The Opto-inflammasome in zebrafish as a tool to study cell and tissue responses to speck formation and cell death

  1. Eva Hasel de Carvalho
  2. Shivani S Dharmadhikari
  3. Kateryna Shkarina
  4. Jingwei Rachel Xiong
  5. Bruno Reversade
  6. Petr Broz
  7. Maria Leptin

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Abstract

The inflammasome is a conserved structure for the intracellular detection of danger or pathogen signals. As a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control experimentally on a single-cell level using canonical triggers. We constructed Opto-ASC, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce ASC inflammasome (speck) formation in individual cells of the skin. We find that cell death resulting from ASC speck formation is morphologically distinct from apoptosis in periderm cells but not in basal cells. ASC-induced PCD can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb and triggers a strong Ca 2+ signaling response in nearby cells.

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  1. Version published to 10.7554/elife.86373 on eLife
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    Reply to the reviewers

    We are obviously very pleased with the general support expressed by the referees, and appreciate their critical comments. We detail below how we propose to respond to their suggestions and queries.

    In view of the fact that my lab is no longer in existence, I will have to rely on the kind generosity of my colleagues at EMBL to host former team members (the two first authors) for a limited period to come back to Heidelberg to carry out any further experimental work that may be needed. This means we will have to limit the work we can do to those experiments with the highest priority. However, we are optimistic that we will be able to obtain indicative results.

    We will also follow most of the referees’ other suggestions and requests for additional data and quantifications, as outlined (or already included) below.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: ASC is the Pyrin/CARD-containing adapter protein that functions as a core component of inflammasome signaling complexes. ASC functions downstream of various NLR- and ALR-inflammasome initiator proteins and upstream of the inflammatory caspases that function as inflammasome effector enzymes. This study uses a novel chimeric construct (Opto-ASC) comprising the Arabidopsis photo-oligomerizable cryptochrome 2 (Cry2-olig) protein with zebrafish ASC to generate transgenic zebrafish larvae wherein ASC oligomerization can be rapidly, dynamically and spatially induced by blue light illumination of either the entire larva or single cells within discrete tissues of an intact larva. Induction of these "opto-inflammasome" complexes is coupled with state-of-the-art, live-cell optical imaging of multiple single cell and integrative tissue parameters to assay various modes of regulated cell death within the peridermal and basal cellular layers of the larval skin. This experimental model was further combined with genetic manipulation of the expression of various zebrafish inflammatory or apoptotic caspases, as well as the two zebrafish members of the gasdermin family of pore-forming proteins which can mediate disruption of plasma membrane permeability without (pre-lytic) or with (pyroptosis) progression to lytic cell death.

    The main results of the study are: 1) introduction of a novel experimental system for dynamic and spatially resolved ASC oligomerization and speck formation within the cells of intact epithelial tissues of a living organism; 2) the ability of these optically induced ASC oligomers/specks to drive multiple modes of regulated cell death which exhibit some (but not all) features of lytic pyroptosis or non-lytic apoptosis depending on cell type and tissue location; 3) the ability of the dying epithelial cells containing optically-induced ASC specks to coordinate rapid adaptive responses in adjacent non-dying cells to maintain integrity/ continuity of skin epithelial barrier; and 4) unexpectedly, no obvious role for either of the two zebrafish gasdermins in the regulated cell death responses.

    Major Comments:

    1. Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them? The major goal of this MS is to present a new experimental model (optogenetic activation of ASC oligomerization in transgenic zebrafish) that has the potential to provide new insights regarding the multiple mechanisms by which ASC can regulate inflammasome/ cell death signaling responses in the context of an intact organism. As noted above, some of the observed results are unexpected (e.g., lytic cell death independent of the zebrafish gasdermins in particular epithelial cells) and may reflect mechanisms unique to zebrafish as a non-mammalian vertebrate model versus the mammalian experimental systems (murine and human) that have informed most of our current understanding of how ASC coordinates inflammasome and cell death responses. However, the authors have used rigorous genetic approaches to rule out trivial explanations for the unexpected observations. Thus, no major additional experiments are required to support the claims and conclusions presented in the MS.

    2. Are the suggested experiments realistic in terms of time and resources? Yes. It would help if you could add an estimated time investment for substantial experiments: A few weeks.

    3. Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate? Yes.

    4. Are the experiments adequately replicated and statistical analysis adequate? Yes.

    Minor comments

    1. Specific experimental issues that are easily addressable:

    There's a significant concern with the use of LDC7559 (line 387) as a putative small molecule inhibitor of gasdermin D function to test roles (or lack thereof) of the zebrafish gasdermins in the ASC-triggered lytic cell death responses. A recent study (Amara et al. 2021. Cell. PMID34320407) has reported that LDC7559 does not inhibit gasdermin D (and possibly other gasdermins) but rather acts as an allosteric activator of PFKL (phosphofructosekinase-1 liver type) in neutrophils and thereby suppress generation of the NADPH required for the phagocytic oxidative burst and consequent NETosis. Thus, use of LDC7559 as a presumed gasdermin inhibitor in the current MS is problematic and should be deleted. As an alternative pharmacological approach to suppress gasdermin function, the authors might consider the use of either disulfiram (Hu et al. 2020. Nature Immunology. PMID32367036) and/or dimethylfumarate (Humphries et al. Science. 2020. PMID32820063). These would be straightforward additional experiments.

    We have ordered the reagents to do these experiments. We are optimistic that we will obtain data that will strengthen this part of the ms and be a pointer for future studies by others.

    We propose to keep the information on LDC7559 included, but to discuss the reservations the referee lists above - otherwise, others might ask why we did not even try this inhibitor. .

    Are prior studies referenced appropriately? there are some problems; see below. 2a. One paper is cited twice in lines 724-726 and 727-729. 2b. Another paper is cited twice in lines 790-792 and 793-795. 2c. No journal is included for the referenced study by Shkarina et al in lines 827-828. 2d. No journal is included for the referenced study by Stein et al in lines 831-832. 2e. No journal is included for the referenced study by Masumoto et al in lines 793-795. 2f. No journal is included for the referenced study by Kuri et al in lines 774-775.

