Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

Abstract

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (ϳ1:24 Myosin-Va:tubulin; K d ‫؍‬ 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca 2؉ , ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 M Ca 2؉ , ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments. INTRODUCTIONMyosin-Va (Myo5a) is the best characterized of three class V myosins identified in vertebrates (Berg et al., 2001). Myo5a is a two-headed motor, consisting of two heavy chains and multiple light chains (reviewed in Reck-Peterson et al., 2000). The N-terminal half of each heavy chain consists of a head or motor domain followed by a neck domain composed of six IQ motifs, each of which binds a calmodulin light chain although chicken Myo5a also contains a pair of myosin-II essential light chains . The C-terminal tail domain consists of a proximal dimerization stalk of largely coiled-coil forming alpha helix followed by a globular domain that has been shown to interact with numerous presumed "cargo" molecules (Reck-Peterson et al., 2000;Karcher et al., 2002;Langford, 2002). The tail domain also contains at least a pair of the low-molecular-weight light chain, LC8 or PIN, first identified as a light chain of the microtuble (MT) motor, dynein, but subsequently shown to interact with numerous other proteins . Biochemical studies on both tissue-purified and baculovirus-expressed Myo5a (reviewed in Reck-Peterson et al., 2000;Mehta, 2001) have shown that it is a Ca 2ϩ -regulated, barbed-end directed, processive motor that takes large, ϳ36-nm steps in a hand-overhand manner (Forkey et al., 2003; Yildiz et al., 2003) along the actin filament.Insights into the potential functions for Myo5a have come from several lines of investigation, including identification of organelles and proteins that interact with Myo5a (ReckPeterson et al., 2000;Karcher et al., 2002;Langford, 2002). The most powerful strategy has been the phenotypic characterization of lethal alleles of the dilute (Myo5a lethal mutant) mouse and cells derived from them. The dilute mice develop severe seizures and die within 3 wk after birth. The primary neuronal defect thought to be responsible is the absence of smooth endoplasmic reticulum (SER) within the dendritic spines of Purkinje neurons (Takagishi et al., 1996). Anal...

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