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

Siyuan Wanga, Jeffrey R. Moffitta, Graham T. Dempseya, X. Sunney Xiea, and Xiaowei Zhuanga,b,c,1

Departments of aChemistry and Chemical Biology and bPhysics, Harvard University, cHoward Hughes Medical Institute, Cambridge, MA 02138

Contributed by Xiaowei Zhuang, April 10, 2014 (sent for review February 7, 2014)

Photoactivatable fluorescent proteins (PAFPs) have been widely photons, a higher photon budget leads to higher localization used for superresolution imaging based on the switching and precision and hence higher image resolution (7, 9). (ii) The localization of single molecules. Several properties of PAFPs second property is the on–off switching rate ratio (on–off ratio), strongly influence the quality of the superresolution images. defined as the ratio between the on-switching (activation) and These properties include (i) the number of photons emitted per off-switching or photobleaching rates under the illumination of switching cycle, which affects the localization precision of individ- the imaging light only (7). Even in the absence of activation light, ual molecules; (ii) the ratio of the on- and off-switching rate con- the imaging light itself can also switch on the PAFPs, albeit at stants, which limits the achievable localization density; (iii) the a low rate. Thus, the ratio between the on-switching and off- dimerization tendency, which could cause undesired aggregation switching rates under this condition determines the lower bound of target proteins; and (iv) the signaling efficiency, which de- of the fraction of PAFP molecules in the on-state at any given termines the fraction of target–PAFP fusion proteins that is de- time. The presence of activation light would increase this frac- tectable in a cell. Here, we evaluated these properties for 12 tion. When the product of this fraction and the density of fluorescent labels reaches approximately one fluorophore per commonly used PAFPs fused to both bacterial target proteins, diffraction-limited volume, it becomes difficult to resolve and H-NS, HU, and Tar, and mammalian target proteins, Zyxin and precisely localize the activated fluorophores. Hence the on–off Vimentin. Notably, none of the existing PAFPs provided optimal ratio limits the density of fluorescent labels that can be localized, performance in all four criteria, particularly in the signaling effi- which in turn affects the effective image resolution based on the ciency and dimerization tendency. The PAFPs with low dimeriza- Nyquist sampling theorem (10). (iii) The third property is the tion tendencies exhibited low signaling efficiencies, whereas dimerization tendency. Many PAFPs have a weak tendency to mMaple showed the highest signaling efficiency but also a high form dimers; this could even be true for the PAFPs that are dimerization tendency. To address this limitation, we engineered reported as being monomeric. When these proteins are fused to two new PAFPs based on mMaple, which we termed mMaple2 and target proteins that also tend to polymerize, they may cause mMaple3. These proteins exhibited substantially reduced or unde- undesired aggregation of the target proteins and distort the na- tectable dimerization tendencies compared with mMaple but main- tive distribution of the protein of interest. (iv) The fourth tained the high signaling efficiency of mMaple. In the meantime, property is the signaling efficiency, defined as the ratio between these proteins provided photon numbers and on–off switching rate the number of detectable PAFP-fusion molecules per cell and ratios that are comparable to the best achieved values among PAFPs. the expression level of the fusion protein. Fluorescent proteins do not necessarily fold with 100% efficiency. Among the folded STORM | PALM | fPALM | photoconvertible | photoswitchable molecules, not all of them will become mature at the time of imaging. Among the matured PAFP molecules, only a subset can hotoactivated localization microscopy, stochastic optical re- be photoactivated and imaged. Because of these deficiencies, the construction microscopy, and related imaging methods take number of fusion molecules detected could be substantially P lower than the expression level of the fusion protein. PAFPs with advantage of photoswitching and imaging of single molecules to higher signaling efficiencies will lead to higher localization circumvent the diffraction limit of spatial resolution in light – densities for a given target protein, which will in turn increase the microscopy (1 3). In these methods, only a subset of the fluo- effective image resolution. rescent labels in the sample is switched on at any given time such In this work, we measured the above properties of 12 commonly that the positions of individual fluorophores can be localized used PAFPs, including PAGFP (11), Dendra2 (12, 13), mEos2 from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image Significance 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 Photoactivatable fluorescent proteins (PAFPs) are important probes for superresolution fluorescence microscopy, which allows the to another are widely used for such imaging approaches (4, 5). – Although photoactivatable fluorescent proteins (PAFPs) are spatial organization of proteins in living cells to be probed with sub generally dimmer than photoswitchable dyes (6, 7) and hence diffraction-limit resolution. Here, we compare four properties of give lower image resolution, the ease and high specificity of la- PAFPs that are critical for superresolution imaging and report two beling protein targets in living cells with fluorescent proteins new PAFPs that exhibit excellent performance in all four properties. makes PAFPs highly appealing probes for imaging the dynamics Author contributions: S.W., J.R.M., and X.Z. designed research; S.W., J.R.M., and G.T.D. of cellular structures (4, 8). performed research; S.W. and J.R.M. contributed new reagents/analytic tools; S.W., J.R.M., For single-molecule–based superresolution imaging methods, X.S.X., and X.Z. analyzed and interpreted data; and S.W., J.R.M., G.T.D., X.S.X., and X.Z. several properties of PAFPs are particularly important for the wrote the paper. image quality. Here, we focus on four such key properties. (i) Conflict of interest statement: A US provisional patent application has been filed for the The first property is the photon budget, defined as the average new fluorescent proteins developed in this work. number of photons emitted in each switching event. Given that 1To whom correspondence should be addressed. E-mail: [email protected]. the error of localizing an individual fluorophore approximately This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scales with the inverse square root of the number of detected 1073/pnas.1406593111/-/DCSupplemental.

