Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC.
J Cell Biol. 2018 Feb 5;217(2):779-793. doi: 10.1083/jcb.201705190. Epub 2017 Dec 19.
We developed a novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.
我们开发了一种新型的光遗传学工具，即 SxIP 改良的光诱导二聚体（iLID），以使用蓝光依赖末端结合蛋白的方式将因子可逆地募集到微管（MT）末端。我们证明 SxIP-iLID 可以跟踪 MT 末端，并在蓝光激活时募集 tgRFP-SspB。我们使用该系统研究了 S2 细胞中 MT 末端和 F-肌动蛋白的交联作用，以深入了解 spectrin 功能和机制。我们表明，SxIP-iLID 可用于暂时募集 F-肌动蛋白结合结构域到 MT 末端并交联 MT 和 F-肌动蛋白网络。交联会降低 MT 的生长速度并产生外周 MT 排斥区。SxIP-iLID 促进特定因子与 MT 末端的一般募集，具有时间控制能力，使研究人员能够系统地调节 MT 末端动力学，并在许多生物过程中探测 MT 末端功能。
