Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
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Evaluation Summary:
This work develops a new method to probe protein-protein interactions using proximity-assisted photoactivation, 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. While the new method bears the potential to expand the tool kit for probing protein-protein interactions, further characterizations of its photoactivation properties and comparisons with existing methods would be needed to inform researchers interested to apply this method to their own systems.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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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.
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Author Response
Reviewer 3
This is work by an internationally recognized group with strong expertise in sophisticated single-molecule microscopy assays in cells. They present here a single-molecule fluorescence-based assay for proximity in the nanometer range.
It has long been reported that cyanine dyes such as Cy3, Cy5 and derivatives such as AF555, AF647 can undergo a photoswitching mechanism by which the shorter wavelength dye when being excited can switch the longer wavelength dye which is in a dark state back into the bright state. And it has furthermore been reported that this switching mechanism is not based on FRET, as the distance requirement is more stringent (up to ~ 2 nm). However, this mechanism has not been fully explored for the investigation of molecular interactions yet.
The authors in the present work present a …
Author Response
Reviewer 3
This is work by an internationally recognized group with strong expertise in sophisticated single-molecule microscopy assays in cells. They present here a single-molecule fluorescence-based assay for proximity in the nanometer range.
It has long been reported that cyanine dyes such as Cy3, Cy5 and derivatives such as AF555, AF647 can undergo a photoswitching mechanism by which the shorter wavelength dye when being excited can switch the longer wavelength dye which is in a dark state back into the bright state. And it has furthermore been reported that this switching mechanism is not based on FRET, as the distance requirement is more stringent (up to ~ 2 nm). However, this mechanism has not been fully explored for the investigation of molecular interactions yet.
The authors in the present work present a similar mechanism for a different class of rhodamine-based fluorophores, specifically JF549 and JFX650. They describe the discovery of this mechanism in dual-color labeling of a pentameric protein and initial characterization to distinguish it from UV-light-mediated recovery from a pumped dark state as reported for (d)STORM-like measurements. They extend their observation to TMR, JF529 as lower wavelength "senders" and JF646 and JFX646 as longer wavelength "receivers" that can become reactivated into the ground state upon illumination of a nearby "sender". The authors then test activation pulse length and distance dependence and find that longer pulses lead to more recovery and that PAPA of JF549/JFX650 has unlike previously observed for the Cy3/Cy5 pair a smaller distance dependence than FRET of the same fluorophore pair. The authors then move on to use both the UV-light mediated direct reactivation "DR" and proximityassisted photoactivation "PAPA" to activate different molecules that are either double-labeled for PAPA or singly labeled with JFX650 for DR. They succeeded in four different cases to identify clear population shifts to distinguish molecules of different mobility.
Overall, I think the authors made an interesting discovery and characterizing this previously poorly characterised interaction for cellular single-molecule experiments is certainly an important effort. The authors make an honest and quite complete effort to work out the practical details of this interaction and designed experiments that convincingly highlight the basic capabilities this technique offers to the detection of verified interactions and the mobility of interacting molecules in cells.
The weakness is that these capabilities do not seem to be as clear-cut as the reviewer hoped for when starting to read this manuscript. It remains unclear to this reviewer, to what extant PAPA molecules can be separated from DR molecules. In all but the last diffusion experiment(s) in Figure 4, PAPA molecules seem to be significantly perturbed by DR molecules, casting doubt on the usefulness in real experiments. Similarly, in Figure 5, a difference is seen but does not allow for quantification. This certainly is not the case for other methods of sensing as well, but maybe the authors could more specifically compare their efforts and the dynamic range to other sensors for example in Figure 5? This would make it easier for the reader to make up their mind if the assay is worthwhile adopting for their system.
We agree that a problem with PAPA at present is that although PAPA trajectories are significantly enriched for double-labeled complexes, they are still “contaminated” with singlelabeled molecules. As we described in the Discussion (and as pointed out by Reviewer 1), we think that one major contribution to this background arises from chance proximity of sender and receiver molecules independent of direct physical interaction. Additionally, some background is expected from continual spontaneous (a.k.a. “thermal”) reactivation of molecules from the dark state.
In response to the reviewers’ comments, we have tried to quantify more precisely how much PAPA enriches for one population over another by fitting the diffusion spectra of 2-component mixtures to linear combinations of the corresponding individual components (Figure 4–figure supplement 4). We estimate that the fold enrichment of double-labeled molecules ranged from 3.7 to 37-fold between different 2-component mixtures.
