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 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.

[1]  P. Hordijk,et al.  Jcb: Article Introduction , 2002 .

[2]  K. Nozaki,et al.  The Rho-mDia1 Pathway Regulates Cell Polarity and Focal Adhesion Turnover in Migrating Cells through Mobilizing Apc and c-Src , 2006, Molecular and Cellular Biology.

[3]  S. Narumiya,et al.  Rho effectors and reorganization of actin cytoskeleton , 1997, FEBS letters.

[4]  Krister Wennerberg,et al.  Rho and Rac Take Center Stage , 2004, Cell.

[5]  D. Bar-Sagi,et al.  Redox-dependent downregulation of Rho by Rac , 2003, Nature Cell Biology.

[6]  J. Hartwig,et al.  FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling , 2006, Nature Cell Biology.

[7]  Alan Hall,et al.  Rho GTPases: biochemistry and biology. , 2005, Annual review of cell and developmental biology.

[8]  M. Schwartz,et al.  Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK , 2000, The EMBO journal.

[9]  K. Hahn,et al.  Spatiotemporal dynamics of RhoA activity in migrating cells , 2006, Nature.

[10]  K. Hahn,et al.  Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. , 2002, Nature Reviews Molecular Cell Biology.

[11]  Miguel Vicente-Manzanares,et al.  Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner , 2008, Nature Cell Biology.

[12]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[13]  G. Gundersen,et al.  mDia mediates Rho-regulated formation and orientation of stable microtubules , 2001, Nature Cell Biology.

[14]  C. Waterman-Storer,et al.  Conserved microtubule–actin interactions in cell movement and morphogenesis , 2003, Nature Cell Biology.

[15]  Gaudenz Danuser,et al.  Fluctuations of intracellular forces during cell protrusion , 2008, Nature Cell Biology.

[16]  A. Hall,et al.  Cell migration: Rho GTPases lead the way. , 2004, Developmental biology.

[17]  K. Hahn,et al.  Activation of Endogenous Cdc42 Visualized in Living Cells , 2004, Science.

[18]  M. Matsuda,et al.  Localized RhoA activation as a requirement for the induction of membrane ruffling. , 2005, Molecular biology of the cell.

[19]  W. Arthur,et al.  RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity. , 2001, Molecular biology of the cell.

[20]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[21]  K. Hahn,et al.  Biosensors for Characterizing the Dynamics of Rho Family GTPases in Living Cells , 2010, Current protocols in cell biology.

[22]  K. Hahn,et al.  Localized Rac activation dynamics visualized in living cells. , 2000, Science.

[23]  Biochemistry and Biology , 1933, Nature.

[24]  K. Rottner,et al.  Interplay between Rac and Rho in the control of substrate contact dynamics , 1999, Current Biology.

[25]  K. Hahn,et al.  Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. , 2006, Methods in enzymology.

[26]  Hai-Tao He,et al.  Dynamics in the plasma membrane: how to combine fluidity and order , 2006, The EMBO journal.

[27]  Z. Kam,et al.  Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells , 2003, Journal of Cell Science.

[28]  G. Danuser,et al.  Morphodynamic profiling of protrusion phenotypes. , 2006, Biophysical journal.

[29]  E. Schaefer,et al.  Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics , 2006, The Journal of cell biology.