Photoswitchable cyan fluorescent protein for protein tracking

Chudakov, Dmitriy M.; Verkhusha, Vladislav V.; Staroverov, Dmitry B; Souslova, Ekaterina A.; Lukyanov, Sergey; Lukyanov, Konstantin A.

doi:10.1038/nbt1025

Cited by 343 publications

(270 citation statements)

References 24 publications

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“…Here, we compare four properties of PAFPs that are critical for superresolution imaging and report two new PAFPs that exhibit excellent performance in all four properties. (14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18), PAtagRFP (19), mMaple (20), PSCFP2 (13,21), Dronpa (22), and mGeosM (23). From this screen, we found that none of these PAFPs was simultaneously optimal in all four criteria described above.…”

mentioning

confidence: 88%

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

328

382

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Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

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mentioning

confidence: 88%

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

328

382

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“…Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable [11][12][13] can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths.…”

Section: A R T I C L E Smentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

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“…They can have emission spectra from cyan at less than 470 nm (Chudakov et al 2004) to red at greater than 600 nm peak emission (Matz et al 1999;Wiedenmann et al 2002;Schnitzler et al 2008). They can also act as chromoproteins (Lukyanov et al 2000), which absorb light to cause a visible colour without producing appreciable fluorescence.…”

Section: Introductionmentioning

confidence: 99%

A photoactivatable green-fluorescent protein from the phylum Ctenophora

2009

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Genes for the family of green-fluorescent proteins (GFPs) have been found in more than 100 species of animals, with some species containing six or more copies producing a variety of colours. Thus far, however, these species have all been within three phyla: Cnidaria, Arthropoda and Chordata. We have discovered GFP-type fluorescent proteins in the phylum Ctenophora, the comb jellies. The ctenophore proteins share the xYG chromophore motif of all other characterized GFP-type proteins. These proteins exhibit the uncommon property of reversible photoactivation, in which fluorescent emission becomes brighter upon exposure to light, then gradually decays to a non-fluorescent state. In addition to providing potentially useful optical probes with novel properties, finding a fluorescent protein in one of the earliest diverging metazoans adds further support to the possibility that these genes are likely to occur throughout animals.

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Photoswitchable cyan fluorescent protein for protein tracking

Cited by 343 publications

References 24 publications

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

A photoactivatable green-fluorescent protein from the phylum Ctenophora

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