    We are embarrassed about these omissions and mistakes and have corrected them..

    Are the text and figures clear and accurate? Generally, yes but with a few exceptions noted below: 3a. line 28: "morphological distinct" should read "morphologically distinct" 3b. line 161: this sentence contains in parentheses "for how long?" I think this was a comment by one author that wasn't removed from the final submitted MS 3c. line 945: spelling "balck" > "black" 3d. line 268: "whereas showed a delayed speck formation": the authors need to specify what model/ condition showed a delayed speck formation 3e. line 262: spelling "egnerated" > "generated"

    Thank you, all corrected.

    CROSS-CONSULTATION COMMENTS I also agree with the comments of the other 2 reviewers. Between the 3 sets of comments and suggestions, the aggregate review will provide the authors with a suitable range of feasible recommendations that will improve an already strong MS.

    Reviewer #1 (Significance (Required)):

    1. General assessment: As noted above, this the major goal of this MS is to present a new experimental model (optogenetic activation of ASC oligomerization in transgenic zebrafish) that has the potential to provide new insights regarding the multiple mechanisms by which ASC can regulate inflammasome/ cell death signaling responses in the context of an intact organism. The authors have used rigorous genetic approaches to rule out trivial explanations for the unexpected observations. In general, the MS describes an elegant model system that will provide a platform for identifying new mechanisms of ASC-dependent inflammasome signaling and regulated cell death.

    2. Advance: This MS describes a highly novel experimental model. Zebrafish are increasingly being used as a genetically tractable model for inflammasome signaling within integrated tissues of intact organism. As noted above, the advances are technical but also conceptual. Future application of this novel model is likely to yield identification of new mechanisms for ASC function in innate immunity and regulated cell death within the context of tissue homeostasis and host defense.

    3. Audience: Basic research and discovery.

    4. Please define your field of expertise with a few keywords to help the authors contextualize your point of view: My group investigates multiple aspects of inflammasome signaling biology at the cellular level with an emphasis on cell-type specific roles for gasdermins in coordinating downstream innate immune responses to inflammasome activation in various myeloid leukocytes (macrophages, dendritic cells, neutrophils, eosinophils, mast cells).

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Programmed cell death is critical for host defense and tissue homeostasis. How dead cells initiate cellular responses in the microenvironment with neighbouring cells in vivo is still largely unknown. The authors have chosen a Zebrafish model to tackle this question, given that this model shows advantages for imaging and addresses these pathways in a complex in vivo setting. Their recent development of light-induced activation of caspases (published in JEM) enabled them to investigate cellular responses to a specific type of cell death in vivo at a single cell resolution. In this study, the author further developed a light-induced activation of ASC to specifically look at inflammasome activation-mediated cell death in vivo. The authors have successfully established this system in zebrafish and also observed that Opto-Asc-induced cell death showed different phenotypes as compared to Opto-caspase-a/b-induced cell death. However, it is not really clear why. I have a few specific comments to be addressed or discussed.

    1. In Fig.3 and Fig.4, the majority of Opto-Asc localizes to the plasma membrane but not endogenous Asc. It seems that tagging affects its localization, which could potentially explain its slow kinetics in oligomerization.

    That is an interesting suggestion. The membrane enrichment is indeed reproducible and we have no full explanation for it. However, ASC itself seems to have some affinity for the cell cortex as seen by its association with the apical actin ridges in keratinocytes in the resting state (see e.g. figure 3A). Affinity of ASC for actin is also documented in the literature:(F-actin dampens NLRP3 inflammasome activity via flightless-1 and LRRFIP2 OPEN; https://doi.org/10.1038/srep29834).

    Perhaps the fusion to the optogenetic module somehow enhances the affinity through the initial dimerization. But we can only speculate and have no further evidence that would allow reliable conclusions.

    In Fig.7, the authors showed that deletion of Caspb, but not Caspa, affected the apical extrusion, without affecting cell death. This may indicate that other caspases, like Caspase-8 or/and caspase-3 were involved. This could be addressed through deletion of Caspase-8 or/and caspase-3.

    These are experiments we had in fact done. Unfortunately, they did not allow us to address the question, because the deletions resulted in embryonic lethality. We have added this information to the text.

    It is very surprising that Opto-Asc-mediated cell death is not dependent on Gasdermins, at least in Caspb-dependent apically extruded dead cells.

    Indeed – but see comment by and our response to reviewer 1. We hope to be able to provide additional data.

    CROSS-CONSULTATION COMMENTS I agree with the other two reviewers and don't have further comments.

    Reviewer #2 (Significance (Required)):

    The Opto-Asc zebrafish model developed in this study will enable us to specifically look at inflammasome-mediated cell death in vivo. This model is more physiologically relevant compared to Opto-caspase1 model.

    Audience interested in physiological function of inflammasome activation, but it is questionable whether such a tool will address mechanisms in mammalian cells. Eventually, more evidence for the latter could be provided.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this article, de Carvahlo and colleagues describe a novel optogenetic tool allowing single cell and temporally controlled induction of ASC clusters in vivo (in zebrafish), a central adaptator protein of the inflammasome complex which is involved in the induction of pyroptosis. This alternative mode of programmed cell death is involved in pathogen response and promote cell swelling and the release of pro-inflammatory factors. Previous works have shown that the inflammasome activation is associated with the formation of a large cluster of ASC protein (called speck) which promotes then the recruitment and the activation of caspase 1. Specks were previously characterised by the same group in vivo (in zebrafish larvae) and could be induced by the overexpression of ASC protein. This however was not compatible with fine spatio-temporal control of speck formation, thus preventing very refined characterisation of the dynamics and the distinction of the cell autonomous and non-cell autonomous effects.

    By fusing ASC to the blue-light sensitive oligomerising protein Cry2-olig under the control of a heat shock promoter, they could induce time controlled induction of speck at the single cell level, which is then followed by cell extrusion and cell death both in the periderm and the basal cell of the skin of zebrafish larvae. Doing so, they could characterise the dynamics of speck formation as well as key paramters affecting its dynamics and the subsequent extrusion. While ASC activation led to apical or basal extrusion in the periderm layer followed by non-apoptotic cell death, it triggers basal extrusion and apoptosis in the basal layer. Importantly, periderm cell elimination does not seem to strictly follow all the features of pyroptosis as it does not require GSDM, and relies on Caspb (not Caspa). It is also associated with strong Calcium release both in the dying and neighbouring cells.

