Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells
Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells
复制标题
确定细胞内肌动蛋白结合蛋白 transgelin-2 的结构域水平反应扩散特性
DOI：
10.1016/j.yexcr.2021.112619
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发表时间：
2021
影响因子：
3.7
通讯作者：
Deguchi Shinji
中科院分区：
文献类型：
--
作者：
Saito Takumi;Matsunaga Daiki;Matsui Tsubasa S.;Noi Kentaro;Deguchi Shinji
细胞中的蛋白质与其他分子反复结合，从而降低了它们在细胞内扩散的表观程度。虽然已经做出了很多努力来分析解耦纯扩散和化学结合的这些组合效应，但是用常规方法很难将测量的量归因于蛋白质的特定结构域的性质。在细胞信号研究的共同目标，旨在确定负责特定的分子间相互作用的域的动机，在这里，我们描述了一个框架，用于确定与固定支架相关的细胞蛋白质的局部理化性质。为了验证这种新方法，我们将其应用于transgelin-2，肌动蛋白结合蛋白，其细胞内动力学仍然难以捉摸。我们开发了一个基于荧光漂白后恢复（FRAP）的框架，在该框架中创建了结构域缺失突变体的综合组合，并分析了它们在FRAP响应中的差异。我们证明，transgelin-2在肌动蛋白应力纤维（SFs）与F-肌动蛋白通过两个独立的域相互作用，并确定为各个域的化学性质。它的纯扩散性能独立的协会F-肌动蛋白也得到。因此，我们的方法将是有用的，在这里介绍transgelin-2，在解决与SF相关的细胞蛋白的信号转导机制。
Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.