Limits...
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.

Show MeSH
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172791&req=5

fig2: 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.

Mentions: In addition to GEFs and GAPs, most Rho-family GTPases are regulated by another class of proteins, RhoGDI. To assess the activity of RhoGDI by monitoring the level of endogenous GTP-RhoA, we prepared another type of probe consisting of an RBD of effectors sandwiched by YFP and CFP (Fig. 2 A). We expected that the binding of endogenous GTP-RhoA to RBD in the probe displaced YFP and CFP, thereby decreasing the FRET efficiency. Again, we tested RBDs of mDia, Rhotekin, Rhophilin, and PKN for the optimization. We also tested several monomeric mutants of YFP and CFP (Zacharias et al., 2002) to improve the sensitivities of the probes. For the sake of simplicity, we will limit ourselves to a description of the best probe derived from the many trials, Raichu-1502 (hereafter used as Raichu-RBD), which consisted of monomeric YFP-L222K/F224R, the RBD of Rhotekin, and CFP. As shown in Fig. 2 B, the FRET efficiency of Raichu-RBD was decreased in the presence of wild-type RhoA or constitutively active RhoA, but not in the presence of dominant-negative RhoA, indicating that the decrease in FRET correlated with the binding to RhoA. Surprisingly, wild-type RhoA decreased FRET of the probe as efficiently as did the constitutively active RhoA. This may be explained by the property of Raichu-RBD that the FRET efficiency of Raichu-RBD–expressing cells correlated with the net amount of GTP-RhoA, but not with the GTP/GDP ratio on RhoA. In the overexpression system, the wild-type RhoA might provide a saturating amount of GTP-RhoA. Furthermore, we also examined the spectrogram of Raichu-RBD in living HeLa cells by using a flat field imaging spectrograph and obtained a similar result (Fig. 2 C). The difference in the emission ratio between Fig. 2 B and Fig. 2 C arose mostly from the difference in the filter sets used to dissect the fluorescences of YFP and CFP. These observations supported the idea that the FRET efficiency of Raichu-RBD reflected its binding to the endogenous RhoA. We also prepared a probe named Raichu–RBD–X, wherein carboxy terminus of RhoA was fused to Raichu-RBD. By placing the probe only to the membrane, we could reduce the background from the probe in the cytoplasm.


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-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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: In addition to GEFs and GAPs, most Rho-family GTPases are regulated by another class of proteins, RhoGDI. To assess the activity of RhoGDI by monitoring the level of endogenous GTP-RhoA, we prepared another type of probe consisting of an RBD of effectors sandwiched by YFP and CFP (Fig. 2 A). We expected that the binding of endogenous GTP-RhoA to RBD in the probe displaced YFP and CFP, thereby decreasing the FRET efficiency. Again, we tested RBDs of mDia, Rhotekin, Rhophilin, and PKN for the optimization. We also tested several monomeric mutants of YFP and CFP (Zacharias et al., 2002) to improve the sensitivities of the probes. For the sake of simplicity, we will limit ourselves to a description of the best probe derived from the many trials, Raichu-1502 (hereafter used as Raichu-RBD), which consisted of monomeric YFP-L222K/F224R, the RBD of Rhotekin, and CFP. As shown in Fig. 2 B, the FRET efficiency of Raichu-RBD was decreased in the presence of wild-type RhoA or constitutively active RhoA, but not in the presence of dominant-negative RhoA, indicating that the decrease in FRET correlated with the binding to RhoA. Surprisingly, wild-type RhoA decreased FRET of the probe as efficiently as did the constitutively active RhoA. This may be explained by the property of Raichu-RBD that the FRET efficiency of Raichu-RBD–expressing cells correlated with the net amount of GTP-RhoA, but not with the GTP/GDP ratio on RhoA. In the overexpression system, the wild-type RhoA might provide a saturating amount of GTP-RhoA. Furthermore, we also examined the spectrogram of Raichu-RBD in living HeLa cells by using a flat field imaging spectrograph and obtained a similar result (Fig. 2 C). The difference in the emission ratio between Fig. 2 B and Fig. 2 C arose mostly from the difference in the filter sets used to dissect the fluorescences of YFP and CFP. These observations supported the idea that the FRET efficiency of Raichu-RBD reflected its binding to the endogenous RhoA. We also prepared a probe named Raichu–RBD–X, wherein carboxy terminus of RhoA was fused to Raichu-RBD. By placing the probe only to the membrane, we could reduce the background from the probe in the cytoplasm.

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