    The authors performed a very careful characterisation of the tools and the optimisation of the condition to form speck and eliminate cells. The experiments are very well performed with all the necessary controls. The results, while to some extend still hard to fully interpret for some aspects, illustrate the plasticity of cell death and cell extrusion, which include several very interesting observations on the direction of extrusion, putative compensatory modes of cell death upon Caspase1 perturbation and the difference of response to ASC clustering depending on the tissue layer. While it is not the main point of this study, the observation that the direction of extrusion can vary very significantly in different genetic backgrounds is also extremely interesting.

    The atypical cell elimination revealed in the system may require further characterisation in the future and suggest that the tools may not be the best to study bona fide pyroptosis. However, I don't believe there is always such strict separation between the modes of cell death and I am sure that it could lead to very interesting insights on inflammasome formation, extrusion and charcaterisation of downstream signalling in vivo, so overall this will be a very interesting resource for the community working on inflammasome, cell death and extrusion.

    I have some suggestions that could help to better characterise the mode of elimination as well as the mechanism of speck formation. I have also some suggestions for comparison with other published results as well as some text editing.

    Main points :

    1. So far, it remains a bit unclear how the authors define precisely speck versus any aggregate and the light induced clusters of Cry2 olig. Is it related to the timescale of formation and/or the lifetime of the aggregates? Is it related to their size?

    There Is no ‘formal’ definition of an inflammatory speck apart from it being the unusually large aggregates that ASC forms once it is activated. Light-induced clusters of Cry2Olig alone, or of Cry2olig fusions with proteins that do not normally oligomerize are much smaller (extensive documentation in the literature).

    A speck is thus a stable aggregate of ASC which is usually around 1 µm in size and is able to activate downstream caspases. But neither of these aspects alone are unique to ASC: prion-like structures can also be large aggregates (indeed ASC-specks have been compared to prions), and much smaller molecular assemblies can activate caspases. Thus ‘speck’ is more an operational definition, and ‘natural’ specks do have both of these properties, but as our experiments show, the properties can actually be separated. I would rather not try to narrow or change the definition, but leave any further discussion to the experts in the field.

    Figure 4E shows a number of variants of ‘speck’-like and other multimers: ASC-mKate and Opto-ASC form large single specks in the presence of endogenous ASC. Opto-ASC specks are only slightly smaller than those formed by endogenously tagged ASC-GFP (see also Supplementary Figure 2E.. Opto-PYD recruits endogenous ASC and becomes incorporated into a speck of approximately the same size, while Opto-CARD does so less efficiently. All of these kill cells. In the absence of endogenous ASC, Opto-ASC forms much smaller specks, and very many in each cell, but these are still functional as seen by the fact that they still kill cells (not the large spot at t = 60 min in the right half of Fig. 4E is not a speck, but the contracted dying cell). Both Opto-PYD and Opto-CARD also form only the small aggregates (quantification will be included), with Opto-PYD still killing the cell by virtue of its ability to recruit caspases via their PYD, whereas Opto-CARD does not.

    Since the authors use most of the time constant blue light illumination, could they also assess how long the speck remains after stopping blue light exposure and quantify their lifetime (relative to the CRY2olig cluster lifetime)?

    Briefly, any speck that contains a functional ASC moiety remains stable and does not disassemble once the blue light is turned off. In skin cells it is not possible to make quantitative measurements because they are killed by the speck. Opto-ASC specks remain stable until they are taken up by macrophages, as originally reported for ASC-GFP specks in Kuri et al. 2017.

    Stability can best be assessed in muscle cells, which do not die upon speck formation. The figure below shows that specks begin to form within minutes of a short pulse of illumination and remain stable (and indeed grow further) for at least 60 min.

    Here is an example:

    __Revisions Figure A: __

    __Stability of __Opto-ASC specks in muscle cells after exposure to a single pulse of blue light

    Specks in muscle cells expressing Opto-AscTg(mCherry-Cry2olig-asc) are induced by a single illumination with blue light (488nm) at t = 0 for 32 seconds. Multiple oligomers begin to form within 6 minutes, continue to gradually increase in number and, and remain until the end of the movie (60 mins).

    Cell outlines in the overlying epithelium labeled by AKT-PH-GFP are faintly visible in the first frame. Scale bar is 20 mm.

    Similarly could they provide some comparison of the size and localisation of CRY2 olig clusters compared to the speck.

    For size, see above. In addition, the size of the Cry2 oligomers as well as of Opto-ASC specks can vary with expression levels.

    For location, Cry2olig clusters are usually distributed throughout the cell, as seen in most of the right panels in Fig 4E, and in earlier work in cultured cells (e.g. Taslimi et al 2014). ASC specks can form anywhere in the cell, while Cry2olig-ASC has a preference for the cell cortex, but this is not absolute. In keratinocytes, but not in basal cells, the speck usually forms close to the lateral membrane. In the absence of endogenous ASC no real speck is formed but Opto-ASC in this case shows no clear localisation of Opto-ASC to the membrane.

    In view of the variation we see, a strict quantification is difficult: what would be the ‘correct’ definition of classes to look at? To make statistically significant statements, we would need an enormous number of examples in which we could control for all of the variation of expression levels, cell size, day to day variation etc, and we currently don’t have these. We hope the qualitative evidence in the micrographs we show represents the differences well, and we will be happy to provide a larger number of images, if the referees feel this would be helpful.

    With the non functional CRY2olig Asc fusion (Cter fusion), do they still see transient olig2 clustering which then reverse when blue light illumination is gone? I think it might be useful to clarify these points in the main text since most of the quantifications are based on speck localisation/numbering, so their characteristics have to be very well defined.

    That would be interesting to work out, but after our initial experiments with this construct, we did not pursue this further, since it was not a pressing issue at the time. If we can fit this into our planned experimental time table, we will re-assess it. However, while of interest, we feel these data would not add substantially to what we know at this point.

