PROJECT ABSTRACT Cell motility is one of the oldest problems in cell biology as the movement of cells was one of the first things noted when the microscope was developed in the 17th century. The last sixty years of work revealed that the molecular underpinnings of motility involve the active control of the cytoskeleton. However, many questions remain unanswered. While we have identified many of the components of the cytoskeleton and know a great deal about their biochemical and structural characteristics, we lack a systematic understanding of how the parts interact to produce coordinated cytoskeletal function such as during cell migration. Perhaps the most important problem in cell motility is understanding how cells perceive various cues in their environment and convert this information into a directed migration response. A deeper understanding of these two inter-related problems will inform higher order biological processes such as embryogenesis, immune response, and wound healing, as well as diseases states such as metastatic cancer. This proposal seeks to extend our work in this area with the renewal of our R35/MIRA grant, R35 GM130312. Using a conditional knockout mouse model where the gene encoding the critical Arpc2 (p34) subunit of Arp2/3 can be deleted on command, we have been dissecting the systematic relationship between Arp2/3-branched actin and non-branched actin in several cellular processes. Our published and preliminary data indicate that the migration substrate has a profound influence on the architecture of branched actin at the leading edge of migrating cells. Furthermore, unpublished proteomic data demonstrate that a tyrosine kinase pathway controlling clathrin-mediated endocytosis is mis-regulated in the absence of Arp2/3. Building on these observations, we propose to study the role of integrin-based cell adhesion on Arp2/3-branched actin regulation and the regulatory network that controls the balance between clathrin pits and flat clathrin lattices. On the question of directed migration, we propose to build on our recently published durotaxis assay to understand the relationship between cell-generated traction force, cell shape and directed whole cell movement. Finally, we propose to use our expertise in cellular optogenetics to develop an optotaxis assay to direct the migration of cell with gradients of light and test the role of polarized signaling pathways in controlling actin architecture, cell shape and directed migration.