8452–8457 | PNAS | June 10, 2014 | vol. 111 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1406593111 Downloaded by guest on September 28, 2021 (14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18), and switching off (or photobleaching) the PAFPs in the presence PAtagRFP (19), mMaple (20), PSCFP2 (13, 21), Dronpa (22), and of imaging light only. By definition, the on-switching rate is the mGeosM (23). From this screen, we found that none of these increment in probability of the on-switching events per unit time. PAFPs was simultaneously optimal in all four criteria described To measure this quantity, we imaged the Zyxin-PAFP–express- above. For example, PAtagRFP and mEos3.2 exhibited the ing cells in the superresolution mode for a short period without highest photon budgets among PAFPs, excellent on–off ratios, and any activation light (with imaging light only). The samples were undetectable dimerization tendencies, but showed poor signaling then imaged to completion with an additional activation light at efficiencies. Alternatively, mMaple provided excellent signaling 405 nm. The ratio of the total number of activation events ac- efficiency and on–off ratio with a photon budget nearly equal to cumulated by a certain time during the period without activation those of PAtagRFP and mEos3.2, but had a substantial di- light over the total number of activation events by the end of the merization tendency. To address this limitation, we developed two imaging process was determined. The slope of this cumulative new PAFPs based on mMaple that exhibited substantially reduced activation probability against time then gave the on-switching or undetectable dimerization tendencies while maintaining the rate (Fig. 2 A and B, and Table S1). The off-switching rate was high signaling efficiency, high photon budget, and low on–off ratio determined from the inverse of the mean lifetime of the on-state of mMaple. These PAFPs will substantially facilitate super- of each PAFP in the presence of the imaging light (Fig. 2 C and resolution imaging of cellular structures. D, and Table S1). The ratios between the on- and off-switching rates were determined for all 12 PAFPs (Table 1). Because both Results on- and off-switching rates scale linearly with the illumination in- Photon Budget of Photoactivatable Fluorescent Proteins. tensity, the on–off ratio should be independent of the imaging light To evalu- − intensity. The on–off ratios were generally very small (10 5 to ate the properties of PAFPs under conditions similar to typical − − superresolution imaging experiments, we fused each PAFP to 10 6) except for PAGFP, Dronpa, and mGeosM, which were ∼10 3. various target proteins and expressed these fusion proteins in From the superresolution images of Zyxin, it is evident that the either mammalian cells or bacteria. To measure the photon bud- PAFP with a smaller on–off ratio gave images with a higher lo- get, we fused the PAFPs to the mammalian focal adhesion protein calization density and hence higher image quality (Fig. 2 E and F). Zyxin, transiently transfected BS-C-1 cells with the fusion con- This correlation can be understood based on the Nyquist sampling structs, and imaged the cells using the superresolution imaging theorem, which suggests that the final resolution of an image is at mode in which individual activated proteins were imaged. The least twice the average distance between the localized probes (10). distributions of the photon numbers detected per activation event were determined for all 12 PAFPs. Four example distributions, Dimerization Tendency of Photoactivatable Fluorescent Proteins. for mEos3.2, mMaple, PSCFP2, and PAGFP, are shown in Fig. Dimerization or oligomerization of fluorescent proteins may 1. The mean photon numbers determined from these dis- cause undesired aggregation of the target proteins. Here, instead tributions are listed in Table 1. of measuring the dimerization affinity of PAFPs in vitro, we di- Fewer photons were detected from the green PAFPs (PAGFP, rectly probed whether the PAFPs could cause aggregation of PSCFP2, Dronpa, and mGeosM) than from the red ones (Dendra2, a target protein using a previously reported method (26). In this mEos2, mEos3.2, tdEos, mKikGR, PAmCherry, PAtagRFP, and approach, the fluorescent proteins are fused to the Escherichia coli mMaple). However, within the same color group, the difference protease ClpP, which itself oligomerizes to form a tetradecameric in photon budget was less than twofold. In addition to the complex. It has been suggested that ClpP proteins tend to ag- above-listed fluorophores, we also imaged rsFastLime (24) and gregate and form a single visible punctum in E. coli when fused to rsEGFP (25). Both of these proteins gave relatively low photon a fluorescent protein with a substantial dimerization tendency, budget (<60 photons per switching event), which would lead to whereas fusion to a fluorescent protein with a weak or no di- relatively poor localization precision. We thus did not further merization tendency tends to display a diffuse localization pattern characterize these proteins. It is, however, worth noting that (26). We thus fused E. coli codon-optimized PAFP sequences these proteins are excellent choices for a different mode of to the chromosomal copy of clpP, and report whether cells superresolution imaging [reversible saturable optical fluorescence exhibit the single-punctum phenotype (Table 1 and Fig. 3A). transitions (RESOLFT)] due to the large number of switching Fusion with mKikGR, mGeosM, mMaple, PAmCherry, cycles that they exhibit before photobleaching (24, 25). PSCFP2, or mEos2 all led ClpP to form a single punctum in at least a subset of cells, suggesting that these PAFPs exhibit ap- On–Off Ratio of Photoactivatable Fluorescent Proteins. To de- preciable dimerization tendency, whereas Dendra2, mEos3.2, termine the on–off ratio, we measured the rates for switching on tdEos, PAtagRFP, PAGFP, and Dronpa did not show high enough dimerization tendency to drive detectable ClpP aggregation (Table 1 and Fig. 3A). As a cautionary note, because different target proteins have different expression levels and 5 5 A x 10 mEos3.2 C x 10 PSCFP2 intrinsic oligomerization or polymerization tendencies, PAFPs 2 Mean = 809 6 Mean = 223 that cause appreciable aggregation of ClpP may not necessarily 1.5 4 cause aggregation of other target proteins, and vice versa. 1 To further illustrate the potential effect of PAFP dimerization Frequency 0.5 Frequency 2 on target proteins, we imaged E. coli nucleoid-associated protein 0 0 E. coli 0 1000 2000 0 1000 2000 H-NS, chemotactic receptor Tar, and mammalian in- Number of photons Number of photons termediate filament protein Vimentin fused to different PAFPs.

