Department of Biological Sciences, Rutgers, The State University of New Jersey, Newark, New Jersey, USA.
Sci Rep. 2016 Jun 30;6:28822. doi: 10.1038/srep28822.
Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.
定量研究特定细胞质隔室内的多分子复合物组装对于理解细胞如何利用这些复合物的组装/拆卸来控制功能至关重要。目前，荧光共振能量转移和荧光相关光谱等生物物理方法可提供直接蛋白质-蛋白质相互作用的定量测量，而亚细胞分级分离和免疫沉淀等传统生化方法仍然是研究多蛋白复合物组装/拆卸动力学的主要方法。在本文中，我们使用荧光共方差分析和激光共聚焦显微镜验证和定量分析了黏着连接多蛋白复合物的原位组装。利用特异性荧光标记的蛋白对，我们定量分析了黏着连接复合物组装的各个阶段，以及新形成的细胞-细胞接触后，调节上皮组织结构和功能的多蛋白复合物。我们的研究结果表明：在核周细胞质中，细胞黏附连接的最小钙黏蛋白-连环蛋白复合物组装，随后定位于细胞-细胞接触区；黏着连接复合物的组装；肌动球蛋白张力介导的锚定；以及新形成的细胞-细胞接触后的黏着连接成熟。最后，我们在表达荧光标记黏着连接复合物蛋白的活细胞中应用荧光共方差分析，还定量分析了上皮细胞单层形成过程中黏着连接复合物的组装动力学。
