Signal Integration in Neutrophil Chemotaxis

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DESCRIPTION (provided by applicant): Directed cell migration is required for single-celled organisms to hunt and mate, enables innate immune cells to seek and destroy pathogens, and is essential for the morphogenesis of multicellular organisms. Misregulation of cell migration is intimately involved in atherosclerosis and defective cardiac development. Though we are beginning to understand some of the key components involved in cell migration, we do not understand how these components act together to organize cell shape and movement. To address this question, we have analyzed the spatial dynamics of a key actin regulator the Scar/WAVE complex, which is required for morphogenesis in both metazoans and plants. We have recently discovered that the Hem-1 component of the Scar/WAVE complex localizes to propagating waves that appear to organize the leading edge of a motile immune cell, the human neutrophil. Curiously, actin is both an output and input to the Scar/WAVE complex: the complex stimulates actin assembly, and actin polymer is also required to remove the complex from the membrane. These reciprocal interactions appear to generate propagated waves of actin nucleation that embody many of the properties of morphogenesis in motile cells such as the ability of cells to flow around barriers and the intricate spatial organization of protrusion at the leading edge. Our central hypothesis is that the interaction between the Hem-1 wave generator and other signaling cues spatially organizes actin polymerization during cell migration. In this proposal, we will dissect the signals that organize Hem-1 wave dynamics and study their relationship to cell morphogenesis and directed motility. Specifically, we will: Quantitate the effect of external gradients on Hem-1 wave dynamics. We will quantitatively analyze Hem-1 wave dynamics during chemotaxis to test two competing hypotheses in the field-- whether generation of new protrusions or selection among existing ones is responsible for directional migration. 2. Dissect the reciprocal interactions between Rac and Hem-1. We are using both micropatterning and small molecule dimerizers to control the spatial and temporal dynamics of Rac and Hem-1 localization in living cells to dissect how these signals interact with one another. 3. Elucidate the role of the actin cytoskeleton in Hem-1 wave propagation. We will use a combination of actin perturbing drugs and targeted mislocalization of actin nucleation factors to investigate how actin polymer interfaces with Hem-1 wave dynamics. 4. Test role of Hem-1 in front/back crosstalk. We are using microfluidics-based drug perfusion and small-molecule based dimerization to spatially manipulate the signals involved in front (Rac/Hem-1) and back (Rho/myosin) organization to determine how these regulators of polarity communicate. PUBLIC HEALTH REVELANCE: Misregulation of actin polymerization and leukocyte migration are causative factors in heart disease. Leukocytes play a central role in atherosclerosis, and the motility circuit that we study is essential for angiogenesis and cardiovascular development. The ability to control cell migration would be a valuable tool for combating atherosclerosis and other pathological processes that occur upon disruption of cellular guidance mechanisms.