A Functional Genomics Approach to Uncover the Mechanisms of Neutrophil Galvanotaxis. - Project Summary/Abstract During acute inflammation our immune cells orchestrate a complex, but coordinated mitigation response. Immune cells are especially good at navigating the complex extracellular environment through dynamic modulation of their actomyosin cytoskeletons, enabling a rapid and effective response throughout the body. The ability of cells to sense a variety of chemical and physical cues that direct their migratory paths is paramount to this action. Migration in response to bioelectric currents has long been demonstrated, leading to clinical applications in wound healing through exogenously applied electric potentials. While also implicated in our response to infections and in the metastatic spread of some cancers, our understanding of this directional cue, referred to as galvanotaxis or electrotaxis, remains limited. The experiments proposed in this application will develop the technology to perform large-scale assays of galvanotaxis and enable a comprehensive genome- wide strategy to identify the genes and cellular mechanisms involved in human neutrophil galvanotaxis. In Aim 1, I will fabricate a device that enables electric field-directed separation of the millions of cells required to perform genome-scale perturbation assays. In collaboration with Dr. Thomas Daniel, I will optimize the device and assay conditions to develop a robust protocol for studying galvanotaxis. Here I will gain training in computational and engineering tools for assay development. In Aim 2, I will apply a genome-wide CRISPRi knockdown screen of galvanotaxis, providing the first comprehensive strategy to identify the key genes involved in this mode of migration. Due to the technical challenges of such assays, several rounds of experiments will be performed to increase our confidence in identified gene candidates. In Aim 3, I will use computational and experimental approaches to gain new insights into the cellular mechanisms of galvanotaxis based on hypotheses generated from the screen work. In the course of this work, I will collaborate with experimentalist Dr. Sean Collins who is an expert in receptor-based signaling and signal transduction. He will provide invaluable guidance in these core components common to most modes of directed cell migration. Throughout Aim 2 and 3, I will also strengthen my experimental training in molecular biology and biochemical techniques through the expertise of the Theriot lab. Importantly, along with these research opportunities, the development award will provide me with additional career training that I currently need to start and manage a lab. It will also provide critical career training in laboratory leadership, teaching, grant writing and scientific communication. My mentor, Dr. Julie Theriot, will provide mentoring that will enable me to successfully transition to independence. This award will therefore provide the crucial training that will enable my longer-term goals of comprehensively understanding neutrophil motility and downstream effector functions.