    1. In all the snapshots of speck formation, there seems to be a relative enrichment of the ASC signal at the cytoplasmic membrane (relative to the cytoplasm) prior to strong speck formation. This seems specific of optoASC as it does not seem to happen for the endogeneous ASC or upon overexpression of ASC-mKate (both in this study and in the previous study published by the same group). Is this apparent membrane enrichment something reproducible? (I see that on pretty much every example of this manuscript). If so what could be the explanation? Is there an actual recruitment at the membrane or is it because the membrane/cortical pool takes longer to be recruited in the speck (hence looking relatively more enriched at intermediate time points).

    See our speculations in response to point 1 of the first referee.

    We too would really like to understand this, but see no easy and efficient way of testing it at this point.

    1. There is also a very distinctive ring accumulation that seems to match with apical constriction and/or a putative actomyosin ring (since this is perfectly round, it could match with a structure with high line tension) (see Figure 1E, Figure 3B, Figure 4D...). Is it something already known? Could the authors comment a bit more on this? This could suggest that ASC accumulates in actomyosin cortex, which would be a very interesting property.

    We see that we had failed to be clear about this.

    There are two types of actin-labelled rings that appear around dying cells. One is formed by the epithelial cells that surround the dying cell. This structure becomes visible as soon as the cell begins to shrink. That it is formed by the surrounding cells is clear from mosaics where the dying cell does not express the actin marker (e.g. suppl. Figure 4A) and the parts of the ring are seen only in the subset of surrounding cells that do express the marker. This ring is also not circular, but follows the polygonal shape of the shrinking cell. We believe that this is the contractile structure that closes the wound, as observed in many other cases of wound healing.

    The other is the one the referee describes here. It is formed within the dying cell, as shown by the fact that it is visible in labelled cells when all the surrounding cells are negative for the marker. The other difference is that it appears only once the dying cell has already contracted considerably and begins to round up and be extruded (most clearly seen in Fig. 1E). The third referee had raised a similar point in relation to the same structure seen in Fig. 6C, and we provide below the requested analysis. It relies on resolution in the y-axis, which is unsatisfactory, but nevertheless, it is clear that this ring is in a plane above the apical surface of the epithelium (marked by the red membrane marker, i.e is present in the detaching cell. It may well simply be actin appearing in the entire cortex of the cell as it rounds up and looking like a ring when seen from above. A completely different method for imaging would have to be set up to document this reliably, but we hope that these explanations help to clarify the confusion we may have created.

    We do not see this accumulation in cells that leave the epithelium towards the interior (see figure in the response to ‘minor points’ below).

    In the end, since cell death can also occur without visible speck formation, I am wondering if they are eventually the most relevant structure to be quantified. Is it because speck can be dissolved upon caspase activation and could it relates to the speed at which caspase are activated (which may not leave enough time for strong aggregation and visible speck formation)? I believe it would help to get more explanation/discussion on this point.

    As already mentioned above, it is indeed not obvious what the significance of the large speck is (and it is extremely puzzling why it is that normally one a single one forms in each cell). We agree that it is not necessarily functionally relevant for the signalling outcome to quantify this property – but nor was this the purpose of this work. Regardless of what kind of aggregate is formed, the optogenetic tool allows the induction of ASC-dependent cell death, and therefore the study of the ensuing cellular events.

    The compensatory mechanisms that lead to cell death/extrusion despite depletion of caspb is very interesting. Could the authors use some pan caspase inhibitor (like zvad FMK) to confirm that this block opto-ASC cell death also in this context? Alternatively could they check the status of effector caspase activation using live probe (nucview) or immunostaining in the context of caspb depletion?

    Those would be interesting avenues to pursue. However, for the reason stated above (Leptin lab closing down, members of fish group no longer at EMBL), we are forced to restrict ourselves to the most important experiments, and think we should prioritize the ones mentioned above.

    1. If I understand well, Figure 7C on the right side suggest that the double KO cells don't extrude (if indeed "no change" mean no extrusion, by the way this nomenclature may deserve some clarification in the legend). I don't think these results are mentioned at any point in the main text, but it would be important to include them (since this is an important control).

    This interpretation is in fact correct, and we have changed the labelling in the figure to ‘no immediate death’

    1. Waves of calcium following cell death and cell extrusion have been previously characterised (Takeushi et al. Curr Biol 2020, Y Fujita group). Interestingly, in this previous article they observed waves of calcium near Caspase8 induced death (in MDCK) as well as near laser induced death in zebrafish, while apparently the authors don't see such Calcium waves upon Caspase8 activation in the zebrafish here. I think it would be important to include a comparison of the authors results with this previous paper in the discussion

    We have included this in our discussion.

    There is also a previous study which characterised the impact of caspase1 on cell extrusion (Bonfim Melo et al. Cell Report 2022, A. Yap lab) which promotes apical extrusion in Caco2 cells. I think it would also be important to include this work in the discussion and to compare with the results obtain here in vivo.

    We have included this in our discussion.

    Other minor points:

    1. Line 439: are the numbers given in percentage? if these are absolute numbers, it is out of how many cells ? Same remark line 445: what are the number of cases representing? (percentage?)

    We have rephrased this to make it unambiguous.

    Figure 5: could the authors show periderm and basal cell extrusion with the same type of markers? (membrane or actin or ZO1)? This would help to really compare accurately the morphology and the remodellings associated.

    We used Utr-mNeonGreen to lable actin both in periderm and basal cells. Actin labeling of extruded periderm cells is shown in figure 6C, actin labeling of a dying basal cells and the overlying periderm cells is shown in supplementary figure 5A.

    Is there any obvious differences in cell size or characteristic cell shape between the classic lab strains (golden, AB, AB2B2) and the WIK and experiment strain used here? I do acknowledge that this is clearly not the focus of this study, but given this striking difference (which is related to an important question in the field of extrusion), it would interesting to mention this if there is anything obvious.

    We will make these measurements and include the data.

    1. Figure 6C: what is exactly the localisation in Z of this strong actin accumulation observed during apical extrusion? Is it apical or is it rather on the basal side of the cell? A lateral view of actin could be useful in this figure for all the different conditions described.

