Fuchs Margit, Boulanger Marie-Chloé, Lambert Herman, Landry Jacques, Lavoie Josée N
Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de médecine, Centre de recherche sur le cancer de l'Université Laval; Oncology, Centre de recherche du CHU de Québec, Université Laval.
Laboratoire d'études moléculaires des valvulopathies (LEMV), Groupe de recherche en valvulopathies (GRV), Quebec Heart and Lung Institute/Research Center; Department of Surgery, Université Laval.
J Vis Exp. 2016 Sep 16(115):54557. doi: 10.3791/54557.
Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3'UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into >75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.
诸如有丝分裂和细胞分化等细胞过程受细胞形状变化的调控，而细胞形状变化在很大程度上依赖于细胞细胞骨架结构的适当重塑。这涉及在特定时间和位置高阶大分子结构的组装-拆卸过程，该过程对蛋白质过表达引起的扰动特别敏感。在广泛的细胞过程中，能够维持蛋白质稳态并保持接近正常细胞形态的方法对于确定感兴趣蛋白质的功能贡献非常有必要。基于RNA干扰的瞬时耗竭-拯救实验是分析蛋白质功能和结构要求的有力方法。然而，以最小程度偏离其生理水平重新引入靶蛋白是一项真正的挑战。在此，我们描述了一种称为腺转染的方法，该方法用于研究分子伴侣和伙伴在分裂细胞正常运作中的作用以及与肌动蛋白重塑的关系。使用靶向3'UTR区域的siRNA双链体使HeLa细胞中的BAG3耗竭。利用与转染试剂偶联的重组腺病毒将绿色荧光蛋白（GFP）标记的BAG3蛋白同时重新引入超过75%的细胞中。腺转染能够在没有应激反应的情况下，在BAG3耗竭的HeLa细胞中以接近生理水平表达BAG3-GFP蛋白。对内源性热休克蛋白伴侣（蛋白质稳态的主要应激诱导调节因子）的水平未观察到影响。此外，通过在细胞转导-转染时添加驱动荧光标记表达的杆状病毒，我们可以通过延时显微镜分析来剖析有丝分裂细胞动力学，同时对正常有丝分裂进程的扰动最小。腺转染也适用于难以感染的小鼠细胞，并且适用于成肌细胞分化为肌管的功能分析。因此，腺转染提供了一种通用方法，可对依赖高阶细胞骨架动力学的敏感生物学过程中涉及的蛋白质进行结构-功能分析。
