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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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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.
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fig4: 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.

Mentions: Using these Raichu probes, we examined the activity of RhoA, Rac1, and Cdc42 in living HeLa cells progressing from G2 to G1 phase (Fig. 4 A; Videos 1–3). In this experiment, the objective lens was focused on the basal plasma membrane before imaging, and was fixed during the experiment. For this and the aforementioned reasons, we show here the results obtained by using the probes with K-Ras4B carboxy-terminal region, although we obtained essentially the same results with probes with the authentic carboxy-terminal regions. To show the time course of activity change more quantitatively, we calculated the emission ratios from the averaged intensities of YFP and CFP and plotted them against the cell cycle stage (Fig. 4 B). On entry into prophase, the level of RhoA activity decreased rapidly, leaving high activity at the periphery of the cells. After reaching its nadir at anaphase, the RhoA activity increased gradually in telophase. Essentially the same result was obtained by using Raichu-RhoA and Raichu-RBD. The activity of Rac1 decreased on the entry into prometaphase and reached its lowest level at early telophase, later than RhoA. After the midbody formation, Rac1 activity increased rapidly in the late telophase. Cdc42 activity changed essentially as did Rac1 activity, although the level of activity change was less remarkable. When we used Raichu–Cdc42/Cdc42–CT, we sometimes observed transient and moderate increase in Cdc42 activity in anaphase; however, we could not appreciate its significance at this moment. No detectable change in the FRET efficiency was observed in Raichu–Pak–Rho-expressing cells. We have obtained at least six similar video images of cell division for each probe.


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)

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.
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Related In: Results  -  Collection

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fig4: 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.
Mentions: Using these Raichu probes, we examined the activity of RhoA, Rac1, and Cdc42 in living HeLa cells progressing from G2 to G1 phase (Fig. 4 A; Videos 1–3). In this experiment, the objective lens was focused on the basal plasma membrane before imaging, and was fixed during the experiment. For this and the aforementioned reasons, we show here the results obtained by using the probes with K-Ras4B carboxy-terminal region, although we obtained essentially the same results with probes with the authentic carboxy-terminal regions. To show the time course of activity change more quantitatively, we calculated the emission ratios from the averaged intensities of YFP and CFP and plotted them against the cell cycle stage (Fig. 4 B). On entry into prophase, the level of RhoA activity decreased rapidly, leaving high activity at the periphery of the cells. After reaching its nadir at anaphase, the RhoA activity increased gradually in telophase. Essentially the same result was obtained by using Raichu-RhoA and Raichu-RBD. The activity of Rac1 decreased on the entry into prometaphase and reached its lowest level at early telophase, later than RhoA. After the midbody formation, Rac1 activity increased rapidly in the late telophase. Cdc42 activity changed essentially as did Rac1 activity, although the level of activity change was less remarkable. When we used Raichu–Cdc42/Cdc42–CT, we sometimes observed transient and moderate increase in Cdc42 activity in anaphase; however, we could not appreciate its significance at this moment. No detectable change in the FRET efficiency was observed in Raichu–Pak–Rho-expressing cells. We have obtained at least six similar video images of cell division for each probe.

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
Related in: MedlinePlus