imaging experiments were conducted on an olympus fluoview 1000 laser scanning confocal microscope and an olympus ix81-zdc inverted microscope . simultaneous biosensor imaging and activation of pa - rac was achieved using a mag biosystems frap-3d add - on ( photometrics ) for galvanometer control of laser position .
the precise spatio - temporal dynamics of protein activity are often critical in determining cell behaviour , yet for most proteins they remain poorly understood ; it remains difficult to manipulate protein activity at precise times and places within living cells . 
 protein activity has been controlled by light , through protein derivatization with photocleavable moieties1 or using photoreactive small molecule ligands2 . 
 however , this requires use of toxic uv wavelengths , activation is irreversible , and/or cell loading is accomplished via disruption of the cell membrane ( i.e. through microinjection ) . 
 we have developed a new approach to produce genetically - encoded photo - activatable derivatives of rac1 , a key gtpase regulating actin cytoskeletal dynamics3,4 . 
 rac1 mutants were fused to the photoreactive lov ( light oxygen voltage ) domain from phototropin5,6 , sterically blocking rac1 interactions until irradiation unwound a helix linking lov to rac1 . 
 photoactivatable rac1 ( pa - rac1 ) could be reversibly and repeatedly activated using 458 or 473 nm light to generate precisely localized cell protrusions and ruffling . 
 localized rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement . 
 myosin was involved in rac control of directionality but not in rac - induced protrusion , while pak was required for rac - induced protrusion . 
 pa - rac1 was used to elucidate rac regulation of rhoa in cell motility . 
 rac and rho coordinate cytoskeletal behaviours with seconds and submicron precision7,8 . 
 their mutual regulation remains controversial9 , with data indicating that rac inhibits and/or activates rho10,11 . 
 rac was shown to inhibit rhoa in living cells , with inhibition modulated at protrusions and ruffles . a pa - rac crystal structure and modelling revealed lov - rac interactions that will facilitate extension of this photoactivation approach to other proteins .