Mechanisms of septin-actin cytoskeletal crosstalk

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PROJECT SUMMARY This proposal addresses how the cellular functions of two major cytoskeletal polymer systems, septins and actin, are coordinated and influence each other. To address this question, I will use the budding yeast S. cerevisiae, where septins were first discovered, and where I am readily able to combine a ‘bottom up’ in vitro reconstitution and single molecule imaging approach with a ‘top down’ genetics and live imaging approach. The proposal builds off of recent discoveries made in the Goode lab, which reveal that septins are organized at the yeast bud neck into 8-10 evenly-spaced bars, or “pillars”, which co-align with F-actin cables used for intracellular transport. While work in a number of model organisms has closely linked the in vivo functions of septins and actin, we still have only a limited understanding of the molecular mechanisms underlying this septin-actin crosstalk. The goal of this proposal is to define these mechanisms. S. cerevisiae express 5 different septin proteins, which co-polymerize into filaments and are further organized into higher order structures. My preliminary data show that one of the septins (Shs1) mediates direct binding to F-actin in vitro, and that loss of SHS1 disrupts actin cable architecture and function in vivo. In Aim1, I will use targeted mutagenesis to generate new shs1 separation-of-function mutants disrupting F-actin binding. I will then use these mutants to investigate how Shs1-mediated F-actin binding contributes to the alignment of actin cables with septin pillars in vivo, and intracellular transport of secretory vesicles. In Aim2, I will reconstitute purified septin oligomers and filaments decorated with actin- nucleating formins (Bnr1) and formin-regulatory proteins (e.g., Gin4, Bud6, and the Mlc1-Iqg1-Hof1 complex), and define their effects on F-actin assembly and organization by TIRF microscopy and single molecule imaging. These experiments will test several important hypotheses, including: (1) whether septins and Gin4 activate full- length Bnr1 from autoinhibition to promote actin assembly; (2) whether Iqg1 has regulatory effects on F-actin and Bnr1, like its human counterpart IQGAP1 (based on a recent study from the Goode lab; Hoeprich et al., 2022); and (3) whether septin oligomers/filaments themselves (via Shs1) directly influence F-actin bundling and dynamics. In parallel to these in vitro experiments, I will acutely deplete the same proteins in vivo (using degron tags) to determine how each contributes to the assembly and alignment of actin cables at the bud neck. My preliminary data already point to an exciting new role for Iqg1 in controlling actin cable formation during polarized cell growth. Together, the in vitro and in vivo work outlined in this proposal will: (i) clarify how septins, formins, and formin-regulatory proteins work in concert to shape actin networks, (ii) define new subunit-specific roles for septins in actin regulation, (iii) lay a strong foundation for launching my own lab focused on septin-actin crosstalk, and (iv) provide new leads that will allow me to extend this work in the future (on my own and via collaboration) into other systems.