    See response to ‘main point 3’ above.

    The images that show this are below. However, even from these images it is hard to appreciate the locations. They are in fact much easier to see by following the movies over time, and through the z-sections at any given time point. We will of course submit the movies with the manuscript.

    Revisions figure B:

    Localization of actin in the yz and xz planes in Opto-Asc-induced cell death and Opto-caspase-8-induced apoptosis

    Orthogonal projections of images of apically (A) and basally (B, C) extruded cells at four time points from time lapse recordings. Each time point shows the x-z plane and the orthogonal yz and and xz planes, in which the apical sides of the epithelium faces the x-z image.

    Actin is labeled with mNeonGreen-UtrCH (cyan), plasma membranes and internal membranes by lyn-tagRFP (magenta). Actin is initially concentrated in the apical cortical ridges of periderm cells.

    1. Apically extruded cell after death is induced by Opto-Asc. As the cell dies actin is lost from the apical ridges and accumulates in the cell cortex in a plane above the original apical surface of the epithelium
    2. Basally extruded cell after death is induced by Opto-Asc. Actin is retained in the apical ridges as the cell shrinks and moves below the epithelium within the dying cell.
    3. Basally extruded cell after death is induced by Opto-Caspase 8. The apical surfaces forms a transient dome in which the actin ridges remain intact before the dying cell is internalized. .

    Figure S3B: could the authors show the utrophin-neonGreen channel separatly? Is there a ring of actin in the dying cell? Also are the membrane protrusion formed more basally? (I suspect this is a z projection, but this would need to be specified in the legend).

    1. Figure 4A legend: I guess the authors meant red arrowheads rather than frame ? This has been corrected

    2. I list below a number of typos I could find in the main text

    Thanks for noticing these, we have corrected all of these, as well as further typos we found.

    Line 29: in Line 30: but Line 151 : from the ...[...] (tissue ?) Line 161: there is most likely a text commenting that was not removed (for how long?) Line 262: generated (egnrtd) Line 268: whereas showed a delay (the subject is missing) Line 269: a point is missing Line 362: which the lack Line 368: a point is missing Line 400: a space is lacking "cellsdepending" Line 438: shrinkwe (space) Line 459 : or I infections Line 525: there is a point missing.

    CROSS-CONSULTATION COMMENTS I generally agree with all the comments raised by the other reviewers which partially overlap with comments I had (see for instance referee two for the role of other caspases and the membrane localisation of the probe).

    Reviewer #3 (Significance (Required)):

    In this article, de Carvahlo and colleagues describe a novel optogenetic tool allowing single cell and temporally controlled induction of ASC clusters in vivo (in zebrafish), a central adaptator protein of the inflammasome complex which is involved in the induction of pyroptosis. This alternative mode of programmed cell death is involved in pathogen response and promote cell swelling and the release of pro-inflammatory factors. Previous works have shown that the inflammasome activation is associated with the formation of a large cluster of ASC protein (called speck) which promotes then the recruitment and the activation of caspase 1. Specks were previously characterised by the same group in vivo (in zebrafish larvae) and could be induced by the overexpression of ASC protein. This however was not compatible with fine spatio-temporal control of speck formation, thus preventing very refined characterisation of the dynamics and the distinction of the cell autonomous and non-cell autonomous effects.

    By fusing ASC to the blue-light sensitive oligomerising protein Cry2-olig under the control of a heat shock promoter, they could induce time controlled induction of speck at the single cell level, which is then followed by cell extrusion and cell death both in the periderm and the basal cell of the skin of zebrafish larvae. Doing so, they could characterise the dynamics of speck formation as well as key paramters affecting its dynamics and the subsequent extrusion. While ASC activation led to apical or basal extrusion in the periderm layer followed by non-apoptotic cell death, it triggers basal extrusion and apoptosis in the basal layer. Importantly, periderm cell elimination does not seem to strictly follow all the features of pyroptosis as it does not require GSDM, and relies on Caspb (not Caspa). It is also associated with strong Calcium release both in the dying and neighbouring cells.

    The authors performed a very careful characterisation of the tools and the optimisation of the condition to form speck and eliminate cells. The experiments are very well performed with all the necessary controls. The results, while to some extend still hard to fully interpret for some aspect, illustrate the plasticity of cell death and cell extrusion, which include several very interesting observations on the direction of extrusion, putative compensatory modes of cell death upon Caspase1 perturbation and the difference of response to ASC clustering depending on the tissue layer. While it is not the main point of this study, the observation that the direction of extrusion can vary very significantly in different genetic backgrounds is also extremely interesting.

    The atypical cell elimination revealed in the system may require further characterisation in the future and suggest that the tools may not be the best to study bona fide pyroptosis. However, I don't believe there is always such strict separation between the modes of cell death and I am sure that it could lead to very interesting insights on inflammasome formation, extrusion and charcaterisation of downstream signalling in vivo, so overall this will be a very interesting resource for the community working on inflammasome, cell death and extrusion.

    My expertise are in cell extrusion, optogenetics, apoptosis and epithelial mechanics. I am not a specialist of the inflammasome and pyroptosis.

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    Referee #3

    Evidence, reproducibility and clarity

    In this article, de Carvahlo and colleagues describe a novel optogenetic tool allowing single cell and temporally controlled induction of ASC clusters in vivo (in zebrafish), a central adaptator protein of the inflammasome complex which is involved in the induction of pyroptosis. This alternative mode of programmed cell death is involved in pathogen response and promote cell swelling and the release of pro-inflammatory factors. Previous works have shown that the inflammasome activation is associated with the formation of a large cluster of ASC protein (called speck) which promotes then the recruitment and the activation of caspase 1. Specks were previously characterised by the same group in vivo (in zebrafish larvae) and could be induced by the overexpression of ASC protein. This however was not compatible with fine spatio-temporal control of speck formation, thus preventing very refined characterisation of the dynamics and the distinction of the cell autonomous and non-cell autonomous effects.

