Photoactivatable fluorescent proteins

- Light-induced high-contrast photoconversion
- Easy detection by flow cytometry or fluorescence microscopy
- No cofactors, substrates, or chemical staining required
- Suitability for long-term tracking of cellular events

Photoactivatable fluorescent proteins (PAFPs) represent a novel effective tool for monitoring cellular events. PAFPs change spectral properties in response to irradiation with specific light. This provides unique possibilities for the optical labelling and tracking living cells, organelles, and intracellular molecules in a spatio-temporal manner. Moreover, PAFPs open new possibilities to study cell physiology, in particular, they allow careful determination of protein half-life.

Applications

Tracking movement of cells, organelles, and proteins
PAFPs allow a more precise, direct, and less damaging study movement of cells and proteins than photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence loss in photobleaching (FLIP). An individual cell, a cellular organelle, or a protein fraction tagged by PAFP can be photoconverted using a beam of focused light. Then, direct visualization of the activated objects within living tissues becomes available [Patterson and Lippincott-Schwartz, 2002; Chudakov et al., 2004; Chapman et al., 2005; Gurskaya et al., 2006; Chudakov et al., 2007].

Protein degradation study
PAFPs allow careful determination of protein half-life [Zhang et al., 2007]. Cells are transfected with a construct coding for target protein fused with a PAFP. A steady-state concentration of the fusion protein and corresponding fluorescent signal depends on protein synthesis and maturation rates as well as protein degradation rate. After photoconversion of the PAFP in a whole cell, a pool of distinct fluorescent molecules appears, which is independent on the synthesis and maturation of the new PAFP molecules. Thus, the decay of the activated fluorescence directly corresponds to the degradation of the PAFP-tagged protein. Time-lapse imaging of the activated signal allows for quantification of degradation process in real-time at the single cell level.

Available proteins