Geometry-dependent assembly of the septin cytoskeleton

摘要/摘要细胞形状是功能所不可或缺的，可以用等离子体来描述 膜曲率。晶胞曲率在微米尺度上发生了许多变化，但 蛋白质的尺寸是纳米级的，这就提出了一个问题，即细胞如何感知、控制和 使用微米级几何体。隔膜蛋白是一种保守的、形成细丝的家族 优先聚集在微米尺度的膜曲率位置的蛋白质。胡萝卜素 聚集在许多曲面上，包括细胞动态沟、树突刺、 细胞内细菌以及纤毛和鞭毛底部周围的膜泡。考虑到这些 不同的细胞环境，间隔蛋白的功能障碍与多种人类疾病有关，包括 许多癌症、神经病和不孕不育。在微米尺度的膜曲率处， 葡聚糖素可以影响蛋白质在膜上的扩散，起到支架的作用 聚集在一起的信号蛋白，并影响细胞皮质的刚性。如何弯曲隔板 组装形成并募集信号蛋白到局部细胞膜是理解 间隔蛋白如何将细胞几何形状与反应联系起来。Septin细丝组装通过 在脂质双层或其他细胞骨架网络上对短的(~24-32 nm)低聚物棒进行热处理。 我们假设细胞调节膜的亲和力、长度、密度和几何形状 以依赖于曲率的方式排列隔膜。这项提议的目标是 识别引导隔膜在曲面上组装的机制，并测量如何 弯曲的组件调节信令网络。我们将结合各种成像技术 高分辨率荧光、扫描电子显微镜和高速原子力等方法 显微镜(HS-AFM)、建模、蛋白质组学和分子遗传学。基于初步的 数据，我们假设依赖于曲率的间隔蛋白组装涉及工作机制 在几个长度尺度上。这项工作将由三个目标指导：(1)分析 曲率传感中的膜相互作用；(2)测定Septin的生物物理性质 使曲率感应成为可能的细丝；(3)确定弯曲的隔膜组件如何招募 特定的信号蛋白。从拟议的实验中，我们将了解到纳米 长度尺度机制有助于感测微米尺度的新出现的中尺度过程。 缩放曲率。这些研究还将揭示Septin支架作为一种 局部曲率函数。这项拟议研究的长期目标是确定如何 间隔蛋白识别微米级的曲率，然后使用形状信息来调制 细胞功能。

Summary/abstract Cell shape is integral to function and can be described in terms of plasma membrane curvature. Many changes in cell curvature occur on the micrometer scale but proteins are nanometers in size, raising the question as to how cells can perceive, control and use micrometer-scale geometry. The septins are a conserved, filament-forming family of proteins that preferentially assemble at sites of micrometer-scale membrane curvature. Septins assemble on many curved surfaces including at the cytokinetic furrow, dendritic spines, membrane blebs, around intracellular bacteria and bases of cilia and flagella. Given these diverse cell contexts, malfunction of septins is linked to diverse human diseases including many cancers, neuropathies and infertility. At sites of micrometer-scale membrane curvature, septins can influence the diffusion of proteins in the membrane, act as scaffolds to bring together signaling proteins, and impact the rigidity of the cell cortex. How curved septin assemblies form and recruit signaling proteins to the local membrane is critical to understand how septins link cell geometry to responses. Septin filament assembly occurs through annealing of short (~24-32nm) oligomeric rods on lipid bilayers or other cytoskeletal networks. We hypothesize that cells modulate the membrane affinity, length, density, and geometrical arrangement of septins in a curvature-dependent manner. The goal of this proposal is to identify the mechanisms directing assembly of septins on curved surfaces and to measure how curved assemblies regulate signaling networks. We will combine a variety of imaging approaches including high-resolution fluorescence, SEM and high-speed atomic force microscopy (HS-AFM), modeling, proteomics, and molecular genetics. Based on preliminary data, we hypothesize that curvature-dependent septin assembly involves mechanisms at work on several length scales. This work will be directed by three aims: (1) Analyze septin membrane interaction in curvature sensing; (2) Determine the biophysical properties of septin filaments that enable curvature sensing; (3) Identify how curved septin assemblies recruit specific signaling proteins. From the proposed experiments, we will learn how nanometer length scale mechanisms contribute to the emergent mesoscale process of sensing micron- scale curvature. These studies will also reveal how septin scaffolding may change as a function of local curvature. The long-term goal of this proposed study is to identify how septins recognize micrometer-scale curvature and then use shape information to modulate cellular functions.