Dissecting the Mechanism of Arp2/3 Complex Activation by Actin Filament Binding and the Regulation and Function of JMY in Cells
The cellular functions of the actin cytoskeleton require precise regulation of the polymerization and organization of actin filaments. Actin nucleation is one of the key control points in this regulation and is accelerated by the action of actin nucleating proteins. Mammalian cells express a diverse set of actin nucleating proteins, each of which has a distinct molecular mechanism of action and mode of regulation.
One of the major actin nucleating proteins in cells it the Arp2/3 complex, which nucleates new filaments from the sides of existing ones to generate Y-branched actin networks. To investigate the mechanism of Arp2/3 complex activation by actin filament binding, we mutated amino acid residues within the predicted actin binding surfaces of the ARPC2 and ARPC4 subunits of the complex and examined the biochemical properties of mutant complexes. Using this approach, we defined sites on ARPC2 and ARPC4 that are required for high-affinity binding to actin filaments. Biochemical characterization of the actin binding mutants revealed that actin binding is crucial for actin nucleation and Y-branch stability.
The junction-mediating and regulatory protein (JMY) was recently discovered as a new actin nucleating protein that is unique among such proteins because it nucleates actin through both Arp2/3-complex-dependent and Arp2/3-complex-independent mechanisms. To investigate the mechanism of JMY regulation, we examined the activity of full-length JMY in actin assembly in vitro and in cells. We found that full-length recombinant JMY and the truncated WWWCA region have comparable actin nucleating and Arp2/3-complex-activating abilities in vitro. In contrast, the ability of full-length JMY to polymerize actin is somewhat inhibited in cells, suggesting autoinhibition and posttranslational modifications as potential mechanisms for JMY regulation. We also showed that JMY localizes primarily to the cytosol, in addition to its localization to the nucleus, and induces formation of actin filament clusters in cytosol consistent with its in vitro activity. Finally, we discovered a new function for JMY in neuritogenesis, as a negative regulator of neurite outgrowth.