Photoactivatable fluorescent proteins are capable of dramatic changes in fluorescent properties in response to specific light irradiation. For example, they can be converted from cyan to green, or from green to red, or from nonfluorescent to a brightly fluorescent state. Several types of such proteins were developed recently, and some of them are already becoming popular tools to study protein mobility. Here we provide detailed recommendations on application of the monomeric green-to-red photoconvertible fluorescent protein Dendra2 for protein tracking in living cultured cells.
Green fluorescent protein (GFP) and its homologs from jellyfishes, anthozoans, and copepods represent a unique protein family capable of self-catalyzed formation of a chromogenic group within the protein globule. GFP-like proteins become fluorescent or colored after protein translation, requiring no external enzymes or cofactors except molecular oxygen. Perhaps, 99% of GFP-related works utilize fluorescent protein markers allowing in vivo visualization of diverse cellular processes (1). In particular, photobleaching of a fluorescent protein fused to a protein of interest is a widely used technique for studies of protein mobility by tracking the photobleached and nonphotobleached protein exchange rate (2).
A few years ago, several laboratories in the world (including ours) began to work on the development of light-controlled fluorescent proteins, whose spectral properties can be directly changed by a pulse of light. Of course, fluorescence that can be switched on by light represents the strongest practical interest. A number of so-called photoactivatable fluorescent proteins (PAFP) capable of a marked increase in fluorescence brightness in response to irradiation with light of specific wavelength and intensity were developed (3) (Table 1).
PAFPs allow one to photolabel and track the movement of living cells, organelles, and intracellular molecules. Probably the most productive use of PAFPs is tracking of the redistribution of fusion protein(s) of interest. Here, a protein of interest, tagged with a PAFP, can be precisely photolabeled and tracked in a spatially and temporally controlled manner. By timing the motion, one could determine rates and preferred directions of protein movement inside living cells. The approach can be widely applied to estimate protein movement rates and to compare these rates as cells respond to various stimuli. PAFPs can also be targeted to particular organelles, to measure and compare the rates of protein movement in various compartments. They also allow one to study protein exchange between compartments, as well as fission and fusion of organelles. Fusion of membrane-associated proteins or short peptides with PAFPs allows researchers to study their lateral diffusion in, and the viscosity of, membranes.
A number of novel techniques have been developed using PAFPs for precise cellular studies. These include: (i) monitoring of protein turnover by tracking of degradation of the irreversibly photoactivated protein (4); (ii) monitoring of dynamic protein interactions with photoquenching fluorescence resonance energy transfer (FRET) (5); (iii) fast protein tracking by repeated reversible photoactivation and averaging of tracking series (6); and (iv) high-resolution imaging techniques, based on reversible (7) or irreversible (8) photoactivation.
Several types of PAFPs can be distinguished (Table 1). The first type includes photoactivatable GFP (PA-GFP) (9), photoswitchable cyan fluorescent protein (PS-CFP) (10) and its enhanced version PS-CFP2 (Evrogen, Moscow, Russia). All these PAFPs are mutant variants of natural GFPs from Aequorea victoria and Aequorea coerulescens. In these proteins, the chromophore initially exists exclusively in a neutral state with an absorption maximum at about 400 nm. These initial forms are fluorescent, and excitation by a weak 400 nm light can be used for the preliminary visualization of PA-GFP (green emission peaked at 515 nm), PS-CFP, and PS-CFP2 (cyan emission peaked at 468 nm). Irradiation with more intense UV or violet light (350–420 nm) leads to the irreversible chromophore transition from a neutral to an anionic state, which results from light-driven decarboxylation of an inner glutamate residue (Glu222) (11). This transition is associated with 100- to 400-fold increase in absorption-excitation peak at about 500 nm, with green emission at about 515 nm. Compared to PA-GFP, PS-CFP2 provides easier primary visualization and higher photoactivation contrast.
The second PAFP type includes Anthozoa-derived green-to-red convertible proteins and their enhanced variants. In the dark, these proteins mature (fold and form chromophore) up to the green fluorescent state, while irradiation with UV-violet light results in their irreversible transition into a red fluorescent state. The first member of this group was named Kaede (12). Later, EosFP and mEosFP (13), KikGR (14), Dendra (15), and its enhanced variant Dendra2 (Evrogen) were developed. These PAFPs are characterized with high contrasting ratiometric photoswitching (i.e., expressed increase of the red fluorescent signal is accompanied by an expressed decrease of the green fluorescence) and allow reliable visualization of the initial (nonactivated) protein form and tracking of both forms’ redistribution after the activation.