Plasma Membrane Repair Mechanisms - PROJECT SUMMARY Although the plasma membrane of animal cells demonstrates considerable structural and functional plasticity, it is also susceptible to mechanical disruption and perforation by various enzymes, chemicals, and pore-forming proteins. To counteract this vulnerability, evolution has equipped cells with robust repair mechanisms that rapidly reseal the membrane, preventing cell death and restoring homeostasis. It is well established that calcium influx through membrane disruptions serves as a key signal to activate repair. However, the downstream molecular mechanisms responsible for executing this process remain incompletely understood. To address this gap, we investigated one of the most potent membrane-damaging agents: bacterial pore-forming toxins known as cholesterol-dependent cytolysins (CDCs). These toxins are major virulence factors of Gram- positive pathogens, including Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium perfringens, and Bacillus anthracis, which cause severe diseases worldwide. Our research focuses on the repair of cells injured by listeriolysin O (LLO) and pneumolysin (PLY), the CDCs respectively produced by L. monocytogenes and S. pneumoniae. We discovered that septins play a critical role in repairing cells injured by LLO and PLY, and by mechanical wounding. This finding supports that septin-mediated repair mechanisms are relevant to infections and other diseases characterized by excessive cell injury, abnormal, or impaired plasma membrane repair. Septins are a conserved family of multifunctional cytoskeletal proteins that bind phospholipids and F-actin, playing key roles in shaping and regulating the plasma membrane. We will assess the role of septins across multiple cell lines and primary cells, including epithelial cells, macrophages, and cardiomyocytes, in response to different forms of plasma membrane injury, such as mechanical disruption and CDC-induced perforation. In Aim 1, we will investigate the spatiotemporal dynamics of the newly identified septin repair domains, from injury to resealing. We will define the spatiotemporal relationships between septin repair domains and other known repair pathways, and if septins function as scaffolds to recruit and organize repair effectors in space and time. Using super-resolution and FRET microscopy, we will establish the molecular architectures of the septin repair domains. To elucidate the mechanisms by which septins mediate plasma membrane repair, we will test several, non-mutually exclusive, repair mechanisms in Aim 2. Additionally, we will employ an unbiased proteomic approach to define the septin interactome during plasma membrane repair. Finally, we will define the role of the identified repair pathways in host cell invasion by L. monocytogenes and pathogenesis using a murine model of infection. This work will provide fundamental insights into the mechanisms of plasma membrane repair, with broad implications for understanding physiological and pathological processes and developing therapeutic strategies against infections and other diseases involving excessive plasma membrane damage or dysregulated repair.