5 4 In an earlier paper (27), we have reported that H-NS appears as x 10 mMaple x 10 PAGFP B D a few large and discrete clusters in cells when fused to mEos2, Mean = 798 Mean = 313 10 BIOPHYSICS AND 4 a previously reported monomeric version of the Eos fluorescent

protein (14, 16). We also observed similar results when H-NS COMPUTATIONAL BIOLOGY 2 5 was fused to PAmCherry (27), another previously reported mo- Frequency Frequency nomeric PAFP. Fusion with these PAFPs did not appear to 0 0 0 1000 2000 0 1000 2000 perturb the functional activity of H-NS (27). Given the residual Number of photons Number of photons dimerization tendency of mEos2 and PAmCherry detected by Fig. 1. Photon number measurements of PAFPs. The histograms are ex- the ClpP assay (Table 1 and Fig. 3A), we extended the study of ample photon number distributions of mEos3.2 (A), mMaple (B), PSCFP2 (C), H-NS using other PAFPs here (Fig. 3B). Notably, H-NS generally and PAGFP (D) measured by imaging individual Zyxin-PAFP fusion proteins in appeared as large and discrete clusters in cells when fused to live BS-C-1 cells. The mean photon numbers are indicated. PAFPs that exhibited appreciable dimerization tendency in the

Wang et al. PNAS | June 10, 2014 | vol. 111 | no. 23 | 8453 Downloaded by guest on September 28, 2021 Table 1. Properties of PAFPs Preactivation/postactivation On–off switching No. of localizations Maturation † ‡ PAFP emission wavelength, nm* Photon no. rate ratio ClpP clustering per cell time, min§

− Dendra2 507/573 686 4.2 × 10 6 − 1,810 38 − mEos2 519/584 745 2.9 × 10 6 + 1,290 340 mEos3.2 516/580 809 2.6 × 10−6 − 1,950 330 tdEos 516/581 774 3.2 × 10−6 − 1,800 330 − mKikGR 515/591 599 4.1 × 10 6 + 3,800 31 − PAmCherry —/595 706 7.8 × 10 6 + 4,200 61 PAtagRFP —/595 906 5.7 × 10−6 − 760 200 − mMaple 505/583 798 1.9 × 10 6 + 24,000 48 − mMaple2 506/582 783 1.0 × 10 6 + 21,000 62 mMaple3 506/583 675 6.2 × 10−7 − 12,300 49 PAGFP —/517 313 1.3 × 10−3 − <10 − PSCFP2 468/511 223 8.1 × 10 6 + − Dronpa —/517 262 5.8 × 10 4 − 25 mGeosM —/514 248 4.9 × 10−4 +<10

A 405-nm laser was used for photoactivation. The photon number and on–off switching rate ratio were measured in live BS-C-1 cells. The ClpP clustering, number of localizations per cell, and maturation time were measured in live E. coli cells. *The mMaple2 and mMaple3 emission wavelengths were measured in this work with purified proteins. The other wavelengths are cited from refs. 4 and 16. †The “+” indicates that ClpP-PAFP exhibits clustered distributions in at least a subset of cells, whereas “−” indicates that ClpP-PAFP does not exhibit clustered distributions in any cells. The results on Dendra2, Dronpa, and mEos2 are consistent with a previous report (26). ‡ The number of HU-PAFP localizations per E. coli cell. §Maturation time is defined as the half-life of the immature state.

