Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.
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.
Yoshizaki
Hisayoshi
H
Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Japan.
Ohba
Yusuke
Y
Kurokawa
Kazuo
K
Itoh
Reina E
RE
Nakamura
Takeshi
T
Mochizuki
Naoki
N
Nagashima
Kazuo
K
Matsuda
Michiyuki
M
D000256:Adenoviridae; D002455:Cell Division; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D015335:Molecular Probes; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D015335:Molecular Probes; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein