Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components

  1. Gabriel Muciño-Hernández
  2. Pilar Sarah Acevo-Rodríguez
  3. Sandra Cabrera-Benitez
  4. Adán Oswaldo Guerrero
  5. Horacio Merchant-Larios
  6. Susana Castro-Obregón

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Abstract

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

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  1. Version published to 10.1242/jcs.260563
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    Reply to the reviewers

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

    This paper examines the formation and repair of micronuclei in non-cancerous cells, specifically in mouse embryonic fibroblasts. This work was performed completely in culture and used a combination of western blot, confocal and superresolution microscopy to assess the contents of micronuclei over a repair period of 5 hours after 2 hours of induction of double strand breaks by treatment with etoposide. The authors found that the bodies colocalised with LC3, Beclin 1 and lysosomes suggestive of autophagy. However no evidence of autophagic flux has been demonstrated.

    Major issues are as follows:

    Figure 2

    A - Any sense of the autophagic flux? LC3B - I and LC3B - II seem to be in equal quantities most of the time. Maybe using the tandem LC3 in this system could provide further insight. Also remove the violin plots from this graph and from G and H, as there are too few data points.

    Thank you for your comment. We have evidence of a functional autophagic flux, since we observed an increasing number of acidic vesicles stained with Lysotracker in response to DNA damage, which were reduced after DNA repair. Some of the micronuclei were also co-stained with Lysotracker, suggesting their lysosomal degradation. We reorganized the data in the revised figure 2A to communicate better these observations. We reproduce here the dynamic of Lysotracker stain, please notice an increase in the abundance of acidic vesicles after 2h of DNA damage. A further evidence of activation of functional autophagy is the dynamic intracellular distribution of both LC3 and BECN1, indicative of autophagy induction. Please notice in revised Figure 2A that LC3 surrounding vesicles increases after 2h of DNA damage and diminish when DNA is repaired. BECN1 in control MEFs is highly concentrated inside the nucleus, predominantly at the nucleolus, and after DNA damage it redistributes towards the cytoplasm. Finally after DNA repair, BECN1 appears highly concentrated at the nucleus again. These dynamic changes correlate with autophagosomes formation and successful fusion with lysosomes. In the revised manuscript we removed the violin plot as suggested. Since the elimination of nuclear components occurs in a subset of cells, the role of the autophagic machinery needs to be analyzed cell by cell. We considered better to eliminate also the Western blot, as an analysis of the whole population does not provide information relevant for this study.

    Can you reduce the brightness in the merge image, as I cannot see DAPI nor a convincing Beclin-1/LC3 co-localisation.

    Thank you for the observation. We improved the quality of the images and reorganized Figure 2 to convincingly show BECN1 and LC3 co-localization, together with Lysotracker, in nuclear alterations (buds and micronuclei). We modified the results text accordingly.

    Although the data is convincing, It would be clearer if the brightness of the merge image was reduced.

    Thank you for your comment. We improved the images shown, these data is now integrated in new Figure 2A.

    Is the significant result the difference between 5h R Control si and 5h R Atg7? if so, there is no significant change in micronuclei as the same time point, can you explain this disconnect? are the buds being degraded prior to becoming micronuclei?

    That is correct, we found no statistical significant difference in the number of micronuclei formed silencing Atg7, although there was a trend to reduce them. To consolidate the role of autophagy in nuclear buds and micronuclei formation, we studied Atg4-/- MEFs. We confirmed a statistical significant reduction of buds formation when autophagy is impaired (new Figure 2G). However, we observed that the number of micronuclei increased after 2h of DNA damage in Atg4-/- MEFs, suggesting that autophagy does not contribute to micronuclei formation but elimination. Together, our results suggest that the origin of buds and micronuclei are mechanistically different. A difference in the biogenesis of buds and micronuclei has been previously suggested studying cells cultured under strong stress conditions that induce DNA amplification, as well as in cells under folic acid deficiency. While interstitial DNA without telomere was more prevalent in buds than in micronuclei, telomeric DNA was more frequently observed in micronuclei (Fennech et al. 2011, Mutagenesis 26:125-132). We agree with the reviewer, it seems that not all the buds become micronuclei.

    Figure 3 A - nice microscopy showing the co-localisation of TOP2A and LC3-GFP. I'm interested in DAPI being on some bodies and not others. Do you have any sense of the dynamics of this?

    Thank you for the interesting question. Since removal of nuclear alterations as nuclear buds and micronuclei is a very dynamic process, we detect nuclear damaged material in the cytoplasm are at different degradation stages. Nucleases could be degrading DNA in micronuclei. Another possibility to the lack of DAPI signal in some micronuclei containing TOP2A and GFP-LC·is that TOP2A could be expelled from the nucleus with undetectable fragments of DNA or even without DNA, as a renewal process. We believe that nuclear buds can form without extruding DNA in some cases, perhaps to modulate proteostasis in addition to protect genome stability. In the revised manuscript we discuss this possibility further.

    G - c shows a strand of mostly TOP2B coming from the nucleus. Is there any evidence that this occurs using either confocal microscopy or super resolution approaches. Could you try Z-stack to find these?

    Thank you for the suggestion, we analyzed Z-stack images and tried to observe it also by immunofluorescence. We could detect some tubular signal connecting the nucleolus with a micronucleus containing TOP2B and BECN1 (arrow head in Fig 3B reproduced below), although we cannot be certain we are detecting the same nuclear extrusion mechanism by Electron Microscopy than by immunofluorescence.

    Figure 4 C - is there a significant increase in FBL negative bodies, this would make sense if FBN is being degraded in the micronuclei during the repair process

    We found that the number of micronuclei without FBL increased with statistical significant difference by Two-way-ANOVA followed by Dunnett´s multiple comparison test (P=0.463 comparing cells with 2h of DNA damage with control cells; P=0.0017 comparing cells after 5h of DNA repair with untreated cells; n=5). We agree with the reviewer, a possible explanation is that FBL is being degraded in micronuclei during the repair process. Although it could also be possible that nucleolar is less sensitive to Etoposide poisoning, or that nucleolar DDR is mechanistically different.

    Would it be possible to increase the n of these experiments to confirm either no change in FBL/LC3 co-loc, or evidence of increase?

    Thank you for the suggestion. We repeated the experiment two more times to increase the n to 5. We found no statistical difference in the number of nuclear buds or micronuclei containing both FBL and LC3 during DNA damage and repair. Therefore it seems that the release of nucleolar components is not enhanced by Etoposide-induced DSB, suggesting that nucleolar DDR is a unique response, independent of DDR elsewhere in the genome (reviewed in Nucleic Acids Research, 2020, Vol. 48, No. 17 9449–9461 doi: 10.1093/nar/gkaa713).

    Minor issues:

    Figure 4 and 5 legends are in a different font.

    Thank you. We correct the font in the current manuscript.

    Reviewer #1 (Significance (Required)):

    There is little specific data on the role of autophagy in clearing micronuclei in cancer cells, so this may be suggestive of a new mechanism that occur during normal cellular homeostasis. There are known links between lamin A defects and the formation of micronuclei, but not explicitly that the micronuclei are also Lamin A positive. it is likely that analogous processes occur in both cancer and non-cancer, so the impact of these data is not clear to me. This paper may be of interest to researchers interested in nuclear structure and DNA damage, but based on the data presented the significance is limited.

    The significance of the present work is to discover that autophagy is relevant both during physiological DNA damage and in response to an exogenous DNA damaging agent, to extrude damaged DNA, TOP2cc and Fibrillarin from the nucleus. This knowledge is relevant since insufficiencies on autophagy imply a risk of genomic instability, which in turn could drive the cell into a senescent or malignant state. We present data showing that autophagy regulates the dynamic formation and elimination of nuclear buds and micronuclei in a mechanistically differentiated way. While autophagy contributes to nuclear buds formation, it is necessary for micronuclei elimination. Our data suggest that nucleophagy could be also a mechanism to alleviate basal nucleolar stress. As the reviewer noticed, some micronuclei did not have DNA. It is conceivable then that nuclear buds and micronuclei form also for a proteostatic function, not necessarily involving DNA damage elimination. We believe the significance of our work contributes to our understanding of the cell, as well as to cancer research. Whether common mechanisms between cancerous and normal cells occur is relevant to know, to consider the specificity of potential therapeutic approaches.

