Measurement and Analysis of In Vitro Actin Polymerization
Measurement and Analysis of In Vitro Actin Polymerization
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DOI：
10.1007/978-1-62703-538-5_16
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Padrick, Shae B.
中科院分区：
文献类型：
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作者：
Doolittle, Lynda K.;Rosen, Michael K.;Padrick, Shae B.
肌动蛋白的聚合是许多细胞过程中力产生的基础。虽然肌动蛋白聚合可以自发地发生，但细胞通过阻止肌动蛋白丝成核，然后通过几种调节因子刺激聚合和伸长来保持对这一重要过程的控制。肌动蛋白聚合、受调节的成核和受控的延伸活性可以在体外重建，并用于探测细胞用于控制肌动蛋白聚合发生的时间和地点的信号级联。引入芘荧光团允许通过芘荧光的增加来检测细丝形成。这一方法已使用多年，并因其简单灵活而继续得到广泛使用。在这里，我们描述了如何执行和分析这些在体外肌动蛋白聚合测定，重点是从动力学数据中提取有用的描述性参数。
The polymerization of actin underlies force generation in numerous cellular processes. While actin polymerization can occur spontaneously, cells maintain control over this important process by preventing actin filament nucleation and then allowing stimulated polymerization and elongation by several regulated factors. Actin polymerization, regulated nucleation, and controlled elongation activities can be reconstituted in vitro, and used to probe the signaling cascades cells use to control when and where actin polymerization occurs. Introducing a pyrene fluorophore allows detection of filament formation by an increase in pyrene fluorescence. This method has been used for many years and continues to be broadly used, owing to its simplicity and flexibility. Here we describe how to perform and analyze these in vitro actin polymerization assays, with an emphasis on extracting useful descriptive parameters from kinetic data.