ClpP assay, such as mEos2, PAmCherry, and mMaple. H-NS also by effectively decreasing the labeling density. Here, we compared appeared as large clusters when fused to tdEos, even though tdEos the relative signaling efficiency of different PAFPs by determining did not show sufficient dimerization to drive ClpP aggregation. the number of single-molecule localizations per cell of the PAFPs However, fusion of H-NS to Dendra2, mEos3.2, and PAtagRFP, fused to a common target protein under endogenous expression. which did not exhibit detectable dimerization tendency by the ClpP We fused the PAFPs to the E. coli gene hupA, which encodes assay, appeared much more dispersed in the nucleoids. We also a subunit of the nucleoid-associated protein HU, at its endoge- observed the more dispersed phenotype with immunofluores- nous chromosomal locus and determined the total number of cence imaging of H-NS, but did not present this comparison HU-PAFP localizations per cell (Fig. 4 and Table 1). The four green here because fixation of the bacterial nucleoid is in general PAFPs were too dim to be detected as single molecules in E. coli, discouraged. It is known that H-NS oligomerizes/clusters even whereas the eight red PAFPs showed similar photon budget to those without fusion to fluorescent proteins (28, 29), and this intrinsic measured in mammalian cells (data not shown). Among the red H-NS clustering effect is important for its in vivo function as PAFPs, mMaple provided by far the largest number of HU a transcriptional silencer (29) and brings gene loci in different localizations, which was 6- to 32-fold higher than those of other chromosome regions into spatial proximity in wild-type cells HU-PAFP fusion proteins (Fig. 4). (27). However, fusion with mEos2, PAmCherry, mMaple, and The difference in the number of HU-PAFP localizations for tdEos may have exaggerated the H-NS clustering effect. Without different PAFPs may be attributed to multiple reasons. One of prior knowledge of the residual dimerization tendency of these the possible reasons could be the difference in the number of “monomeric” PAFPs, we previously interpreted the large clusters blinking (switching) events per fluorophore. We measured the of H-NS-mEos2 and H-NS-PAmCherry earlier as a property of average number of blinking events in fixed HU-PAFP–express- H-NS (27). Results here suggest that this interpretation is likely ing cells (Table S2). The average number of blinking events inaccurate because the clustering of H-NS is likely enhanced by the spanned only a small range from 1.7 to 3.3, which are similar to the underappreciated dimerization effect of mEos2 and PAmCherry. results derived from purified PAFP in vitro (Fig. S1). The locali- Although it is formally possible that the more monomeric PAFPs zation numbers divided by the average numbers of blinking events could disrupt intrinsic H-NS clustering by discouraging PAFP yielded the numbers of imaged molecules per cell (Table S2), which self-interactions, we consider this a less likely possibility. is still much bigger in the case of HU-mMaple–expressing cells (4- As another example, Tar preferentially clustered at cell poles to 36-fold higher than in other HU-PAFP–expressing cells). when fused to mEos2 and mKikGR (Fig. 3C) (30), but spread A second possible reason for the difference in localization out more evenly along the envelope of E. coli when fused to the numbers could be the difference in HU-PAFP expression levels. more monomeric mEos3.2 (Fig. 3C), suggesting that the pref- However, quantitative Western blot experiments showed that erential cell pole distribution might have been exaggerated by most HU-PAFP constructs are expressed at similar levels (Table the dimerization of mEos2 and mKikGR. Such aggregation ef- S2), except for tdEos. We divided the number of imaged mole- fect could also apply to eukaryotic systems. For example, fusion to cules per cell by the number of expressed fusion proteins per cell mEos2 and mKikGR caused Vimentin filaments to cluster into to calculate the percentage of PAFP imaged (Table S2). The thick bundles in mammalian cells, whereas Vimentin-mEos3.2 percentage for mMaple imaged was still 5- to 22-fold higher than appeared as thin filaments that were similar to immunofluores- the other PAFPs. Therefore, neither the number of blinking cence images of Vimentin in untransfected cells (Fig. 3D). events nor the expression level is a major contributing factor to the difference in the observed localization numbers. Signaling Efficiency of Photoactivatable Fluorescent Proteins. Even A third potential reason is the difference in the fluorescent when the PAFP is fused to the target protein at its endogenous protein maturation time. Because E. coli has a relatively short chromosomal locus, the number of detectable fluorescent proteins doubling time under our growth conditions, a substantial fraction does not necessarily reflect the expression level of the fusion of the PAFP molecules may not have sufficient time to mature and protein. This deficiency not only prevents quantitative analysis of become fluorescent. To test this idea, we measured the in vivo the target protein, but may also reduce the resolution of the image maturation time of several PAFPs by using kanamycin to block

8454 | www.pnas.org/cgi/doi/10.1073/pnas.1406593111 Wang et al. Downloaded by guest on September 28, 2021 A PSCFP2 C PSCFP2 E PSCFP2 dimerization tendency. To this end, we engineered two new 0.03 1 PAFPs by introducing point mutations into mMaple designed to Mean lifetime destabilize the dimerization of this protein. 0.02 = 0.54 We first took inspiration from the two mutations that make 0.5 mEos3.2 more monomeric than mEos2: I102N and Y189A (15). 0.01

