This thesis presents work toward understanding the biophysical mechanisms that control polarized cell movement.  Many cells move in response to external cues, and this directed movement occurs during development, axon guidance, wound healing, tumor progression, and the immune response.  Cells must interpret external signals through signal transduction networks and engage actin polymerization machinery to generate mechanical force for membrane protrusion at the leading edge of the cell.  Our work focuses specifically on the interface between the cell's signaling guidance system and actin nucleation machinery.

In the first project, we analyzed the protein dynamics of a major cytoskeleton regulator called the WAVE complex in a human neutrophil-like cell line in response to gradient and uniform agonist stimulations (Chapter 3). We demonstrated that biased generation and selection of WAVE-complex activity drives the development of morphological polarity and we discovered that actin polymer sculpts the localization of the WAVE-complex.

In the second project, we developed a biochemical system to study the WAVE complex, discovered a GTPase exchange factor likely involved in WAVE-complex activation, searched for other WAVE-complex binding proteins through immunoprecipitation, and examined cell behavior in response to temporal changes in agonist (Chapter 4).

In the third project, we studied the single-molecule behavior of cytoskeletal proteins in XTC cells using epifluorescence and TIRF microscopy. We showed that the WAVE complex intercalates into the actin network during retrograde grade flow. Additionally, we observed lateral diffusion of both the WAVE complex and the Arp2/3 complex and captured the transition of the Arp2/3 complex from diffusion to network incorporation.