Decision letter: Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Methods Appendix 1 Appendix 2 Data availability References Decision letter Author response Article and author information Metrics Abstract Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the ‘sender’) can reactivate a second fluorophore (the ‘receiver’) from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein–protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells. Editor's evaluation This work develops a new method to probe protein–protein interactions using proximity-assisted photo activation, in which a receiver fluorophore (longer wavelength) can be photoactivated by the excitation of a nearby sender fluorophore (shorter wavelength). This new method is validated through in-depth characterization, comparison with FRET, and application to known systems of protein–protein interactions. It will expand the tool kit for probing protein–protein interactions. https://doi.org/10.7554/eLife.76870.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest A human body is made up of trillions of cells, each containing millions of proteins working to keep our bodies going. Since the invention of the microscope four hundred years ago, scientists have made large strides in visualizing cells and even single protein molecules within cells. To do this, proteins of interest are labeled with fluorescent dyes that absorb – or are ‘excited’ by – light of one color, and then give off light of a different color. The labeled proteins are excited by a powerful laser, and a sensitive camera detects the light emitted by single molecules of dye. This technique is called single-particle tracking (SPT), and it can reveal how proteins move around inside a cell. Because most proteins work together in teams or complexes, it would be useful to track the movement of proteins while at the same time observing their interactions. Unfortunately, SPT does not typically allow scientists to watch how proteins interact with each other. Graham et al. accidentally discovered how to do precisely this. First, they labeled proteins with two different colored dyes. Then, the dyes were excited using alternating red and green lasers. Repeated excitation destroys the fluorescent dye molecules, and sure enough, red-excited dye molecules went dark over time. Unexpectedly, however, molecules of the dye that had been excited with red light reappeared after exciting the second dye with green light. The fluorescent molecules were not dead, just sleeping. ‘Resuscitating’ one dye with the other required that they be close together, and therefore this process was called proximity-assisted photoactivation (PAPA for short). PAPA was able to detect interactions between proteins labeled with different dyes in live human cells, and combining PAPA with SPT allowed Graham et al. to distinguish protein molecules labeled with two different dyes from those labeled with a single dye. Finally, Graham et al. labeled molecules of the androgen receptor protein with two different dyes to monitor how they responded to testosterone. Combining PAPA and SPT measurements successfully detected the pairing of androgen receptor molecules, as well as increased binding of these paired androgen receptor molecules to DNA. This new way of observing how proteins interact will be useful for studying where and how fast these interactions happen in living cells. Understanding how teams of proteins work together under normal conditions will also shed light on how they misbehave in diseases. Introduction Most proteins function by interacting with other proteins, yet we lack tools to study these potentially transient interactions at single-molecule resolution in live cells. Single-particle tracking (SPT) is a valuable approach for monitoring the motions of individual protein molecules (Chen et al., 2021; Hansen et al., 2018; Heckert et al., 2022; Nguyen et al., 2021), but it does not distinguish compositionally and functionally distinct complexes of the same protein. Two-color SPT can infer interactions between proteins when both partners are so dilute that they can be fully labeled while still resolving single molecules (Asher et al., 2021; Sotolongo Bellón et al., 2022; Wilmes et al., 2020). For most proteins, however, detection of single molecules requires sparse labeling, which makes double-labeled complexes exceedingly rare. Single-molecule Förster resonance energy transfer (smFRET), though powerful for monitoring intra-molecular conformational changes, is not in general a practical way to detect protein–protein interactions in live cells due to a similar requirement for sparse double-labeling, challenges with spectral crosstalk, and the large size of genetically encoded tags relative to the working distance of FRET (see Appendix 1; Quast and Margeat, 2021). Fluorescence cross-correlation spectroscopy (FCCS) can detect bulk molecular interactions, yet it does not provide spatial trajectories for individual molecules, which are useful for