    I don't have sufficient expertise to evaluate the super resolution microscopy beyond assessing the images.

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

    Peer review of the manuscript with the number RC-2021-01181 by Muciño-Hernandez G et. al. at Review Commons and with the tittle "Nucleophagy contributes to genome stability 1 though TOP2cc and nucleolar components degradation"

    Summary Muciño-Hernandez G et. al. show in this manuscript that mouse embryonic fibroblasts (MEFs) have basal levels of nuclear buds and micronuclei, which are indicators of genomic DNA damage. These basal levels of nuclear buds and micronuclei in MEFs increased after Etoposide treatment, which is known to induce DNA Double stranded Breaks (DSD). Interestingly, the nuclear buds and micronuclei co-localize with makers for nucleophagy (BECN1 and LC3) and acidic vesicles, suggesting that they are cleared by nucleophagy. The authors propose that basal levels of nucleophagy clear basal levels of genomic DNA damage that occurs as result from DNA-dependent biological processes in the cell nucleus, thereby contributing to nuclear stability of MEFs under physiological conditions. These basal levels of nucleophagy increase after the action of factors that induce DNA damage and nuclear stress. The concepts proposed by Muciño-Hernandez G et. al. are novel, since most of the current published data on nucleophagy related to DNA damage have been obtained under pathological conditions, e.g. implementing cancer cells.

    The authors use in their manuscript various molecular biology techniques to obtain data that support their claims, including Western Blot analysis of protein extracts from MEFs, immunostaining on MEFs and neutral comet assays, complemented with state of the art imaging techniques, such as confocal microscopy, immunoelectron microscopy and super resolution microscopy. The quality of the data is sound. The structure of the manuscript support the understanding of the reader. However, I would like to suggest several improvements that will help to increase the quality of the manuscript, in order that fits to the standards of articles recently published in journals affiliated to Review Commons, such as the Journal of Cell Biology, the EMBO Journal or eLife.

    1. Major comments

    2.1 The authors have to improve the description of the results. Especially the description of those Figure panels containing plots that were generated using data from several experiments has to be improved.

    One example is the description of the Figure 1D, which is in the lanes 137-151 of the current version of the manuscript. Whereas the authors describe in lanes 137-147 observations related to representative pictures of confocal microscopy after immunostaining presented in Figure 1D (left), the description of the quantification from 9 independent experiments presented in the plots in Figure 1D (right) comes relatively short in lanes 147-150 without mentioning any of the values implemented for creating the plots.

    "Interestingly, while the frequency of nuclear buds gradually increased after DNA damage and during DNA repair, the frequency of micronuclei also increased after DNA damage, but diminished upon DNA repair."

    The other plots presented in the different figure panels across the manuscript are described in a similar manner. I would like to suggest to the authors to improve their manuscript by including during the description of their results the values that were implemented for the degeneration of the plots presented in the manuscript. For example, in the specific case of Figure 1D above:

    "Interestingly, the percentage of MEFs with nuclear buds gradually increased from XY% ({plus minus} XY SD) in control non-treated (Ctrl) MEFs to XY% ({plus minus} XY SD; P=XY) after 2 h Etoposide-induced DSB in MEFs and XY% ({plus minus} XY SD; P=XY) after DNA repair take place in MEFSs 5 h upon stop of Etoposide treatment (Figure 1D, right). In contrast, the percentage of MEFs with micronuclei significantly increased from XY% ({plus minus} XY SD) in Ctrl MEFs to XY% ({plus minus} XY SD; P=XY) after 2 h Etoposide-induced DSB, whereas it was reduced to XY% ({plus minus} XY SD; P=XY) 5 h after stop of Etoposide treatment (Figure 1D, right)."

    Descriptions of the plots as mentioned above will make the text more intuitive for the reader, and they will make possible to read the Results Section without switching to the Figure Legends or the Material and Methods Section or to Supplementary Files. Even though the representative pictures from different microscopy techniques presented in the manuscript are of good quality and support the claims of the authors, it is important to mention that the quantifications presented in the plots demonstrate the statistical significance of these representative pictures. Thus, the authors should consistently include in the manuscript during the description of theirs results all the information (mean values, standard error of the means, P values, n values, etc.) that support their interpretation of the results and demonstrate the statistical significance of their claims.

    Thank you for your clear and valuable advice. We followed it and in the revised manuscript we included the data in the results section.

    2.2 Following a similar line of argumentation as in the previous point, the authors should provide as Supplementary Material an Excel file containing a statistical summary, including all statistical relevant information from each one of the plots presented in each Figure panel, such as n values, P values, Test implemented, values used for the plots, numbers of experiments, etc. The information could be organized in the Excel file in different data sheets according to the Figure panels, in order that the reader can easily navigate through the data. In the current version of the manuscript, one cannot find the values used for the generation of the plots presented in the manuscript in any of the submitted files.

    Thank your for this suggestion. We have included in Table S1 an Excel file with a data sheet for each Figure panel, containing all the data collected and the statistical analysis performed.

    Minor comments

    3.1 In general, prior studies were appropriately referenced. Only few references has to be added.

    Line 48: Add to the already included reference "Dobersch et al., 2021" also the reference Singh et al., 2015 PMID 26045162.

    Thank you, we added this reference.

    Line 53: Add the corresponding reference after the word "respectively".

    We added the corresponding reference.

    Line 82: Add the corresponding reference after the word "them".

    We added the corresponding reference.

    Line 125: Add the corresponding reference after the word "cells".

    We added the corresponding reference.

    Line 130: The expression "...by analyzing the recruitment of the phosphorylated histone γH2AX..." is the first time that the authors mention in the manuscript the DNA damage maker γH2AX. I suggest that is better introduced as " ... by analyzing the recruitment of the DNA damage marker γH2AX (histone variant H2A.X phosphorylated a serine 139, Rogakou EP, et al., 1998, PMID 9488723) to DSB sites."

    Thank you very much for your suggestion. In the revised manuscript we corrected the text as suggested.

    Line 199: Add the corresponding reference after the word "formation".

    We added the corresponding reference.

    Line 205: Add the corresponding reference after the word "cells".

    We added the corresponding reference.

    3.2 The use of the English language is appropriate throughout the manuscript. However, there are minor errors in the use of punctuation marks, in the use of prepositions and typos. I will list some of them below. However, I would like to recommend that manuscript is corrected by an English native speaker.

    Thank you for your careful review of our manuscript. We corrected all the errors listed. A college proficient in English has reviewed the revised manuscript.

    Line 41: "...and reproductive systems; genome instability also..." the semicolon can be replaced by a period.

    √

    Line 43: "Since early in development DNA is under constant endogenous..." between "development" and "DNA" there should a comma.

    √

    The sentence in lanes 53-55 has to be rephrased.

    √

    Lines 62-63: the expression "...throughout life." should be substituted.

    √

    Line 70: The abbreviation "rDNA" has to be explained the first time that is used.

    √

    Lines 81-82: It has to be explained for the scientist that is not specialized in the field of nucleophagy, how the integrity of the genome is threatened by micronuclei and nuclei-derived material.

    √ Lines 106-110: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences.

    √

    Lines 121-122: The subtitle should be rephrased.

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    Lines 132-138: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences, e.g. with a period before the word "hence".

    √

    Lines 143-144: "... in a subpopulation of healthy, untreated cells...". The interpretation of "healthy" might be subjective. I would like to suggest substituting in the complete manuscript the word "healthy" by "control".

    √

    Line 163: The abbreviation for γH2AX was already introduced in line 130.

    √

    Line 182: A comma after "cell lines" is missed.

    √

    Line 183: delete "either". √ Lines 190-194: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences, e.g. with a period after the word "decreased" in line 191.

    √

    Line 218: I assume that instead of "bus", it should be "buds".

    √

    Line 220: I assume that instead of "iRNA", it should be "siRNA". In addition, it is the first time that the abbreviation is used. Thus, I suggest introducing it as "...was silenced by specific small interfering RNA (siRNA) previous to ..."

    √

    Line 327: delete the word "chronic".

    √

    Line 344: I assume that instead of "(figures 4C)", it should be "(Figure 4D)".