    • By fusing ASC to the blue-light sensitive oligomerising protein Cry2-olig under the control of a heat shock promoter, they could induce time controlled induction of speck at the single cell level, which is then followed by cell extrusion and cell death both in the periderm and the basal cell of the skin of zebrafish larvae. Doing so, they could characterise the dynamics of speck formation as well as key paramters affecting its dynamics and the subsequent extrusion. While ASC activation led to apical or basal extrusion in the periderm layer followed by non-apoptotic cell death, it triggers basal extrusion and apoptosis in the basal layer. Importantly, periderm cell elimination does not seem to strictly follow all the features of pyroptosis as it does not require GSDM, and relies on Caspb (not Caspa). It is also associated with strong Calcium release both in the dying and neighbouring cells.

    • The authors performed a very careful characterisation of the tools and the optimisation of the condition to form speck and eliminate cells. The experiments are very well performed with all the necessary controls. The results, while to some extend still hard to fully interpret for some aspects, illustrate the plasticity of cell death and cell extrusion, which include several very interesting observations on the direction of extrusion, putative compensatory modes of cell death upon Caspase1 perturbation and the difference of response to ASC clustering depending on the tissue layer. While it is not the main point of this study, the observation that the direction of extrusion can vary very significantly in different genetic backgrounds is also extremely interesting.

    • The atypical cell elimination revealed in the system may require further characterisation in the future and suggest that the tools may not be the best to study bona fide pyroptosis. However, I don't believe there is always such strict separation between the modes of cell death and I am sure that it could lead to very interesting insights on inflammasome formation, extrusion and charcaterisation of downstream signalling in vivo, so overall this will be a very interesting resource for the community working on inflammasome, cell death and extrusion.

    I have some suggestions that could help to better characterise the mode of elimination as well as the mechanism of speck formation. I have also some suggestions for comparison with other published results as well as some text editing.

    Main points:

    1. So far, it remains a bit unclear how the authors define precisely speck versus any aggregate and the light induced clusters of Cry2 olig. Is it related to the timescale of formation and/or the lifetime of the aggregates ? Is it related to their size ? Since the authors use most of the time constant blue light illumination, could they also assess how long the speck remains after stoping blue light exposure and quantify their lifetime (relative to the CRY2olig cluster lifetime) ? Similarly could they provide some comparison of the size and localisation of CRY2 olig clusters compared to the speck. With the non functional CRY2olig Asc fusion (Cter fusion), do they still see transient olig2 clustering which then reverse when blue light illumination is gone ? I think it might be useful to clarify these points in the main text since most of the quantifications are based on speck localisation/numbering, so their characteristics have to be very well defined.

    2. In all the snapshots of speck formation, there seems to be a relative enrichment of the ASC signal at the cytoplasmic membrane (relative to the cytoplasm) prior to strong speck formation. This seems specific of optoASC as it does not seem to happen for the endogeneous ASC or upon overexpression of ASC-mKate (both in this study and in the previous study published by the same group). Is this apparent membrane enrichment something reproducible ? (I see that on pretty much every example of this manuscript). If so what could be the explanation ? Is there an actual recruitment at the membrane or is it because the membrane/cortical pool takes longer to be recruited in the speck (hence looking relatively more enriched at intermediate time points).

    3. There is also a very distinctive ring accumulation that seems to match with apical constriction and/or a putative actomyosin ring (since this is perfectly round, it could match with a structure with high line tension) (see Figure 1E, Figure 3B, Figure 4D...). Is it something already known ? Could the authors comment a bit more on this ? This could suggest that ASC accumulates in actomyosin cortex, which would be a very interesting property.

    4. In the end, since cell death can also occur without visible speck formation, I am wondering if they are eventually the most relevant structure to be quantified. Is it because speck can be dissolved upon caspase activation and could it relates to the speed at which caspase are activated (which may not leave enough time for strong aggregation and visible speck formation) ? I believe it would help to get more explanation/discussion on this point.

    5. The compensatory mechanisms that lead to cell death/extrusion despite depletion of caspb is very interesting. Could the authors use some pan caspase inhibitor ( like zvad FMK) to confirm that this block opto-ASC cell death also in this context ? Alternatively could they check the status of effector caspase activation using live probe (nucview) or immunostaining in the context of caspb depletion ?

    6. If I understand well, Figure 7C on the right side suggest that the double KO cells don't extrude (if indeed "no change" mean no extrusion, by the way this nomenclature may deserve some clarification in the legend). I don't think these results are mentioned at any point in the main text, but it would be important to include them (since this is an important control).

    7. Waves of calcium following cell death and cell extrusion have been previously characterised (Takeushui et al. Curr Biol 2020, Y Fujita group). Interestingly, in this previous article they observed waves of calcium near Caspase8 induced death (in MDCK) as well as near laser induced death in zebrafish, while apparently the authors don't see such Calcium waves upon Caspase8 activation in the zebrafish here. I think it would be important to include a comparison of the authors results with this previous paper in the discussion

    8. There is also a previous study which characterised the impact of caspase1 on cell extrusion (Bonfim Melo et al. Cell Report 2022, A. Yap lab) which promotes apical extrusion in Caco2 cells. I think it would also be important to include this work in the discussion and to compare with the results obtain here in vivo.

    Other minor points:

    1. Line 439: are the numbers given in percentage ? if these are absolute numbers, it is out of how many cells ? Same remark line 445 : what are the number of cases representing ? (percentage ?)

    2. Figure 5: could the authors show periderm and basal cell extrusion with the same type of markers ? (membrane or actin or ZO1) ? This would help to really compare accurately the morphology and the remodellings associated.

    3. Is there any obvious differences in cell size or characteristic cell shape between the classic lab strains (golden, AB, AB2B2) and the WIK and experiment strain used here ? I do acknowledge that this is clearly not the focus of this study, but given this striking difference (which is related to an important question in the field of extrusion), it would interesting to mention this if there is anything obvious.

    4. Figure 6C: what is exactly the localisation in Z of this strong actin accumulation observed during apical extrusion ? Is it apical or is it rather on the basal side of the cell ? A lateral view of actin could be useful in this figure for all the different conditions described.

