- 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 unique tool for monitoring cellular events. PAFPs change spectral properties in response to irradiation with specific light. In recent years PAFPs have been utilized for the development of novel methods for the optical labeling and tracking living cells, organelles, and intracellular molecules in a spatio-temporal manner. PAFPs became indispensible tools for super-resolution imaging techniques. Moreover, PAFPs opened new possibilities to study cell physiology, in particular, they allow careful determination of protein half-life.
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]. In recent years PAFPs became indispensible tools for super-resolution imaging techniques [Subach et al., 2010].
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.
|* Brightness is a product of extinction coefficient and quantum yield, divided by 1000.|
Ext.coeff. – extinction coefficient.
|Fluorescence color||cyan / green
||NO / red
||NO / red
|Excitation max (nm)||400 / 490
||580 / 580
||- / 562
|Emission max (nm)||468 / 511
||600 / 600
||- / 595
|Quantum yield||0.20 / 0.23
||<0.001 / 0.07
||nd / 0.38
|Ext. coeff. (M-1cm-1)||43 000 / 47 000
||123 000 / 59 000
||nd / 66 000
|Brightness*||8.6 / 10.8
||0 / 4.1
||0 / 25.1
|Activating light||UV-violet (e.g. 405 nm)
||green (530-560 nm)
||UV-violet (e.g. 390-420 nm)
|Calculated contrast, fold||up to 2000
|Cell toxicity||not observed
|Maturation rate at 37°C||fast
|Molecular weight (kDa)||27
- Chapman et al. (2005) Curr Opin Plant Biol. 8(6):565-573.
Chudakov et al. (2004) Nat Biotechnol. 22(11):1435-1439.
Chudakov et al. (2007) Nat Protocols 2 (8):2024-32.
- Gurskaya et al. (2006) Nat Biotechnol. 24(4):461-465.
- Patterson and Lippincott-Schwartz (2002) Science 297 (5588):1873-1877.
- Subach et al. (2010) J Am Chem Soc. 132 (18):6481-91
- Zhang et al. (2007) BioTechniques 42:446-450.