Fox Helen M, Gallop Jennifer L
Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, Cambridge CB2 1QN, United Kingdom.
Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, Cambridge CB2 1QN, United Kingdom
Cold Spring Harb Protoc. 2019 Feb 1;2019(2):pdb.prot100545. doi: 10.1101/pdb.prot100545.
The actin cytoskeleton comprises many different architectures of filaments, including branched networks, parallel bundles and antiparallel fibers. A current challenge is to elucidate how the diverse array of actin regulators, which controls the growth, assembly and turnover of actin filaments, is used to orchestrate cytoskeletal organization and in turn cell shape and movement. Long observed to assemble at cell membranes, actin in egg extracts recapitulates membrane-triggered assembly at specific lipid and membrane environments. The use of egg extracts has contributed greatly to identifying how constitutively autoinhibited regulatory pathways are activated, which converge on activation of the Arp2/3 complex. Here we describe a protocol for making parallel actin bundles using egg extracts from supernatants prepared by high-speed centrifugation. These filopodia-like actin bundles emanate from clusters of actin regulators that self-assemble at phosphatidylinositol (4,5)-bisphosphate-containing supported lipid bilayers. Forming a plasma membrane-mimicking bilayer on glass allows easy, optimizable, high signal-to-noise microscopy at high spatial and temporal resolution. The use of egg extracts yields large quantities of active material that can be flexibly tailored to address specific questions, for example, by dilution, addition of fluorescent proteins, antibodies or protein fragments, immunodepletion, addition of small molecule inhibitors, or biochemical fractionation.
肌动蛋白细胞骨架由许多不同结构的细丝组成，包括分支网络、平行束和反平行纤维。当前的一个挑战是阐明各种各样的肌动蛋白调节因子是如何被用来协调细胞骨架组织进而调控细胞形状和运动的，这些调节因子控制着肌动蛋白丝的生长、组装和周转。长期以来人们观察到肌动蛋白在细胞膜处组装，卵提取物中的肌动蛋白在特定的脂质和膜环境中重现了膜触发的组装过程。卵提取物的使用对于确定组成型自抑制调节途径是如何被激活的起到了很大作用，这些途径最终汇聚于Arp2/3复合物的激活。在这里，我们描述了一种使用高速离心制备的上清液中的卵提取物来制作平行肌动蛋白束的方法。这些丝状伪足样的肌动蛋白束从在含磷脂酰肌醇（4,5）-二磷酸的支持脂质双层上自组装的肌动蛋白调节因子簇中发出。在玻璃上形成模拟质膜的双层允许在高空间和时间分辨率下进行简便、可优化、高信噪比的显微镜观察。卵提取物的使用产生了大量的活性物质，可以灵活地进行调整以解决特定问题，例如通过稀释、添加荧光蛋白、抗体或蛋白片段、免疫去除、添加小分子抑制剂或进行生化分级分离。
