Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

  1. Sridevi Challa
  2. Keun W Ryu
  3. Amy L Whitaker
  4. Jonathan C Abshier
  5. Cristel V Camacho
  6. W Lee Kraus

  • Curated by eLife

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    Evaluation Summary:

    Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

    (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 #1 agreed to share their name with the authors.)

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Abstract

ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as ‘readers’ for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)—optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems.

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  1. Version published to 10.7554/elife.72464 on eLife
  2. eLife

    Author Response:

    Evaluation Summary

    Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

    We appreciate the positive and encouraging words from the reviewer. We also appreciate the helpful comments, criticisms, and suggestions, which we have endeavored to address fully.

    Reviewer 1 (Public Review):

    While these tools are more sensitive than existing tools, it is unclear whether a dynamic range of 6-fold (GFP) and 3-fold (luciferase) provide sufficient sensitivity for properly understanding the PAR dynamics (which was thought to increase as much as 100-fold in DNA damage settings). In addition, it is unclear whether the fold increases in both fluorescence and luminescence linearly correlate with the traditional measures by western blot.

    We are pleased that the reviewer found our sensors to potentially useful. The reviewer provided a number of excellent comments and suggestions that have served as a useful guide for improving our paper. We have carefully considered all of the comments, insights, and suggestions from the reviewer and revised the manuscript accordingly. We think this has strengthened our conclusions and improved the paper considerably. We thank the reviewer for the careful and thorough review of our paper.

    Figure 1F indicates on the western blot that there was a precipitous drop of PARylation after 5 min, but the GFP signal indicated a linear drop. It will be important to quantify the signals on western blots and test how correlate their data with the GFP/luciferase data in scatter plots for their various sets of data. Would this system under-estimate the changes and be not sensitive enough to subtle changes that may be 1-2 fold measured by traditional means

    We agree with the reviewer that a comparison with existing PAR detection technologies will improve the manuscript. We now performed a comparative analysis of ELISA, Western blot, and immunofluorescence assays with live cell imaging using PAR-T GFP (Figures 6A, 6C, 6D). The results indicate that the detection range of PAR-T ddGFP is comparable to the established PAR detection assays. In addition, we also compared the live cell luciferase assays using PAR-T NanoLuc to Western blotting (Figure 6B) and found that these two assays are able to detect PAR changes at comparable levels. We would also like to emphasize that these sensors were developed to improve our ability to detect PAR changes in living cells and animals, which the existing techniques are not capable of doing.

    Similarly, how is their quantitation in Figure 2 compared with traditional immunofluorescence?

    We performed this comparison and observed that the changes in PAR levels as detected by live cell imaging using PAR-T ddGFP are comparable to the changes detected in immunofluorescence assays using the WWE-Fc reagent (Figure 6D and 6E).

    Lastly, for the luciferase signal in Figure 3B and C, the corresponding signal in western blots are missing. Therefore, it is difficult to estimate the background signal. If Niraparib, as in other figures, eliminates PAR signals on western blot, these data would indicate half of the basal signal are background, which is rather high. Having said that, tool development is an evolution process. These tools will provide a good foundation for future development. Therefore, understanding these limitations (dynamic range, quantitative sensitivity correlation, and background) will provide a better assessment of the utility of these new tools for investigating PAR biology.

    We appreciate the reviewer’s concern about the high background signal in Niraparibtreated samples. To answer this concern, we compared the dynamic range of PAR-T NanoLuc to Western blotting (Figure 6B) and found that the results from live cell luciferase assays using PAR-T NanoLuc are comparable to Western blotting using WWE-Fc. Of note, we were able to detect decreases in PAR levels with Niraparib using live cell luciferase assays using PAR-T NanoLuc, but not Western blotting. Based on these analyses, we can conclude that the changes in PAR levels at the basal level are very minimal, leading to only 50% decrease in PAR-T NanoLuc signal with Niraparib treatment (Figure 6B, Figure 5A-5C). Note that the decrease in PAR-T NanoLuc signal is greater when UV-treated cells were pre-treated with Niraparib, which is consistent with the results from Western blot analysis (Figure 5A).

