Regulation of epithelial cell migration and macropinocytosis by septin GTPases

Epithelial cancers, or carcinomas, account for approximately 90% of all cancers. Unregulated cellular proliferation drives tumor growth and expansion, while metastatic behavior, or the motility of tumor cells to distant sites, underlies the majority of cancer-related deaths. Thus, better understanding of epithelial cell biology will provide novel anti-cancer therapeutics. Septin GTPases are a novel component of the mammalian cytoskeleton. Human septins are comprised of thirteen genes that are divided into four groups based on sequence similarity. Septins bind and hydrolyze GTP in order to assemble into heteropolymers and higher-order structures such as bundles, ring and patches. Septins bind directly to the actin and microtubule cytoskeleton and cell membranes. Through these interactions, septins are posited to regulate their organization and dynamics by acting as scaffolds and diffusion barriers. Importantly, septins are frequently overexpressed in carcinomas, but their role in tumorigenesis and metastasis is unknown. In the first part of this thesis, I investigated how septins function in renal epithelial cell migration. Epithelial cell migration is driven by the actin cytoskeleton, which assembles into a network of stress fibers that exert contractile forces at focal adhesions with the extracellular matrix (ECM). Here, I discovered that septin 9 (SEPT9) crosslinks directly actin filaments and controls focal adhesions stability, which is essential for productive epithelial migration. Importantly, I found that SEPT9 expression is upregulated during epithelial-to-mesenchymal transition (EMT), a critical step during tumor cell invasion, and SEPT9 overexpression promotes renal carcinoma and renal epithelial cell migration. These results are among the first evidence that septins are bona fide actin crosslinking and suggest that septin overexpression may promote tumor cell invasion. In the second part of this thesis, I investigated the molecular interaction between SEPT9 and actin filaments. I identified that a basic region in the SEPT9 N-terminus is sufficient to crosslink actin filaments. In addition, SEPT9 binds to a common site on the actin filament and interferes with the binding of the myosin motor domain and the actin-severing protein cofilin. By inhibiting myosin binding and cofilin-mediated severing, SEPT9 may protect nascent actin filaments from depolymerization. These results provide the first evidence that septins regulate spatially the binding of actin-binding proteins along the actin filament. In the last part of this thesis, I identified a novel role of septins in epithelial macropinocytosis, a form of clathrin-independent endocytosis that is frequently upregulated in cancer and provides essential molecular intermediates for carbon metabolism during tumor cell proliferation. Here, I found that septins localize to macropinosome contact sites with endosomes in a phosphoinositide-specific manner. Significantly, septins regulate the delivery of macropinocytic cargo to the lysosome by promoting membrane fusion. These results provide the first evidence of a role for septins in a clathrin-independent endocytic pathway and endocytic vesicle fusion. Collectively, these findings advance our knowledge of septin cell biology and provide new evidence for how septin overexpression may promote tumor growth and metastasis through two different cellular pathways.