Haar Lauren L, Lawrence David S, Hughes Robert M
Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, United States.
Department of Chemistry, University of North Carolina, Chapel Hill, NC, United States; Department of Pharmacology, University of North Carolina, Chapel Hill, NC, United States.
Methods Enzymol. 2019;622:309-328. doi: 10.1016/bs.mie.2019.02.020. Epub 2019 Mar 12.
Optogenetic tools provide a level of spatial and temporal resolution needed to shed new light on dynamic intercellular processes. In this chapter we outline specific protocols for applying these tools to cell motility (optogenetic cofilin), apoptosis [optogenetic Bcl-like protein 4 (Bax)], and protein kinase-mediated signaling pathways [optogenetic cAMP-dependent protein kinase (PKA)]. The activity of these optogenetic species is regulated by the light-mediated dimerization of a cryptochrome/Cib protein pair, which controls the intracellular positioning of the protein of interest. The light induced recruitment of cofilin to the cytoskeleton is utilized for directed migration studies and filopodial dynamics. Light-triggered migration of Bax to the outer mitochondrial membrane induces cellular collapse and eventual apoptosis. Finally, the light-mediated movement of PKA to specific intracellular compartments offers the means to assess the consequences of PKA activity in a site-specific fashion via phosphoproteomic analysis.
光遗传学工具提供了一定水平的空间和时间分辨率，有助于为动态细胞间过程带来新的认识。在本章中，我们概述了将这些工具应用于细胞运动（光遗传学肌动蛋白结合蛋白）、细胞凋亡[光遗传学Bcl样蛋白4（Bax）]以及蛋白激酶介导的信号通路[光遗传学环磷酸腺苷依赖性蛋白激酶（PKA）]的具体方案。这些光遗传学物种的活性由隐花色素/Cib蛋白对的光介导二聚化调节，该二聚化控制着目标蛋白在细胞内的定位。光诱导肌动蛋白结合蛋白募集到细胞骨架用于定向迁移研究和丝状伪足动力学研究。光触发Bax迁移到线粒体外膜会诱导细胞崩溃并最终导致细胞凋亡。最后，可以通过磷酸化蛋白质组分析，以位点特异性方式评估PKA活性的后果，而光介导的PKA向特定细胞内区室的移动提供了实现这一目的的手段。
