细胞质肌动蛋白：纯化及单分子组装分析
Cytoplasmic actin: purification and single molecule assembly assays.
作者信息
Hansen Scott D, Zuchero J Bradley, Mullins R Dyche
机构信息
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA.
出版信息
Methods Mol Biol. 2013;1046:145-70. doi: 10.1007/978-1-62703-538-5_9.
The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy.
肌动蛋白细胞骨架对所有真核细胞都至关重要。除了发挥重要的结构作用外，肌动蛋白组装成丝还为多种细胞过程提供动力，包括细胞运动、胞质分裂和内吞作用。肌动蛋白聚合受到其众多辅助因子的严格调控，这些辅助因子控制着肌动蛋白的时空组装以及这些丝的物理性质。从纯化成分建立肌动蛋白聚合的体外模型，使得在确定这些蛋白质对肌动蛋白细胞骨架的影响方面取得了巨大进展。在这里，我们描述如何使用芘肌动蛋白组装测定法来直接或通过成核或封端因子等蛋白质介导来确定一种蛋白质对肌动蛋白组装动力学的影响。其次，我们展示如何使用荧光标记的鬼笔环肽来可视化体外形成的丝，以深入了解蛋白质如何调节肌动蛋白丝结构。最后，我们描述一种使用全内反射荧光（TIRF）显微镜观察单个肌动蛋白丝和荧光标记的肌动蛋白结合蛋白动态组装和解聚的方法。
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