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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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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.
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fig5: 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.

Mentions: Because the most drastic activity changes occurred after metaphase, we concentrated our efforts on the period from anaphase to telophase and performed experiments by continuously focusing at the middle depth of the cells (Fig. 5). In early telophase, the RhoA activity gradually increased at the plasma membrane, including the cleavage furrow. This increase was less pronounced when we used Raichu–RhoA/RhoA–CT (Fig. 5 A), but was more clear by using Raichu–RhoA/K-Ras4B–CT (Fig. 5 C) or by TPEM (Fig. 5 D). The increase in RhoA activity at the plasma membrane was confirmed by using Raichu-RBD (Fig. 5 B). In contrast to RhoA, the activity of Rac1 was suppressed at the center and started increasing from the polar sides of the plasma membrane at late telophase after the abscission of daughter cells (Fig. 5, A and C). Suppression of Rac1 activity at the cleavage furrow was more clearly depicted by TPEM (Fig. 5 D). The activity of Cdc42 was high at the intracellular membrane compartments and did not change remarkably from anaphase to telophase; however, we noticed that the activity always reached its nadir at the time of abscission of daughter cells (Fig. 5 A).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172791&req=5

fig5: 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.
Mentions: Because the most drastic activity changes occurred after metaphase, we concentrated our efforts on the period from anaphase to telophase and performed experiments by continuously focusing at the middle depth of the cells (Fig. 5). In early telophase, the RhoA activity gradually increased at the plasma membrane, including the cleavage furrow. This increase was less pronounced when we used Raichu–RhoA/RhoA–CT (Fig. 5 A), but was more clear by using Raichu–RhoA/K-Ras4B–CT (Fig. 5 C) or by TPEM (Fig. 5 D). The increase in RhoA activity at the plasma membrane was confirmed by using Raichu-RBD (Fig. 5 B). In contrast to RhoA, the activity of Rac1 was suppressed at the center and started increasing from the polar sides of the plasma membrane at late telophase after the abscission of daughter cells (Fig. 5, A and C). Suppression of Rac1 activity at the cleavage furrow was more clearly depicted by TPEM (Fig. 5 D). The activity of Cdc42 was high at the intracellular membrane compartments and did not change remarkably from anaphase to telophase; however, we noticed that the activity always reached its nadir at the time of abscission of daughter cells (Fig. 5 A).

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