    Reviewer 2 (Public Review):

    In this study, the authors attempted to extend their own work and that of others in the field in developing probes to detect the signaling molecule, poly-ADPribose (PAR) that can be used in the test tube, in cells and in tumor models. Major strengths include the development of a set of probes with data demonstrating utility and efficacy. Further, the authors show the assay to be useful in cell models and tumor models. Some weaknesses include what appears to be a high level of background in the assay. Further, regarding methods, the exact probes (sequences) being evaluated are not defined. This is one of several new PAR probes being developed over the last few years but may have widespread utility due to the quantitative nature of the bioluminescent assay.

    We thank the reviewer for these thoughtful and encouraging comments, as well as the interesting, thought-provoking, and constructive criticisms that have prompted us to dig deeper and provide more evidence to support our claims

    Reviewer 3 (Public review):

    The major drawback is that, while the authors demonstrated some applications of these PAR trackers (PAR-T) in both culture cells and in animals, the data of PAR-T ddGFP on cancer cells and the data of PAR-T Nano luciferase may not be sufficient to support the authors' claim that the new tool can detect spatial and temporal dynamics of PAR in cells and in animals. That said, the new tools can potentially expand the capability of cell biologists to visualize and study the PAR production process in both normal and disease states with improved sensitivity and tissue compatibility.

    We thank the reviewer for appreciating the potential utility of the PAR-T sensors, as well as the detailed and constructive criticisms that have prompted us to provide more evidence to support our claims. Addressing these comments has helped us to improve the paper.

    One of the major issues of this manuscript is the lack of time-course data for PAR-T luminescent sensors to demonstrate temporal monitoring of PAR levels in animals. If the binding of two split Nano Luciferase parts is irreversible, the application might be limited. However, according to the literature (Scientific Reports volume 11, Article number: 12535 (2021)), the split Nanoluc technology should be able to detect dynamic changes. Either way, a set of time-course data would be necessary. The authors need to provide evidence to support their statement "The high sensitivity and low signal to noise ratios of the PAR-Trackers described here enable spatial and temporal monitoring of PAR levels in cells and in animals.

    We agree with the reviewer’s comment that the original manuscript did not demonstrate that the PAR-T sensors can be used to detect spatio-temporal changes in PAR. To demonstrate that PAR-T NanoLuc can be used to detect time-dependent changes in PAR levels, we performed a time course of UV-mediated PARP-1 activation (Figure 5D). The results from this assay demonstrated that the dynamic changes in PAR in live cells, in response to DNA damage, can be recaptured using the PAR-T NanoLuc sensors. In addition, we also measured PARGi-mediated PAR accumulation in vivo in xenograft tumors (Figure 8 - figure supplement 1B-1D). We found that PAR can be detected readily in breast cancer cells when injected into mice. Upon treatment with PARGi, the luminescence from PAR-T NanoLuc increased significantly by 6 hours and then diminished by 24 hours. These data demonstrate that PAR-T NanoLuc can be used to track dynamic changes in PAR levels both in cells and in animals. While not in vivo, our work with spheroids also addresses this concern. See our response to the next comment below.

    Figure 2- figure supplement 2. For the detection of spatial dynamics of PAR signals in cancer spheroids, the authors did not provide sufficient evidence as only static images of different spheroids in different conditions were provided. And 2 out of 3 fields of view only include one spheroid. In addition, there is no time-course image data showing the spatial patterns of PAR in cancer cells are dynamic.