measuring such properties as chromatin residence time and anomalous diffusion (Hansen et al., 2020; Hansen et al., 2017; Izeddin et al., 2014; McSwiggen et al., 2019; Nguyen et al., 2021). Bimolecular fluorescence complementation (BiFC) detects molecular interactions based on the reconstitution of a fluorescent protein or HaloTag from two split halves fused to interacting partners (Ghosh et al., 2000; Hu et al., 2002; Kerppola, 2008; Makhija et al., 2021; Shao et al., 2021). While BiFC can be combined with single-molecule imaging (Mao et al., 2021; Nickerson et al., 2014; Shao et al., 2021), a drawback of this approach is that the extremely strong association of split proteins perturbs the binding equilibrium of their interacting partners (Kerppola, 2008; Kodama and Hu, 2012; Nickerson et al., 2014), making it impossible to accurately measure dynamic interactions. An alternative in vitro proximity sensor to smFRET was devised by Bates, Blosser, and Zhuang, who observed that exciting one cyanine dye can reactivate a nearby cyanine dye from a dark state (Bates et al., 2005). Although photoswitching of cyanine dye pairs enabled early implementations of STORM imaging (Rust et al., 2006), its application as a proximity sensor has been limited by the short inter-fluorophore distance required (≤2 nm), the poor cell permeability of cyanine dyes, and the need for high thiol concentrations and an oxygen-scavenging system (Chen et al., 2016; Geertsema et al., 2015). To our knowledge, it has not been reported whether a similar process of reactivation can occur for pairs of non-cyanine dyes. However, many fluorophores—notably rhodamine dyes—can enter a dark state and be directly reactivated by short-wavelength (e.g., 405 nm) light (van de Linde et al., 2011). This phenomenon, which has been employed for direct STORM (dSTORM) imaging in both live and fixed cells (Grimm et al., 2015; Heilemann et al., 2008; Tang et al., 2021), is thought to involve conversion of excited triplet-state fluorophores to reduced species whose absorbance is shifted to shorter wavelengths (Bates et al., 2005; Dempsey et al., 2009; Gidi et al., 2020; Heilemann et al., 2008; van de Linde et al., 2011; Vaughan et al., 2012). The development of bright, cell-permeable Janelia Fluor (JF) dyes, based on rhodamine and silicon-rhodamine chemical scaffolds, has transformed single-molecule imaging in live cells (Grimm et al., 2015). Here, we show that Janelia Fluor X 650 (JFX650; Grimm et al., 2021) and similar fluorophores can be reactivated from a dark state by excitation of a nearby fluorophore such as Janelia Fluor 549 (JF549), a phenomenon which we term proximity-assisted photoactivation (PAPA). In contrast to cyanine dye reactivation, PAPA of JF dyes occurs under physiological conditions in live cells and requires neither an oxygen-scavenging system nor exogenous thiols. While PAPA requires proximity between the two fluorophores, its effective distance range extends beyond that of FRET, making it a potentially more versatile interaction sensor. Most importantly, PAPA provides a new way to detect protein interactions in live cells at single-molecule resolution. We show that PAPA can be used to detect the formation of protein dimers and that it can enrich for double-labeled molecules within defined mixtures, albeit not with perfect selectivity (see ‘Discussion’ and Appendix 2). As a further proof of concept, we combined SPT with PAPA to analyze the increase in chromatin binding induced by self-association of androgen receptor. By enabling the previously elusive detection of protein–protein interactions, PAPA will provide a new dimension of information in live-cell single-molecule imaging. Results PAPA of JF dyes We fortuitously discovered PAPA while imaging an oligomeric protein labeled with two different JF dyes. U2OS cells expressing Halo-tagged NPM1 (a pentameric nucleolar protein; Heckert et al., 2022) were labeled with a low concentration of Janelia Fluor X 650 HaloTag ligand (JFX650-HTL; Grimm et al., 2021) to track single molecules, together with a higher concentration of Janelia Fluor 549 HaloTag ligand (JF549-HTL) to visualize nucleoli. When we alternately excited JFX650 with red light (633 nm) and JF549 with green light (561 nm), we noticed that some JFX650 molecules that had gone dark during red illumination suddenly reappeared after a brief, 7 ms pulse of green light (green vertical lines in Figure 1ai and green box in Figure 1b, Figure 1—video 1). Consistent with previous work (Grimm et al., 2015), we also observed reactivation of JFX650 by violet light, both with and without JF549-HTL (violet vertical lines in Figure 1ai,ii and violet box in Figure 1b). However, reactivation of JFX650 by green light required co-labeling with JF549 (compare Figure 1ai and ii), implying that reactivation results not from direct absorption of green light by dark-state JFX650 but indirectly due to excitation of JF549. Green illumination of cells labeled with JF549-HTL alone did not produce