Espenel et al. describe how a FRET-based biosensor can be used to track the activity of a kinesin motor protein in living cells.
The kinesin KIF17 promotes the polarization of epithelial cells by stabilizing microtubules, perhaps by coupling their plus ends to the cell cortex. How KIF17’s activity is regulated is unknown, however, so Espenel et al. monitored the kinesin’s activity in vivo by constructing a version of the protein with GFP and mCherry tags at either end. Inactive KIF17 folds in on itself so that the two fluorescent tags are close enough to undergo Förster resonance energy transfer (FRET). Active KIF17, on the other hand, adopts an extended conformation with the fluorophores too far apart to generate a strong FRET signal.
Espenel et al. used fluorescence lifetime imaging to detect the biosensor’s FRET signal and analyzed their data using the “phasor” method so that they could resolve the localization of active and inactive KIF17 molecules within epithelial cells. Active, extended KIF17 concentrated on stable microtubules at the cell periphery. Protein kinase C, part of the microtubule stabilization pathway in other cell types, promoted the kinesin’s activation, and the microtubule plus end–tracking protein EB1 recruited active KIF17 to the end of dynamic microtubules to initiate stabilization.
Senior author Geri Kreitzer now wants to follow how KIF17’s activity changes as epithelial cells form intercellular adhesions and polarize. Because kinesins control membrane trafficking as well as cytoskeletal dynamics, she also hopes to use similar kinesin biosensors to study many other cellular processes.
Text by Ben Short