On−rate Probability Based on a sequence alignment between mEos2 and mMaple, we Cumulative on−probability = 1.49e−05 made the comparable mutations, I111N and Y198A, in mMaple. 0 0 0 1000 0 5 We then tested the properties of this protein, which we termed Frames Lifetime (frames) mMaple2, by fusing it to Zyxin, ClpP, and HupA and performing B PAGFP D PAGFP F PAGFP measurements on the photon budget, on–off ratio, dimerization 0.03 tendency, and signaling efficiency as described above. mMaple2 0.8 Mean lifetime exhibited a similar photon budget, on–off ratio, and signaling 0.02 0.6 = 0.70 efficiency as those of mMaple (Fig. 5 A–C, Table 1, and Tables 0.4 S2 and S3). ClpP-mMaple2 proteins still formed puncta in some 0.01 On−rate Probability cells. However, the percentage of cells showing single punctum Cumulative 0.2 on−probability = 0.00188 was significantly reduced in comparison with ClpP-mMaple, 0 0 0 10 20 30 0 5 suggesting a lower dimerization tendency of mMaple2 (Fig. 5D). Frames Lifetime (frames) To further reduce the residual dimerization tendency of mMaple2, Fig. 2. On–off switching rate ratio measurements of PAFPs. Sample data are we screened a series of mutations to residues that are predicted presented for PSCPF2 (A, C,andE)andPAGFP(B, D,andF). (A and B) Cumu- to be solvent-exposed near residues 111 and 198. We focused on lative on-switching probability as a function of time without activation light. charged residues and switched them to the opposite charge. One of The slope of the line gives the on-switching rate. (C and D) Distribution of the the derivatives, with mutations E82R, D83K, and D197K, in ad- on-state lifetime. The blue bars represent measured data,R which were fitted dition to I111N and Y198A, exhibited undetectable dimerization n − x with a binned exponential function (magenta dots) P = ð1=mÞe m dx,to n n−1 tendency when fused to ClpP (Fig. 5D and Table 1). This derivative, take into account that lifetimes are rounded to the next larger integer. m is the which we termed mMaple3, again had a similar photon budget to mean lifetime, the inverse of which gives the off-switching rate. The on–off that of mMaple and even a lower on–off ratio compared with switching rate ratio is defined as the on-rate divided by the off-rate. Each frame A B corresponds to 16 ms. (E and F) Representative superresolution images of Zyxin- mMaple (Fig. 5 and , and Table 1). Its signaling efficiency was PSCFP2 and Zyxin-PAGFP in live BS-C-1 cells. only moderately reduced from those of mMaple or mMaple2 (Fig. 5C, Table 1, and Tables S2 and S3).

protein synthesis in E. coli cells expressing HU-PAFP, and then recording the increase in cellular fluorescence due to PAFP maturation (Fig. S2 and Table 1). Indeed, compared with some of A mEos2 mEos3.2 mMaple PAtagRFP the PAFPs with low signaling efficiencies, mMaple has a substantially faster maturation time. However, the difference in maturation time was substantially smaller than and hence not sufficient to account for the observed difference in the localization numbers (SI Note and Fig. S3). 1 µm Other potential contributing factors include the fraction of properly folded PAFPs at equilibrium, the fraction of folded B Dendra2 mEos3.2 PAtagRFP tdEos mEos2 PAmCherry mMaple PAFPs that can ultimately mature, the fraction of folded and mature PAFPs that can be photoactivated, and the fraction of activated PAFPs that are sufficiently bright to image. Given that different red PAFPs tend to have similar photoactivation efficiencies (31) and photon budgets (Table 1), the last two factors 1 µm are less likely explanations for the observed range of signaling efficiencies. More experiments are required to test these possibilities. C mEos2 mKikGR mEos3.2 1 µm Finally, we measured the signaling efficiency of the PAFPs in mammalian cells by transient transfection of A549 cells with the zyxin::PAFP constructs. For fast data collection, conventional fluorescent microscopy was used and cells were imaged until the PAFPs were fully bleached. The number of imaged molecules in mEos2 mKikGR mEos3.2 IM each cell was estimated by dividing the total photon count from D the cell by the product of the average photon number per switching event (Table 1) and the average number of blinking events for each PAFP (Table S3). The results indicate that mMaple again offers much higher signaling efficiency (7- to 70- 1 µm fold higher) than the other red PAFPs, even in mammalian cells. Among the green PAFPs, PSCFP2 offers a comparable signaling efficiency to that of mMaple (Table S3). Fig. 3. Dimerization tendency of PAFPs and its effect on the distributions of BIOPHYSICS AND target proteins. (A) Phase contrast (Left) and conventional fluorescent images COMPUTATIONAL BIOLOGY New PAFPs with High Signaling Efficiency and Low Dimerization (Right) of live E. coli cells expressing ClpP-PAFP fusions. PAFPs with substantial dimerization tendencies (mEos2, mMaple) result in the formation of ClpP Tendency. The above results show that mMaple has a much puncta. PAFPs with little to no dimerization tendencies (mEos3.2, PAtagRFP) higher signaling efficiency than all of the other tested red PAFPs. produce a diffusive ClpP distribution. (B) Overlaid superresolution (magenta) However, the dimerization tendency of mMaple could lead to and phase-contrast (gray) images of live E. coli cells expressing H-NS-PAFP aggregation effects on the target proteins. Dendra2, mEos3.2, fusions. (C) Overlaid superresolution and phase-contrast images of fixed E. coli and PAtagRFP exhibit undetectable dimerization tendency, cells expressing Tar-PAFP fusions. (D) Superresolution images of fixed Cos-7 but have low signaling efficiency. It is thus desirable to develop cells expressing Vimentin-PAFP fusions or fixed untransfected Cos-7 cells with a new PAFP that has both high signaling efficiency and low immunofluorescent labeling of Vimentin (IM). (Scale bars: 1,000 nm.)

