Journal Article

Coordination of Rho GTPase activities during cell protrusion

Nalbant, Perihan, Abell, Amy, Hodgson, Louis, Welch, Christopher, Johnson, Gary L., Elliott, Hunter, Machacek, Matthias, Pertz, Olivier, Danuser, Gaudenz, Hahn, Klaus M.

2009

Overview

Rho GTPases during cell protrusion The Rho GTPase family acts in concert to regulate cyoskeletal dynamics during processes such as cell motility. In this study, Danuser and colleagues study the coordination of RhoA, Rac1 and Cdc42 during cell migration by simultaneously visualizing two molecules using complementary biosensor designs, and by computationally defining the relationships between individual molecules visualized in separate cells. The latter approach demonstrates that different biosensors, imaged separately, can be freely combined to produce maps of relative signalling activities with seconds and single-micron resolution. These technologies pave the way to defining the dynamics of many proteins in large signal transduction networks. The Rho GTPase family is involved in the control of cytoskeleton dynamics, but the spatiotemporal coordination of each element (Rac1, RhoA and Cdc42) remains unknown. Here, GTPase coordination in mouse embryonic fibroblasts is examined both through simultaneous visualization of two GTPase biosensors and using a computational approach. 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 μm 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Animals / Biological and medical sciences / Biosensing Techniques / Biosensors / cdc42 GTP-Binding Protein - metabolism / Cell Movement / Cell physiology / Cell Shape / Cell Surface Extensions - metabolism / Cells / Correlation coefficient / Embryo, Mammalian - cytology / Enzyme Activation / Fibroblasts - cytology / Fibroblasts - enzymology / Fundamental and applied biological sciences. Psychology / G proteins / Guanosine triphosphatase / Humanities and Social Sciences / letter / Measurement / Methods / Mice / Molecular and cellular biology / Motility and taxis / multidisciplinary / Neuropeptides - metabolism / Noise / Physiological aspects / Protein Transport / Proteins / rac GTP-Binding Proteins - metabolism / rac1 GTP-Binding Protein / rho GTP-Binding Proteins - metabolism / rhoA GTP-Binding Protein / Science / Time Factors / Velocity