A new type of fluorescent biosensor makes it possible to visualize enzymatic and cell-signaling activities occurring at highly specific locations in live cells.
Such activities often occur at 100-nm-sized sites and observing them is currently difficult or impossible. For example, the diffraction limit of visible light prevents light microscopy from capturing dynamic events at sites smaller than 200 to 250 nm.
Superresolution techniques such as SOFI (stochastic optical fluctuation imaging) break the diffraction limit of light microscopes. But they can image only static structures in cells, not dynamic bioactivities.
Now, Jin Zhang of the University of California, San Diego, and coworkers have developed biosensors that light up cellular processes in a new way and are SOFI-detectable, down to a resolution of about 100 nm (Nat. Methods 2017, DOI: 10.1038/nmeth.4221).
They discovered a new biosensing phenomenon called FLINC—Fluorescence fLuctuation INcrease by Contact—in which fluorescence fluctuations speed up when two fluorescent proteins are in close contact. FRET (fluorescence resonance energy transfer) and BiFC (bimolecular fluorescence complementation) have mechanisms reminiscent of FLINC. But FRET is not easily compatible with superresolution imaging, and BiFC is a one-time, irreversible fluorescence-generation process that can’t track dynamic bioactivity.
Zhang and coworkers discovered serendipitously that a fluorescent protein, Dronpa, significantly increases the rate of fluorescence fluctuations of another protein, TagRFP-T, when the two are in close proximity. The team created biosensors in which these proteins are placed at either end of a peptide sequence that an enzyme or signaling molecule can recognize and modify. Normally, the biosensors have extended conformations in which the two proteins remain far apart. But when an enzyme modifies the peptide sequence—for example, by phosphorylation—the biosensor changes to a compact shape. This brings the proteins into close proximity and turns on a FLINC signal that can be imaged. Zhang and coworkers used the biosensors with SOFI to visualize kinase activity in cell microdomains at superresolution.
FLINC “is an important step forward that will be useful within the community,” comments John D. Scott of the University of Washington School of Medicine, an expert on cell signaling. “Only time will tell as to the generality and utility of this approach, but it’s a promising start.”
Zhang’s group “is pitching FLINC as a biosensor technique, but any processes that involve proximity between molecules, such as protein-protein interactions, could be studied using this strategy,” says biomedical optics specialist Xiaolin Nan of Oregon Health & Science University.