Regulated actin polymerization is crucial for diverse biological processes such as cytokinesis, cell migration and organogenesis. Disruption of actin polymerization results in diseases that include immunologic and cardiovascular disorders, as well as increased cancer metastasis. A key actin nucleator is the Arp2/3 complex, which forms Y-branched networks in the cell. Despite the essential role of branched networks in activities such as cell migration, the regulation of Arp2/3 complex Y-branch stability is not understood. Intrinsic factors, including Arp2/3 complex actin filament binding, ATP binding and hydrolysis, as well as extrinsic factors, such as cortactin, coronin and ADF/cofilin, likely contribute to Arp2/3 complex Y-branch dynamics, however the roles of each of these remain to be determined. The long-term goals of this project are to determine how Arp2/3 complex Y-branch stability is controlled in diverse cellular processes. To understand how activities intrinsic to the Arp2/3 complex contribute to Y-branch dynamics, in Aim 1 I will test the hypothesis that actin filament and ATP binding are required for Y-branch formation, that ATP hydrolysis is necessary for Y-branch disassembly, and that surfaces of each subunit mediate filament interactions. Mutant Arp2/3 complexes will be measured for their ability to nucleate actin and form Y-branches in vitro. To determine whether these activities of Arp2/3 complex are also important for its function in vivo, in Aim 2 I will test the hypotheses that actin filament and ATP binding are required for Y-branch formation, and that ATP hydrolysis is necessary for network dissociation in lamellipodia of cells. Quantitative fluorescence microscopy (qFSM) and phase contrast microscopy will be used to analyze lamellipodia dynamics in cells containing mutant Arp2/3 complexes. To determine the role of extrinsic factors in Arp2/3 complex Y-branch stability, in Aim III I will test whether cortactin stabilizes Y-branches and coronin and ADF/cofilin de-stabilize Y- branches in lamellipodia. Cells depleted for Arp2/3 complex regulators will be analyzed using phase contrast microscopy and qFSM. The experiments in this proposal will provide valuable insight into how Arp2/3 complex Y-branches are regulated in the cell. The mis-regulation of actin polymerization has severe consequences for immune system function and cancer cell migration. A better understanding of Y-branch formation and regulation may someday lead to new diagnostics or treatments for diseases such as cardiovascular disease and cancer.