    √

    3.3 The structure of the Figures is ok for the peer review process and it might be optimized during editing of the manuscript. Nevertheless, I would like to suggest to the authors to increase the lettering size throughout all the figures. It will make the figures more intuitive.

    Thank you for the suggestion. We increase the font size of the figures.

    Reviewer #2 (Significance (Required)):

    Significance

    The work presented by Muciño-Hernandez G et. al. will be clearly a significant contribution to the scientific community working on autophagy, DNA damage repair and cancer, among others. It will be of interest to a broad spectrum of scientists, as I will elaborate in the following lines. The authors propose that MEFs have basal levels of genomic DNA damage under physiological conditions, which are cleared by basal levels of nucleophagy. On one hand, these findings are in line with various publications demonstrating that DNA-dependent biological processes in the cell nucleus, such as transcription, replication, recombination, and repair, involve intermediates with DNA breaks that may compromise the integrity of DNA. Thus, there must be mechanisms that ensure the integrity of the genome during these processes under physiological conditions, one of them seems to be nucleophagy. This perspective might explain the fact that proteins and histone modifications that were initially characterized during DNA repair also play a role during transcription, recombination, and replication. For example, phosphorylated H2AX at S139 (γH2AX) is often used as a marker for DNA-DSB [PMID 9488723]. However, accumulating evidences suggest additional functions of this histone modification [PMIDs 19377486; 22628289; 23382544]. In addition, McManus et al. [PMID 16030261] analyzed the dynamics of γH2AX in normal growing mammalian cells and found γH2AX in all phases of cell cycle with a maximum during M phase, suggesting that γH2AX may contribute to the fidelity of the mitotic process, even in the absence of ectopic- induced DNA damage. Further, Singh et al [PMID 26045162] and Dobersch et al [PMID 33594057] report that γH2AX plays a role in transcriptional activation in response to TGFB-signaling. Moreover, classical DNA-repair complexes have been linked to DNA demethylation and transcriptional activation [PMIDs 17268471; 28512237; 25901318], and DNA-DSB is known to induce ectopic transcription that is essential for repair, supporting a tight mechanistic correlation between transcription, DNA damage, and repair [PMID 24207023]. Perhaps, the authors might consider introducing several of the aspects and the citations written above into the Discussion section of the revised version of their manuscript. On the other hand, most of the published data related to nucleophagy have been obtained from cancer cells. Muciño-Hernandez G et. al. obtained their data implementing MEFs to demonstrate that the proposed mechanisms take also place under non-pathological conditions, what is one of the novel aspects of the present work.

    I hope that my suggestions help the authors to improve their manuscript, thereby reaching the standards of manuscripts recently published in journals affiliated to Review Commons AND increasing the impact of their contribution to the scientific community.

    Thank you very much for your suggestions. They helped us to present now a much-improved manuscript. We hope the revised work is now suitable for publication in the Journal of Cell Science.

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

    In this manuscript, Muciño-Hernández and colleagues suggest that basal formation of nuclear buds and micronuclei increases in primary mouse embryonic fibroblasts following etoposide-induced double strand breaks (DSBs). The study combines the use of biochemical methodologies with confocal and super resolution microscopy in an effort to explore the contribution of nucleophagy to genome stability. The authors provide evidence that autophagy is induced upon etoposide treatment. They detected GFP-LC3 and BECN1 signals in nuclear buds and micronuclei even in untreated control and to a higher extent in etoposide-treated cells. Then, the authors examined whether nucleophagy is required for the removal of nuclear buds and micronuclei, by treating fibroblasts with control and Atg7 siRNA. The authors claim that the percentage of cells with micronuclei or nuclear buds decrease upon Atg7 knockdown, suggesting that components of the autophagy machinery induce the formation of these nuclear abnormalities. Moreover, Type II DNA Topoisomerases (TOP2A and TOP2B) and the ribosomal protein fibrillarin were detected in nuclear buds and micronuclei in fibroblasts treated or not with etoposide. Again in this case, GFP-LC3 was detected in fibrillarin-containing nuclear alterations. Based on these observations, the authors suggest that nucleophagy contributes to the elimination of chromosomal fragments or nucleolar bodies exiting the nucleus under DNA damage -inducing conditions. Specifically, they propose a key role for nucleophagy in maintaining genome stability by eliminating Type II DNA Topoisomerase cleavage complex (TOP2cc) and nucleolar components such as fibrillarin.

    While it seems that there is a relationship between nuclear-extruded TOP2 with endogenous BECN1 and GFP-LC3 suggesting autophagic engagement, inconsistencies of fluorescent images between different figures indicate possible technical problems/limitations (please see specific comments, below), compromising authors' claims. LC3 immunoblotting and GFP-LC3 localization results appear over-interpreted (comments below). Neither TOP2 nor Fibrillarin have been shown to be actual autophagic substrates. Also, the link between genomic stability, micronuclei formation and autophagy has been previously reported (Zhao et al., PMID: 33752561).

    An additional major concern is relates to nucleophagy being a selective type of autophagy. As such it requires efficient recognition and sequestration of the nuclear material destined to be degraded. Cargo specificity is mediated by receptor proteins, but no evidence for such receptors is provided in this study. Moreover, there is no real mechanistic insight on how nucleophagy mediates genome stability and how this can be interpreted in terms of cell survival under physiological and stress conditions. In other words, the biological significance of the findings presented has not been addressed.

    Specific comments are summarized below:

    The authors suggest that autophagy is induced after etoposide treatment and during the DNA repair process. However, the Western blot presented in Fig. 2A is not convincing and quantification does not support a significant autophagy induction in any of these cases. Autophagy appears to be induced 1h after etoposide removal, as evidenced LC3II/LC3 I increase (Fig. 2A and S2A). Nevertheless, all these changes should be more rigorously assessed.

    Thank you for the observation. We removed the analysis of LC3II/LC3I by Western blot in the revised manuscript because a basal and induced elimination of nuclear components by the autophagic machinery occurs only in a subset of cells. It needs to be analyzed cell by cell. Pooling together all the cells dilutes the observation. Nevertheless, the dynamic intracellular distribution of both LC3 and BECN1 indicate autophagy induction. Please notice in revised Figure 2A that LC3 surrounding vesicles increases after 2h of DNA damage and diminish when DNA is repaired. BECN1 in control MEFs is highly concentrated inside the nucleus, at the nucleolus as it co-localized with Fibrillarin (new Figure 4E), and after DNA damage it redistributes towards the cytoplasm. Finally after DNA repair, BECN1 appears highly concentrated at the nucleus again. A further evidence of a functional autophagic flux, is the observation of an increasing number of acidic vesicles stained with Lysotracker in response to DNA damage, which were reduced after DNA repair. Some of the micronuclei were also co-stained with Lysotracker, suggesting their lysosomal degradation.

    Line 190 and Fig. 2A: It is totally unclear whether "autophagy activation" takes place during the two waves described. There is no LC3B-I to LC3B-II conversion to initially suggest "autophagy activation". It rather suggests that autophagy is stalled. Fig. 2F shows that GFP-LC3 is strongly fluorescent into the lysotracker-stained lysosomes, further pointing to possible functional or technical problems.

    As pointed out by reviewer 1, the images presented in original Figure 2F were over-exposed. In the current version we replaced those images with new images of better quality. We also reorganized the presentation of the data, and in revised Figure 2A we present photos where more convincingly can be observed a co-localization of BECN1 with LC3, with o without Lysotracker signal in nuclear buds and micronuclei. We also performed immunolocalization of endogenous LC3 (new Figure 2D) to rule out a possible misinterpretation of GFP-LC3 aggregates. As explained before, we removed original Figure2A.

    Fig. 2B and Sup. Fig. 2B: BECN1 staining looks problematic. There is extreme BECN1 accumulation in the nucleus. Are those nuclear patterns of endogenous BECN1 and GFP-LC3 normal (see also minor comment 6 and 7)? Is there literature supporting such a distribution?

    Yes, it has been documented BECN1 localization in the nucleus during development and in response to DNA damage stimuli such as ionizing radiation, and with a function related to DNA repair alternative to autophagosome formation (Fei Xu, et al. 2017, Scientific Reports | 7:45385 | DOI: 10.1038/srep45385). In the current manuscript we also detected endogenous LC3, to avoid a possible artifact with GFP-LC3 expression. We observed endogenous LC3 also localized in the nucleus (new Figure 2D).