We fully agree that it is critical that researchers who use PAPA be aware of its limitations, so that they do not fallaciously assume that all green-reactivated localizations are protein complexes. To avoid committing a bait-and-switch against our readers, we now state explicitly in the Introduction that PAPA in its current form enriches for complexes but does not provide perfect selectivity. In Appendix 2, we now discuss the problem of background reactivation in more detail and outline what we think will be required to correct quantitatively for this background. Though we believe that such corrections will ultimately be possible, at least in some cases, figuring out how to do this rigorously will require substantial additional development of experimental and computational methods, which we hope the editor and reviewers agree is beyond the scope of the current paper.
At the end of Appendix 2, we briefly mention another technical problem that we have noticed with SNAP ligand background staining. While this background was negligible for the experiments described in this paper, which involved highly expressed SNAPf transgenes, it may pose a more significant problem for SNAPf-tagged proteins with lower expression levels. We think it is worth mentioning this problem to make readers aware of it and hopefully to motivate the development of better orthogonal pairs of self-labeling tags.
While there are obviously limitations to PAPA, we think this should not overshadow the fact we have identified a novel photophysical property of commonly used fluorophores and harnessed it to detect molecular interactions in live cells. Our initial proof-of-concept study provides a foot in the door of this new biophysical approach, which we and others will continue to refine. Immediate applications of PAPA could include disambiguation of peak assignments in complex diffusion spectra, confirmation of proposed interactions between proteins (and subsequent investigations into the molecular mechanisms supporting such interactions), or integration into SPT-based high-throughput screening (https://www.eikontx.com/technology) to provide a useful additional readout for each experimental condition.
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Evaluation Summary:
This work develops a new method to probe protein-protein interactions using proximity-assisted photoactivation, 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. While the new method bears the potential to expand the tool kit for probing protein-protein interactions, further characterizations of its photoactivation properties and comparisons with existing methods would be needed to inform researchers interested to apply this method to their own systems.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive …
Evaluation Summary:
This work develops a new method to probe protein-protein interactions using proximity-assisted photoactivation, 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. While the new method bears the potential to expand the tool kit for probing protein-protein interactions, further characterizations of its photoactivation properties and comparisons with existing methods would be needed to inform researchers interested to apply this method to their own systems.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Monitoring protein-protein interactions dynamically at the single-molecule level in live cells is of great importance for understanding various molecular mechanisms. Several approaches, including two-color single-particle tracking (SPT), smFRET, BiFC, and fluorescence cross-correlation spectroscopy (FCCS) have been developed for measuring protein-protein interactions in the living cell. In this work, the authors took a clever use of photoactivation properties of rhodamine dyes and developed a new approach to spotlight protein-protein interactions in live cells. The detected signal is based on proximity-assisted photoactivation (PAPA), in which excitation of a "sender" fluorophore (e.g., JF549) reactivates a nearby "receiver" fluorophore (e.g., JFX650) from a dark state. The work is very well done in terms of …
Reviewer #1 (Public Review):
Monitoring protein-protein interactions dynamically at the single-molecule level in live cells is of great importance for understanding various molecular mechanisms. Several approaches, including two-color single-particle tracking (SPT), smFRET, BiFC, and fluorescence cross-correlation spectroscopy (FCCS) have been developed for measuring protein-protein interactions in the living cell. In this work, the authors took a clever use of photoactivation properties of rhodamine dyes and developed a new approach to spotlight protein-protein interactions in live cells. The detected signal is based on proximity-assisted photoactivation (PAPA), in which excitation of a "sender" fluorophore (e.g., JF549) reactivates a nearby "receiver" fluorophore (e.g., JFX650) from a dark state. The work is very well done in terms of inspiration by accidental observations, experimental design and data analysis for method validation, as well as proof of concept applications.