    We have now performed a quantitative analysis of multiple spheroids. As indicated in Figure 3B, we observed a significantly higher GFP fluorescence signal in spheroids derived Challa et al. (Kraus) – Rebuttal February 2, 2022 10 from PAR-T ddGFP expressing cells compared to those expressing ddGFP or those treated with Niraparib. To address the reviewer’s concern about using PAR-T ddGFP for spatio-temporal changes in cells, we included a video for live cell imaging of H2O2-mediated increase in PAR-T ddGFP (Figure 2 - figure supplement 2, video). We also developed an analysis approach that allows us to quantify the signals from the core of the spheroids separately from the periphery of the spheroids. We also performed a time course in 3D cancer spheroids to visualize the spatio-temporal changes in PAR levels (Figure 3C and 3D). The results from this experiment demonstrate that the PAR levels in cells at the core of the spheroids are relatively resistant to Niraparib treatment, as the PAR levels in cells at the core of the spheroid decrease at a lower rate when compared to PAR in the cells at the outer layer of the spheroid.

    In the caption of Figure 2 -figure supplement 1 (B and C), it states "Immunofluorescence assay to track PAR formation in response to H2O2.", but there is no evidence showing any antibodies were used there.

    We thank the reviewer for pointing out this error. It should have been written as live cell imaging, not immunofluorescence assay. We made this correction.

    It seems that Figure 3 B and C does not support the statement "we observed specific detection of firefly luciferase with D-Luciferin and NanoLuc with furimazine with no cross-reactivity" And it is unclear why the authors refer Fig. 3B and C after that statement as those data seems not supporting this claim. Similarly, the statement "Moreover, the luminescence of PAR-T Luc is only 30-fold lower than intact firefly luciferase." Was not supported by Fig. 3B. In fact, the differences between PAR-T Luc and intact firefly luciferase were ~1000 fold in vivo, judging from Fig 5B. It is also unclear which data of the construct was used to plot Fig. 3C.

    We thank the reviewer for this comment. We changed the scale bar to represent the true scale for the luminescence from Nano luciferase and Firefly luciferase. This indicates that the brightness of PAR-T NanoLuc is 30-fold lower than intact firefly luciferase. In Figure 3C, we plotted the ratio of PAR-T NanoLuc to firefly luciferase.

    Fig. 4C, it seems that Firefly luciferase was consistently brighter with PARGi, and I wonder if such difference is statistically significant. The authors did not perform a twoway ANOVA test for the firefly luciferase dataset.

    We included the statistics to indicate that these changes were not significant.

    The statement "Moreover, none of these sensors can detect PAR accumulation in vivo." seems to lack support. Have the authors proved that with evidence? I would recommend using the following statement instead: "Moreover, none of these sensors has yet demonstrated detection of PAR accumulation in vivo

    We made this change.

    For the in vivo experiment, it is unclear about the benefits of normalizing the PAR-T radiance to the Firefly luciferase since the signals from Firefly luciferase did not overlap well with that from the PAR-T nano luciferase, which may cause bigger variations.

    We thank the reviewer for raising this point. We normalize the luminescence from PAR-T NanoLuc to that from firefly luciferase to account for the variability in tumor size between the mice. We think this is an important control in the analysis. The luminescence from firefly luciferase represents the differences in tumor size between the mice. Hence, that signal is greater than the signal from PAR-T NanoLuc and is spread over a larger area.

    Judging from the data of Fig 3 supplement 1E, the signal intensity from the split firefly luciferase-based PAR-T sensors was ~10000 fold less than intact firefly luciferase, not ~1000 fold. It makes more sense to give up the split firefly luciferase for ~10000 fold differences since the signal intensity from the split nano luciferase was ~1000 fold less than intact firefly luciferase (Fig 5B).

    We noted the reviewers concern about the split firefly luciferase PAR-T. We agree with the reviewer that the split nano luciferase is brighter than the split firefly luciferase (Figure 4C and Figure 4 - figure supplement 1E). Although split nano luciferase is 1000-fold dimmer than the intact firefly luciferase in vivo (Figure 8B and Figure 8 - figure supplement 1A), this difference is only 30-fold in in vitro assays (Figure 4C). Hence, the comparison of sensors based on split firefly luciferase to split nano luciferase highlights our efforts to make a brighter sensor. Moreover, we included the split firefly luciferase data to compare the performance of WWE vs macrodomain in the development of the PAR-T NanoLuc sensor. Since firefly luciferase is frequently used for sensor development, we believe that it is important to include the results obtained from this sensor.

