Signalling proteins are under tight control both temporally and spatially to ensure that they carry out their roles correctly. For example, the small GTPase Cdc42 functions in processes such as cell motility, proliferation and apoptosis, so its activation at specific subcellular locations needs to be strictly regulated. It has been difficult to visualize Cdc42 activation dynamics in vivo, because of the limitations of present approaches. But, in Science, Hahn and colleagues now report the development of a biosensor that allows unlabelled, endogenous Cdc42 activation to be visualized in living cells.

They covalently labelled a domain from Wiskott–Aldrich syndrome protein (WASP) — a Cdc42 effector protein — with a dye that specifically reports protein interactions and protein conformational changes in living cells. The dye did not significantly perturb the Cdc42–WASP interaction, and it showed a threefold increase in fluorescence intensity on binding to Cdc42–GTPγS (GTPγS is a non-hydrolysable analogue of GTP). No increase was observed in the presence of Cdc42–GDP. The biosensor could also distinguish between Cdc42 and the related RhoA and Rac GTPases.

Although this biosensor could be used to measure Cdc42 activation in cell lysates, Hahn and co-workers developed a ratiometric imaging approach that allowed Cdc42 activation to be visualized in living cells. The final biosensor was named Mero-CBD — a merocyanine dye plus a Cdc42-binding domain.

The authors used this biosensor to monitor Cdc42 activation during cell adhesion and spreading. They found that Cdc42 is activated at the cell periphery, which extends filopodia, but not in actual filopodia. In addition, using specific inhibitors, they showed that Cdc42 activation at the cell periphery is microtubule dependent. They also found that Cdc42 is activated at the trans-Golgi apparatus: this indicates that it regulates the directional sorting/trafficking of polarity signals or that microtubules mediate the transport of activated Cdc42 to the cell periphery. Finally, they showed that increases and decreases in Cdc42 activity are precisely coordinated spatially and temporally with cell extension and retraction.

So, Hahn and colleagues have developed a biosensor that gives us a natural view of Cdc42 dynamics — it allows the activation of endogenous protein to be detected at physiological concentrations in living cells, and does not require Cdc42 to be modified with a fluorescent label. This sensitive methodology could therefore be extended to “...proteins that cannot be derivatized or overexpressed for live cell studies”, and could allow us to carry out a “...detailed kinetic analysis of rapid cellular processes”.