Neutrophils are professional migrators within our body, moving from the bloodstream through dense forests of extracellular matrix to reach sites of infection and initiate an immune response. While we have long appreciated the role of biochemical signals in neutrophil recruitment, increasing attention is being given to the role of physical signals during this process. For example, membrane tension and membrane shape changes arise as neutrophils navigate complex 3D environments in vivo, which can impact the path cells take through tissue. However, how this information is relayed back to the cytoskeleton has remained largely unexplored. In this thesis we probe the link between biochemical and physical inputs to better understand how neutrophils integrate environmental signals to ensure robust directed migration.

In Chapter 2 we establish how intrinsic protein self-organization and external membrane curvature work together to pattern the actin nucleation promoting factor WAVE. Using super-resolution microscopy, we find that WAVE forms rings at the saddle-shaped necks of membrane invaginations in the absence of actin polymer. Dual inputs from WAVE’s propensity to oligomerize and curvature sensitivity explain this enrichment pattern and inform our understanding of WAVE propagation in the presence of actin. Specifically, we find that WAVE localizes to the saddle-shaped lagging regions of the leading edge to support a uniformly advancing cell front. Elucidation of the mechanisms underpinning WAVE organization has provided initial insights into the integration of internal biochemical signals with external cell shape in organizing the actin cytoskeleton.

In Chapter 3 we continue on this theme and highlight how WASP, another actin nucleation promoting factor, uses a modification of these behaviors to link cell shape and cell polarity during neutrophil migration. Unlike WAVE, WASP persistently enriches to substrate-induced invaginations in the presence of actin. Additionally, WASP assembles into focal structures rather than linear structures. We find that these properties allow WASP to link sites of local membrane deformation to the cytoskeleton by promoting actin polymerization and aligned migration in textured environments. Strikingly, this property requires concomitant inputs from cell shape and cell polarity. WASP only engages with curved membranes at the cell front, which supports forward advance. We show that WASP is essential for integration of substrate features with directed migration, which could have implications for migration through collagen meshworks in vivo.

Finally, Chapter 4 presents unpublished work exploring the molecular mechanisms of cell polarity through GTPase regulation by Rho GEFs and GAPs using HEK293T cells as a model system. Using CRISPR we endogenously label highly expressed GEFs and GAPs. We describe a system for polarizing HEK293T cells and report candidate GEFs and GAPs that enrich to induced cell protrusions. Finally, we supply a protocol for phenotyping the migration of HEK293T GEF or GAP knockout cell lines and provide a positive control in the form of Rac1 knockout. Ultimately, GEFs and GAPs exhibiting loss of function phenotypes would be followed up in the consitituively polarized, highly motile neutrophil cell line HL-60.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl₂, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.