    Therefore, developing tools to measure ADPR dynamics in cells and in vivo is critical for better understating the various biological processes mediated by ADPR". "understating" should be "understanding".

    We corrected this error.

  3. eLife

    Evaluation Summary:

    Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

    (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 #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Challa and Ryu et al aimed to develop a series of sensors that can detect poly(ADP-ribose) (PAR)-a nucleotide-like protein modification important in various diseases-in cell lysates, living cells and animals, with greater sensitivity with temporal and spatial precision. One major strength of the manuscript is that they have successfully developed two split-protein systems (fluorescence- and luciferase-based) to monitor PAR levels after systematic evaluation of various combinations of ADP-ribose-binders. They have demonstrated the specificity using appropriate controls (e.g., inhibitors that reduces or increase PAR levels), and tested in various physiological conditions (such as DNA damage and adipogenesis). In addition, they have demonstrated that the system works in cell lysates and living cells using both systems and living animals using the luciferase system with a sensitivity of 1000 cells. Therefore, compared with previous attempts to make PAR sensors, these new tools are more sensitive. These tools may thus be useful for investigating PAR dynamics in cellular or animal models, which is important for various physiological and pathological settings.

    While these tools are more sensitive than existing tools, it is unclear whether a dynamic range of 6-fold (GFP) and 3-fold (luciferase) provide sufficient sensitivity for properly understanding the PAR dynamics (which was thought to increase as much as 100-fold in DNA damage settings). In addition, it is unclear whether the fold increases in both fluorescence and luminescence linearly correlate with the traditional measures by western blot. For example, Figure 1F indicates on the western blot that there was a precipitous drop of PARylation after 5 min, but the GFP signal indicated a linear drop. It will be important to quantify the signals on western blots and test how correlate their data with the GFP/luciferase data in scatter plots for their various sets of data. Would this system under-estimate the changes and be not sensitive enough to subtle changes that may be 1-2 fold measured by traditional means? Similarly, how is their quantitation in Figure 2 compared with traditional immunofluorescence? Lastly, for the luciferase signal in Figure 3B and C, the corresponding signal in western blots are missing. Therefore, it is difficult to estimate the background signal. If Niraparib, as in other figures, eliminates PAR signals on western blot, these data would indicate half of the basal signal are background, which is rather high.

    Having said that, tool development is an evolution process. These tools will provide a good foundation for future development. Therefore, understanding these limitations (dynamic range, quantitative sensitivity correlation, and background) will provide a better assessment of the utility of these new tools for investigating PAR biology.

  5. eLife

    Reviewer #2 (Public Review):

    In this study, the authors attempted to extend their own work and that of others in the field in developing probes to detect the signaling molecule, poly-ADP-ribose (PAR) that can be used in the test tube, in cells and in tumor models.

    Major strengths include the development of a set of probes with data demonstrating utility and efficacy. Further, the authors show the assay to be useful in cell models and tumor models.

    Some weaknesses include what appears to be a high level of background in the assay. Further, regarding methods, the exact probes (sequences) being evaluated are not defined.

    This is one of several new PAR probes being developed over the last few years but may have widespread utility due to the quantitative nature of the bioluminescent assay.

