Role of the Septin Cytoskeleton in Plasma Membrane Repair - Project Abstract The plasma membrane (PM) forms a biophysical barrier that protects the cell from its extracellular environment and regulates its functions. Mammalian cells are regularly exposed to stressors that can damage their PM. Therefore, conserved molecular mechanisms repair the PM to maintain cell viability. Dysregulation of PM repair mechanisms is involved in chronic or acute pathologies such as muscular dystrophy, ischemia-reperfusion, heart failure, chronic inflammation, and neurodegenerative diseases. PM repair mechanisms can be exploited by intracellular pathogens to successfully infect their host mammalian cells. The bacterial pathogen Listeria monocytogenes produces the pore-forming toxin listeriolysin O (LLO) that perforates the cytoplasmic and endocytic membranes to invade multiple cell types including epithelial cells, endothelial cells, cardiomyocytes, macrophages, and neurons. The PM repair mechanism(s) of LLO-perforated cells are poorly understood. In a preliminary screen, we identified the cytoskeletal protein, septin 7, as a protein required for the PM repair of cells damaged by LLO. The septins are a family of highly conserved cytoskeletal proteins that act as scaffolds for protein recruitment and vesicular trafficking. Septins are known to interact with the PM and the actin and microtubule cytoskeletons to regulate numerous cellular functions. During PM repair of LLO-treated cells we found that the septin filaments redistribute into subplasmalemmal loop structures that contain F-actin and annexin A2 in a Ca2+-dependent manner. Until recently there was no known role of the septins in plasma membrane repair. We plan to dissect the mechanisms used by the septins to repair the plasma membrane. We will test the central hypothesis that septins play a general role in PM repair by controlling the spatiotemporal organization of PM repair domains. Using our expertise in quantitative fluorescence microscopy and the development of inducible cell lines, electron microscopy, and proteomics, we will elucidate the role of septins in PM repair. We expect to determine how the septins regulate PM repair and what molecules septin 7 interacts with during this mechanism. Together, these studies will provide novel insights into septin biology and plasma membrane repair and provide new targets for diseases involving plasma membrane repair.
