Mechanism of cortical septin heteropolymerization/dissociation

尽管Septin杂聚物在体内的皮质定位和它们在体外与磷脂膜的直接相互作用，它们的行为和作用仍然难以捉摸。在这个项目中，我们的特点是在哺乳动物组织培养细胞中发现的主要皮质隔蛋白大会的荧光恢复后光漂白（FRAP）分析。GFP标记的隔蛋白亚基表现出较慢的营业额相比，其他皮质蛋白质分析。肌动蛋白周转的扰动延缓了皮质隔蛋白周转，而RNAi引起的隔蛋白耗竭并不影响皮质肌动蛋白周转。这些现象被解释为septins的选择性协会与肌动蛋白为基础的膜骨架的一个子集，揭示了速冻深蚀刻免疫电子显微镜。我们应用该测定系统来测试septins对其生理结合伴侣的假定支架功能。通过RNAi介导的亚基耗竭引起的隔蛋白丝不稳定促进了GLAST的周转，而隔蛋白稳定药物氯吡脲抑制了不可交换部分中的更多GLAST。因此，皮质隔蛋白杂聚物是肌动蛋白为基础的膜骨架的组成部分，提供支架，其相互作用的合作伙伴，阻碍他们的横向扩散。我们预测，在体内发现的不同的submembranous隔蛋白集群可以作为特定的膜结合蛋白的支架或储备池。

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.