    5. Figure S3B: could the authors show the utrophin-neonGreen channel separatly ? Is there a ring of actin in the dying cell ? Also are the membrane protrusion formed more basally ? (I suspect this is a z projection, but this would need to be specified in the legend).

    6. Figure 4A legend : I guess the authors meant red arrowheads rather than frame ?

    7. I list below a number of typos I could find in the main text

    Line 29: in

    Line 30: but

    Line 151 : from the ...[...] (tissue ?)

    Line 161: there is most likely a text commenting that was not removed (for how long?)

    Line 262: generated (egnrtd)

    Line 268: whereas showed a delay (the subject is missing)

    Line 269: a point is missing

    Line 362: which the lack

    Line 368: a point is missing

    Line 400: a space is lacking "cellsdepending"

    Line 438: shrinkwe (space)

    Line 459 : or I infections

    Line 525: there is a point missing.

    CROSS-CONSULTATION COMMENTS

    I generally agree with all the comments raised by the other reviewers which partially overlap with comments I had (see for instance referee two for the role of other caspases and the membrane localisation of the probe).

    Significance

    In this article, de Carvahlo and colleagues describe a novel optogenetic tool allowing single cell and temporally controlled induction of ASC clusters in vivo (in zebrafish), a central adaptator protein of the inflammasome complex which is involved in the induction of pyroptosis. This alternative mode of programmed cell death is involved in pathogen response and promote cell swelling and the release of pro-inflammatory factors. Previous works have shown that the inflammasome activation is associated with the formation of a large cluster of ASC protein (called speck) which promotes then the recruitment and the activation of caspase 1. Specks were previously characterised by the same group in vivo (in zebrafish larvae) and could be induced by the overexpression of ASC protein. This however was not compatible with fine spatio-temporal control of speck formation, thus preventing very refined characterisation of the dynamics and the distinction of the cell autonomous and non-cell autonomous effects.

    • By fusing ASC to the blue-light sensitive oligomerising protein Cry2-olig under the control of a heat shock promoter, they could induce time controlled induction of speck at the single cell level, which is then followed by cell extrusion and cell death both in the periderm and the basal cell of the skin of zebrafish larvae. Doing so, they could characterise the dynamics of speck formation as well as key paramters affecting its dynamics and the subsequent extrusion. While ASC activation led to apical or basal extrusion in the periderm layer followed by non-apoptotic cell death, it triggers basal extrusion and apoptosis in the basal layer. Importantly, periderm cell elimination does not seem to strictly follow all the features of pyroptosis as it does not require GSDM, and relies on Caspb (not Caspa). It is also associated with strong Calcium release both in the dying and neighbouring cells.

    • The authors performed a very careful characterisation of the tools and the optimisation of the condition to form speck and eliminate cells. The experiments are very well performed with all the necessary controls. The results, while to some extend still hard to fully interpret for some aspect, illustrate the plasticity of cell death and cell extrusion, which include several very interesting observations on the direction of extrusion, putative compensatory modes of cell death upon Caspase1 perturbation and the difference of response to ASC clustering depending on the tissue layer. While it is not the main point of this study, the observation that the direction of extrusion can vary very significantly in different genetic backgrounds is also extremely interesting.

    • The atypical cell elimination revealed in the system may require further characterisation in the future and suggest that the tools may not be the best to study bona fide pyroptosis. However, I don't believe there is always such strict separation between the modes of cell death and I am sure that it could lead to very interesting insights on inflammasome formation, extrusion and charcaterisation of downstream signalling in vivo, so overall this will be a very interesting resource for the community working on inflammasome, cell death and extrusion.

    • My expertise are in cell extrusion, optogenetics, apoptosis and epithelial mechanics. I am not a specialist of the inflammasome and pyroptosis.

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Programmed cell death is critical for host defense and tissue homeostasis. How dead cells initiate cellular responses in the microenvironment with neighbouring cells in vivo is still largely unknown. The authors have chosen a Zebrafish model to tackle this question, given that this model shows advantages for imaging and addresses these pathways in a complex in vivo setting. Their recent development of light-induced activation of caspases (published in JEM) enabled them to investigate cellular responses to a specific type of cell death in vivo at a single cell resolution. In this study, the author further developed a light-induced activation of ASC to specifically look at inflammasome activation-mediated cell death in vivo. The authors have successfully established this system in zebrafish and also observed that Opto-Asc-induced cell death showed different phenotypes as compared to Opto-caspase-a/b-induced cell death. However, it is not really clear why. I have a few specific comments to be addressed or discussed.

    1. In Fig.3 and Fig.4, the majority of Opto-Asc localizes to the plasma membrane but not endogenous Asc. It seems that tagging affects its localization, which could potentially explain its slow kinetics in oligomerization.

    2. In Fig.7, the authors showed that deletion of Caspb, but not Caspa, affected the apical extrusion, without affecting cell death. This may indicate that other caspases, like Caspase-8 or/and caspase-3 were involved. This could be addressed through deletion of Caspase-8 or/and caspase-3.

    3. It is very surprising that Opto-Asc-mediated cell death is not dependent on Gasdermins, at least in Caspb-dependent apically extruded dead cells.

    CROSS-CONSULTATION COMMENTS

    I agree with the other two reviewers and don't have further comments.

    Significance

    The Opto-Asc zebrafish model developed in this study will enable us to specifically look at inflammasome-mediated cell death in vivo. This model is more physiologically relevant compared to Opto-caspase1 model.

    Audience interested in physiological function of inflammasome activation, but it is questionable whether such a tool will address mechanisms in mammalian cells. Eventually, more evidence for the latter could be provided.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    ASC is the Pyrin/CARD-containing adapter protein that functions as a core component of inflammasome signaling complexes. ASC functions downstream of various NLR- and ALR-inflammasome initiator proteins and upstream of the inflammatory caspases that function as inflammasome effector enzymes. This study uses a novel chimeric construct (Opto-ASC) comprising the Arabidopsis photo-oligomerizable cryptochrome 2 (Cry2-olig) protein with zebrafish ASC to generate transgenic zebrafish larvae wherein ASC oligomerization can be rapidly, dynamically and spatially induced by blue light illumination of either the entire larva or single cells within discrete tissues of an intact larva. Induction of these "opto-inflammasome" complexes is coupled with state-of-the-art, live-cell optical imaging of multiple single cell and integrative tissue parameters to assay various modes of regulated cell death within the peridermal and basal cellular layers of the larval skin. This experimental model was further combined with genetic manipulation of the expression of various zebrafish inflammatory or apoptotic caspases, as well as the two zebrafish members of the gasdermin family of pore-forming proteins which can mediate disruption of plasma membrane permeability without (pre-lytic) or with (pyroptosis) progression to lytic cell death.

