Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8

  1. Saeko Aoyama-Ishiwatari
  2. Koki Nakamura
  3. Takahiro Nagao
  4. Yusuke Hirabayashi

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Abstract

Cells adapt to changes in nutrient conditions by reorganizing organelle activities. Mitochondria-endoplasmic reticulum contact sites (MERCS) have been proposed to play an essential role in this adaptive response. However, the lack of suitable tools for detecting these contact sites in living cells has hindered the studies of the mechanisms regulating MERCS in response to nutritional changes. Here, we establish a novel cell line, MERCdRED, which expresses a ddFP-based fluorescent MERCS sensor. Using correlative light-electron microscopy, we show that the MERCdRED signal accurately labels the contact sites as defined by electron microscopy. Live imaging of MERCdRED cells revealed that large MERCS are more stable than smaller ones. Furthermore, in combination with knockout of the ER-mitochondria tethering protein PDZD8, we revealed that nutrient deprivation reduces MERCS area in a PDZD8-dependent manner. This new tool, together with our findings, will contribute to a better understanding of the molecular basis of cellular metabolism.

Summary Statement

We established a novel cell line enabling live imaging of MERCS, which revealed their dynamic nature and demonstrated nutrient deprivation-induced MERCS reduction along with its underlying mechanisms.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    General Statements

    We would like to thank all reviewers assigned by Review Commons for their thoughtful and constructive feedback, which helped us to further improve the quality and clarity of our manuscript. In this study, we developed a novel fluorescence-based live-cell imaging platform for detecting mitochondria-endoplasmic reticulum contact sites (MERCS), which we named MERCdRED. This system enables quantitative analysis of MERCS dynamics in living cells by combining stable gene expression of dimerization-dependent fluorescent proteins with single-cell cloning. Using this tool, we uncovered a nutrient-dependent regulatory mechanism of MERCS formation mediated by the ER-localized tethering protein PDZD8. We appreciate that all the reviewers acknowledged the methodological robustness of this work. In response to reviewers' comments, we will significantly improve the manuscript by adding the live-cell imaging to assess the reversible propertyof MERCdRED, and investigating the physiological impacts of MERCS remodeling in regulating metabolism in response to nutrient starvation. We believe that both the methodological advance and the biological findings presented in this study will be of broad interest to the cell biology community.

    1. Description of the planned revisions

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

    Summary: In this study, the authors successfully established a stable cell line expressing MERCdRED, a dimerization-dependent fluorescent protein (ddFP)-based sensor for monitoring mitochondrial-ER contact sites (MERCS). Through light and electron microscopy analyses, they demonstrated that MERCS formation is regulated by nutrient availability and requires PDZD8. While the work is technically sound and well-presented, the biological implications of nutrient-dependent MERCS regulation remain underexplored.

    Major Concerns: Although the manuscript is methodologically robust and suitable for a Methods-type article, its biological significance is limited. The findings primarily serve as proof-of-concept for the MERCdRED tool, without substantially advancing our understanding of MERCS regulation.

    We appreciate the reviewer for acknowledging the methodological robustness of our study. We would like to respectfully emphasize that, using the MERCdRED cell system, we uncovered the distinct features of MERCS dynamics by comparing structures of various sizes (Figure 4A-D). Furthermore, we discovered an unexpected biological finding: nutrient starvation leads to a reduction in MERCS formation, which contrasts with previous reports using cell lines (former Figure 4E-H). Additionally, we revealed that PDZD8 mediates nutrient-dependent MERCS regulation (former Figure 4E-H).

    To clarify these findings, we have now separated the former Figure 4 into two distinct figures (now Figure 4 and 5). Furthermore, to assess the functional relevance of PDZD8-mediated MERCS regulation upon nutritional change, we will perform rescue experiments by overexpressing PDZD8 in starved cells, along with a metabolomic analysis in these conditions. We will add these new data in Figure 6.

