Abstract：
Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.
摘要：
大多数细胞可以感知并改变其形状以执行基本的细胞过程。在许多真核生物中，septin细胞骨架是协调形状变化如胞质分裂的一个组成部分，极化生长,和移民。Septins是细丝形成蛋白，组装形成不同的高级结构，在许多情况下,在质膜的不同区域发现，最值得注意的是在微米尺度的正曲率区域。细胞中光学显微镜的局限性阻碍了体内隔膜组装过程的监测，以及与细胞膜和细胞骨架元素相互作用的复杂性，使得难以量化生命系统中的隔膜动力学。幸运的是,在过去的十年中，在无细胞系统中重建septin细胞骨架以在高时空分辨率下剖析控制septin组装的机制方面取得了实质性进展。septin组装的核心步骤包括septin杂低聚物与膜的缔合和解离，聚合成长丝，以及通过细丝之间的相互作用形成高阶结构。这里,我们提出了三种在不同情况下观察隔膜组装的方法：平面双层，球形支撑，和杆支撑。这些方法可用于在组装的不同阶段确定隔膜的生物物理参数：作为结合膜的单个八聚体，作为细丝，作为细丝的集合。我们使用这些参数与曲率采样和优先吸附的测量值配对，以了解曲率感测如何在各种长度和时间尺度上运行。
