Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan.
Methods Mol Biol. 2025;2872:101-113. doi: 10.1007/978-1-0716-4224-5_7.
Photoswitchable compounds are potent tools for elucidating molecular functions in dynamic cellular processes. Photoswitchable inhibitors targeting various mitotic spindle factors have been developed. In this chapter, we describe experimental methods for photo-controlling mitotic chromosome dynamics using a recently developed photoswitchable inhibitor of mitotic kinesin, CENP-E. This inhibitor undergoes reversible photoisomerization to a more inhibitory trans or less inhibitory cis state by visible or UV light irradiation, respectively, enabling photoswitching of CENP-E function both in vitro and in vivo. First, we explain the procedures used to optimize the experimental condition for efficient photoswitching of CENP-E functionality in cultured cells. We then describe how to conduct de novo photo-control of mitotic chromosome motion using the inhibitor under a microscope.
光致变色化合物是阐明动态细胞过程中分子功能的有力工具。已经开发出针对各种有丝分裂纺锤体因子的光致变色抑制剂。在本章中，我们描述了使用最近开发的有丝分裂驱动蛋白 CENP-E 的光致变色抑制剂来控制有丝分裂染色体动力学的实验方法。该抑制剂通过可见光或紫外光照射分别可逆地异构化为更具抑制性的反式或抑制性较低的顺式状态，从而使 CENP-E 功能在体外和体内均可进行光切换。首先，我们解释了优化培养细胞中 CENP-E 功能有效光切换实验条件的步骤。然后，我们描述了如何在显微镜下使用抑制剂对有丝分裂染色体运动进行从头光控制。
