1. Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA
2. Departments of Pharmacology, Medicinal Chemistry and Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
3. Department of Molecular Cell Biology, Center for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany
4. These authors contributed equally to this work.
5. Present addresses: Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel, Switzerland (M.M.); Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave, Bronx, New York 10461, USA (L.H.); Department of Biomedicine, University of Basel, Mattenstrasse 28, CH-4058 Basel, Switzerland (O.P.).

Correspondence to: Klaus M. Hahn²Gaudenz Danuser¹ Correspondence and requests for materials should be addressed to K.M.H. (Email: khahn@med.unc.edu) or G.D. (Email: gdanuser@scripps.edu).

Abstract

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics^{1, 2, 3}. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells^{4, 5, 6, 7}, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory^{9, 10, 11, 12}. 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 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.

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