    It is hard to imagine how BECL1 is implicated in a (here hypothetical) nuclear lamina degradation event driven by LC3-lamin B1 direct interaction (Dou et al., 2015). BECL1 is an upstream to LC3 component and is a subunit of the PI3K complex catalyzing the local PI3P generation. The above should cause recruitment of the downstream autophagic machinery. Other subunits of the same complex or downstream effectors should be identified at the same spots to support authors' claims.

    Our proposal that BECN1 is contributing to nucleophagy is supported by its co-localization with LC3 and Lysotracker stained vesicles (new figure 2A), as well as with TOP2 (Figure 3A-C). We appreciate the interesting idea of the reviewer; we certainly did not analyze the presence of BECN1 interacting partners. We agree, further studies analyzing their localization could complement our current findings. Supporting our work, others have observed UVRAG in the nucleus, specifically in centromeric regions, and it also has a role in DNA repair through its interaction with DNA-PK (Dev Cell. 2012 May 15; 22(5): 1001–1016. doi: 10.1016/j.devcel.2011.12.027). Given the anti-tumorigenic role of several autophagic molecules, it is tempting to speculate that several of them could have triple roles in the nucleus: directly interacting with DNA repair machinery, eliminating unrepairable DNA damaged and preventing excessive protein accumulation in the nucleus. Further experiments are necessary to probe this hypothesis, but are beyond the scope of the present manuscript.

    U, 2h D and 5h R images of whole cells are necessary. The authors should also provide representative images of cells under different conditions i.e. control, etoposide-treatment and during DNA repair. Along similar lines, untreated control cells are not included in Fig. 2E and F. These images are needed for a better comparison between normal and DNA damage-inducing conditions.

    The reviewer is right. In the revised Figure 2 we included representative images of control cell, Etoposide-treatment and during DNA repair cells. Images of whole cells are now shown in supplementary Figure 2S.

    The authors state that autophagy is required for nuclear buds and micronuclei formation. However, the data shown in Fig. 2G and H are hardly convincing given that the statistical difference between cells treated with control and Atg7 siRNA is not strong (for example, *p˂0.5, 5h after etoposide removal). To provide further support to this notion, they should use cells from autophagy defective mutants and examine the appearance of nuclear abnormalities across different conditions compared to control cells.

    We agree with the reviewer and followed his/her suggestion. We established collaboration with Dr. Sandra Cabrera, who kindly shared with us *Atg4b-/- *mice from which we isolated MEFs to compare side by side with WT MEFs the appearance of nuclear abnormalities. We confirmed a statistical significant reduction in the formation of nuclear buds in both conditions: silencing the expression of Atg7 by siRNA and in *Atg4b-/- *MEFs, suggesting that the autophagic machinery contributes to buds formation (new Figure 2F-G). Interestingly, we observed a different result analyzing micronuclei. While we found no statistical significant difference in the percentage of cells with micronuclei silencing the expression of Atg7 by siRNA, we found a statistical significant increment of cells with micronuclei in *Atg4b-/- *MEFs (new Figure 2F-G). This apparently discrepant result suggests that nuclear buds and micronuclei have a different mechanistic origin. A difference in the biogenesis of buds and micronuclei has been previously suggested studying cells cultured under strong stress conditions that induce DNA amplification, as well as in cells under folic acid deficiency. While interstitial DNA without telomere was more prevalent in buds than in micronuclei, telomeric DNA was more frequently observed in micronuclei (Fennech et al. 2011, Mutagenesis 26:125-132).

    Lines 223-228: The role of autophagic machinery in the formation of nuclear buds is not supported and furthermore hard to conceptualize. How the components of autophagy are implicated during the nuclear buds and micronuclei formation? Colocalization of autophagic proteins might mean that autophagy is engaged at some point after or during the above formation. The causal, mechanistic and temporal aspects of the above budding and nucleophagic events need experimental support and/or more accurate interpretation.

    We agree with the reviewer, and now we expressed our interpretation with more caution. The role of autophagic machinery in the formation of nuclear buds is supported by the following findings: a) the localization of LC3 and BECN1 in nuclear buds; b) the inhibition of Atg7 expression by specific siRNAs reduced the number of cells with buds and c) *Atg4b-/- *MEFs had reduced number of cells with buds (new Figure 2G). How the components of autophagic machinery are implicated in nuclear buds formation is an interesting question and deserves further investigation, beyond the scope of the present manuscript.

    The authors claim that nucleophagy eliminates topoisomerase cleavage complex because TOP2A and TOP2B appear to more extensively co-localize with GFP-LC3 and BECN1 after etoposide-induced DSBs. However, the quantification presented in Fig. 3D-F to support this statement does not, in general, show a statistically significant difference in fibroblasts across different conditions (normal, etoposide treatment, etoposide removal).

    Autophagic elimination of TOP2 protein is supported by the following findings: 1) both BECN1 and LC3 were detected in micronuclei in acidic vesicles (labeled with Lysotracker), which is indicative of the autolysosomal nature of the cytoplasmic compartment containing TOP2 (Figure 2A); 2) TOP2B was found by electron microscopy in some cells exiting the nucleus surrounded by LC3 (Figure 3G); 3) TOP2B accumulated in cells lacking ATG4, as expected if it is degraded by autophagy (Figure 3H).

    Why would BECLIN colocalise with TOP2B in Figure 3g, given that beclin is involved in the initiation process?

    We think that BECN1 is involved in additional functions to the initiation process of bud formation. For example, it has been shown by others that BECN interacts with TOP2 (Dev Cell. 2012 May 15; 22(5): 1001–1016. doi: 10.1016/j.devcel.2011.12.027). It could be working as an autophagic receptor targeting TOP2cc to buds and micronuclei. We are aware that further studies are necessary to test this hypothesis, but they are beyond the scope of this manuscript.

    Fig. 4A and B: There is no enrichment of GFP-LC3 in "the nuclear alterations containing Fibrillarin" as stated in lines 341-343 comparing to the rest of the cellular GFP fluorescence.

    It is true that there is not a local enrichment of GFP-LC3 as those normally reported as LC3 puncta in response to autophagy induction by starvation, for example. Nevertheless we are confident of the specificity of the observation, as not every nuclear alteration was found having GFP-LC3. We detected GFP-LC3 in 72% (mean ± 3.61 SD) of the nuclear alterations containing Fibrillarin in untreated cells, in 65.7% (mean ± 1.97 SD) of cells with 2h of DNA damage and in 90.33% (mean ±6.36 SD) after 5 h of DNA repair (in 5 independent experiments).

    Moreover, there is no statistical significance in Fig. 4C and D measurements limiting the safety of authors' conclusions in lines 341-346.

    We agree with reviewer´s observation. We repeated these experiments two more times and did not find a statistical significant difference in the percentage of cells with nuclear lesions containing Fibrillarin and GFP-LC3 after DNA damage nor after DNA repair. These results suggest that nucleolar DDR is a particular response, independent of DDR elsewhere in the genome, as has been suggested (reviewed in Nucleic Acids Research, 2020, Vol. 48, No. 17 9449–9461; doi: 10.1093/nar/gkaa713). An alternative is that the release of nucleolar components is not enhanced by Etoposide at the dose and time used in this work.

    Lines 368-370: As discussed by the authors and reported in previous publication (Xu et al., 2017), "BECN1 interacts directly with TOP2B, which leads to the activation of DNA repair proteins, and the formation of NR and DNA-PK repair complexes", independent of its role in autophagy. Currently, there are no rigorous findings supporting the contribution of BECN1 (as a functional constituent of the core autophagic machinery) to nuclear damaged material extrusion (lines 382-384).

    We agree with the reviewer in that we did not perform an assay to demonstrate that BECN1 is contributing to TOP2 nuclear extrusion as a functional constituent of the core autophagic machinery. Nevertheless, the following data support the proposal of an autophagic elimination of TOP2cc: 1) TOP2B was detected in micronuclei containing BECN1 (Figure 3B); 2) BECN1 was found in micronuclei containing LC3 and in an acidic vesicle (labeled with Lysotracker), indicative of the autolysosomal nature of the compartment (Figure 2A); 3) TOP2 was found in some cells exiting the nucleus surrounded by LC3 (Figure 3G); d) TOP2 accumulated in cells lacking ATG4, suggesting its autophagic degradation (Figure 3H).