localizations in the JFX650 channel, demonstrating that this effect is not due to JF549 photochromism (Figure 1aiii). Figure 1 with 4 supplements see all Download asset Open asset Proximity-assisted photoactivation (PAPA) of JFX650 by JF549. (a) Green and violet light reactivate JFX650 through distinct JF549-dependent and JF549-independent mechanisms. Left column: schematic of NPM1 pentamers in heterozygously tagged NPM1-Halo U2OS cells labeled with JF549 (orange) and/or JFX650 (red). Right column: average number of localizations in the JFX650 channel as a function of frame number. JFX650 molecules were excited with red (633 nm) light, interspersed with 7 ms pulses of violet (405 nm) and green (561 nm) light (violet and green vertical lines). Reactivation of JFX650 by green light required labeling with JF549 (compare black arrows in i and ii). (b) Sample images of a single cell in the JFX650 channel. Leftmost panel: first movie frame prior to fluorophore bleaching/shelving. Green and violet boxes: maximum-intensity projection of all frames immediately before and after green and violet stimulation pulses, showing reactivation of molecules from the dark state. Image dimensions are 24 µm x 24 µm. (c) Average fluorescence intensity in the JFX650 channel as a function of frame number in cells expressing a Halo-SNAPf fusion with a flexible linker (top panel; N = 40 cells) or a tandem P2A-T2A self-cleaving peptide between Halo and SNAPf (PT2A; bottom panel; N = 20 cells). Halo was labeled with JF549-HTL and SNAPf with JFX650-STL. Reactivation by violet light pulses (violet lines) occurred in both cases, but reactivation by green light pulses (green lines) was mostly eliminated by the self-cleaving peptide (compare black arrows). Raw intensity traces are displayed without background subtraction. Because double-labeling of NPM1-Halo pentamers is expected to bring JF549 and JFX650 close together (Figure 1ai, right panel), we asked whether proximity of the dyes is required for reactivation. To test this, we expressed fusions of Halo and SNAPf separated by either a short flexible linker (Halo-SNAPf) or a tandem P2A-T2A self-cleaving peptide (Halo-PT2A-SNAPf; Liu et al., 2017) in U2OS cells (Figure 1c); labeled cells with JF549-HTL and JFX650 SNAP tag ligand (JFX650-STL); and imaged JFX650 with red light interspersed with alternating short pulses of violet and green light. While violet reactivation was similar for both constructs, green reactivation was substantially greater for Halo-SNAPf than for Halo-PT2A-SNAPf, implying that proximity of the two dyes facilitates reactivation by green light (Figure 1c). Thus, we term this phenomenon proximity-assisted photoactivation (PAPA). We will call the dye that undergoes reactivation the ‘receiver’ and the dye whose excitation induces reactivation the ‘sender.’ Also, we will adopt the terms ‘shelving’ for conversion of the receiver into the dark state (Bretschneider et al., 2007; Grimm et al., 2015) and ‘direct reactivation’ (DR) for reactivation by violet light (Dempsey et al., 2009). Conjugating JFX650 to SNAPf instead of Halo led to more efficient shelving in the dark state, as evidenced by a faster decline in fluorescence during red illumination and greater subsequent reactivation by violet light (Figure 1—figure supplement 1a). This accords with the previous observation that JF549 is more photostable when bound to Halo than when bound to SNAP (Presman et al., 2017). Ensemble and single-molecule kinetic measurements indicate that about 10% of JFX650-SNAPf molecules enter the dark state under our experimental conditions and can be reactivated by either DR or PAPA (Figure 1—figure supplements 1b and 3). DR by violet light precluded subsequent PAPA by green light, and vice versa, implying that both wavelengths reactivate the same dark state (Figure 1—figure supplement 1c and d). We tested other fluorophore pairs and found that PAPA occurred when tetramethylrhodamine (TMR), Janelia Fluor X 549 (JFX549), or Janelia Fluor 526 were used as the sender, or when JF646 or JFX646 were used as the receiver (Figure 1—figure supplement 2). Distance dependence of PAPA To investigate how PAPA depends on sender–receiver distance, we generated fusion transgenes in which Halo and SNAPf were separated by zero, one, three, five, or seven repeats of the titin I91 Ig domain (Scholl et al., 2016). The distance distribution between the two dyes was estimated for each fusion protein by simulating an ensemble of conformations using PyRosetta (Figure 2—figure supplement 1a, b; Chaudhury et al., 2010; Ferrie and Petersson, 2020). U2OS cells were stably transfected with each transgene, and fluorescence-activated cell sorting (FACS) was used to obtain pools of cells with similar low expression levels of each protein (Figure 2—figure supplement 1c and d; see Appendix 3, Supplementary note 1). Cells were labeled with a mixture of JF549-HTL and JFX650-STL and imaged as described above with red light interspersed with alternating