Top: Irradiation of photoactivatable Rac1 in a living fibroblast (red circle, left image) leads to localized cell protrusion and translocation of PAK kinase (red in the right hand image).

Bottom: The new Rac1 biosensor used for multiplex imaging shows Rac1 activation at protrusions in a living fibroblast (warmer colours indicate greater activation).

Images courtesy of Dr. Klaus Hahn, University of North Carolina, NC, USA and Dr. Gaudenz Danuser, The Scripps Research Institute, CA, USA.

The GTPases RhoA, Rac1 and Cdc42 control the actin cytoskeleton dynamics that provide the force for cell motility. All three GTPases are activated at the cell front and regulate one another; however, their fine spatio-temporal coordination and mutual regulation are not well characterised. Two studies published in Nature now report the development of technologies that allow the activity of Rho GTPases to be precisely controlled and monitored.

Klaus Hahn and colleagues developed a photoactivatable version of Rac1 (PA-Rac1) by fusing residues 404-547 from Avena sativa Phototropin¹ to the amino terminus of constitutively active Rac1. The phototropin fragment contains a flavin-binding LOV domain and a helical extension that adopt a closed conformation in the dark, blocking the binding of effectors to Rac1. Upon illumination, the helix unwinds and releases steric inhibition, thus leading to Rac1 activation. This protein fusion has advantages over previous light-controlled systems in that it is fully genetically encoded, uses non-toxic wavelengths (458 nm), is reversible (with 43 sec half-life of dark recovery), and allows the subcellular location of activation to be precisely controlled. This is due to the fact that residual activation does not exceed 7.5% at 10 m from an irradiated spot.

Irradiating 20 m spots at the cell edge of mouse embryo fibroblasts (MEFs) stably expressing PA-Rac1 induced localised phosphorylation of the Rac1 effector PAK, actin polymerization and protrusion of the leading edge while the opposite side of the cell retracted. Conversely, irradiating dominant-negative photoactivatable Rac1 led to local retraction and protrusion in other areas, suggesting that localized Rac1 activation or deactivation is sufficient to induce polar movement. Furthermore, Klaus Hahn and colleagues used PA-Rac1 together with a RhoA biosensor to show that Rac1 activation leads to immediate local inhibition of RhoA in vivo.

Gaudenz Danuser and colleagues used two methods to study the temporal and spatial coordination of GTPase activity relative to the movements of the cell edge during constitutive protrusion/retraction cycles (that were not stimulated by external factors). In their 'computational multiplexing' approach, the activity of Cdc42, Rac1 and RhoA was measured in separate experiments. Signals for each of the three GTPase biosensors were measured every 10 sec in 40–80 sampling windows that moved with the leading edge. Edge velocities were also sampled and the data analyzed so that the timing of GTPase activation was coupled with protrusion or retraction. Using these correlation functions, the authors showed that the GTPases are activated over a fixed time interval relative to the dynamics of the leading edge. The correlation analysis was then repeated in sampling windows at various distances from the cell edge to obtain spatial information. Together these analyses showed that RhoA activation occurs at the cell edge synchronously with protrusion, whereas Rac1 and Cdc42 are activated 2 m behind the edge with a delay of 40 sec. This suggests RhoA might initiate protrusions, and Cdc42 and Rac1 might stabilise them.

In a complementary approach, biosensors for both RhoA and Cdc42 were expressed in a single cell simultaneously to visualise their activation using four-channel imaging. This method, which gives unprecedented spatial and temporal resolution, showed that Cdc42 is activated after RhoA and confirmed the signalling relationships inferred by the computational multiplexing.

Together the studies of Hahn and Danuser groups provide powerful methods that enable the immediate coupling between signalling pathway states and their morphological outputs to be visualized.

Author: Kim Baumann - Copyright © 2009 Nature Publishing Group, a division of MacMillan Publishers Limited; used with permission