Friday, May 3, 2024
5/3/2024
ABSTRACT: In humans, septins are essential for the proper development and function of nearly every major organ system in the body. At the molecular scale, septins form scaffolds for cell signaling in fundamental processes like cytokinesis, cell polarization, and membrane remodeling. In many cases, septin functions rely on their ability to recognize and bind lipid membranes, as this step concentrates septins and promotes self-assembly. However, what drives septin localization to membranes of different lipid compositions and membrane shapes is poorly understood. While septins are capable of binding to a range of membrane shapes (from curved to flat), they preferentially localize to shallow, convexly curved membranes in vitro and in vivo. Septins have two proposed membrane binding motifs: one that is essential for curvature sensitivity in vitro and in vivo as well as another potentially responsible for septins' preference for anionic lipids. Interestingly, these motifs are located on opposite sides of the septin heterooligomer and are likely not the only components of the septin-membrane interface(s) – raising the questions, how do septins recognize and bind lipid membranes? What membrane properties control septin assembly? I hypothesize that septin localization is modulated by both membrane composition and shape, and that distinct membrane-binding motifs are responsible for directing septin assembly in different contexts. Aim 1: Determine role of lipid packing in septin localization. Lipid packing is directly related to membrane curvature and lipid composition. I hypothesize that septin localization is directed via lipid packing defects that arise from membrane curvature and/or lipid composition, depending on the context. Using a combination of molecular dynamics simulations, in vitro reconstitution assays, and whole-cell manipulations, I will measure how septin assembly changes with the degree of lipid packing in both synthetic and cellular membranes. Aim 2: Identify specific septin residues required for membrane interactions. I hypothesize that septins use different amino acid residues to bind membranes of different shapes. Using cryogenic electron microscopy, I will determine structures of septins bound to liposomes and nanodiscs to emulate curved and flat membranes, respectively. I will map septin-membrane interface(s) and design mutants to experimentally validate key residues via in vitro membrane binding assays and in vivo complementation. This work will determine how septins interact with membranes of different shapes and compositions by directly monitoring these associations using state-of-the-art fluorescence microscopy and cryo-EM approaches. The ways that septins distinguish different lipid bilayers directly impacts how septins self-assemble into scaffolds that organize cell signaling for many different processes.
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