    The main results of the study are:

    1. introduction of a novel experimental system for dynamic and spatially resolved ASC oligomerization and speck formation within the cells of intact epithelial tissues of a living organism;

    2. the ability of these optically induced ASC oligomers/specks to drive multiple modes of regulated cell death which exhibit some (but not all) features of lytic pyroptosis or non-lytic apoptosis depending on cell type and tissue location;

    3. the ability of the dying epithelial cells containing optically-induced ASC specks to coordinate rapid adaptive responses in adjacent non-dying cells to maintain integrity/ continuity of skin epithelial barrier; and

    4. unexpectedly, no obvious role for either of the two zebrafish gasdermins in the regulated cell death responses.

    Major Comments:

    1. Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them? The major goal of this MS is to present a new experimental model (optogenetic activation of ASC oligomerization in transgenic zebrafish) that has the potential to provide new insights regarding the multiple mechanisms by which ASC can regulate inflammasome/ cell death signaling responses in the context of an intact organism. As noted above, some of the observed results are unexpected (e.g., lytic cell death independent of the zebrafish gasdermins in particular epithelial cells) and may reflect mechanisms unique to zebrafish as a non-mammalian vertebrate model versus the mammalian experimental systems (murine and human) that have informed most of our current understanding of how ASC coordinates inflammasome and cell death responses. However, the authors have used rigorous genetic approaches to rule out trivial explanations for the unexpected observations. Thus, no major additional experiments are required to support the claims and conclusions presented in the MS.

    2. Are the suggested experiments realistic in terms of time and resources? Yes. It would help if you could add an estimated time investment for substantial experiments: A few weeks.

    3. Are the data and the methods presented in such a way that they can be reproduced? Are the experiments adequately replicated and statistical analysis adequate? Yes.

    4. Are the experiments adequately replicated and statistical analysis adequate? Yes.

    Minor comments:

    1. Specific experimental issues that are easily addressable:

    There's a significant concern with the use of LDC7559 (line 387) as a putative small molecule inhibitor of gasdermin D function to test roles (or lack thereof) of the zebrafish gasdermins in the ASC-triggered lytic cell death responses. A recent study (Amara et al. 2021. Cell. PMID34320407) has reported that LDC7559 does not inhibit gasdermin D (and possibly other gasdermins) but rather acts as an allosteric activator of PFKL (phosphofructosekinase-1 liver type) in neutrophils and thereby suppress generation of the NADPH required for the phagocytic oxidative burst and consequent NETosis. Thus, use of LDC7559 as a presumed gasdermin inhibitor in the current MS is problematic and should be deleted. As an alternative pharmacological approach to suppress gasdermin function, the authors might consider the use of either disulfiram (Hu et al. 2020. Nature Immunology. PMID32367036) and/or dimethylfumarate (Humphries et al. Science. 2020. PMID32820063). These would be straightforward additional experiments.

    1. Are prior studies referenced appropriately? there are some problems; see below.

    2a. One paper is cited twice in lines 724-726 and 727-729.

    2b. Another paper is cited twice in lines 790-792 and 793-795.

    2c. No journal is included for the referenced study by Shkarina et al in lines 827-828.

    2d. No journal is included for the referenced study by Stein et al in lines 831-832.

    2e. No journal is included for the referenced study by Masumoto et al in lines 793-795.

    2f. No journal is included for the referenced study by Kuri et al in lines 774-775.

    1. Are the text and figures clear and accurate? Generally, yes but with a few exceptions noted below:

    3a. line 28: "morphological distinct" should read "morphologically distinct"

    3b. line 161: this sentence contains in parentheses "for how long?" I think this was a comment by one author that wasn't removed from the final submitted MS

    3c. line 945: spelling "balck" > "black"

    3d. line 268: "whereas showed a delayed speck formation": the authors need to specify what model/ condition showed a delayed speck formation

    3e. line 262: spelling "egnerated" > "generated"

    CROSS-CONSULTATION COMMENTS

    I also agree with the comments of the other 2 reviewers. Between the 3 sets of comments and suggestions, the aggregate review will provide the authors with a suitable range of feasible recommendations that will improve an already strong MS.

    Significance

    1. General assessment:

    As noted above, this the major goal of this MS is to present a new experimental model (optogenetic activation of ASC oligomerization in transgenic zebrafish) that has the potential to provide new insights regarding the multiple mechanisms by which ASC can regulate inflammasome/ cell death signaling responses in the context of an intact organism. The authors have used rigorous genetic approaches to rule out trivial explanations for the unexpected observations. In general, the MS describes an elegant model system that will provide a platform for identifying new mechanisms of ASC-dependent inflammasome signaling and regulated cell death.

    1. Advance:

    This MS describes a highly novel experimental model. Zebrafish are increasingly being used as a genetically tractable model for inflammasome signaling within integrated tissues of intact organism. As noted above, the advances are technical but also conceptual. Future application of this novel model is likely to yield identification of new mechanisms for ASC function in innate immunity and regulated cell death within the context of tissue homeostasis and host defense.

    1. Audience:

    Basic research and discovery.

    1. Please define your field of expertise with a few keywords to help the authors contextualize your point of view:

    My group investigates multiple aspects of inflammasome signaling biology at the cellular level with an emphasis on cell-type specific roles for gasdermins in coordinating downstream innate immune responses to inflammasome activation in various myeloid leukocytes (macrophages, dendritic cells, neutrophils, eosinophils, mast cells).

  6. Version published to 10.1101/2022.10.19.512883v2 on bioRxiv
  7. Version published to 10.1101/2022.10.19.512883v1 on bioRxiv