    Lines 435-441 and Fig. 5: The current findings do not support the proposed model. It is hard to support and conceptualize the statement "proteasome and nucelophagy function in a dynamic way inside the nucleus".

    The reviewer is right. We made a mistake integrating an interpretation within the summary of the actual findings of this work. We correct the text in the current version.

    In Fig. 5, LC3 appears to decorate inner nuclear membrane and probably to interact with some of the other proteins depicted, which is misleading.

    We agree with the reviewer. We removed the scheme in the current manuscript.

    Beclin-1 appears to interact with Fibrillarin (Nucleolus).

    This is correct. We observed by immunofluorescence a co-localization of BECN1 with Fibrillarin (new Figure E), and demonstrated by co-immunoprecipitation that they are constituents of a complex (new Figure F).

    Most of the differences in Sup. Fig. 3 lack statistical significance compromising the authors' claims.

    We agree with the reviewer. To perform a separated statistical analysis of the percentage of cells with nuclear buds or micrnonuclei did not provide further information. We eliminated this analysis in the current version.

    Many conclusions are drawn by colocalisation-immunofluorescence analysis. Co-immunoprecipitation experiments should also be performed to show that TOP2B and fibrillarin interact with LC3/autophagic machinery.

    Thank you for your suggestion. We performed immunoprecipitation analysis and confirmed an interaction of Fibrillarin with BECN1, this result is now presented in Figure 4F. We found no co-immunoprecipitation of LC3 with either Fibrillarin or TOP2A, nor of TOP2B with BECN1.

    Additionally, colocalisation analysis should be performed using tools such as Pearson's correlation and is an initial indication of nucleophagy. In the case of fibrillarin, immunofluorescence images do not indicate colocalisation, they need to be repeated.

    The transport of Fibrillarin out of the nucleus by micronuclei formation and its autophagic degradation implies that both proteins are contained in the same vesicular compartment, it does not necessarily requires a direct interaction of Fibrillarin with LC3. Therefore, a co-localization detected by Pearson´s analysis is not a necessary confirmation of the nucelophagic degradation of Fibrillarin. Actually, Fibrillarin does not seem to interact with LC3, since we could not detect both proteins by co-immunoprecipitation. Nevertheless, we observed a nucleolar localization of BECN1 overlapping with Fibrillarin (new Figure 4E), and we confirm by co-immunoprecipitation the presence of both BECN1 and Fibrillarin in a complex (new Figure 4F). Following reviewer´s advice, we repeated two more times the analysis of Fibrillarin immunolocalization. We corroborated its localization in micronuclei and nuclear buds in 5.86% (mean ± 5.03 SD) of untreated cells, indicating a basal level of nucleolar material exclusion from the nucleus. Interestingly, the percentage of cells with Fibrillarin in nuclear alterations did not increased with statistical significance with Etoposide treatment. At 2 h of DNA damage we observed only a slight increase to 6.8% (mean ± 4.03 SD) of cells having nuclear buds and micronuclei with Fibrillarin, while the number of cells with nuclear lesions increased to 30.6% (mean ± 4.2 SD). Similarly, the proportion of cells having Fibrillarin in nuclear lesions after 5 h of DNA repair increased only to 7.66 % (mean ±6.08 SD), while the total number of cells having nuclear buds and micronuclei increased to 38.42% (mean ± 9.3SD). These results suggest that nucleolar components are constantly sent out of the nucleus as a homeostatic process, and not significantly in response to Etoposide-induced DSB.

    Measurement of LC3/fibrillarin positive puncta should be performed, under basal conditions, genotoxic, and nucleolar stress under control and Atg7 knockdown conditions.

    Since we observed no statistical significant change in the number of micronuclei with Fibrillarin under Etoposide-induced DSB nor DNA repair, we did not perform the suggested experiment.

    Moreover, if nuclear proteins described are substrates of autophagy, then their levels would decrease upon autophagic induction i.e. starvation or in this case DNA damage and nucleolar stress. Thus, western blot analysis of relative protein levels can be performed.

    Thank you for the suggestion. Since only 5% of the cells have micronuclei with Fibrillarin, and this proportion did not increased significantly in response to DNA damage, it is unlikely to detect a difference in the amount of Fibrillarin in response to autophagy manipulation performing a population analysis (as it is in a Western blot). Nevertheless, we compared Fibrillarin abundance by Western blot in WT MEFs vs. Atg4-/- MEFs untreated (U), treated for 2 h with Etoposide (D) and after 5 h of DNA repair (5) shown in the top panel of the follow figure. As expected, we found no statistical significant difference determined by 2way-ANOVA followed by Sidak´s multiple comparisons test (n=3). Ajusted P values are shown for each comparison (left graph).

    On the other hand, since the percentage of cells with TOP2B in micronuclei and nuclear buds increased in response to DNA damage and during DNA repair, it was possible to detect a statistical significant accumulation of TOP2B in cells lacking ATG4 after 5h of DNA repair (bottom panel and right graph in the figure above). This observation is now included in new Figure 3H. Supporting our finding, TOP2A is reduced in cancerous cells grown under glucose deprivation (Alchanati, I., et al. 2009. PLoS One. 4:e8104).

    Endogenous LC3 nuclear buds should also be detected to verify nucleophagy as GFP-LC3 has been shown to aggregate, causing artifacts under certain conditions.

    We agree with the reviewer. We detected endogenous LC3 by immunofluorescence. This result is now included in Figure 2D.

    Minor comments

    In the Discussion section, the paragraph focused on the role of the ubiquitin-proteasome system is not substantiated by the data presented in the manuscript. Along similar lines, formation of aggresomes following etoposide treatment and their subsequent removal has not been monitored.

    We apologized for the confusion, we corrected the text to now clearly distinguish which are our findings and which are published data that we just attempt to relate.

    Western blots of better quality should be provided with assigned markers of protein size.

    The Western blots shown have markers of protein size.

    There are several language errors in the text that need to be corrected. Several sentences are too long and confusing or must be re-phrased. For example, see the lines: 123-125, 209-210,212, 218,221-222.

    We apologize for our language errors. We corrected all errors indicated and asked colleges proficient in English to review our text.

    Fig. 1B. Place "μm" into parenthesis.

    √

    Sup. Fig. 1B: Replace "gH2AX" with "γH2AX".

    √

    Fig. 1D: Separate DAPI and γH2AX channel images would be informative.

    We now show also separated channels.

    Fig. 2E: Enlarged separate DAPI, GFP-LC3 and lamin A/C channel images would be informative.

    We now show also separated channels.

    Line 218: Replace "bus" with "buds".

    √

    Fig. 2B, 2E, 2F, 3A and probably Sup. Fig. 2B represent MEFs treated for 2h with etoposide. The pattern of GFP-LC3 in 2B looks extensively nuclear and almost absent from cytoplasm.

    We confirmed our finding detecting endogenous LC3.

    In addition, Fig. 2B and 3B represent MEFs treated for 2h with Etoposide. The pattern of endogenous BECN1 in Fig. 2B looks extensively nuclear and almost absent from cytoplasm. In Fig. 3B the pattern is notably different.

    BECN1 pattern of distribution is rather similar, predominantly in the nucleolus. We demonstrate it further by detecting BECN1 overlapping localization with Fibrillarin (new Figure 4E) and co-immunoprecipitation (new Figure 4F).

    Sup. Fig. 2C: Index box is not properly aligned.

    Thank you. We reviewed the alignment of each index box and reorganized the figure in the revised manuscript to add the whole blots of the new experiments we performed to analyze MEFs Atg4-/-.

    Lines 154, 343 and 837: Replace "DBS" with "DSB".

    Thank you, we corrected these typos.

    Fig. 4 panels are not clearly cited at the text.

    We apologize, we reviewed that they are clearly cited now.

    Line 220: siRNA

    Thank you, we corrected the text.

    Lines 373-374: References "Lenain et al., 2015" and "Li et al., 2019" are missing.

