the three biosensors were made and used as originally described 4 - 6 , with modifications where noted in the text . the cdc42 biosensor was modified for multiplex imaging by removing egfp and replacing it with an alexa750 dye . the new cdc42 biosensor was fused to maltose binding protein to enhance solubility . for control studies a new dual chain rhoa biosensor was constructed by eliminating the linker in the published rhoa biosensor ( see supplementary material ) . biosensors were imaged as previously described26,27 . in order to obtain sufficient signal / noise for multiplexing , cells with biosensor were filmed at 10 s intervals , using a 40/1.3 na objective and binning , resulting in an effective pixel size in object space of 330 nm . raw images were aligned and noise - filtered to allow ratiometric correction of volume effects , bleed - through and variation in sensor concentration . corrected biosensor images were segmented and cell edge displacements tracked as in13 . sampling windows of 0.9 m depth and 1.8 - 3m width were constructed to follow morphological changes at a fixed distance from the cell edge . for each window , biosensor activation time courses were recorded . for windows placed at the cell edge , a time course of protrusion coupling of two activity time courses was analyzed per window by pearson 's cross - correlation function . subsequently , for a cell the per - window correlation functions were averaged over all windows following the edge at a specific distance d ( fig . per - cell correlation functions were averaged over multiple cells and statistically analyzed by bootstrap sampling to determine the significance and time lag of the coupling between two activities . the three biosensors were made and used as originally described 4 - 6 , with modifications where noted in the text . the cdc42 biosensor was modified for multiplex imaging by removing egfp and replacing it with an alexa750 dye . the new cdc42 biosensor was fused to maltose binding protein to enhance solubility . for control studies a new dual chain rhoa biosensor was constructed by eliminating the linker in the published rhoa biosensor ( see supplementary material ) . biosensors were imaged as previously described26,27 . in order to obtain sufficient signal / noise for multiplexing , cells with biosensor were filmed at 10 s intervals , using a 40/1.3 na objective and binning , resulting in an effective pixel size in object space of 330 nm . raw images were aligned and noise - filtered to allow ratiometric correction of volume effects , bleed - through and variation in sensor concentration . corrected biosensor images were segmented and cell edge displacements tracked as in13 . sampling windows of 0.9 m depth and 1.8 - 3m width were constructed to follow morphological changes at a fixed distance from the cell edge . for each window , biosensor activation time courses were recorded . for windows placed at the cell edge , a time course of protrusion coupling of two activity time courses was analyzed per window by pearson 's cross - correlation function . subsequently , for a cell the per - window correlation functions were averaged over all windows following the edge at a specific distance d ( fig . correlation functions were averaged over multiple cells and statistically analyzed by bootstrap sampling to determine the significance and time lag of the coupling between two activities .
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 m 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 .