  6. eLife

    Reviewer #3 (Public Review):

    In this manuscript, Challa et al. developed a set of PAR Trackers (PAR-Ts) based on dimerization-dependent fluorescent protein and split-protein reassembly of luciferase for both in vitro and in vivo studies. The PAR-Ts contain a PAR-binding domain WWE fused to both parts of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc) with LSSmOrange. The ddGFP version (PAR-T GFP) allows for real-time assessment of dynamic PAR production in vitro and in living cells, while the split NanoLuc version (PAR-T Luc) allows detection of PAR production in tissues in living mammals (though the authors implied PAR-T Luc can monitor the dynamics of PAR level in animals). The major drawback is that, while the authors demonstrated some applications of these PAR trackers (PAR-T) in both culture cells and in animals, the data of PAR-T GFP on cancer cells and the data of PAR-T Nano luciferase may not be sufficient to support the authors' claim that the new tool can detect spatial and temporal dynamics of PAR in cells and in animals. That said, the new tools can potentially expand the capability of cell biologists to visualize and study the PAR production process in both normal and disease states with improved sensitivity and tissue compatibility.

    Detailed comments:
    1. One of the major issues of this manuscript is the lack of time-course data for PAR-T luminescent sensors to demonstrate temporal monitoring of PAR levels in animals. If the binding of two split Nano Luciferase parts is irreversible, the application might be limited. However, according to the literature (Scientific Reports volume 11, Article number: 12535 (2021)), the split Nanoluc technology should be able to detect dynamic changes. Either way, a set of time-course data would be necessary. The authors need to provide evidence to support their statement "The high sensitivity and low signal to noise ratios of the PAR-Trackers described here enable spatial and temporal monitoring of PAR levels in cells and in animals."
    2. Figure 2- figure supplement 2. For the detection of spatial dynamics of PAR signals in cancer spheroids, the authors did not provide sufficient evidence as only static images of different spheroids in different conditions were provided. And 2 out of 3 fields of view only include one spheroid. In addition, there is no time-course image data showing the spatial patterns of PAR in cancer cells are dynamic.
    3. In the caption of Figure 2 -figure supplement 1 (B and C), it states "Immunofluorescence assay to track PAR formation in response to H2O2.", but there is no evidence showing any antibodies were used there.
    4. It seems that Figure 3 B and C does not support the statement "we observed specific detection of firefly luciferase with D-Luciferin and NanoLuc with furimazine with no cross-reactivity" And it is unclear why the authors refer Fig. 3B and C after that statement as those data seems not supporting this claim. Similarly, the statement "Moreover, the luminescence of PAR-T Luc is only 30-fold lower than intact firefly luciferase." Was not supported by Fig. 3B. In fact, the differences between PAR-T Luc and intact firefly luciferase were ~1000 fold in vivo, judging from Fig 5B. It is also unclear which data of the construct was used to plot Fig. 3C.
    5. Fig. 4C, it seems that Firefly luciferase was consistently brighter with PARGi, and I wonder if such difference is statistically significant. The authors did not perform a two-way ANOVA test for the firefly luciferase dataset.
    6. The statement "Moreover, none of these sensors can detect PAR accumulation in vivo." seems to lack support. Have the authors proved that with evidence? I would recommend using the following statement instead: "Moreover, none of these sensors has yet demonstrated detection of PAR accumulation in vivo"
    7. For the in vivo experiment, it is unclear about the benefits of normalizing the PAR-T radiance to the Firefly luciferase since the signals from Firefly luciferase did not overlap well with that from the PAR-T nano luciferase, which may cause bigger variations.
    8. Judging from the data of Fig 3 supplement 1E, the signal intensity from the split firefly luciferase-based PAR-T sensors was ~10000 fold less than intact firefly luciferase, not ~1000 fold. It makes more sense to give up the split firefly luciferase for ~10000 fold differences since the signal intensity from the split nano luciferase was ~1000 fold less than intact firefly luciferase (Fig 5B).
    9. "Therefore, developing tools to measure ADPR dynamics in cells and in vivo is critical for better understating the various biological processes mediated by ADPR". "understating" should be "understanding".

  7. Version published to 10.1101/2021.08.02.454729v2 on bioRxiv
  8. Version published to 10.1101/2021.08.02.454729v1 on bioRxiv