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

Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M - J. Cell Biol. (2003)

Bottom Line: Cell.Biol. 22:6582-6591).The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Japan.

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.

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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 32Pi. 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.
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fig1: 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 32Pi. 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.

Mentions: As a follow-up to our previous probes for Ras-superfamily GTPases, here we developed the probes for RhoA, which generally consisted of truncated RhoA (aa 1–189), the RhoA-binding domain (RBD) of effectors, and a pair of GFP mutants, YFP and CFP (Fig. 1 A). In these probes, the intramolecular binding of GTP-RhoA to the effector protein was expected to bring CFP in closer proximity to YFP, resulting in an increase in FRET from CFP to YFP. We chose mDia, Rhotekin, Rhophilin, and PKN for the RhoA effector proteins, and tested probes with either of the configurations YFP–RhoA–effector–CFP or YFP–effector–RhoA–CFP. The Rac1/Cdc42-binding domain of PAK was used as a negative control. As a typical example, the emission profile of Raichu–RhoA–1237X, which will be described later in detail, is shown in Fig. 1 B. Because FRET was most clearly observed as an increase in an emission peak of YFP at 527 nm and a decrease in an emission peak of CFP at 475 nm, the emission ratio of YFP/CFP is used to demonstrate the FRET efficiency hereafter.


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

Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M - J. Cell Biol. (2003)

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 32Pi. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172791&req=5

fig1: 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 32Pi. 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.
Mentions: As a follow-up to our previous probes for Ras-superfamily GTPases, here we developed the probes for RhoA, which generally consisted of truncated RhoA (aa 1–189), the RhoA-binding domain (RBD) of effectors, and a pair of GFP mutants, YFP and CFP (Fig. 1 A). In these probes, the intramolecular binding of GTP-RhoA to the effector protein was expected to bring CFP in closer proximity to YFP, resulting in an increase in FRET from CFP to YFP. We chose mDia, Rhotekin, Rhophilin, and PKN for the RhoA effector proteins, and tested probes with either of the configurations YFP–RhoA–effector–CFP or YFP–effector–RhoA–CFP. The Rac1/Cdc42-binding domain of PAK was used as a negative control. As a typical example, the emission profile of Raichu–RhoA–1237X, which will be described later in detail, is shown in Fig. 1 B. Because FRET was most clearly observed as an increase in an emission peak of YFP at 527 nm and a decrease in an emission peak of CFP at 475 nm, the emission ratio of YFP/CFP is used to demonstrate the FRET efficiency hereafter.

Bottom Line: Cell.Biol. 22:6582-6591).The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Japan.

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.

Show MeSH