pulses of violet light to induce DR and green light to induce PAPA. The ratio of the increase in fluorescence intensity in response to green and violet pulses (the ‘PAPA/DR ratio’) provides a normalized measure of PAPA efficiency, which corrects for cell-to-cell variability in the labeled protein concentration. For sufficiently short reactivation pulses, the PAPA/DR ratio increased linearly with the green pulse duration (with the violet pulse duration held constant), making it possible to measure relative rate constants by linear fitting (Figure 2a, b, left panel). In parallel, fluorescence lifetime imaging (FLIM) was used to measure FRET between JF549 and JFX650 for the same fusion proteins (Figure 2b, right panel; see Appendix 3, Supplementary note 2). Figure 2 with 1 supplement see all Download asset Open asset Comparison of distance dependence of proximity-assisted photoactivation (PAPA) and Förster resonance energy transfer (FRET). (a) PAPA/direct reactivation (DR) ratio vs. green pulse duration for Halo-SNAPf fusions with a short, flexible linker or linkers containing different numbers of tandem Ig domains. Curves are linear fits (y = ax). Error bars, ±2 * SE. PT2A, tandem P2A-T2A self-cleaving peptide. (b) Left panel: relative rates of reactivation by PAPA (slope of fits in a divided by the slope of the short linker construct). Right panel: FRET efficiency measured using fluorescence lifetime imaging (FLIM). As predicted by our simulations (Figure 2—figure supplement 1b), FRET efficiency between JF549 and JFX650 declined sharply with increasing spacer length, from 0.271 ± 0.010 (95% CI) for the short linker to 0.124 ± 0.006 for a single Ig repeat and 0.020 ± 0.010 for three Ig repeats (Figure 2b, right panel). FRET was essentially undetectable for five or seven Ig repeats and for the PT2A self-cleaving peptide linker (Figure 2b, right panel). In contrast, PAPA was observed for the 3×, 5×, and 7× Ig linker constructs (Figure 2a and b). The rate of photoactivation by green light declined gradually with increasing linker length yet was distinguishable from the background rate of the PT2A self-cleaving linker. These results indicate that PAPA has a less stringent dependence on average inter-fluorophore distance than FRET. Detection of inducible protein–protein interactions using PAPA Based on the above results, we reasoned that PAPA could be used to detect interaction of two different proteins labeled with SNAPf-JFX650 and Halo-JF549. As a test case, we monitored the rapamycin-inducible interaction of the proteins FRB and FKBP. U2OS cells expressing Halo-FRB and SNAPf-FKBP were labeled with JF549-HTL and JFX650-STL and imaged with alternating green and violet photostimulation as described above (Figure 3a, Figure 3—figure supplement 1a). Addition of rapamycin caused a dramatic increase in the ratio of PAPA (green reactivation) to DR (violet reactivation), consistent with ligand-induced dimerization of Halo-FRB and SNAPf-FKBP bringing together JF549 and JFX650 (Figure 3b and c, Figure 3—figure supplement 1b, Figure 3—video 1). Figure 3 with 2 supplements see all Download asset Open asset Detection of inducible dimerization using proximity-assisted photoactivation (PAPA). (a) Halo-FRB was labeled with the sender fluorophore (JF549) and SNAPf-FKBP with the receiver fluorophore (JFX650). After shelving JFX650 with red light, direct reactivation (DR) and PAPA were alternately induced with pulses of violet and green light, respectively. Midway through the experiment, cells were treated with rapamycin (1 µM final concentration) to induce FRB-FKBP dimerization or with dimethylsulfoxide (DMSO) solvent as a negative control. (b) Ratio of fluorescence increase due to PAPA (green reactivation) and DR (violet reactivation) as a function of time after rapamycin addition. Blue, rapamycin. Brown, DMSO solvent-only control. Individual data points represent single cells; solid lines show a 2-min moving average. (c) Average PAPA/DR ratio before (-) and after (+) addition of rapamycin (Rapa) or DMSO. Total number of cells: 75 before and 74 after rapamycin, 30 before and 30 after DMSO. Error bars, ± 2 * SEM. Statistical significance was calculated using a two-tailed t-test. PAPA optically enriches a subset of molecules in defined two-component mixtures We next asked whether PAPA can be used to spotlight a subpopulation of receiver molecules close to sender molecules. As a simple test case, we analyzed defined mixtures of two proteins—one labeled with JFX650 only, and a second labeled with both JFX650 and JF549—and investigated whether PAPA could optically enrich the double-labeled component to distinguish its properties in single-molecule imaging. First, we co-expressed SNAPf-tagged histone H2B (SNAPf-H2B), which is predominantly chromatin-bound, with a Halo-SNAPf fusion with a which is mostly (Figure Hansen et al., 2018; Heckert et al., Cells were with JFX650-STL and JF549-HTL to with both JFX650 and JF549 and with JFX650 alone (Figure Figure supplement 