    Thank you for noticing it, we added the missing references. We use EndNote X9, we did not expect it to fail.

    Lines 400-401 and 407: Probably the second "Latonen, 2011" reference needs "et al".

    It is correct. We now cite this paper properly.

    Line 427: Do authors refer to Fig. 1E rather than Fig. 2B?

    Yes, we are sorry for this mistake. Thank you for pointing it out.

    Line 434: Correct "clearance" spelling.

    Thank you, we corrected it.

    Reviewer #3 (Significance (Required)):

    The authors suggest that nucleophagy contributes to the elimination of chromosomal fragments or nucleolar bodies exiting the nucleus under DNA damage -inducing conditions. Specifically, they propose a key role for nucleophagy in maintaining genome stability by eliminating Type II DNA Topoisomerase cleavage complex (TOP2cc) and nucleolar components such as fibrillarin.

    However, neither TOP2 nor Fibrillarin have been shown to be actual autophagic substrates. Also, the link between genomic stability, micronuclei formation and autophagy has been previously reported (Zhao et al., PMID: 33752561).

    We found nuclear buds and micronuclei with markers of different stages of the autophagic pathway, suggesting an active role of autophagy proteins in buds formation, and micronuclei removal. We detected TOP2 and Fibrillarin in micronuclei and propose their elimination by nucleophagy by the following findings: 1) both BECN1 and LC3 were detected in micronuclei in acidic vesicles (labeled with Lysotracker), which is indicative of autolysosomes (Figure 2A); 2) TOP2B was found by electron microscopy in some cells exiting the nucleus surrounded by LC3 (Figure 3G); 3) TOP2B accumulated in cells lacking ATG4, as expected if it is degraded by autophagy (Figure 3H); 4) BECN1 has a dynamic cytoplasmic-nucelar traffic in response to DNA damage; 5) BECN1co-localized with Fibrillaron in nucleolus and both proteins were co-immunoprecupitated.

    The link between genomic stability, micronuclei formation and autophagy has been previously reported only in cancerous cells. Considering that physiological DNA damage occurs constantly in the cell, basal nucleophagy is potentially fundamental to maintain cells healthy.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, Muciño-Hernández and colleagues suggest that basal formation of nuclear buds and micronuclei increases in primary mouse embryonic fibroblasts following etoposide-induced double strand breaks (DSBs). The study combines the use of biochemical methodologies with confocal and super resolution microscopy in an effort to explore the contribution of nucleophagy to genome stability. The authors provide evidence that autophagy is induced upon etoposide treatment. They detected GFP-LC3 and BECN1 signals in nuclear buds and micronuclei even in untreated control and to a higher extent in etoposide-treated cells. Then, the authors examined whether nucleophagy is required for the removal of nuclear buds and micronuclei, by treating fibroblasts with control and Atg7 siRNA. The authors claim that the percentage of cells with micronuclei or nuclear buds decrease upon Atg7 knockdown, suggesting that components of the autophagy machinery induce the formation of these nuclear abnormalities. Moreover, Type II DNA Topoisomerases (TOP2A and TOP2B) and the ribosomal protein fibrillarin were detected in nuclear buds and micronuclei in fibroblasts treated or not with etoposide. Again in this case, GFP-LC3 was detected in fibrillarin-containing nuclear alterations. Based on these observations, the authors suggest that nucleophagy contributes to the elimination of chromosomal fragments or nucleolar bodies exiting the nucleus under DNA damage -inducing conditions. Specifically, they propose a key role for nucleophagy in maintaining genome stability by eliminating Type II DNA Topoisomerase cleavage complex (TOP2cc) and nucleolar components such as fibrillarin.

    While it seems that there is a relationship between nuclear-extruded TOP2 with endogenous BECN1 and GFP-LC3 suggesting autophagic engagement, inconsistencies of fluorescent images between different figures indicate possible technical problems/limitations (please see specific comments, below), compromising authors' claims. LC3 immunoblotting and GFP-LC3 localization results appear over-interpreted (comments below). Neither TOP2 nor Fibrillarin have been shown to be actual autophagic substrates. Also, the link between genomic stability, micronuclei formation and autophagy has been previously reported (Zhao et al., PMID: 33752561).

    An additional major concern is relates to nucleophagy being a selective type of autophagy. As such it requires efficient recognition and sequestration of the nuclear material destined to be degraded. Cargo specificity is mediated by receptor proteins, but no evidence for such receptors is provided in this study. Moreover, there is no real mechanistic insight on how nucleophagy mediates genome stability and how this can be interpreted in terms of cell survival under physiological and stress conditions. In other words, the biological significance of the findings presented has not been addressed.

    Specific comments are summarized below:

    The authors suggest that autophagy is induced after etoposide treatment and during the DNA repair process. However, the Western blot presented in Fig. 2A is not convincing and quantification does not support a significant autophagy induction in any of these cases. Autophagy appears to be induced 1h after etoposide removal, as evidenced LC3II/LC3 I increase (Fig. 2A and S2A). Nevertheless, all these changes should be more rigorously assessed.

    Line 190 and Fig. 2A: It is totally unclear whether "autophagy activation" takes place during the two waves described. There is no LC3B-I to LC3B-II conversion to initially suggest "autophagy activation". It rather suggests that autophagy is stalled. Fig. 2F shows that GFP-LC3 is strongly fluorescent into the lysotracker-stained lysosomes, further pointing to possible functional or technical problems.

    Fig. 2B and Sup. Fig. 2B: BECN1 staining looks problematic. There is extreme BECN1 accumulation in the nucleus. Are those nuclear patterns of endogenous BECN1 and GFP-LC3 normal (see also minor comment 6 and 7)? Is there literature supporting such a distribution? It is hard to imagine how BECL1 is implicated in a (here hypothetical) nuclear lamina degradation event driven by LC3-lamin B1 direct interaction (Dou et al., 2015). BECL1 is an upstream to LC3 component and is a subunit of the PI3K complex catalyzing the local PI3P generation. The above should cause recruitment of the downstream autophagic machinery. Other subunits of the same complex or downstream effectors should be identified at the same spots to support authors' claims. U, 2h D and 5h R images of whole cells are necessary. The authors should also provide representative images of cells under different conditions i.e. control, etoposide-treatment and during DNA repair. Along similar lines, untreated control cells are not included in Fig. 2E and F. These images are needed for a better comparison between normal and DNA damage-inducing conditions.

    The authors state that autophagy is required for nuclear buds and micronuclei formation. However, the data shown in Fig. 2G and H are hardly convincing given that the statistical difference between cells treated with control and Atg7 siRNA is not strong (for example, *p˂0.5, 5h after etoposide removal). To provide further support to this notion, they should use cells from autophagy defective mutants and examine the appearance of nuclear abnormalities across different conditions compared to control cells.

    Lines 223-228: The role of autophagic machinery in the formation of nuclear buds is not supported and furthermore hard to conceptualize. How the components of autophagy are implicated during the nuclear buds and micronuclei formation? Colocalization of autophagic proteins might mean that autophagy is engaged at some point after or during the above formation. The causal, mechanistic and temporal aspects of the above budding and nucleophagic events need experimental support and/or more accurate interpretation.

    The authors claim that nucleophagy eliminates topoisomerase cleavage complex because TOP2A and TOP2B appear to more extensively co-localize with GFP-LC3 and BECN1 after etoposide-induced DSBs. However, the quantification presented in Fig. 3D-F to support this statement does not, in general, show a statistically significant difference in fibroblasts across different conditions (normal, etoposide treatment, etoposide removal). Why would BECLIN colocalise with TOP2B in Figure 3g, given that beclin is involved in the initiation process?

    Fig. 4A and B: There is no enrichment of GFP-LC3 in "the nuclear alterations containing Fibrillarin" as stated in lines 341-343 comparing to the rest of the cellular GFP fluorescence. Moreover, there is no statistical significance in Fig. 4C and D measurements limiting the safety of authors' conclusions in lines 341-346.

    Lines 368-370: As discussed by the authors and reported in previous publication (Xu et al., 2017), "BECN1 interacts directly with TOP2B, which leads to the activation of DNA repair proteins, and the formation of NR and DNA-PK repair complexes", independent of its role in autophagy. Currently, there are no rigorous findings supporting the contribution of BECN1 (as a functional constituent of the core autophagic machinery) to nuclear damaged material extrusion (lines 382-384).