Wang et al. PNAS | June 10, 2014 | vol. 111 | no. 23 | 8455 Downloaded by guest on September 28, 2021 A they are fused. Among the 12 PAFPs tested here, the majority Dendra2 mEos2 mEos3.2 tdEos appeared to exhibit a substantial propensity toward aggregation or bundling of target proteins. The exceptions are Dendra2, mEos3.2, PAtagRFP, PAGFP, and Dronpa, which did not show a detectable aggregation effect for the target proteins tested here. It is important to note that these results do not imply that such aggregation will not happen when these PAFPs are fused to other target proteins with strong intrinsic clustering or polymerization mKikGR PAmCherry PAtagRFP mMaple tendency. As another cautionary note, it is also possible that monomeric fluorescent proteins that discourage self-interactions may disrupt natural oligomerization of the target proteins. Thus, it is a good practice to verify the spatial organization derived from PAFPs with alternative approaches, such as immunohistochemistry or by using SNAP/CLIP/HALO or short peptide tags to label

B 4 x 10 3

AB−6 2 x 10 1000 6 1 4 0 Number of localizations Dendra2 mEos2 mEos3.2 tdEos mKikGR PAmCherryPAtagRFP mMaple 500 2

Fig. 4. Signaling efficiency comparison among PAFPs. (A) Superresolution On−off ratio images of live E. coli cells expressing HU-PAFP fusions (magenta) overlaid with 0

Mean photon number 0 phase-contrast images (gray). Superresolution images were acquired until all of mMaple mMaple2mMaple3 mMaple mMaple2 mMaple3 the PAFP molecules in the field of view were bleached. (B) Numbers of observed 4 CDx 10 localizations per cell for different HU-PAFP fusions. (Error bars represent SEMs.) 3 60 We further tested the aggregation effects of mMaple2 and 2 40 mMaple3 on H-NS, Tar, and Vimentin. H-NS-mMaple3 showed substantially more spread out H-NS distributions than H-NS- 1 mMaple, although H-NS-mMaple2 still appeared as a few discrete 20 clusters in each cell (Fig. 5E). Tar-mMaple2 and Tar-mMaple3

0 % of cells with ClpP foci HU localizations per cell 0 were substantially less concentrated at the cell poles than mMaple mMaple2 mMaple3 mMaple mMaple2 mMaple3 Tar-mMaple (Fig. 5F). Vimentin-mMaple2 and Vimentin-mMaple3 filaments were much less bundled than Vimentin-mMaple fila- E ments (Fig. 5G). All of these observations are consistent with the reduced dimerization tendency of mMaple2 and the undetectable dimerization tendency of mMaple3. 1 µm The excitation and emission spectra of mMaple2 and mMaple3 were similar to those of mMaple (Table 1 and Fig. S4). H-NS-mMaple H-NS-mMaple2 H-NS-mMaple3 F Discussion In this work, we characterized four properties of PAFPs that are important for superresolution imaging based on single-molecule switching and localization. First, the photon budgets are not 1 µm substantially different for different PAFPs within the same color Tar-mMaple Tar-mMaple2 Tar-mMaple3 group, but the red PAFPs provide substantially more photons G (600–900 photons per activation event) than the green PAFPs (200–300 photons). Even the red PAFPs’ photon budgets are substantially lower than those of the bright photoswitchable/ photoactivatable dyes (several thousand to one million photons) (7, 32). Given that the localization precision scales approximately linearly with the inverse square root of the photon numbers, the PAFPs thus give substantially lower localization precision than photoswitchable dyes. Second, the on–off ratios are also not substantially different for different PAFPs within the 1 µm same color group, but the red PAFPs tend to show much lower Vimentin-mMaple Vimentin-mMaple2 Vimentin-mMaple3 − − − on–off ratio (10 5 to 10 6) than the green PAFPs (∼10 3). PSCFP2 is a noticeable exception with an on–off ratio similar to Fig. 5. mMaple2 and mMaple3 exhibit both high signaling efficiency and low – dimerization tendency. (A–D)Photonbudget(A), on–off switching rate ratio the red PAFPs. The on off ratios of the red PAFPs are sub- (B), signaling efficiency (C), and dimerization tendency (D) of mMaple2 and stantially lower than those of the popularly used photoswitchable −3 −4 mMaple3 in comparison with mMaple. (Insets in D) Sample fluorescent images dyes (10 to 10 ) (7), and hence PAFPs can provide sub- of ClpP-mMaple2– and ClpP-mMaple3–expressing E. coli. Error bars are SEMs stantially higher localization density. Third, natural fluores- and are too small to be visualized in A.(E) H-NS appears more spread out in E. cent proteins tend to dimerize or tetramerize. Although mutant coli cells when fused to mMaple3 in comparison with the mMaple and fluorescent proteins have been made to reduce dimerization, mMaple2 fusion proteins. (F) Tar-mMaple2 and Tar-mMaple3 appear more many of the so-called monomeric fluorescent proteins still have evenly distributed along the cell envelope and less concentrated at the polar some residual dimerization tendency and thus can cause undesired caps than Tar-mMaple. (G) Vimentin-mMaple2 and Vimentin-mMaple3 aggregation and mislocalization of the target proteins to which are less bundled than Vimentin-mMaple. (Scale bars: 1,000 nm.)

