Myosin I, the Arp2/3 Complex, and Actin Dynamics: Fusion proteins containing the SH3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116kDa protein (p116), plus nine other proteins identified by microsequencing as the seven-member Arp2/3 complex, CapZ alpha and CapZ beta. Immunoprecipitations performed using wild type and mutant cell extracts indicate that myoB and myoC are present in a complex with p116, Arp2/3, and CapZ in vivo, that their SH3 domains are required for this interaction, and, together with other experiments, that p116 acts as a scaffold, binding myosin I, CapZ, and the Arp2/3 complex at independent sites. Cloning of p116 reveals a leucine-rich- repeat domain, a WASP-like WH2/acidic domain, and proline-rich sequences which we show contain the SH3 domain binding site, and indicates that p116 is a Dictyostelium homolog of Acan 125. p116 colocalizes extensively with Arp3, actin, coronin, myoB and myoC in dynamic actin-rich cellular extensions, especially the leading edge of migrating cells and dorsal, cup-like, macropinocytic extensions (crowns). p116 knockout cells exhibit a profound defect in the formation of these macropinocytic structures, and a concomitant reduction in the rate of fluid phase endocytosis (~ 50% of WT). MyoB/myoC double mutants also exhibit a striking defect in crown formation (but with interesting differences) and partially mislocalize Arp3. These results identify a complex linking the major actin filament nucleator and barbed-end capper to a ubiquitous barbed-end-directed motor, indicate that this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes are due at least in part to defects in the function of Arp2/3 and CapZ.Myosin V and Melanosome Dynamics: Previous studies have shown that myosin V localizes to melanosomes in mouse melanocytes, and that it cooperates with microtubule motors to drive the peripheral accumulation of melanosomes characteristic of mammalian melanocytes. The domain of myosin V that is responsible for the interaction with the melanosome has been mapped and is now being used to search for the myosin V receptor on the melanosome surface. - Arp2/3 complex, myosin I, actin dynamics, myosin V, melanosomes, melanocytes, mouse, Dictyostelium
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