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Reviewer #2 (Public Review):
Assessing protein-protein interactions in live cells and in particular measuring dynamic properties of complexed proteins over a background of isolated proteins is challenging. Graham et al. demonstrate that a rhodamine dye in a dark state can be photoactivated by exciting another, low-wavelength fluorophore if this is nearby. The authors characterize this photoactivation process, which they call PAPA, in terms of distance dependence and find the process occurs over larger distances than FRET. Moreover, they demonstrate that PAPA enables detecting inducible dimerization and measuring distinct diffusion dynamics of slow and fast diffusing molecular species in single-molecule experiments. While reporting several highly useful applications of PAPA, the authors only provide limited insight into the physical …
Reviewer #2 (Public Review):
Assessing protein-protein interactions in live cells and in particular measuring dynamic properties of complexed proteins over a background of isolated proteins is challenging. Graham et al. demonstrate that a rhodamine dye in a dark state can be photoactivated by exciting another, low-wavelength fluorophore if this is nearby. The authors characterize this photoactivation process, which they call PAPA, in terms of distance dependence and find the process occurs over larger distances than FRET. Moreover, they demonstrate that PAPA enables detecting inducible dimerization and measuring distinct diffusion dynamics of slow and fast diffusing molecular species in single-molecule experiments. While reporting several highly useful applications of PAPA, the authors only provide limited insight into the physical nature of the photoactivation process. Moreover, they do not compare PAPA with previously published methods able to yield similar single-molecule insight into protein-protein interactions. Yet, the experiments are rigorously performed, outcomes critically tested and procedures comprehensively described. Thus, the application of the new method by other researchers should be straightforward and promises to yield hitherto inaccessible mechanistic insight.
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Reviewer #3 (Public Review):
This is work by an internationally recognized group with strong expertise in sophisticated single-molecule microscopy assays in cells. They present here a single-molecule fluorescence-based assay for proximity in the nanometer range.
It has long been reported that cyanine dyes such as Cy3, Cy5 and derivatives such as AF555, AF647 can undergo a photoswitching mechanism by which the shorter wavelength dye when being excited can switch the longer wavelength dye which is in a dark state back into the bright state. And it has furthermore been reported that this switching mechanism is not based on FRET, as the distance requirement is more stringent (up to ~ 2 nm). However, this mechanism has not been fully explored for the investigation of molecular interactions yet.
The authors in the present work present a …
Reviewer #3 (Public Review):
This is work by an internationally recognized group with strong expertise in sophisticated single-molecule microscopy assays in cells. They present here a single-molecule fluorescence-based assay for proximity in the nanometer range.
It has long been reported that cyanine dyes such as Cy3, Cy5 and derivatives such as AF555, AF647 can undergo a photoswitching mechanism by which the shorter wavelength dye when being excited can switch the longer wavelength dye which is in a dark state back into the bright state. And it has furthermore been reported that this switching mechanism is not based on FRET, as the distance requirement is more stringent (up to ~ 2 nm). However, this mechanism has not been fully explored for the investigation of molecular interactions yet.
The authors in the present work present a similar mechanism for a different class of rhodamine-based fluorophores, specifically JF549 and JFX650. They describe the discovery of this mechanism in dual-color labeling of a pentameric protein and initial characterization to distinguish it from UV-light-mediated recovery from a pumped dark state as reported for (d)STORM-like measurements. They extend their observation to TMR, JF529 as lower wavelength "senders" and JF646 and JFX646 as longer wavelength "receivers" that can become reactivated into the ground state upon illumination of a nearby "sender". The authors then test activation pulse length and distance dependence and find that longer pulses lead to more recovery and that PAPA of JF549/JFX650 has unlike previously observed for the Cy3/Cy5 pair a smaller distance dependence than FRET of the same fluorophore pair. The authors then move on to use both the UV-light mediated direct reactivation "DR" and proximity-assisted photoactivation "PAPA" to activate different molecules that are either double-labeled for PAPA or singly labeled with JFX650 for DR. They succeeded in four different cases to identify clear population shifts to distinguish molecules of different mobility.
Overall, I think the authors made an interesting discovery and characterizing this previously poorly characterised interaction for cellular single-molecule experiments is certainly an important effort. The authors make an honest and quite complete effort to work out the practical details of this interaction and designed experiments that convincingly highlight the basic capabilities this technique offers to the detection of verified interactions and the mobility of interacting molecules in cells.
The weakness is that these capabilities do not seem to be as clear-cut as the reviewer hoped for when starting to read this manuscript. It remains unclear to this reviewer, to what extant PAPA molecules can be separated from DR molecules. In all but the last diffusion experiment(s) in Figure 4, PAPA molecules seem to be significantly perturbed by DR molecules, casting doubt on the usefulness in real experiments. Similarly, in Figure 5, a difference is seen but does not allow for quantification. This certainly is not the case for other methods of sensing as well, but maybe the authors could more specifically compare their efforts and the dynamic range to other sensors for example in Figure 5? This would make it easier for the reader to make up their mind if the assay is worthwhile adopting for their system.
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