Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Strengths:
The manuscript utilizes a previously reported misfolding-prone reporter to assess its behaviour in ER in different cell line models. They make two interesting observations:
(1) Upon prolonged incubation, the reporter accumulates in nuclear aggregates.
(2) The aggregates are cleared during mitosis. They further provide some insight into the role of chaperones and ER stressors in aggregate clearance. These observations provide a starting point for addressing the role of mitosis in aggregate clearance. Needless to say, going ahead understanding the impact of aggregate clearance on cell division will be equally important.
Weaknesses:
The study almost entirely relies on an imaging approach to address the issue of aggregate clearance. A complementary biochemical approach would be more insightful. The intriguing observations pertaining to aggregates in the nucleus and their clearance during mitosis lack mechanistic understanding. The issue pertaining to the functional relevance of aggregation clearance or its lack thereof has not been addressed. Experiments addressing these issues would be a terrific addition to this manuscript.
We have performed protein blotting and proteomics to characterize ER-FlucDM-eGFP expressing cells. We have also provided evidence to support the role of ER reorganization in regulating aggregate clearance. Our proteomic analysis provided a global view of the cellular state of cells expressing ER-FlucDM-eGFP, which potentially revealed functional relevance of ER-FlucDM-eGFP. Details are explained in the following comments.
Reviewer #2 (Public Review):
Summary:
The authors provide an interesting observation that ER-targeted excess misfolded proteins localize to the nucleus within membrane-entrapped vesicles for further quality control during cell division. This is useful information indicating transient nuclear compartmentalization as a quality control strategy for misfolded ER proteins in mitotic cells, although endogenous substrates of this pathway are yet to be identified.
Strengths:
This microscopy-based study reports unique membrane-based compartments of ERtargeted misfolded proteins within the nucleus. Quarantining aggregating proteins in membrane-less compartments is a widely accepted protein quality control mechanism. This work highlights the importance of membrane-bound quarantining strategies for aggregating proteins. These observations open up multiple questions on proteostasis biology. How do these membrane-bound bodies enter the nucleus? How are the singlelayer membranes formed? How exactly are these membrane-bound aggregates degraded? Are similar membrane-bound nuclear deposits present in post-mitotic cells that are relevant in age-related proteostasis diseases? Etc. Thus, the observations reported here are potentially interesting.
Weaknesses:
This study, like many other studies, used a set of model misfolding-prone proteins to uncover the interesting nuclear-compartment-based quality control of ER proteins. The endogenous ER-proteins that reach a similar stage of overdose of misfolding during ER stress remain unknown.
We have included a previous study that showed accumulation of BiP aggregates in the nucleus upon overexpression of BiP (Morris et al., 1997; DOI: 10.1074/jbc.272.7.4327) in the discussion (Line 299).
The mechanism of disaggregation of membrane-trapped misfolded proteins is unclear. Do these come out of the membrane traps? The authors report a few vesicles in living cells. This may suggest that membrane-untrapped proteins are disaggregated while trapped proteins remain aggregates within membranes.
We initially made mStayGold-Sec61β to image the ER structures and ER-FlucDM-eGFP aggregates. However, we could not obtain convincing time-lapse images to show the release of ER-FlucDM-eGFP aggregates from the ER membrane as there are abundant ER structures present close to the aggregates during mitosis, preventing the differentiation of the membrane encapsulating aggregates from the ER structures.
The authors figure out the involvement of proteasome and Hsp70 during the disaggregation process. However, the detailed mechanisms including the ubiquitin ligases are not identified. Also, is the protein ubiquitinated at this stage?
We performed cycloheximide chase experiments in cells released from the G2/M and found that ER-FlucDM-eGFP protein level did not fluctuate significantly when cells progressed through mitosis and cytokinesis. Thus, we did not consider protein ubiquitination and degradation of ER-FlucDM-eGFP as a major mechanism for its clearance. We have included this observation in the results (Figure S7A; Line 266) and in the discussion (Line 324) of the revised manuscript.
This paper suffers from a lack of cellular biochemistry. Western blots confirming the solubility and insolubility of the misfolded proteins are required. This will also help to calculate the specific activity of luciferase more accurately than estimating the fluorescence intensities of soluble and aggregated/compartmentalized proteins.
We performed solubility test in cells expressing ER-FlucDM-eGFP and detected insoluble ERFlucDM-eGFP after heat stress (Figure S1E; Line 102). We have also performed protein blotting to detect ER-FlucDM-eGFP to normalize the luciferase activity (Line 609). We have updated the method section for luciferase measurement (Line 494).
Microscopy suggested the dissolution of the membrane-based compartments and probably disaggregation of the protein. This data should be substantiated using Western blots. Degradation can only be confirmed by Western blots. The authors should try time course experiments to correlate with microscopy data. Cycloheximide chase experiments will be useful.