8456 | www.pnas.org/cgi/doi/10.1073/pnas.1406593111 Wang et al. Downloaded by guest on September 28, 2021 proteins with dyes (33). The fourth property that we probed is the consider when other experimental requirements need to be taken signaling efficiency, which determines the number of detectable into account. For example, spectral properties are essential for molecules of the PAFP fusion protein for a given target protein. multicolor imaging. For two-color imaging, green PAFPs are When the signaling efficiency is low, the number of localizations is particularly useful (when paired with a red PAFP) even though not sufficient to map out the fine organization of the target protein. they generally give lower photon numbers and higher on–off ra- Notably, among the eight red PAFPs tested, mMaple has the highest tios. Dark-to-fluorescent PAFPs are also more favorable than signaling efficiency, which is about one order of magnitude higher color-changing PAFPs as the former take a smaller spectral space. than the other PAFPs. The downside of mMaple is, however, its The number of switching cycles is another important parameter to relatively high dimerization tendency. consider. For quantifying the stoichiometry of the target proteins, To overcome this problem, we developed two new PAFPs, it is desirable to have PAFPs that can be switched on only once, mMaple2 and mMaple3, which largely maintained the superb although almost all PAFPs tend to blink more than once (31). signaling efficiency of mMaple but exhibited substantially reduced dimerization tendency. In particular, mMaple3 exhibited However, for mapping of protein spatial organization, a larger no detectable dimerization tendency. The photon numbers and number of switching cycles per molecule would allow the target on–off ratios of both are similar to those of mMaple, which are structure to be sampled more times, which is advantageous when it among the best for PAFPs. Based on the above results, we rec- comes to time-lapsed imaging for probing dynamics. For a differ- ommend researchers to label target proteins of interest with ent superresolution imaging method, RESOLFT, reversibly mMaple3 when performing single-molecule–based superresolution switching fluorescent proteins with a large number of switching imaging using fluorescent proteins. mMaple3 provides excellent cycles (such as rsEGFP) is actually required (25). performance in all four key properties described here, including high signaling efficiency, low dimerization tendency, high photon Methods budget, and low on–off ratio. In the cases that the localization Plasmids were constructed with PCR and isothermal assembly (34). E. coli number provided by mMaple3 is suboptimal, one should also chromosomal insertions were created by lambda RED recombination (35). A consider labeling with mMaple2, which will give substantially list of the plasmids and strains is provided in Table S4. E. coli cells were higher localization numbers, but it is important to check whether grown to exponential phase in M9 minimal media before imaging. Mam- fusion with mMaple2 has led to undesired aggregation effect. The malian cell lines were transfected with purified plasmids using nucleofection large number of localizations provided by mMaple2 and mMaple3 or lipofection, and incubated for 24–26 h before imaging. Phase-contrast, will not only facilitate mapping out the fine spatial distribution of superresolution, or conventional fluorescence images were collected on an the target protein, but will also allow the localizations to be divided Olympus IX-71 inverted microscope with 405-, 488-, and 561-nm laser lines. into more snapshots to facilitate time-lapsed imaging of the dy- Images were analyzed with custom-written software. See SI Text for details. namics of cellular structures. When localization number is less of a concern, mEos3.2 and PAtagRFP are excellent choices for their ACKNOWLEDGMENTS. We thank Michael Davidson, Stefan Hell, Stefan high photon budget and undetectable dimerization tendency. Jakobs, Alice Ting, Antoine van Oijen, Ethan Garner, Sara Jones, and Chongyi The four properties measured in this work are key proper- Chen for providing plasmids and strains, and David Liu for helpful discussions on the design of PAFPs. S.W. is supported by a Jane Coffin Childs Fellowship. J.R.M. ties of PAFPs to consider when imaging the spatial organization is supported in part by a Helen Hay Whitney Fellowship. This work is supported of a target protein with single-molecule–based superresolution by National Institutes of Health Grants GM096450 and GM068518 (to X.Z.) and imaging. However, they are not the only important properties to GM096450 (to X.S.X.). X.Z. is a Howard Hughes Medical Institute Investigator.

1. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical 19. Subach FV, Patterson GH, Renz M, Lippincott-Schwartz J, Verkhusha VV (2010) Bright reconstruction microscopy (STORM). Nat Methods 3(10):793–795. monomeric photoactivatable red fluorescent protein for two-color super-resolution 2. Betzig E, et al. (2006) Imaging intracellular fluorescent proteins at nanometer reso- sptPALM of live cells. J Am Chem Soc 132(18):6481–6491. lution. Science 313(5793):1642–1645. 20. McEvoy AL, et al. (2012) mMaple: A photoconvertible fluorescent protein for use in 3. Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluores- multiple imaging modalities. PLoS One 7(12):e51314. cence photoactivation localization microscopy. Biophys J 91(11):4258–4272. 21. Chudakov DM, et al. (2004) Photoswitchable cyan fluorescent protein for protein 4. Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for tracking. Nat Biotechnol 22(11):1435–1439. diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11):555–565. 22. Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling 5. Nienhaus K, Ulrich Nienhaus G (2014) Fluorescent proteins for live-cell imaging with observed by reversible protein highlighting. Science 306(5700):1370–1373. – super-resolution. Chem Soc Rev 43(4):1088 1106. 23. Chang H, et al. (2012) A unique series of reversibly switchable fluorescent proteins with 6. Jones SA, Shim SH, He J, Zhuang X (2011) Fast, three-dimensional super-resolution beneficial properties for various applications. Proc Natl Acad Sci USA 109(12):4455–4460. – imaging of live cells. Nat Methods 8(6):499 508. 24. Stiel AC, et al. (2007) 1.8 A bright-state structure of the reversibly switchable fluorescent 7. Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluo- protein Dronpa guides the generation of fast switching variants. Biochem J 402(1):35–42. rophores for optimal performance in localization-based super-resolution imaging. 25. Grotjohann T, et al. (2011) Diffraction-unlimited all-optical imaging and writing with Nat Methods 8(12):1027–1036. a photochromic GFP. Nature 478(7368):204–208. 8. Lukyanov KA, Chudakov DM, Lukyanov S, Verkhusha VV (2005) Innovation: Photo- 26. Landgraf D, Okumus B, Chien P, Baker TA, Paulsson J (2012) Segregation of molecules activatable fluorescent proteins. Nat Rev Mol Cell Biol 6(11):885–891. at cell division reveals native protein localization. Nat Methods 9(5):480–482. 9. Thompson RE, Larson DR, Webb WW (2002) Precise nanometer localization analysis 27. Wang WQ, Li GW, Chen CY, Xie XS, Zhuang X (2011) Chromosome organization by for individual fluorescent probes. Biophys J 82(5):2775–2783. a nucleoid-associated protein in live bacteria. Science 333(6048):1445–1449. 10. Shroff H, Galbraith CG, Galbraith JA, Betzig E (2008) Live-cell photoactivated locali- 28. Smyth CP, et al. (2000) Oligomerization of the chromatin-structuring protein H-NS. zation microscopy of nanoscale adhesion dynamics. Nat Methods 5(5):417–423. Mol Microbiol 36(4):962–972. 11. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective 29. Ueguchi C, Seto C, Suzuki T, Mizuno T (1997) Clarification of the dimerization domain photolabeling of proteins and cells. Science 297(5588):1873–1877. and its functional significance for the Escherichia coli nucleoid protein H-NS. J Mol 12. Gurskaya NG, et al. (2006) Engineering of a monomeric green-to-red photo- – activatable fluorescent protein induced by blue light. Nat Biotechnol 24(4):461–465. Biol 274(2):145 151. 13. Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular protein move- 30. Greenfield D, et al. (2009) Self-organization of the Escherichia coli chemotaxis net- ments using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat Protoc work imaged with super-resolution light microscopy. PLoS Biol 7(6):e1000137.

2(8):2024–2032. 31. Durisic N, Laparra-Cuervo L, Sandoval-Álvarez A, Borbely JS, Lakadamyali M (2014) BIOPHYSICS AND 14. McKinney SA, Murphy CS, Hazelwood KL, Davidson MW, Looger LL (2009) A bright Single-molecule evaluation of fluorescent protein photoactivation efficiency using an COMPUTATIONAL BIOLOGY and photostable photoconvertible fluorescent protein. Nat Methods 6(2):131–133. in vivo nanotemplate. Nat Methods 11(2):156–162. 15. Zhang M, et al. (2012) Rational design of true monomeric and bright photo- 32. Vaughan JC, Jia S, Zhuang X (2012) Ultrabright photoactivatable fluorophores cre- activatable fluorescent proteins. Nat Methods 9(7):727–729. ated by reductive caging. Nat Methods 9(12):1181–1184. 16. Wiedenmann J, et al. (2004) EosFP, a fluorescent marker protein with UV-inducible 33. Fernández-Suárez M, Ting AY (2008) Fluorescent probes for super-resolution imaging green-to-red fluorescence conversion. Proc Natl Acad Sci USA 101(45):15905–15910. in living cells. Nat Rev Mol Cell Biol 9(12):929–943. 17. Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008) mKikGR, 34. Gibson DG, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred a monomeric photoswitchable fluorescent protein. PLoS One 3(12):e3944. kilobases. Nat Methods 6(5):343–345. 18. Subach FV, et al. (2009) Photoactivatable mCherry for high-resolution two-color 35. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Es- fluorescence microscopy. Nat Methods 6(2):153–159. cherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640–6645.

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