    Lines 435-441 and Fig. 5: The current findings do not support the proposed model. It is hard to support and conceptualize the statement "proteasome and nucelophagy function in a dynamic way inside the nucleus". In Fig. 5, LC3 appears to decorate inner nuclear membrane and probably to interact with some of the other proteins depicted, which is misleading. Beclin-1 appears to interact with Fibrillarin (Nucleolus).

    Most of the differences in Sup. Fig. 3 lack statistical significance compromising the authors' claims.

    Many conclusions are drawn by colocalisation-immunofluorescence analysis. Co-immunoprecipitation experiments should also be performed to show that TOP2B and fibrillarin interact with LC3/autophagic machinery. Additionally, colocalisation analysis should be performed using tools such as Pearson's correlation and is an initial indication of nucleophagy. In the case of fibrillarin, immunofluorescence images do not indicate colocalisation, they need to be repeated. Measurement of LC3/fibrillarin positive puncta should be performed, under basal conditions, genotoxic, and nucleolar stress under control and Atg7 knockdown conditions. Moreover, if nuclear proteins described are substrates of autophagy, then their levels would decrease upon autophagic induction i.e. starvation or in this case DNA damage and nucleolar stress. Thus, western blot analysis of relative protein levels can be performed.

    Endogenous LC3 nuclear buds should also be detected to verify nucleophagy as GFP-LC3 has been shown to aggregate, causing artifacts under certain conditions.

    Minor comments

    In the Discussion section, the paragraph focused on the role of the ubiquitin-proteasome system is not substantiated by the data presented in the manuscript. Along similar lines, formation of aggresomes following etoposide treatment and their subsequent removal has not been monitored.

    Western blots of better quality should be provided with assigned markers of protein size.

    There are several language errors in the text that need to be corrected. Several sentences are too long and confusing or must be re-phrased. For example, see the lines: 123-125, 209-210,212, 218,221-222.

    Fig. 1B. Place "μm" into parenthesis.

    Sup. Fig. 1B: Replace "gH2AX" with "γH2AX".

    Fig. 1D: Separate DAPI and γH2AX channel images would be informative.

    Fig. 2E: Enlarged separate DAPI, GFP-LC3 and lamin A/C channel images would be informative.

    Line 218: Replace "bus" with "buds".

    Fig. 2B, 2E, 2F, 3A and probably Sup. Fig. 2B represent MEFs treated for 2h with etoposide. The pattern of GFP-LC3 in 2B looks extensively nuclear and almost absent from cytoplasm.

    In addition, Fig. 2B and 3B represent MEFs treated for 2h with Etoposide. The pattern of endogenous BECN1 in Fig. 2B looks extensively nuclear and almost absent from cytoplasm. In Fig. 3B the pattern is notably different.

    Sup. Fig. 2C: Index box is not properly aligned.

    Lines 154, 343 and 837: Replace "DBS" with "DSB".

    Fig. 4 panels are not clearly cited at the text.

    Line 220: siRNA

    Lines 373-374: References "Lenain et al., 2015" and "Li et al., 2019" are missing.

    Lines 400-401 and 407: Probably the second "Latonen, 2011" reference needs "et al".

    Line 427: Do authors refer to Fig. 1E rather than Fig. 2B?

    Line 434: Correct "clearance" spelling.

    Significance

    The authors suggest that nucleophagy contributes to the elimination of chromosomal fragments or nucleolar bodies exiting the nucleus under DNA damage -inducing conditions. Specifically, they propose a key role for nucleophagy in maintaining genome stability by eliminating Type II DNA Topoisomerase cleavage complex (TOP2cc) and nucleolar components such as fibrillarin.

    However, neither TOP2 nor Fibrillarin have been shown to be actual autophagic substrates. Also, the link between genomic stability, micronuclei formation and autophagy has been previously reported (Zhao et al., PMID: 33752561).

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Peer review of the manuscript with the number RC-2021-01181 by Muciño-Hernandez G et. al. at Review Commons and with the tittle "Nucleophagy contributes to genome stability 1 though TOP2cc and nucleolar components degradation"

    1. Summary

    Muciño-Hernandez G et. al. show in this manuscript that mouse embryonic fibroblasts (MEFs) have basal levels of nuclear buds and micronuclei, which are indicators of genomic DNA damage. These basal levels of nuclear buds and micronuclei in MEFs increased after Etoposide treatment, which is known to induce DNA Double stranded Breaks (DSD). Interestingly, the nuclear buds and micronuclei co-localize with makers for nucleophagy (BECN1 and LC3) and acidic vesicles, suggesting that they are cleared by nucleophagy. The authors propose that basal levels of nucleophagy clear basal levels of genomic DNA damage that occurs as result from DNA-dependent biological processes in the cell nucleus, thereby contributing to nuclear stability of MEFs under physiological conditions. These basal levels of nucleophagy increase after the action of factors that induce DNA damage and nuclear stress. The concepts proposed by Muciño-Hernandez G et. al. are novel, since most of the current published data on nucleophagy related to DNA damage have been obtained under pathological conditions, e.g. implementing cancer cells.

    The authors use in their manuscript various molecular biology techniques to obtain data that support their claims, including Western Blot analysis of protein extracts from MEFs, immunostaining on MEFs and neutral comet assays, complemented with state of the art imaging techniques, such as confocal microscopy, immunoelectron microscopy and super resolution microscopy. The quality of the data is sound. The structure of the manuscript support the understanding of the reader. However, I would like to suggest several improvements that will help to increase the quality of the manuscript, in order that fits to the standards of articles recently published in journals affiliated to Review Commons, such as the Journal of Cell Biology, the EMBO Journal or eLife.

    2. Major comments

    2.1 The authors have to improve the description of the results. Especially the description of those Figure panels containing plots that were generated using data from several experiments has to be improved.

    One example is the description of the Figure 1D, which is in the lanes 137-151 of the current version of the manuscript. Whereas the authors describe in lanes 137-147 observations related to representative pictures of confocal microscopy after immunostaining presented in Figure 1D (left), the description of the quantification from 9 independent experiments presented in the plots in Figure 1D (right) comes relatively short in lanes 147-150 without mentioning any of the values implemented for creating the plots.

    "Interestingly, while the frequency of nuclear buds gradually increased after DNA damage and during DNA repair, the frequency of micronuclei also increased after DNA damage, but diminished upon DNA repair."

    The other plots presented in the different figure panels across the manuscript are described in a similar manner. I would like to suggest to the authors to improve their manuscript by including during the description of their results the values that were implemented for the degeneration of the plots presented in the manuscript. For example, in the specific case of Figure 1D above:

    "Interestingly, the percentage of MEFs with nuclear buds gradually increased from XY% ({plus minus} XY SD) in control non-treated (Ctrl) MEFs to XY% ({plus minus} XY SD; P=XY) after 2 h Etoposide-induced DSB in MEFs and XY% ({plus minus} XY SD; P=XY) after DNA repair take place in MEFSs 5 h upon stop of Etoposide treatment (Figure 1D, right). In contrast, the percentage of MEFs with micronuclei significantly increased from XY% ({plus minus} XY SD) in Ctrl MEFs to XY% ({plus minus} XY SD; P=XY) after 2 h Etoposide-induced DSB, whereas it was reduced to XY% ({plus minus} XY SD; P=XY) 5 h after stop of Etoposide treatment (Figure 1D, right)."

    Descriptions of the plots as mentioned above will make the text more intuitive for the reader, and they will make possible to read the Results Section without switching to the Figure Legends or the Material and Methods Section or to Supplementary Files. Even though the representative pictures from different microscopy techniques presented in the manuscript are of good quality and support the claims of the authors, it is important to mention that the quantifications presented in the plots demonstrate the statistical significance of these representative pictures. Thus, the authors should consistently include in the manuscript during the description of theirs results all the information (mean values, standard error of the means, P values, n values, etc.) that support their interpretation of the results and demonstrate the statistical significance of their claims.