We performed cycloheximide chase experiments in cells released from the G2/M and found that ER-FlucDM-eGFP protein level did not fluctuate significantly when cells progressed through mitosis and cytokinesis (Figure S7A to S7C). Also, live-cell imaging of cells released from the G2/M indicated no significant change of total fluorescence intensity of ER-FlucDMeGFP (Figure S7D). Thus, we do not think that protein degradation of ER-FlucDM-eGFP is the major mechanism for its clearance.
The cell models express the ER-targeted misfolded proteins constitutively that may already reprogram the proteostasis. The authors may try one experiment with inducible overexpression.
We have re-transduced fresh MCF10A cells with lentiviral particles to induce expression of ER-FlucDM-eGFP. The aggregates started to form after 24 h post-transduction. We made similar observations as described in the manuscript (e.g. aggregate clearance) two days after re-transduction.
It is clear that a saturating dose of ER-targeted misfolded proteins activates the pathway.
The authors performed a few RT-PCR experiments to indicate the proteostasis-sensitivity.
Proteome-based experiments will be better to substantiate proteostasis saturation.
We have performed proteomic analysis in cells expressing ER-FlucDM-eGFP and observed up-regulation of multiple proteins involved in the ER stress response, indicating that cells expressing ER-FlucDM-eGFP experience proteostatic stress (Figure S4A; Line 179).
The authors should immunostain the nuclear compartments for other ER-membrane resident proteins that span either the bilayer or a single layer. The data may be discussed.
We have co-expressed ER-FlucDM-mCherry and mStayGold-Sec61β and detected mStayGold- Sec61β around ER-FlucDM-mCherry aggregates (Figure 1B).
All microscopy figures should include control cells with similarly aggregating proteins or without aggregates as appropriate. For example, is the nuclear-targeted FlucDM-EGFP similarly entrapped? A control experiment will be interesting. Expression of control proteins should be estimated by western blots.
We targeted FlucDM-eGFP to the nucleus by expressing NLS-FlucDM-eGFP (Figure S1A). We found that the nuclear FlucDM-eGFP did not co-localize with the ER-FlucDM-mCherry aggregates (Figure S1B; Line 96). We have also determined the expression levels of NLSFlucDM-eGFP and ER-FlucDM-mCherry (Figure S1C and S1D).
There are few more points that may be out of the scope of the manuscript. For example, how do these compartments enter the nucleus? Whether similar entry mechanisms/events are ever reported? What do the authors speculate? Also, the bilayer membrane becomes a single layer. This is potentially interesting and should be discussed with probable mechanisms. Also, do these nuclear compartments interfere with transcription and thereby deregulate cell division? What about post-mitotic cells? Similar deposits may be potentially toxic in the absence of cell division. All these may be discussed.
Thank you for interesting suggestions for our study. We speculated that ER-FlucDM-eGFP aggregates may derive from the invagination of the inner nuclear membrane given that the aggregates are in close proximity to the inner nuclear membrane in interpase cells (Line 299). We have included a previous study that reported a similar aggregate upon BiP overexpression (Morris et al., 1997; DOI: 10.1074/jbc.272.7.4327; Line 300). Our proteomic analysis showed that cells expressing ER-FlucDM-eGFP have several up-regulated proteins related to cell cycle regulation (Figure S4A; Line 346).
Reviewer #3 (Public Review):
Summary:
This paper describes a new mechanism of clearance of protein aggregates occurring during mitosis.
The authors have observed that animal cells can clear misfolded aggregated proteins at the end of mitosis. The images and data gathered are solid, convincing, and statistically significant. However, there is a lack of insight into the underlying mechanism. They show the involvement of the ER, ATPase-dependent, BiP chaperone, and the requirement of Cdk1 inactivation (a hallmark of mitotic exit) in the process. They also show that the mechanism seems to be independent of the APC/C complex (anaphase-promoting complex). Several points need to be clarified regarding the mechanism that clears the aggregates during mitosis:
• What happens in the cell substructure during mitosis to explain the recruitment of BiP towards the aggregates, which seem to be relocated to the cytoplasm surrounded by the ER membrane.
We have included images to show that BiP co-localizes with ER-FlucDM-eGFP aggregates in interphase cells (Figure S5C). We think that BiP participates in the formation of ER-FlucDMeGFP during interphase instead of getting recruited to the aggregates during mitosis.
• How the changes in the cell substructure during mitosis explain the relocation of protein aggregates during mitosis.
We provided evidence to show that clearance of ER-FlucDM-eGFP aggregates involves the ER remodeling process. We depleted ER membrane fusion proteins ATL2 and ATL3 to perturb the distribution of ER sheets or tubules and found that cells were defective in clearing the aggregates (Figure 7A and B; Line 278).
• Why BiP seems to be the main player of this mechanism and not the cyto Hsp70 first described to be involved in protein disaggregation.
In our proteomic analysis, we found that BiP (HSPA5) but not other Hsp70 family members were up-regulated in cells expressing ER-FlucDM-eGFP (Line 352; Figure S4A). This explains why BiP is the main player of the ER-FlucDM-eGFP aggregate clearance.
