Coordination of Rho GTPase activities during cell protrusion

Abstract

The GTPases Rac1, RhoA and Cdc42 act in concert to control cytoskeleton dynamics1-3. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells4-7 and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac18, and Rac1 and RhoA are mutually inhibitory9-12. However, their spatiotemporal coordination, at the seconds and single micron dimensions typical of individual protrusion events, remains unknown. Here, we examine GTPase coordination 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 sec. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA plays a role in the initial events of protrusion, while Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…All three proteins are involved in actin polymerization and have been shown to be localized in the cell leading edge during protrusion formation [18][19][20][21][22]. We found both increasing (V12Rac1) and lowering (N17Rac1) activity resulted in defective heterotypic CIL.…”

“…While RhoA activation occurs as a band 2 lm from the leading edge, Cdc42 and Rac1 peak 1.8 lm behind the leading edge of the protrusion close to the sites of maturing adhesions. Hence, the authors suggest the latter two GTPases may regulate adhesion dynamics [20]. If similar spatiotemporal coordination occurs between RhoA and Rac1 in the protrusions of NIH3T3 cells, increased RhoA activity could be affecting heterotypic CIL by disrupting the retraction phase of the cycle.…”

“…Lowering Rac1 activity resulted in an increase in RhoA activity and we found increased RhoA activity (V14RhoA) also gave rise to defective heterotypic CIL. RhoA was thought to be predominantly involved in tail retraction of migrating cells [24], however, recent studies suggest RhoA may participate in both membrane protrusion and retraction at the leading edge [20,25]. Cells expressing dominant active mDia became more invasive, possibly because mDia activates Rac1 [26].…”

“…In mouse embryo fibroblasts during spontaneous cell motility the periodicity of the protrusion and retraction cycle at the cell edge is approximately 100 s [20]. RhoA is activated during the protrusion phase whereas Rac1 and Cdc42 activation lags edge protrusion, reaching their peak activation 40 s after protrusion has commenced.…”

Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

“…There is evidence that Rac1 can stabilize cortical actin to promote junctional stability in endothelial cells 22, 23. In addition, coordinated, cyclic activity of Rac1 and RhoA are known to control the protrusion and withdrawal of cell protrusions 83. Recently, we reported that treatment of endothelial cells with either thrombin or a selective inhibitor of Rac1 can decrease the protrusion frequency of small, local lamellipodia in conjunction with disruption of the endothelial barrier 17.…”