1a). JFX650 fluorophores were using a pulse of red light, after which JFX650 was imaged with red light interspersed with pulses of green and violet light. After and tracking single molecules, we separated trajectories after a green pulse (PAPA from those after a violet pulse and a state SPT et al., 2022) to infer the distribution of diffusion for each of trajectories for short). As diffusion two one to bound molecules = the in the state and one to molecules = PAPA trajectories were for molecules to DR as expected PAPA double-labeled molecules (Figure PAPA and DR trajectories from individual cells were using a with bound = and = Consistent with the ensemble PAPA trajectories had a bound than DR trajectories in cell (Figure The same is from comparison of and trajectories (Figure supplement 2a, b). Figure 4 with supplements see all Download asset Open asset of defined two-component mixtures using proximity-assisted photoactivation (PAPA). Left schematic of different defined mixtures of two labeled proteins, in which one protein is labeled with JFX650 and the other is labeled with both JFX650 and JF549. In and each of the is fused to SNAPf or though one is displayed for (b) diffusion of PAPA and direct reactivation (DR) trajectories from 20 cells or cells (c) Right column: bound or of PAPA and DR trajectories from individual cells, from fits to a or two-tailed of the in and all to be with 1 and 4 respectively. To the that of molecules from a in our we the with the mixture of and (Figure As the was PAPA trajectories were in bound molecules to DR both an ensemble of cells and at the single-cell (Figure and Figure supplement and d). As a further we analyzed cells expressing double-labeled or As PAPA and DR trajectories displayed diffusion for these individual (Figure supplement To test whether PAPA can also distinguish a mixture of we co-expressed Halo-SNAPf with a SNAPf-tagged protein that et al., 2016; Figure As diffusion had two to and (Halo-SNAPf) (Figure to DR trajectories (violet PAPA trajectories (green were in the consistent with reactivation of the double-labeled Halo-SNAPf protein by green light (Figure The same was observed in single-cell and trajectories (Figure Figure supplement and The of the was not as a was still observed PAPA trajectories (Figure green see As SNAPf and Halo-SNAPf labels the both at the ensemble and single-cell (Figure Figure supplement and Figure 1). of the by PAPA is in this (compare green and violet in Figure which reactivation of a receiver by of sender molecules within a To the of double-labeled molecules by the in Figure 4 were to a linear of the for individual For the was for DR trajectories with a mixture of and while the for PAPA trajectories was with and (Figure supplement The estimated ratio of to from DR trajectories to PAPA an of double-labeled molecules by PAPA. that PAPA over by (Figure supplement Halo-SNAPf over by (Figure supplement and over SNAPf by (Figure supplement together, these results that PAPA can be used to enrich a subpopulation of molecules in which a receiver fluorophore (e.g., is in proximity to a sender fluorophore (e.g., the distinct properties of this subpopulation at both the ensemble and single-cell level. While this was and for different defined was not and it is not to PAPA trajectories as a of interacting molecules (see ‘Discussion’ and Appendix 2). the properties of androgen receptor and dimers in single cells As a proof-of-concept we tested whether PAPA could be used to detect ligand-induced self-association of androgen receptor and distinguish the properties of and First, we stably co-expressed SNAPf and Halo fusions of in U2OS cells et al., labeled the two proteins with a mixture of JFX650-STL and JF549-HTL (Figure supplement and measured the PAPA/DR ratio by in JFX650 fluorescence intensity in response to alternating green and violet stimulation as described As with the androgen led to an increase in the ratio of PAPA to DR over the of (Figure Figure supplement 1b), consistent with the two fluorophores together by ligand-induced interaction between and Figure with 1 supplement see all Download asset Open asset of androgen receptor using proximity-assisted tracking (a) of dimerization of and (b) PAPA/direct reactivation (DR) ratio as a function of time relative addition. (c) of PAPA and DR (c) addition of N = cells. After addition of to a final concentration of N = cells. bound was by the of each = we combined PAPA with single-molecule imaging to how self-association diffusion and chromatin binding by Consistent with previous biochemical and live-cell imaging experiments, addition of caused an increase in the bound of (Figure and d; et al., 2005; van et al., 2007; van et al., 2012). PAPA trajectories had a higher bound than DR both before and after addition of (Figure d). This is consistent with an increase in the of for PAPA that a subset of molecules and bound chromatin with even prior to addition of exogenous
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