Mechanism of cortical septin heteropolymerization/dissociation

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Despite the cortical localization of septin heteropolymers in vivo and their direct interaction with phospholipid membranes in vitro, their behavior and roles remain elusive. In this project, we characterized the major cortical septin assembly found in mammalian tissue culture cells by fluorescence recovery after photobleaching (FRAP) analysis. GFP-tagged septin subunits exhibited slower turnover compared with other cortical proteins analyzed. Perturbation of actin turnover retarded the cortical septin turnover, while septin depletion by RNAi did not affect cortical actin turnover. These phenomena are interpreted by septins' selective association with a subset of actin-based membrane skeleton, as revealed by quick-freeze deep-etch immunoelectron microscopy. We applied the assay system to test septins' presumptive scaffold function on their physiological binding partners. Septin filament destabilization by RNAi-mediated subunit depletion facilitated the turnover of GLAST, while a septin-stabilizing drug forchlorfenuron restrained more GLAST in the unexchangeable fraction. Thus, cortical septin heteropolymers are components of the actin-based membrane skeleton, providing scaffolds for their interacting partners by impeding their lateral diffusion. We predict that diverse submembranous septin clusters found in vivo may serve as scaffolds or reserve pools for specific membrane-bound proteins.