Strengths:
Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting.
Weaknesses:
Weak mechanistic insight to explain the process of protein disaggregation, particularly the interconnection between what happens in the cell substructure during mitosis to trigger and drive clearance of protein aggregates.
In our revised manuscript, we now provided evidence to show that ER-FlucDM-eGFP aggregate clearance involved remodeling of the ER structures during mitotic exit. This is added as a new Figure 7 in the revised manuscript and is described in the result section (Line 278) and in the discussion section (Line 323). We believe that this addition has provided mechanistic insights into ER-FlucDM-eGFP aggregate clearance.
Recommendations for the authors:
Reviewing Editor comments:
I have read these reviews in detail and would like to recommend that the authors perform the experiments according to the reviewers' suggestions, as well as provide the appropriate controls raised by the reviewers.
I think there are not that many requests and they all seem very reasonable and easily doable. I would recommend that the authors carry out the suggested experiments to develop a stronger story where the evidence transitions from being incomplete presently to a "more complete" standard.
We have addressed questions raised by three reviewers and updated our manuscript (labeled in red in the main text).
Reviewer #1 (Recommendations For The Authors):
The manuscript makes exciting observations about the accumulation of reporter protein aggregates in the nucleus and its clearance during mitosis. It also provides some insight into the role of chaperons in aggregate clearance. These observations provide a good platform to perform in-depth analysis of the underlying mechanism and its functional relevance which perhaps the authors will plan over the long term. However, the below suggestions will help improve the current version of the manuscript:
(1) Although it is assumed that the aggregates are cleared by the protein degradation mechanism, clear evidence supporting this assumption in the author's experiments is lacking and needs to be provided. Is it possible that mitosis induces disassembly of these aggregates instead of degradation?
We performed two experiments to verify whether ER-FlucDM-eGFP aggregates are cleared by the protein degradation mechanism. In the first experiment, we treated cells expressing ER-FlucDM-eGFP released from the G2/M boundary with cycloheximide (CHX) and found that ER-FlucDM-eGFP did not decrease in protein abundance in cells progressing through mitosis (Figure S7A to S7C). In the second experiment, we measured the intensity of ERFlucDM-eGFP in early dividing cells and late dividing cells after release from the G2/M boundary and found that there was no significant difference between early and late dividing cells (Figure S7D). Thus, we concluded that protein degradation of ER-FlucDM-eGFP is not the primary mechanism of its clearance during cell division (Line 324). Furthermore, we included new data to show that the ER-FlucDM-eGFP aggregate clearance depends on ER reorganization during cell division, so mitotic exit induces disassembly of the aggregates instead of protein degradation.
(2) It is intriguing that the aggregates are nuclear. Is the nuclear localization mediated by localization to ER? A time course analysis would reveal this and would provide credence to the idea that the reporter was originally expressed in the ER. It is currently unclear if the reporter ever gets expressed in ER.
We showed that in interphase cells, ER-FlucDM-eGFP co-localizes with mStayGold-Sec61β, which labels the ER structures (Figure 1B). So, ER-FlucDM-eGFP is expressed and present in the ER network and invaginates into the inner nuclear membrane as aggregates. We attempted to image ER-FlucDM-eGFP for its formation; however it was technically challenging as the aggregates appeared very small and not too visible after clearance under our microscopy system.
(3) It would be expected that the persistence of these aggregates would impact cell division and cellular health. An experiment addressing this hypothesis would be very useful in establishing the functional relevance of this observation in the context of the current study.
We have performed proteomic analysis on cell expressing ER-FlucDM-eGFP and found that multiple proteins involved in the ER stress response were up-regulated (Figure S4A). Additionally, proteins related to cell cycle regulation were up-regulated upon expression of ER-FlucDM-eGFP (Figure S4A). The increase of these proteins may indicate a perturbed cellular health (Line 344).
(4) A recent report (PMID: 34467852) identified the role of ER tubules in controlling the size of certain misfolded condensates. Would specific ER substructures affect the nuclear localization and/or clearance of the FlucDM aggregates? This is tied to point#2 and would provide insights into the connection between ER and the nuclear aggregates.
Thank you for your suggestions. We perturbed the ER remodeling process by knocking down ATL2 and ATL3, which are ER membrane fusion proteins, and found that clearance of ER-FlucDM-eGFP aggregates was affected (Figure 7A and B). Hence, perturbation of the distribution of ER tubules and ER sheets affects ER-FlucDM-eGFP aggregate clearance. We have also added the recent paper about ER tubule size in regulating the sizes of misfolded condensates in the discussion (Line 321)
Reviewer #2 (Recommendations For The Authors):
I expect that the images indicate z-sections. Should be indicated in legends as applicable.
We have indicated whether the images are Z-stack or single Z-slices in the figure legends.
Small point: the control region (outside inclusion) that was bleached in 2c may be clearly indicated.
We have added the explanation in the figure legend of Figure 2C.
