Cell migration plays important roles in embryonic development, tissue homeostasis, wound healing, and immune response. Abnormal migratory behaviors can also lead to serious diseases such as autoimmune syndrome and cancer metastasis. Actin polymerization at the lamellipodial leading edge of the cell provides force generation for cell migration. There are two major types of actin nucleators which drive actin polymerization, formin and Arp2/3. Current models postulate that Arp2/3 drives the assembly of a branched lamellipodial filament network at the leading edge while formin is responsible for the nucleation of filopodia and stress fibers. Most existing models of cell migration entirely focus on Arp2/3-mediated actin assembly as the driver of lamellipodial protrusions, despite initial experimental evidence from our lab and others that formin and Arp2/3 coexist with distinct molecular and functional properties in cell protrusions. Therefore, the spatiotemporal coordination of formin and Arp2/3 in vivo as well as the function of their cross-talk in terms of cell migration has not been addressed. The central hypothesis in this project is that cells modulate the activities of formin and Arp2/3 to optimize migration efficiency in different mechanical environments. My working model suggests that these different nucleators serve specific functions in a protrusion event - formin is responsible for initiation and Arp2/3 for reinforcement of assembly against increasing membrane tension. The goal of this research is to establish the mechanism of such coordination of formin and Arp2/3. I plan to use quantitative imaging approaches supported by computational data modeling that will allow us to deconvolve the dynamics of these two different actin nucleation modules. I plan to measure formin and Arp2/3 activation by single molecule imaging in conjunction with quantitative fluorescent speckle microscopy of actin and determine their spatiotemporal relationships. In order to show that the reinforcement of Arp2/3-mediated actin assembly is mechano-responsive, I will investigate how the mechanical environment of different stiffness and adhesiveness of substrates affects Arp2/3 activation. Finally, I propose to establish relationships between the protrusion force output and the coordination between formin and Arp2/3 activities using high-resolution traction force microscopy.
Metastatic cancer cells need to migrate through different mechanical microenvironments on the journey from the primary tumor to the tissue they invade. This project will test the hypothesis that the coordination and adaptive activation of two different actin nucleators, formin and Arp2/3, allows metastatic epithelial carcinoma cells to more effectively migrate in variable microenvironments than non-metastatic cells.