Wu Daniel Z, Lackner Rachel M, Aonbangkhen Chanat, Lampson Michael A, Chenoweth David M
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States.
Lynch Laboratories, Department of Biology, University of Pennsylvania, Philadelphia, PA, United States.
Methods Enzymol. 2019;624:25-45. doi: 10.1016/bs.mie.2019.05.002. Epub 2019 Jun 6.
Protein-protein interactions are highly dynamic biological processes that regulate various cellular reactions. They exhibit high specificity and spatiotemporal control in order to efficiently utilize finite resources in a cellular compartment. Photoactivatable chemically inducible dimerization (pCID) has emerged as an attractive technique in the scientific community, leading to the development of systems that can be activated with various wavelengths of light in order to manipulate processes on biologically relevant scales with molecular specificity. These systems can be modified to control various protein functions with unprecedented precision and spatiotemporal resolution. In this chapter, we describe an optogenetic platform that provides reversible control over dimerization of genetically tagged proteins using orthogonal wavelengths of light. We demonstrate photoactivation and photo-reversal of protein localization and transport. Mitosis is manipulated by activating and silencing the spindle assembly checkpoint through recruitment and release of proteins from kinetochores.
蛋白质-蛋白质相互作用是调节各种细胞反应的高度动态的生物学过程。它们表现出高度的特异性和时空控制,以便在细胞区室中有效利用有限的资源。光激活化学诱导二聚化(pCID)已成为科学界一种有吸引力的技术,促使开发出可以用各种波长的光激活的系统,以便在生物学相关尺度上以分子特异性操纵各种过程。这些系统可以进行修饰,以前所未有的精度和时空分辨率控制各种蛋白质功能。在本章中,我们描述了一个光遗传学平台,该平台使用正交波长的光对基因标记蛋白质的二聚化提供可逆控制。我们展示了蛋白质定位和运输的光激活和光逆转。通过从动粒募集和释放蛋白质来激活和沉默纺锤体组装检查点,从而操纵有丝分裂。