Smith Benjamin A, Gelles Jeff, Goode Bruce L
Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA; Present address: Biogen Idec, Cambridge, Massachusetts, USA.
Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.
Methods Enzymol. 2014;540:95-117. doi: 10.1016/B978-0-12-397924-7.00006-6.
The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks.
肌动蛋白细胞骨架非常动态，在体内受到多种相关蛋白的高度调控。要了解这个蛋白质系统在肌动蛋白网络组装和拆卸过程中的功能，需要采用方法来剖析单个因子以及协同作用的多个因子的活性机制。单丝和单分子荧光成像方法的出现，为发现肌动蛋白调节活性以及直接、定量地洞察调节肌动蛋白网络结构和动力学的分子相互作用途径提供了一种强大的新方法。在这里，我们描述了用于获取和分析单分子数据的技术，这些技术应用于体外研究肌动蛋白相关蛋白的丝状体组装和拆卸活性这一全新挑战。我们讨论了单分子分析在直接可视化分子事件顺序、测量丝状体结合和解离的动力学速率以及研究多个因子之间的协同作用方面的优势。这里描述的方法在阐明重组丝状网络中的肌动蛋白调节机制方面补充了传统的生化方法。
