Lan Tien-Hung, Ambiel Nicholas, Lee Yi-Tsang, Nonomura Tatsuki, Zhou Yubin, Zuchero J Bradley
Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, 77401, USA.
Department of Neurosurgery, Stanford University, Palo Alto, CA, 94304, USA.
Small Methods. 2025 Jul;9(7):e2401522. doi: 10.1002/smtd.202401522. Epub 2025 Jan 31.
The actin cytoskeleton and its nanoscale organization are central to all eukaryotic cells-powering diverse cellular functions including morphology, motility, and cell division-and is dysregulated in multiple diseases. Historically studied largely with purified proteins or in isolated cells, tools to study cell type-specific roles of actin in multicellular contexts are greatly needed. DeActs are recently created, first-in-class genetic tools for perturbing actin nanostructures and dynamics in specific cell types across diverse eukaryotic model organisms. Here, ChiActs are introduced, the next generation of actin-perturbing genetic tools that can be rapidly activated in cells and optogenetically targeted to distinct subcellular locations using light. ChiActs are composed of split halves of DeAct-SpvB, whose potent actin disassembly-promoting activity is restored by chemical-induced dimerization or allosteric switching. It is shown that ChiActs function to rapidly induce actin disassembly in several model cell types and are able to perturb actin-dependent nano-assembly and cellular functions, including inhibiting lamellipodial protrusions and membrane ruffling, remodeling mitochondrial morphology, and reorganizing chromatin by locally constraining actin disassembly to specific subcellular compartments. ChiActs thus expand the toolbox of genetically-encoded tools for perturbing actin in living cells, unlocking studies of the many roles of actin nano-assembly and dynamics in complex multicellular systems.
肌动蛋白细胞骨架及其纳米级组织对于所有真核细胞都至关重要，它驱动着包括形态、运动和细胞分裂在内的多种细胞功能，并且在多种疾病中失调。历史上，对肌动蛋白的研究主要是使用纯化的蛋白质或在分离的细胞中进行，因此非常需要能够在多细胞环境中研究肌动蛋白细胞类型特异性作用的工具。DeActs是最近创建的一类新型遗传工具，用于在多种真核模式生物的特定细胞类型中干扰肌动蛋白纳米结构和动力学。在此，介绍ChiActs，这是新一代的肌动蛋白干扰遗传工具，可在细胞中快速激活，并通过光遗传靶向到不同的亚细胞位置。ChiActs由DeAct-SpvB的拆分部分组成，其强大的促进肌动蛋白解聚的活性通过化学诱导的二聚化或变构转换得以恢复。研究表明，ChiActs能够在几种模型细胞类型中快速诱导肌动蛋白解聚，并能够干扰肌动蛋白依赖性纳米组装和细胞功能，包括抑制片状伪足突出和膜皱褶、重塑线粒体形态以及通过将肌动蛋白解聚局部限制在特定亚细胞区室来重组染色质。因此，ChiActs扩展了用于在活细胞中干扰肌动蛋白的基因编码工具库，开启了对肌动蛋白纳米组装和动力学在复杂多细胞系统中多种作用的研究。
