MYOSIN VA ASSOCIATES WITH MICROTUBULE-RICH DOMAINS IN BOTH INTERPHASEAND DIVIDING CELLS

Class V unconventional myosins are two-headed, nonfilamentous, actin-b ased mechanoenzymes that appear to be expressed ubiquitously. Mice pos sess at least two myosin V heavy chain genes (dilute and and myr6) who se similar to 190 kDa protein products are referred to as myosin Va an d Vb, respectively. Using antibodies that are specific for the Va isof orm and immunofluorescence microscopy, we show here that myosin Va loc alizes to the microtubule organizing center (MTOC) in interphase cells , and to the mitotic asters, spindle, and midbody of dividing cells. T hese associations, which in the case of mitotic cells are characterize d by the concentration of myosin Va in the immediate vicinity of the m icrotubules, were observed in a variety of cell types. including prima ry and immortal mouse melanocytes and fibroblasts, Hela cells, and Cos cells. Importantly. these associations were not observed in melanocyt es and fibroblasts cultured From dilute null mice, indicating that the staining of these microtubule-rich domains was due to the presence of myosin Va, as opposed to another protein(s) containing a shared epito pe(s) with myosin Va. When cells were extracted with detergent prior t o fixation, myosin Va remained associated with each of these microtubu le-rich domains. suggesting that these associations are not due to the possible presence of membranes at these sites, This Fact, and our obs ervation that these microtubule-rich domains contain little if any F-a ctin (based on phalloidin staining), suggest that myosin Va may bind t o microtubules either directly or through a microtubule-associated pro tein. Finally, we found that dilute null fibroblasts in primary cultur e are twice as likely to be binucleate as wild type fibroblasts of the same genetic background (35% vs. 17%). Together, these results indica te that myosin Va associates with microtubule-rich domains in both int erphase and dividing cells, and plays a role in the efficiency of cell division in culture. Cell Motil. Cytoskeleton 40:286-303. 1998. a 199 8 Wiley-Liss. Inc.