (Menetrey et al., 2005(Menetrey et al., , 2007.Because of a predicted coiled-coil sequence in the tail, myosin VI was thought to work as a di...

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“…Myo6 purified from tissue is single headed [Lister et al,2004], although dimerization can be effected by numerous binding interactions including actin binding [Park et al,2006] and cargo binding to the tail domain [Spink et al,2008; Phichith et al,2009]. Single‐headed Myo6 is a relatively high duty ratio motor [De La Cruz et al,2001; O'Connell et al,2007; Sakata et al,2007] and two‐headed Myo6 is processive, with a duty ratio of ∼1 [De La Cruz et al,2001; Rock et al,2001; Nishikawa et al,2002]. Myo6 has a much greater step size than expected for a neck domain/lever arm consisting of just one IQ motif [Rock et al,2001].…”

Section: Introductionmentioning

confidence: 99%

Plus‐end directed myosins accelerate actin filament sliding by single‐headed myosin VI

et al. 2012

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Myosin VI (Myo6) is unique among myosins in that it moves toward the minus (pointed) end of the actin filament. Thus to exert tension on, or move cargo along an actin filament, Myo6 is working against potentially multiple plus (barbed)-end myosins. To test the effect of plus-end motors on Myo6, the gliding actin filament assay was used to assess the motility of single-headed Myo6 in the absence and presence of cardiac myosin II (Myo2) and myosin Va (Myo5a). Myo6 alone exhibited a filament gliding velocities of 60.34 +/− 13.68 nm/s. Addition of either Myo2 or Myo5a, at densities below that required to promote plus-end movement resulted in an increase in Myo6 velocity (~100-150% increase). Movement in the presence of these plus-end myosins was minus-end directed as determined using polarity tagged filaments. High densities of Myo2 or Myo5a were required to convert to plus-end directed motility indicating that Myo6 is a potent inhibitor of Myo2 and Myo5a. Previous studies have shown that two-headed Myo6 slows and then stalls in an anchored state under load. Consistent with these studies, velocity of a two headed heavy mero myosin form of Myo6 was unaffected by Myo5a at low densities, and was inhibited at high Myo5a densities.

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Characterization of Native Myosin VI Isolated from Sea Urchin Eggs

Cited by 6 publications

References 48 publications

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

Coiled-Coil–Mediated Dimerization Is Not Required for Myosin VI to Stabilize Actin during Spermatid Individualization inDrosophila melanogaster

Plus‐end directed myosins accelerate actin filament sliding by single‐headed myosin VI

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