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.

FIG. 1.

Fluorescence profiles of Raichu-Rac and Raichu-Cdc42. (A) Schematic representations of Raichu-Rac and Raichu-Cdc42 bound to GDP or GTP. When Rac1 or Cdc42 is bound to GDP, fluorescence of 475 nm emanates from CFP with excitation at 433 nm. When Rac1 or Cdc42 is bound to GTP, intramolecular binding to PAK1 brings YFP into close proximity to CFP, which causes FRET and fluorescence of 527 nm from YFP. (B) 293T cells expressing Raichu-Rac and Raichu-Cdc42 were lysed and analyzed with a fluorescence spectrometer at an excitation wavelength of 433 nm. WT, V12, N17, and Y40C, wild type, constitutively active mutant, dominant-negative mutant, and effector mutant, respectively. The lysates of the wild type were further treated with proteinase K (PK), which cleaved the probes between YFP and CFP. We used emission intensities of CFP at 475 nm and of YFP at 530 nm to calculate the emission ratio, YFP/CFP (right). Experiments were performed in duplicate, and data are shown with standard deviations. Representative results from three independent experiments are shown.

FIG. 2.

GTP/GDP loading of Raichu-Rac and Raichu-Cdc42. 293T cells expressing Raichu-Rac, Raichu-Cdc42, Flag-tagged Rac, and Flag-tagged Cdc42 were labeled with ³²P_i. The G proteins were precipitated with either anti-GFP rabbit serum or an anti-Flag monoclonal antibody, followed by TLC analysis. GTP and GDP were quantitated with a BAS-1000 image analyzer, and values of GTP/(GTP + GDP) with standard deviations were plotted. Asterisks indicate that no ³²P_i-labeled guanine nucleotides were detected.

FIG. 3.

Imaging of Rac and Cdc42 activities in HT1080 cells. (A) HT1080 cells were transfected with expression vectors as indicated on the left. After 48 h, cells expressing EGFP-Rac or EGFP-Cdc42 were photographed for EGFP and phase contrast (PC) images. Cells expressing Raichu-Rac or Raichu-Cdc42 were imaged for YFP, CFP, and differential interference contrast (DIC). In the IMD mode images (Ratio), eight colors from red to blue are used to represent the YFP/CFP ratio, with the intensity of each color indicating the mean intensity of YFP and CFP. The upper and lower limits of the ratio range are shown on the right. (B) Increase in the CFP emission by YFP photobleaching. HT1080 cells expressing Raichu-Rac or Raichu-Cdc42 were photobleached at an excitation wavelength of 510 nm for 10 min. Emission intensities of CFP and YFP in this cell were monitored every 30 s and are shown at the left. Cell images of pre- and postbleaching are shown at the right.

FIG. 4.

Activity of Rac and Cdc42 in a motile HT1080 cell video imaged by using Raichu-Rac and Raichu-Cdc42. HT1080 cells infected with recombinant adenoviruses coding Raichu probes were replated onto a collagen-coated glass base dish. Beginning 1 h after replating, CFP, YFP, and differential interference contrast (DIC) images were obtained every 2 min with a time-lapse microscope. YFP/CFP ratio images were created to represent FRET efficiency, which correlated with the activities of the G proteins. Representative YFP, CFP, YFP/CFP (Ratio), and DIC images obtained at the indicated time points are shown. Arrows point in the direction of cell migration. The middle panels show magnified FRET images of the leading edges (boxes in the top panels). The upper and lower limits of the ratio range are shown on the right. The original video images are presented on our website (http://www-tv/biken.osaka-u.ac.jp/rei/). Experiments were performed at least five times for each probe, and similar results were obtained.

FIG. 5.

Fluorescence profile of Raichu-CRIB. (A) Schematic representations of Raichu-CRIB and Raichu-CRIB-X with or without GTP-Rac or -Cdc42. The farnesyl moiety was fused only to Raichu-CRIB-X. When the probe is free, fluorescence at 527 nm by FRET is observed. When the probe is bound to GTP-Rac or GTP-Cdc42, YFP is displaced from CFP and FRET is inhibited. (B) 293T cells transfected with pRaichu-CRIB and pEB6-Rac1-Q61L, pEB6-Cdc42-Q61L, or pEB6-RhoA-Q63L were lysed and analyzed with a fluorescence spectrometer at an excitation wavelength of 433 nm (left). The lysates of the wild type were further treated with proteinase K (PK), which cleaved the probes between YFP and CFP. Mean YFP/CFP ratios are shown with standard error bars at the right. Single and double asterisks indicate that the differences between control and Rac1-Q61L and between control and Cdc42-Q61L, respectively, were statistically significant by t test (P < 0.05 and P < 0.01, respectively).

FIG. 6.

Activity of Rac and Cdc42 video imaged with Raichu-CRIB-X. (A) Increase in the CFP emission by YFP photobleaching. HT1080 cells expressing Raichu-CRIB-X were photobleached at an excitation wavelength of 510 nm for 10 min. Emission intensities of CFP and YFP in these cells (left) were monitored every 30 s. Cell images of pre- and postbleaching are also shown (right). (B) HT1080 cells transfected with pRaichu-CRIB-X were replated onto a collagen-coated glass base dish. Beginning 1 h after replating, CFP, YFP, and differential interference contrast (DIC) images were obtained every 2 min with a time-lapse microscope as described in the legend to Fig. 4. Arrows point in the direction of cell migration. The upper and lower limits of the ratio range are shown on the right. The original video images are presented on our website (:http://www-tv/biken.osaka-u.ac.jp/rei/).

FIG. 7.

Analysis of GEF and GAP activities. (A) Schematic representation of GEFs and GAPs used in this study. Abbreviations: PH, pleckstrin homology domain; DH, DH domain; SH2 and SH3, Src homology domains 2 and 3. (B) COS-1 cells plated on glass bottom 96-well plates were transfected with CAAX-negative pRaichu-Rac or pRaichu-Cdc42 and expression vectors for the proteins listed at the center. Cells were imaged, and the YFP/CFP ratio was determined as described in the text. Error bars, standard deviations. Asterisks, samples that show a difference from the control vector with statistical significance by t test (P < 0.01). (C) CAAX-negative pRaichu-Rac or pRaichu-Cdc42 was coexpressed in 293T cells with various quantities of pCAGGS-DOCK180, pIRM21-KIAA0362/DBS, pIRM21-KIAA0053, and pIRM21-KIAA1204. Cells were lysed and examined for emission ratio as for Fig. 1B.