Coordination of Rho GTPase activities during cell protrusion.
The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.
Machacek
Matthias
M
Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Hodgson
Louis
L
Welch
Christopher
C
Elliott
Hunter
H
Pertz
Olivier
O
Nalbant
Perihan
P
Abell
Amy
A
Johnson
Gary L
GL
Hahn
Klaus M
KM
Danuser
Gaudenz
G
D000818:Animals; D015374:Biosensing Techniques; D002465:Cell Movement; D048430:Cell Shape; D022081:Cell Surface Extensions / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D051379:Mice; D009479:Neuropeptides / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D009479:Neuropeptides; C484602:Rac1 protein, mouse; C526380:RhoA protein, mouse; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
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