Myosin-Va Regulates Exocytosis through the Submicromolar Ca²+-dependent Binding of Syntaxin-1A

Abstract

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 M Ca 2؉ . 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 Ca 2؉ 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 Ca 2؉ . INTRODUCTIONMyosin-V, a processive molecular motor, conveys vesicles and other organelles along F-actin (Mercer et al., 1991;Espreafico et al., 1992;Cheney et al., 1993;Reck-Peterson et al., 2000;Vale, 2003). This unconventional myosin is a member of the class-V myosins, which are expressed in all eukaryotic species from yeast to mammals (Reck-Peterson et al., 2000;Matsui, 2003;Vale, 2003). Myosin-V is a dimeric protein (Cheney et al., 1993). Each monomer is composed of a head region, a long neck domain containing six tandem IQ-motifs, and a tail region (Reck-Peterson et al., 2000). The head acts as a plus-end ATPase-dependent molecular motor to move myosin-V along F-actin (Reck-Peterson et al., 2000). The long neck region is thought to act as a lever arm to regulate the motor activity of the head and to maintain the large step size of myosin-V via bound light chains. In higher eukaryotes, the light chains consist mainly of calmodulin (CaM) bound to the IQ-motifs in the neck domain (Cheney et al., 1993;Vale, 2003). In addition, the globular tail of myosin-V interacts with membrane-bound vesicles. In these ways, myosin-V plays a central role in intracellular polarized transport (Reck-Peterson et al., 2000;Matsui, 2003;Vale, 2003).Among the three isoforms of myosin-V in higher vertebrates, myosin-Va is the most abundant, and it is highly enriched in the brain (Espreafico et al., 1992), particularly in the neurons (Tilelli et al., 2003). Several lines of evidence indicate that synaptic vesicles, which undergo the Ca 2ϩ -regulated exocytosis, are one of the most important cargoes for myosin-Va (Prekeris and Terrian, 1997;Bridgman, 1999;Tilelli et al., 2003). In addition, Myo2p, a yeast h...

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

“…MyoVa, munc18, and syntaxin-1 may thus cooperate to mediate SG docking. This hypothesis is consistent with the reported interaction of MyoVa with syntaxin-1 (Watanabe et al, 2005), although this binding occurred at calcium concentrations unlikely to be found under resting conditions. Noteworthy, syntaxin-1 binding involves the neck region of MyoVa and not the motor head domain.…”

“…Another possibility is that MyoVa acts as a factor attaching SGs to the plasma membrane, in addition to, or instead of its role as a motor. This possibility was suggested by the observed interaction between MyoVa and syntaxin-1 (Watanabe et al, 2005).…”

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells

“…It has been shown to be important in retaining these vesicular carriers (melanosomes, secretory granules) in the F-actin-rich cortex (Rudolf et al, 2003;Wu et al, 1998), and recently a role in fusion and exocytosis has also been reported via the interaction of myosin-Va with syntaxin-1A (Watanabe et al, 2005). Our data suggest that under basal conditions, microtubule-dependent transport of Na Intracellular transport is a complex process, driven by at least three types of motors: kinesins and dyneins involved in bidirectional transport along microtubules and myosins that move along actin filaments (King, 2000;Mermall et al, 1998;Woehlke and Schliwa, 2000).…”

Powering membrane traffic in endocytosis and recycling

“…Examples include kinesin-1, which has been proposed to interact directly with amyloid precursor protein 137 ; however, data remain controversial 138 . Myosin Va interacts with syntaxin-1A in a Ca 2+ -dependent manner to regulate exocytosis 106 . In inner ear sensory cells, myosins Ia and VIIa interact with the integral membrane protein pleckstrin-homology domain retinal protein-1 (PHR1) 19 (panel b).…”

“…Actin is known to be crucial for synaptic-vesicle recycling 104 , and synaptic-vesicle movement at presynaptic terminals that are devoid of microtubules is dependent on myosin Va 105 . Myosin Va on synaptic vesicles directly interacts, in a Ca 2+ -dependent manner, with plasma membrane syntaxin-1a through its neck domain, and is thereby proposed to regulate stimulated exocytosis through the supply of readily releasable vesicles 106 . Nevertheless, the data linking myosin V with secretion still need to be confirmed in vivo.…”