A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.

细胞分裂周期蛋白42（Cdc42）在众多细胞形态发生过程中扮演关键角色，其激活动力学特性需受到精确调控，方可介导特定细胞事件的发生。为实现活细胞内Cdc42激活状态的直接观测，我们开发并验证了一款全新的Cdc42激活生物传感器。该生物传感器为基因编码型单链设计，可精准定位于细胞膜区室，且能与上游调控因子（包括鸟苷酸解离抑制因子（guanine nucleotide dissociation inhibitor，GDI））发生相互作用。我们在迁移性小鼠胚胎成纤维细胞中对这款新型生物传感器进行了性能表征，观察到其在细胞前沿伪足处呈现出显著的激活动力学特征，与此前采用有机染料基生物传感器系统对内源性Cdc42的观测结果高度一致。随后我们将Cdc42活性的验证与观测拓展至巨噬细胞体系，证实这款新型生物传感器可检测吞噬作用与细胞因子刺激过程中的差异化激活模式。此外，我们首次观测到巨噬细胞足体（podosome）形成过程中Cdc42存在高度瞬时且局域化的激活现象——该现象此前仅被提出假说，却从未被直接可视化观测证实。