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

Abstract

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...

Investigations of human myosin VI targeting using optogenetically controlled cargo loading

“…Strictly speaking, however, binding of myosin VI to Dab2 pep is not equivalent to activation. Activation requires the release of autoinhibition of full-length myosin VI, allowing it to interact with actin in either a transport (56) or anchoring role (16). To demonstrate activation of myosin VI in vivo, we showed that myosin VI recruited to peroxisomes via our engineered positive control construct LOVDab +ctrl stalls these organelles.…”

“…Dissociation of the CBD from the head both frees the head to bind tightly to actin and exposes dimerization sites throughout the tail domain of myosin VI, allowing it to become a processive dimer (13)(14)(15). In some cases, myosin VI could conceivably function as a monomer, for example when fulfilling its role as a membrane tether during spermatid individualization (16). If this is the case, more work is needed to elucidate the cellular signals that determine its oligomeric state at each site of action.…”

Myosin VI and its cargo adaptors – linking endocytosis and autophagy

“…However, under certain conditions, for example, when it acts to regulate actin stabilisation during spermatid individualisation, myosin VI can fully function when it lacks the ability to dimerise. Under these circumstances it remains bound to actin for long periods, thus functioning as an actin tether (Noguchi et al, 2009;Noguchi et al, 2006). Furthermore, it has been suggested that, as load is increased on myosin VI, it switches from its transport function to an anchoring function (Altman et al, 2004), such as at the base of microvilli, stereocilia and on vesicles in the terminal synaptic bouton (Hertzano et al, 2008;Kisiel et al, 2011;Sakurai et al, 2011;Seiler et al, 2004).…”

Unconventional myosins acting unconventionally

“…Myosin-6 mutants have reduced levels of F-actin in cones and, furthermore, this F-actin is disorganized [38]. Somewhat surprisingly, dimerization of the myosin-6 heavy chain is not required for myosin-6 function in cones [39], which implies that transport activity is not likely to be important because dimerization is required for normal motor function in vitro . This finding is also consistent with the observation that the rate of cone movement does not differ between wild-type and mutant embryos.…”