Curtis Brandy N, Vogt Ellysa J D, Cannon Kevin S, Gladfelter Amy S
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill.
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill.
J Vis Exp. 2022 Jul 28(185). doi: 10.3791/64090.
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.
大多数细胞能够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细胞骨架是协调诸如胞质分裂、极化生长和迁移等形状变化的一个不可或缺的组成部分。Septin是形成细丝的蛋白质，它们组装形成各种高阶结构，并且在许多情况下，存在于质膜的不同区域，最显著的是在微米尺度正曲率的区域。由于细胞中光学显微镜的局限性以及与膜和细胞骨架成分相互作用的复杂性，阻碍了对体内septin组装过程的监测，使得难以量化活细胞系统中septin的动态变化。幸运的是，在过去十年中，在无细胞系统中重建septin细胞骨架以在高空间和时间分辨率下剖析控制septin组装的机制方面取得了重大进展。Septin组装的核心步骤包括septin异源寡聚体与膜的结合和解离、聚合成细丝以及通过细丝之间的相互作用形成高阶结构。在这里，我们展示了三种在不同环境下观察septin组装的方法：平面双层、球形支持物和棒状支持物。这些方法可用于确定septin在组装不同阶段的生物物理参数：作为单个八聚体结合膜时、作为细丝时以及作为细丝组装体时。我们将这些参数与曲率采样和优先吸附的测量结果相结合，以了解曲率感知在各种长度和时间尺度上是如何运作的。
