A bright and photostable photoconvertible fluorescent protein

McKinney, Sean A.; Murphy, Christopher S.; Hazelwood, Kristin L.; Davidson, Michael W.; Looger, Loren L.

doi:10.1038/nmeth.1296

Cited by 512 publications

(523 citation statements)

References 16 publications

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“…In an earlier paper (27), we have reported that H-NS appears as a few large and discrete clusters in cells when fused to mEos2, a previously reported monomeric version of the Eos fluorescent protein (14,16). We also observed similar results when H-NS was fused to PAmCherry (27), another previously reported monomeric PAFP.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinssupporting

confidence: 82%

“…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.…”

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confidence: 88%

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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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Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinssupporting

confidence: 82%

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

View full text Add to dashboard Cite

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“…Given its relatively long absorption maxima and excellent photophysics, we then tested the utility of caged SiRh Q with mEos24a in multicolor localization microscopy. To evaluate the cross‐talk between the two labels, we expressed a fusion protein containing mEos2 and a mitochondrial localization sequence and then stained F‐actin with phalloidin conjugate 17 .…”

mentioning

confidence: 99%

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

et al. 2015

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The rhodamine system is a flexible framework for building small‐molecule fluorescent probes. Changing N‐substitution patterns and replacing the xanthene oxygen with a dimethylsilicon moiety can shift the absorption and fluorescence emission maxima of rhodamine dyes to longer wavelengths. Acylation of the rhodamine nitrogen atoms forces the molecule to adopt a nonfluorescent lactone form, providing a convenient method to make fluorogenic compounds. Herein, we take advantage of all of these structural manipulations and describe a novel photoactivatable fluorophore based on a Si‐containing analogue of Q‐rhodamine. This probe is the first example of a “caged” Si‐rhodamine, exhibits higher photon counts compared to established localization microscopy dyes, and is sufficiently red‐shifted to allow multicolor imaging. The dye is a useful label for super‐resolution imaging and constitutes a new scaffold for far‐red fluorogenic molecules.

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“…The foci were also visible as hotspots using the green-red photoswitchable fluorescent protein mEos2 [60] excited by super-resolution stochastic optical reconstruction microscopy (STORM) (Figure 1(c)), with modeling using 3C structural data of the yeast chromosome [61] and sequence alignment analysis for the location of Mig1 target promoters supporting the hypothesis that the majority of Mig1 clusters were specifically binding to Mig1 target genes.…”

Section: Resultsmentioning

confidence: 79%

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Leake

2018

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High-speed single-molecule fluorescence microscopy in vivo shows that transcription factors in eukaryotes can act in oligomeric clusters mediated by molecular crowding and intrinsically disordered protein. This finding impacts on the longstanding puzzle of how transcription factors find their gene targets so efficiently in the complex, heterogeneous environment of the cell.Abbreviations CDF - cumulative distribution function; FRAP - fluorescence recovery after photobleaching; GFP - Green fluorescent protein; STORM - stochastic optical reconstruction microscopy; TF - Transcription factor; YFP - Yellow fluorescent protein

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A bright and photostable photoconvertible fluorescent protein

Cited by 512 publications

References 16 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

Synthesis of a Far‐Red Photoactivatable Silicon‐Containing Rhodamine for Super‐Resolution Microscopy

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

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