He Yuchen, Faulkner Benjamin M, Hyun Emily, Stains Cliff I
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
University of Virginia Cancer Center, University of Virginia, Charlottesville, VA 22908, USA.
bioRxiv. 2025 Jan 28:2025.01.28.635345. doi: 10.1101/2025.01.28.635345.
Small GTPases are critical signaling enzymes that control diverse cellular functions such as cell migration and proliferation. However, dissecting the roles of these enzymes in cellular signaling is hindered by the lack of a plug-and-play methodology for the direct, temporal control of small GTPase activity using user-defined inputs. Herein, we present a method that pairs split-GTPases with user-defined chemical inducer of dimerization (CID) systems in a plug-and-play manner to directly control small GTPase signaling in living cells. The modularity of split-small GTPase systems allows for the selection of CIDs with minimal off-target effects on the pathway being studied. Our results highlight the ability to obtain consistent pathway activation with varying CID systems for direct control of MAPK signaling, filopodia formation, and cell retraction. Thus, split-small GTPase systems provide a customizable platform for development of temporally gated systems for directly controlling cellular signaling with user-defined inputs.
小GTP酶是关键的信号酶，可控制多种细胞功能，如细胞迁移和增殖。然而，由于缺乏一种即插即用的方法，无法使用用户定义的输入直接、适时地控制小GTP酶的活性，因此剖析这些酶在细胞信号传导中的作用受到阻碍。在此，我们提出了一种方法，即将分裂型GTP酶与用户定义的二聚化化学诱导剂（CID）系统以即插即用的方式配对，以直接控制活细胞中的小GTP酶信号传导。分裂型小GTP酶系统的模块化允许选择对所研究途径具有最小脱靶效应的CID。我们的结果突出了使用不同的CID系统获得一致的途径激活以直接控制MAPK信号传导、丝状伪足形成和细胞收缩的能力。因此，分裂型小GTP酶系统为开发用于通过用户定义的输入直接控制细胞信号传导的时间门控系统提供了一个可定制的平台。
