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Published online 14 July 2003. doi:10.1083/jcb.200212049
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© The Rockefeller University Press, 0021-9525/2003/7/223 $5.00
The Journal of Cell Biology, Volume 162, Number 2, 223-232

Article

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

Hisayoshi Yoshizaki1,2, Yusuke Ohba1,2, Kazuo Kurokawa1, Reina E. Itoh1, Takeshi Nakamura1, Naoki Mochizuki3, Kazuo Nagashima2,4 and Michiyuki Matsuda1

1 Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
2 Core Research for Evolutional Science and Technology, Japan Science and Technology Cooperation, Fukuoka 816-8580, Japan
3 Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan
4 Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, Sapporo 060-8638, Japan

Address correspondence to M. Matsuda, Dept. of Tumor Virology, Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan. Tel.: 81-6-6879-8316. Fax: 81-6-6879-8314. E-mail: matsudam{at}biken.osaka-u.ac.jp

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.

Key Words: fluorescent probes; cytokinesis; rho GTP-binding proteins; rac GTP-binding proteins; Cdc42 GTP-binding protein

The online version of this article includes supplemental material.

* Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; RBD, RhoA-binding domain; TPEM, two-photon excitation fluorescence microscopy.


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