STRUCTURE AND FUNCTION OF UNCONVENTIONAL MYOSINS

Heart, Lung, And Blood Institute

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Abstract

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 116 kDa protein (p116), plus nine other proteins identified as the seven-member Arp2/complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that these components are present together in a complex in vivo, that the SH3 domain of the myosin is required for its presence in this complex, and that p116 acts as the scaffold for complex assembly, binding myosin I, capping protein, and the Arp2/3 complex at independent sites. Cloning of p116 reveals a leucine-rich-repeat domain, a verprolin-like sequence followed by an acidic region, 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 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase endocytosis, and a significant decrease in the efficiency of chemtactic aggregation. These results identify a complex that links key players in the nucleation (Arp2/3) and termination (capping protein) of actin filament assembly with a ubiquitous barbed-end-directed motor, indicate that the protein responsible for the formation of this complex (p116/Acan 125)is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium are due at least in part to defects in the assembly state of actin. We have purifed Acan 125 to homogeneity. It copurifies extensively with capping protein, indicating that their interaction is quite strong. A fragment of Acan 125 containing the verprolin-like and acidic sequences stimulates Arp2/3-dependent actin nucleation. Myosin V, Rab 27a, and Melanosome Dynamics: In previous studies we have shown that the peripheral accumulation of melanosomes characteristic of wild type mouse melanocytes is driven by a cooperative process involving rapid,long-range, bidirectional, microtubule-dependent movements coupled to the myosin V-dependent capture and local movement of melanosomes in the actin-rich periphery. We have now verified this model by demonstrating that the restoration of melanosome position in dilute (myosin V null) melanocytes by reintoduction of myosin V requires the presence of the long range, bidirectional, microtubule-dependent movements. The ability of myosin V to influence melanosome position absolutely requires the presence in it of an alternatively spliced, 27-residue, melanoctye-specific exon. We are currently searching for proteins that interact with this important sequence. The product of the ashen locus was recently shown to be a novel rab, rab 27a. We find that the phenotype of ashen melanocytes, like the coat color defect in ashen mice, is indistinquishable from that of dilute melanocytes. This fact, together with data showing that this GTPase and myosin V colocalize on peripheral melanosomes in wild type melanocytes, suggests that rab 27a enables myosin V-dependent capture in the actin-rich periphery. The results of rescue experiments on ashen melanocytes support this conclusion, and, together with other data, suggest that myosin V and rab 27a are in a complex on the melanosome surface.