How do nucleation promoting factors build different actin networks?

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ABSTRACT Neutrophils migrate across the body to sites of infection to initiate an immune response. During this process actin is organized into lamellipodial and endocytic networks that affect cell migration. Much of this actin structure comes in at the level of nucleation. While we have identified a variety of nucleation promoting factors (NPFs) and their upstream regulators, we do not know how these NPFs are spatially arranged to build their different actin networks. I am particularly interested in WASP family proteins, a class of NPFs that control the formation of branched actin networks through activation of the Arp2/3 complex. Despite following similar pathways, members of this family have a different spatial organization that lead to the formation of actin networks with different geometries, ultimately suited for different biological functions. For example, the WASP family member WAVE forms broad, propagating waves at the leading edge that pattern the flat actin network underlying protrusive, sheet-like lamellipodia. Meanwhile, WASP and N-WASP form punctate structures that polymerize actin perpendicular to the membrane to aide in the scission of endocytic vesicles. Presently, we do not understand how these NPFs build different structures, both in their distinct spatial organization and in the resulting geometry of their actin networks. Understanding what organizes WASP family NPFs on a molecular level is crucial, as their disregulation compromises our ability to mount an immune response and results in multiple pathophysiologies. Specifically, chronic inflammatory disorders such as atherosclerosis and obstructive pulmonary diseases arise from to increased immune cell recruitment by neutrophils. Additionally, when these cells hyper-accumulate, tissue damage occurs, as is seen in ischemia-reperfusion injuries and lung damage in cystic fibrosis. My research will test different models for how the distinct spatial organization of WASP family NPFs is maintained. In Aim 1, I will determine how membrane geometry may act locally to recruit different WASP family members. I will test this by plating cells on nanopatterned substrates with varying curvature. Once I have found, and broken, the curvature-sensing mechanism, I will test whether curvature preference alone is responsible for the spatial organization of NPFs and subsequent modulation of actin polymerization needed for directed migration. Then, in Aim 2 I will establish the role that WASP, WAVE and N-WASP each play in migration and determine if they depend on one another for proper localization. Until recently, WAVE was the only one of these NPFs thought to be involved in leading edge formation. However, recent data shows that WASP is also necessary for proper migration. Additionally, I have observed that WASP knockout results in a loss of WAVE localization at the leading edge, suggesting there may be communication between these NPFs. I will use CRISPR-mediated knockout lines for each NPF to investigate whether an observed defect in migration is due to disrupted leading edge formation or endocytosis or from misregulated communication between NPFs.