Activity of Rho-family GTPases during cell division as visualized with FRET-based probes

Abstract

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Figure 1.

Basic profile of Raichu-RhoA. (A) Schematic representations of Raichu-RhoA bound to GDP or GTP. YFP and CFP denote a yellow- and cyan-emitting mutant of GFP, respectively. RBD indicates the RBD of the effector protein. (B) Emission spectra of Raichu-RhoA expressed in 293T cells at an excitation wavelength of 433 nm (left). WT, wild type; Q63L, GTPase-deficient mutant; T19N, a mutant with reduced affinity to guanine nucleotides. In the sample treated with protease, Raichu–RhoA–WT was cleaved with trypsin and proteinase K before analysis. (C) 293T cells expressing Raichu-RhoA and Flag-tagged RhoA were labeled with ³²P_i. The guanine nucleotides bound to the GTPases were analyzed by TLC, and the average of two samples is shown with error bars. Asterisks indicate that the level of guanine nucleotides was beneath the detectable level. (D) pRaichu-RhoA was cotransfected into 293T cells with varying quantities of expression vectors for p115 RhoGEF and Grit. The emission ratio and GTP level were quantitated as in B and C. The emission intensities of CFP at 475 nm and YFP at 527 nm were used to calculate the emission ratio, YFP/CFP. Bars indicate error ranges. Experiments were repeated at least twice and representative data are shown.

Figure 2.

Basic profile of Raichu-RBD. (A) Schematic representations of Raichu-RBD unbound or bound to GTP-RhoA. RBD indicates the RBD of Rhotekin. (B) Cleared lysates of Raichu-RhoA–expressing 293T cells were used to obtain spectrograms at an excitation wavelength of 433 nm. The emission intensities of CFP at 475 nm and YFP at 527 nm were used to calculate the emission ratio, YFP/CFP (n = 3). Bars indicate SD. (C) Spectrograms of Raichu-RBD with or without Rho proteins were obtained in living HeLa cells. The emission intensities of CFP at 475 nm and YFP at 530 nm were used to calculate the emission ratio, YFP/CFP (n = 5). Bars indicate SD.

Figure 3.

Localization and FRET imaging of Raichu probes. (A) HeLa cells expressing GFP-tagged Rho-family GTPases of the wild-type (WT) or constitutively active form (Active) are presented in the first two columns. The next two columns show HeLa cells expressing Raichu probes with either the authentic carboxy termini (CT) or K-Ras4B CT. A cell image of Raichu-RBD–expressing cells is also shown at the bottom of A. Cells were excited by an Argon laser at a 488-nm wavelength and imaged with a confocal microscope. (B) Ratio imaging by TPEM. HeLa cells expressing the Raichu probes listed on the left were imaged for YFP (535 ± 12 nm) and CFP (480 ± 15 nm) with a 790-nm excitation wavelength. XY planes from the bottom to the top of each cell were stacked, and the YFP/CFP ratio image is used to show the FRET efficiency in the intensity-modulated display mode. XZ sections were prepared along the dotted white lines. White bars indicate 10 μm (A and B). The upper and lower limits of the ratio range are shown at the right of each panel.

Figure 4.

Activities of Rho-family GTPases in HeLa cells progressing from G2 to G1 phase. (A) HeLa cells were infected with recombinant adenoviruses for the expression of Raichu–RhoA/K-Ras–CT, Raichu–Rac1/K-Ras–CT, and Raichu–Cdc42/K-Ras–CT as indicated at left. Raichu–Pak–Rho and Raichu–RBD–X was introduced by lipofection. CFP, YFP, and differential interference contrast images were obtained every 2 min with a time-lapse epifluorescent microscope. A ratio image of YFP/CFP was used to represent FRET efficiency. The stages of cell cycle were determined by the differential interference contrast images. Representative FRET images are shown at each stage of the cell cycle denoted at the top of the figure. The upper and lower limits of the ratio range are shown at the right of each panel. At least six similar images were obtained for each probe, and a representative one is used here. White bars indicate 10 μm. (B) From the images in A, the net intensities of YFP and CFP in each cell were measured to calculate the averaged emission ratio.

Figure 5.

Activity of Rho-family GTPases during cytokinesis. HeLa cells expressing Raichu probes with carboxy termini of the authentic proteins (A) or Ki-Ras4B (C) or expressing Raichu-RBD (B) were photographed as in Fig. 4 A, except that the fluorescent images were focused at the middle depth of the cells and subjected to median filtering to reduce noise. The elapsed time is denoted at the top of the figure. The time zero is set to metaphase. At least six similar images were obtained for each probe, and a representative one is shown here. White bars indicate 10 μm. (D) HeLa cells expressing Raichu probes were imaged at early telophase by TPEM as in Fig. 3 B. Horizontal (X-Y) and vertical (X-Z) sections are shown.