Dimchev Georgi, Rottner Klemens
Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig; Department of Cell Biology, Helmholtz Centre for Infection Research.
Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig; Department of Cell Biology, Helmholtz Centre for Infection Research;
J Vis Exp. 2018 May 12(135):57643. doi: 10.3791/57643.
Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged β-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.
研究蛋白质的时空动态可以揭示它们在各种情况下的功能重要性。在本文中，我们将讨论如何利用光漂白后荧光恢复（FRAP）和光激活技术来研究亚细胞位置中蛋白质的时空动态。我们还将展示这些技术如何能够直接测定与肌动蛋白细胞骨架调节和细胞运动相关的各种参数。此外，本文还额外介绍了细胞显微注射作为一种替代处理方法（可能先于或补充上述光操纵技术），以触发易位蛋白对细胞形态和功能的瞬时效应。诸如蛋白质注射或局部应用细胞膜可渗透药物或细胞骨架抑制剂等显微操作可作为强大工具，在单细胞和亚细胞水平记录给定处理对细胞行为的即时影响。正如25年前所证实的那样，注射重组Rac1蛋白可立即诱导片状伪足细胞边缘突出，以此为例进行说明。此外，我们提供了一个方案，用于测定增强型绿色荧光蛋白（EGFP）-VASP的周转情况，EGFP-VASP是一种肌动蛋白丝聚合酶，主要聚集在B16-F1细胞的片状伪足尖端，该方案采用FRAP并包括相关的数据分析和曲线拟合。我们还给出了估算片状伪足肌动蛋白网络聚合速率的指导方针，以表达EGFP标记的β-肌动蛋白的细胞为例进行说明。最后，说明了如何使用光激活方法研究肌动蛋白单体在细胞质内的移动速率，以及随后肌动蛋白在快速丝组装位点（如突出的片状伪足尖端）的掺入情况。这些方案均不限于肌动蛋白细胞骨架的组分或调节因子，而是可以很容易地扩展，以类似方式探索各种不同亚细胞结构或功能背景下蛋白质的时空动态和功能。
