Watanabe, M. et al. Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A. Mol. Biol. Cell 16, 4519-4530

Division of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan.
Molecular Biology of the Cell (Impact Factor: 4.47). 11/2005; 16(10):4519-30. DOI: 10.1091/mbc.E05-03-0252
Source: PubMed


Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 microM Ca2+. Interference with formation of the syntaxin-1A-myosin-Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca2+ depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca2+.

Download full-text


Available from: Konosuke Kumakura,
  • Source
    • "In other studies, TRPV4 surface expression was enhanced when dynamin-mediated endocytosis was blocked (Cuajungco et al. 2006). Furthermore, vesicle exocytosis was also found to be dependent upon myosin activity (Watanabe et al. 2005; Eichler et al. 2006). However, the role of vesicle transport in TRPV4 activity in lung endothelial cells and the involvement of MLCK in this process have not been extensively investigated. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4αPDD). Mean calcium transients peaked at ∼25 sec and persisted ∼500 sec. The 4αPDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4αPDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4αPDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4αPDD < 4αPDD only < dynasore + 4αPDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 μmol/L 4αPDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4αPDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.
    10/2013; 1(5):e00121. DOI:10.1002/phy2.121
  • Source
    • "For its role in motility, myosin Va interacts with MyRIP and Rab27A linking vesicles to F-actin (Desnos et al. 2005). Furthermore, myosin Va could modulate the activity of syntaxin-1 (Watanabe et al. 2005), and consequently , the possibility of interaction with the fusion machinery itself. "
    Dataset: revcytosk08

  • Source
    • "We have shown herein that MVI is involved in cell migration and proliferation but not in catecholamine secretion in neurosecretory PC12 cells. The latter is in contrast with MV, which was shown in numerous studies to play an important role in catecholamine release (Rose et al. 2003; Rudolf et al. 2003; Watanabe et al. 2005). Our observation that depletion of MVI inhibited PC12 cell migration is in line with the previous studies on Drosophila border cells or malignant human ovarian cancer cells (Geisbrecht and Montell 2002; Yoshida et al. 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.
    Journal of Muscle Research and Cell Motility 11/2011; 32(4-5):291-302. DOI:10.1007/s10974-011-9279-0 · 2.09 Impact Factor
Show more