New fluorescent protein includes handy on-off switch
Michael Eisenstein
A GFP-like protein with sensitive and reversible on-off switching capabilities represents the first of a new generation of specialized fluorescent tools for studying protein movement and trafficking inside cells.
Of course, green fluorescent protein (GFP) and its multicolored fluorescent cousins really need no introduction, being among the most routinely used tools for biological visualization. In recent years, however, interest has grown in developing alternatives to GFP whose fluorescence emissions can be modulated, as a boon to more localized or precise studies of protein movement within a cell. Enter photoactivatable GFP (PA-GFP), a modified version of the wild-type protein with considerably elevated fluorescence and improved optical contrast between its inactivated and activated forms relative to conventional GFP (Patterson and Lippincott-Schwartz, 2002). PA-GFP and other subsequent photoactivatable derivatives enabled new types of studies, where a researcher could selectively activate individual molecules or groups of molecules and easily track their behavior in vivo.
Cell biologist Atsushi Miyawaki and his colleagues have made a considerable career from his efforts to develop reagents for the expansion and refinement of the fluorescent protein toolbox. Having also thrown their hat into the ring with the development of the green-to-red switching protein Kaede (Ando et al., 2002), they now sought to expand the possibilities for photoconversion still further. "Since our report two years ago," says Miyawaki, "we have devoted our efforts to innovation in the field of reversible protein-highlighting technology."
In a recent issue of Science, his team presents the outcome of their efforts, a Pectinidae coral-derived protein that they have dubbed 'Dronpa'. Dronpa is an engineered monomeric form of 22G, an oligomeric fluorescent protein with a major absorption peak at 503 nm and a minor peak at 390 nm at neutral pH. The strength of fluorescence of Dronpa is equivalent to that of enhanced GFP (EGFP), but it also has a unique and striking photosensitivity: the protein is rapidly photobleached after excitation at 490 nm but just as quickly has its powerful fluorescence restored after 400-nm irradiation. This pattern of inactivation and activation can be repeated rapidly and seemingly without limit (Fig. 1), indicating that this protein has truly unique reversible photoswitching properties.
Figure 1. Fluorescence in Hela cells expressing Dronpa can be readily erased at 488 nm, then reactivated by irradiation at 405 nm, as seen in this sequence of images.
As a practical test of Dronpa, the Miyawaki team conducted a series of experiments to study the dynamics of ERK import and export. ERK is a cytosolic protein that undergoes nuclear translocation in response to mitogen signaling; in the nucleus, it activates gene transcription, and is then transported back into the cytoplasm.
Starting with cells expressing an ERK1-Dronpa fusion, they bleached all fluorescence from cytoplasm and nucleus and then reactivated fluorescence in a single cytoplasmic patch. The fluorescence was soon distributed throughout the cytoplasm but barred from the nucleus in unstimulated cells, whereas cells that were treated with the mitogen EGF exhibited rapid nuclear import within the first ten seconds. Similar experiments, where fluorescence was reactivated in the nucleus, demonstrated that nuclear ERK1 was exported to the cytoplasm only after treatment with EGF. These findings proved somewhat surprising, as some investigators had previously believed that ERK1 accumulated in the nucleus mainly as a result of EGF-induced inhibition of nuclear export. Miyawaki's group also used this approach to characterize the EGF-independent import and export of nuclear transport factor importin .
NIH cell biologist Jennifer Lippincott-Schwartz, who was behind the development of the original PA-GFP, applauds the Miyawaki group's findings. "It's really beautiful work that they've done," she says. "I think that it's very similar to photoactivatable GFP,... but I think that what is particularly nice about what they're doing is they can immediately erase a signal again, if they choose to, when they're shifting back and forth to measure the rates in and out of the nucleus... to get very detailed kinetics." She also suggests that PA-GFP and a protein like Dronpa could potentially be combined for certain applications: "Now that we have two different molecules that are photoactivatable, it's conceivable that you could have a photoactivated FRET system."
The one substantial limitation, according to Miyawaki, is that Dronpa's very sensitivity requires rapid imaging. "The relatively fast bleaching rate of Dronpa at 488 nm may be a drawback, because a limited number of images can be acquired after photoactivation," he says. However, owing to the strength of its fluorescence, "bright images were readily obtained multiple times when the excitation light was reasonably attenuated." He adds that his team is now in the process of developing additional photoswitching "brother" proteins to Dronpa with different bleaching and activation sensitivities, some of which may completely bypass this limitation and enable other, new approaches to the study of protein dynamics.
Ando, R. et al. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science306, 1370−1373 (2004). | Article | PubMed | ChemPort |
Patterson, G.H. and Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science297, 1873−1877 (2002). | Article | PubMed | ISI | ChemPort |
Ando, R. et al. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA99, 12651−12656 (2002). | Article | PubMed | ChemPort |
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