Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells

TLDR

Rho family G proteins, including Rac and Cdc42, regulate cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer–based probes to monitor the local balance between guanine nucleotide exchange factor and GTPase‑activating protein activities for Rac1 and Cdc42 at the plasma membrane. The Raichu‑Rac and Raichu‑Cdc42 probes comprise Pak‑binding domains, Rac1 or Cdc42, GFP mutants, and a Ki‑Ras CAAX box, enabling rapid, simple assays of GEFs and GAPs in living cells. Video imaging showed Rac and Cdc42 activities rise toward the leading edge, peak just behind or at the tip respectively, and fall quickly when cells change direction, while the probes identified specific GEFs and GAPs (KIAA0362/DBS, KIAA1256, KIAA0053, KIAA1204) and will accelerate spatiotemporal analysis of Rac and Cdc42.

Abstract

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.