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

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.Ras superfamily G proteins function as molecular switches in a variety of signaling cascades (51). Among them, Rho family G proteins, including Rho, Rac, and Cdc42, are involved in the regulation of a variety of cellular processes, probably through actin cytoskeleton reorganization (1, 9, 13, 48). In a pioneering work by Nobes and Hall, it was shown that Rho regulates the assembly of the actin stress fiber, that Rac induces lamellipodia and membrane ruffles, and that Cdc42 triggers filopodium formation (41).Rho family G proteins are regulated by three classes of protein, guanine nucleotide exchange factor (GEF), GTPaseactivating protein (GAP), and guanine nucleotide dissociation inhibitor (GDI) (51). GEF promotes the exchange of GDP with GTP, which results in the binding of the G proteins to their effector proteins. A typical GEF protein of the Rho family of G proteins consists of a Dbl homology (DH) domain, which exhibits GEF activity, and additional domains that mediate interactions with peptides or lipids. DOCK180, originally isolated as a protein bound to adapter protein Crk (14), also promotes guanine nucleotide exchange of Rac, although it does not contain the DH domain (20). The GTP on the activated Rho family G protein is hydrolyzed in the presence of GAP to resume the GDP-bound inactive state. GDI not only competes with GEF but also holds the Rho family G proteins in the cytoplasm (43). Therefore, the dissociation of GDI is a prerequisite for the membrane association and activation of the Rho family G prot...

Controlled levels of canonical Wnt signaling are required for neural crest migration

“…In vitro analysis of singlecell migration revealed that cell protrusions arise at the front and retract at the back of cells respectively through the localized assembly of the actin controlled by the Rho family of small GTPases ( Fig.1.6.3). At the very tip of the cell front cdc42 GTPase controls actin polymerization, which becomes organized in the parallel bundles and contributes to formation of filopodia and nascent cell-substratum adhesion (Itoh et al, 2002). More backwards, but still at the front Rac1 GTPase regulates actin polymerization, which forms a mesh-like network controlling lamellipodia extension and formation of nascent cell-substratum adhesion.…”

Cell biology of embryonic migration

“…Polarization-In order to move in a certain direction, a cell first needs to polarize in that direction, i.e., to define its front (that will move forward) and its back (that will remain in the rear and retract as the leading edge protrudes). The central regulators of establishing this polarity are Rho family small GTPase Cdc42 (Etienne-Manneville and Hall, 2001;Itoh et al, 2002), Par proteins, and atypical protein kinase (aPKC) (see (Ridley et al, 2003) for overview). Additional signals are provided by a gradient of phosphatidylinositol triphosphate (PIP3) (decreasing from front to rear), produced by the action of phosphoinositide-3 kinase (PI3K), and regulated by PTEN, a PIP3 phosphatase, which is located to the cell rear and controls the lower level of PIP3 at the trailing edge.…”

Involvement of adaptor protein Crk in malignant feature of human ovarian cancer cell line MCAS

“…The decrease of Rac activity seemed to be correlated to the knockdown levels of Crk-II. To confirm the decrease of Rac activation, we utilized FRET-based time-lapse analysis for monitoring the activity of Rac (Itoh et al, 2002). Positive signals were observed after serum stimulation in control cells, but not in Crk knockdown cells (Figure 3b and Supplementary data 5 and 6).…”

“…FRET-based analysis of Rac activity pRachu-Rac-CAAX was transfected into MCAS cells, and 30 h later cells were cultured in DMEM with 2% FBS for 8 h. After 10% FBS stimulation, FRET-based analysis of Rac activity was performed following the methods established by Dr M Matsuda (Osaka University, Japan) (Itoh et al, 2002).…”