    2.2 Following a similar line of argumentation as in the previous point, the authors should provide as Supplementary Material an Excel file containing a statistical summary, including all statistical relevant information from each one of the plots presented in each Figure panel, such as n values, P values, Test implemented, values used for the plots, numbers of experiments, etc. The information could be organized in the Excel file in different data sheets according to the Figure panels, in order that the reader can easily navigate through the data. In the current version of the manuscript, one cannot find the values used for the generation of the plots presented in the manuscript in any of the submitted files.

    3. Minor comments

    3.1 In general, prior studies were appropriately referenced. Only few references has to be added.

    Line 48: Add to the already included reference "Dobersch et al., 2021" also the reference Singh et al., 2015 PMID 26045162.

    Line 53: Add the corresponding reference after the word "respectively".

    Line 82: Add the corresponding reference after the word "them".

    Line 125: Add the corresponding reference after the word "cells".

    Line 130: The expression "...by analyzing the recruitment of the phosphorylated histone γH2AX..." is the first time that the authors mention in the manuscript the DNA damage maker γH2AX. I suggest that is better introduced as " ... by analyzing the recruitment of the DNA damage marker γH2AX (histone variant H2A.X phosphorylated a serine 139, Rogakou EP, et al., 1998, PMID 9488723) to DSB sites."

    Line 199: Add the corresponding reference after the word "formation".

    Line 205: Add the corresponding reference after the word "cells".

    3.2 The use of the English language is appropriate throughout the manuscript. However, there are minor errors in the use of punctuation marks, in the use of prepositions and typos. I will list some of them below. However, I would like to recommend that manuscript is corrected by an English native speaker.

    Line 41: "...and reproductive systems; genome instability also..." the semicolon can be replaced by a period.

    Line 43: "Since early in development DNA is under constant endogenous..." between "development" and "DNA" there should a comma.

    The sentence in lanes 53-55 has to be rephrased.

    Lines 62-63: the expression "...throughout life." should be substituted.

    Line 70: The abbreviation "rDNA" has to be explained the first time that is used.

    Lines 81-82: It has to be explained for the scientist that is not specialized in the field of nucleophagy, how the integrity of the genome is threatened by micronuclei and nuclei-derived material.

    Lines 106-110: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences.

    Lines 121-122: The subtitle should be rephrased.

    Lines 132-138: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences, e.g. with a period before the word "hence".

    Lines 143-144: "... in a subpopulation of healthy, untreated cells...". The interpretation of "healthy" might be subjective. I would like to suggest substituting in the complete manuscript the word "healthy" by "control".

    Line 163: The abbreviation for γH2AX was already introduced in line 130.

    Line 182: A comma after "cell lines" is missed.

    Line 183: delete "either".

    Lines 190-194: The sentence is long. It would be easier to understand for the reader if this sentence is divided into two sentences, e.g. with a period after the word "decreased" in line 191.

    Line 218: I assume that instead of "bus", it should be "buds".

    Line 220: I assume that instead of "iRNA", it should be "siRNA". In addition, it is the first time that the abbreviation is used. Thus, I suggest introducing it as "...was silenced by specific small interfering RNA (siRNA) previous to ..."

    Line 327: delete the word "chronic".

    Line 344: I assume that instead of "(figures 4C)", it should be "(Figure 4D)".

    3.3 The structure of the Figures is ok for the peer review process and it might be optimized during editing of the manuscript. Nevertheless, I would like to suggest to the authors to increase the lettering size throughout all the figures. It will make the figures more intuitive.

    Significance

    4. Significance

    The work presented by Muciño-Hernandez G et. al. will be clearly a significant contribution to the scientific community working on autophagy, DNA damage repair and cancer, among others. It will be of interest to a broad spectrum of scientists, as I will elaborate in the following lines. The authors propose that MEFs have basal levels of genomic DNA damage under physiological conditions, which are cleared by basal levels of nucleophagy. On one hand, these findings are in line with various publications demonstrating that DNA-dependent biological processes in the cell nucleus, such as transcription, replication, recombination, and repair, involve intermediates with DNA breaks that may compromise the integrity of DNA. Thus, there must be mechanisms that ensure the integrity of the genome during these processes under physiological conditions, one of them seems to be nucleophagy. This perspective might explain the fact that proteins and histone modifications that were initially characterized during DNA repair also play a role during transcription, recombination, and replication. For example, phosphorylated H2AX at S139 (γH2AX) is often used as a marker for DNA-DSB [PMID 9488723]. However, accumulating evidences suggest additional functions of this histone modification [PMIDs 19377486; 22628289; 23382544]. In addition, McManus et al. [PMID 16030261] analyzed the dynamics of γH2AX in normal growing mammalian cells and found γH2AX in all phases of cell cycle with a maximum during M phase, suggesting that γH2AX may contribute to the fidelity of the mitotic process, even in the absence of ectopic- induced DNA damage. Further, Singh et al [PMID 26045162] and Dobersch et al [PMID 33594057] report that γH2AX plays a role in transcriptional activation in response to TGFB-signaling. Moreover, classical DNA-repair complexes have been linked to DNA demethylation and transcriptional activation [PMIDs 17268471; 28512237; 25901318], and DNA-DSB is known to induce ectopic transcription that is essential for repair, supporting a tight mechanistic correlation between transcription, DNA damage, and repair [PMID 24207023]. Perhaps, the authors might consider introducing several of the aspects and the citations written above into the Discussion section of the revised version of their manuscript. On the other hand, most of the published data related to nucleophagy have been obtained from cancer cells. Muciño-Hernandez G et. al. obtained their data implementing MEFs to demonstrate that the proposed mechanisms take also place under non-pathological conditions, what is one of the novel aspects of the present work.

    I hope that my suggestions help the authors to improve their manuscript, thereby reaching the standards of manuscripts recently published in journals affiliated to Review Commons AND increasing the impact of their contribution to the scientific community.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    This paper examines the formation and repair of micronuclei in non-cancerous cells, specifically in mouse embryonic fibroblasts. This work was performed completely in culture and used a combination of western blot, confocal and superresolution microscopy to assess the contents of micronuclei over a repair period of 5 hours after 2 hours of induction of double strand breaks by treatment with etoposide. The authors found that the bodies colocalised with LC3, Beclin 1 and lysosomes suggestive of autophagy. However no evidence of autophagic flux has been demonstrated.

    Major issues are as follows:

    Figure 2 A - Any sense of the autophagic flux? LC3B - I and LC3B - II seem to be in equal quantities most of the time. Maybe using the tandem LC3 in this system could provide further insight. Also remove the violin plots from this graph and from G and H, as there are too few data points. B. Can you reduce the brightness in the merge image, as I cannot see DAPI nor a convincing Beclin-1/LC3 co-localisation. F. Although the data is convincing, It would be clearer if the brightness of the merge image was reduced. G. Is the significant result the difference between 5h R Control si and 5h R Atg7? if so, there is no significant change in micronuclei as the same time point, can you explain this disconnect? are the buds being degraded prior to becoming micronuclei?

    Figure 3 A - nice microscopy showing the co-localisation of TOP2A and LC3-GFP. I'm interested in DAPI being on some bodies and not others. Do you have any sense of the dynamics of this? G - c shows a strand of mostly TOP2B coming from the nucleus. Is there any evidence that this occurs using either confocal microscopy or super resolution approaches. Could you try Z-stack to find these?

    Figure 4 C - is there a significant increase in FBL negative bodies, this would make sense if FBN is being degraded in the micronuclei during the repair process D. Would it be possible to increase the n of these experiments to confirm either no change in FBL/LC3 co-loc, or evidence of increase?

    Minor issues:

    Figure 4 and 5 legends are in a different font.

    Significance

    There is little specific data on the role of autophagy in clearing micronuclei in cancer cells, so this may be suggestive of a new mechanism that occur during normal cellular homeostasis. There are known links between lamin A defects and the formation of micronuclei, but not explicitly that the micronuclei are also Lamin A positive. it is likely that analogous processes occur in both cancer and non-cancer, so the impact of these data is not clear to me. This paper may be of interest to researchers interested in nuclear structure and DNA damage, but based on the data presented the significance is limited.

    I don't have sufficient expertise to evaluate the super resolution microscopy beyond assessing the images.

  6. Version published to 10.1101/2021.12.02.471011v1 on bioRxiv