A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

Abstract

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.

Fig. 1.

The spectra and photochemical kinetics of mGeos. (A) The excitation (measured at the emission maximum) and emission (measured at the excitation maximum) spectra of photoactivatable mGeos and mEos2. (B) The absorbance spectra of mGeos and mEos2 in their bright states. (C) Single switching kinetics for His62 mutant mGeos, Dronpa, and PDM1-4. (D) Single switching kinetics for His62 and F173S double mutant mGeos and rsFastLime. The blue solid line is mGeos-C, used as a control. The laser intensity in (C) is 15-fold higher than in (D).

Fig. 2.

Confocal images of mammalian cells expressing mGeos-C (A, E, I, M), mGeos-M (B, F, J, N), mGeos-S (C, G, K, O) and mGeos-F (D, H, L, P) labeled proteins. (A)–(D) mGeos-β-actin (mouse) in HeLa cells; (E)–(H) α-actinin-mGeos (human) in HeLa cells; (I)–(L) mGeos-Mito (human, subunit VIII of human cytochrome C oxidase) in HEK293 cells; and (M)–(P) mGeos-histone H2B (human) in HeLa cells. Scale bars: 10 μm.

Fig. 3.

Comparison between mGeos and Dronpa in superresolution microscopy. (A) Distribution of total photon number per burst in HeLa cells expressing β-actin fused with mGeos-M, mGeos-C, mGeos-F, mGeos-S, and Dronpa. (B)–(C) (F)PALM/STORM images of Dronpa-β-actin (B) and mGeos-M-β-actin (C) in a fixed HeLa cell. (D)–(F) Magnified images of the box 1–3 in (C), respectively. (G) TIRF image of area in (F). Scale: 2 μm in (B)–(C); 500 nm in (D)–(F). (H) Distribution of total photon number in HeLa cells expressing β-actin fused with mGeos-M (mean, 458; median, 387; n = 7) and Dronpa (mean, 299; median, 269; n = 6). (I) Distribution of position error in HeLa cells expressing β-actin fused with mGeos-M (mean, 14 nm; median, 12 nm; n = 7 cells) and Dronpa (mean, 17 nm; median, 15 nm; n = 6 cells). The sum of single molecule bursts analyzed for mGeos-M and Dronpa is 320,903 and 262,838, respectively. ***, P < 0.001 (Student’s t test).

Fig. 4.

Nanostructural distribution analyses of β-actin and α-actinin using two-color (F)PALM/STORM in HeLa cell. (A, B) TIRF images of mGeos-M-β-actin (Green) and PAmCherry1-α-actinin (Red). (C) Merged dual-color TIRF image, diffraction-limited. (D, E) (F)PALM/STORM images of mGeos-M-β-actin (Green) and PAmCherry1-α-actinin (Red), magnified from box in (A) and (B). (F) Merged image of mGeos-M-β-actin (Green) and PAmCherry1-α-actinin (Red). Scale bars: 2 μm in (A)–(C); 500 nm in (D)–(E). (G)–(I) Distribution analysis of total photon number per molecule, background photon number and position precision of mGeos-M-β-actin and α-actinin-PAmCherry1 coexpressed in HeLa cells.