Faulkner Benjamin, He Yuchen, Sitrin Daniel, Ziamanesh Linda, Stains Cliff I
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
University of Virginia Cancer Center, University of Virginia, Charlottesville, VA 22904, USA.
bioRxiv. 2025 Sep 7:2025.09.07.674731. doi: 10.1101/2025.09.07.674731.
Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Despite the essential role these proteins play in maintaining cell homeostasis, there is no generalizable method for directly controlling the activity of these signaling enzymes with subcellular precision. Herein, we report the design and implementation of a modular, optogenetic platform for the spatial control of small GTPase activity within living cells. By combining split-small GTPases with improved Light-Inducible Dimerization (iLID) domains, we obtain spLIT-small GTPase constructs that enable spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42), membrane retraction (spLIT-RhoA), and directed cell migration (spLIT-Rac1). These results establish spLIT-small GTPases as a modular optogenetic platform for spatially controlled activation of small GTPase signaling within living cells and demonstrate the ability to probe spatiotemporal aspects of cell signaling using this approach.
小GTP酶是细胞过程的关键调节因子，如细胞迁移，在人类基因组中由超过167种蛋白质组成一个家族。尽管这些蛋白质在维持细胞内稳态中发挥着重要作用，但目前尚无一种通用方法能够以亚细胞精度直接控制这些信号酶的活性。在此，我们报告了一种模块化光遗传学平台的设计与实现，用于在活细胞内对小GTP酶活性进行空间控制。通过将分裂小GTP酶与改进的光诱导二聚化（iLID）结构域相结合，我们获得了spLIT-小GTP酶构建体，能够对细胞骨架动力学进行空间精确控制，如丝状伪足形成（spLIT-Cdc42）、膜回缩（spLIT-RhoA）和定向细胞迁移（spLIT-Rac1）。这些结果确立了spLIT-小GTP酶作为一种模块化光遗传学平台，用于在活细胞内对小GTP酶信号进行空间控制激活，并证明了使用该方法探究细胞信号时空方面的能力。