    Taken together, we believe that our data provide novel mechanistic insights into how MERCS are modulated and utilized for the regulation of metabolism under physiological stress, thereby contributing to a deeper understanding of the roles and regulation of MERCS beyond the scope of a mere proof-of-concept study.

    Reviewer #1 (Significance (Required)):

    To enhance the impact of the study, the authors could use this sensor to investigate novel biological questions-such as the molecular pathways linking nutrient sensing to MERCS dynamics-or explore downstream activities of nutrient-dependent MERCS formation. Deeper mechanistic insights would significantly strengthen the work's contribution to the field.

    We thank the reviewer for their constructive suggestions. We fully agree that the MERCdRED cell system has great potential for investigating upstream signaling pathways regulating MERCS dynamics, as well as the downstream consequences of nutrient-dependent MERCS modulation. As mentioned above, this study already presents important findings, including the discovery of PDZD8 as a key protein linking the nutrient starvation and MERCS remodeling, and a relationship between MERCS dynamics and contact site size.

    To further assess the biological consequence of the MERCS remodeling, we will perform metabolomics analysis in PDZD8-overexpressing cells under starved conditions.

    Additionally, to further reinforce the utility of MERCdRED and extend the findings presented in this study, we performed live-cell imaging experiments using MERCdRED. The preliminary results demonstrated dynamic and reversible changes in MERCS in response to nutrient starvation and subsequent recovery (Please see the response to Reviewer 3 below, Reviewer-only Figure 1).

    These new data will significantly strengthen the contribution of this study to the field.

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

    This manuscript entitled "Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8" by Saeko Aoyama-Ishiwatari et al., describes a novel methodology for visualizing contacts between mitochondria and the endoplasmic reticulum (MERCs) by fluorescence microscopy. Inter-organelle contacts, defined as membrane proximities below ~30 nm, fall below the diffraction limit of conventional light microscopy. The method developed by Hirabayashi's laboratory leverages dimerization-dependent fluorescence complementation to create a reporter capable of both visualizing and quantifying ER-mitochondria contacts (MERCs).

    Reviewer #2 (Significance (Required)):

    This timely study provides a valuable and innovative approach to overcoming a longstanding technical limitation in the field, enabling dynamic analysis of ER-mitochondria contacts.

    We appreciate the reviewer for recognizing the timeliness and innovation of our work.

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

    In this manuscript, the authors develop a new system to study mitochondrial-ER contact sites in living mouse embryonic fibroblast cells and explore the impact that nutritent starvation has on these contact sites in real time. By stably expressing a bicistroic reporter construct of a dimerization-dependent fluorescent protein that will generate a signal once the two moities, one anchored in the ER by Sec61b, and the other anchored in the outer membrane of mitochondria, via TOM20, comme in close apposition. This cell model is validated using sofisticated CLEM experiments and via the ablation of known regulators of MERCs, such as PDZD8 and FKBP8.

    Major comments

    The authors claim to have developed a new for the study of MERCs. They indeed have benchmarked this system using very sophisticated CLEM approaches and through the ablation of known regulators of MERCs, all of which is very carefully performed and convincing.

    We appreciate the reviewer for acknowledging our efforts in the development and validation of the MERCdRED system presented in this study.

    They argue that the generation of a stable cell line via lentiviral delivery is an improvement over the transient transfection approaches that have been applied in the past (see cited references), which I would generally agree. However, they have not contrasted or compared their system to the widly-used SLPICs system from the Tito Cali group (Vallese, F. et al. An expanded palette of improved SPLICS reporters detects multiple organelle contacts in vitro and in vivo. Nat. Commun. 11, 6069 (2020)) which measures bi and tri-partite interactions with other membrane contacts, including mitochondria and ER at two specific distances, which in my opinion has been more extensivley used to study cell and tissue physiology. They accurately point out that the reversability of this and other systems is challenging and it would be important to define highlight whether the current system allows the study of reversible MERCs. It does not appear as though the reversability of MERCs has been explored in this study.

    We thank the reviewer for these thoughtful suggestions and agree that further investigation into the reversibility of MERCS using the MERCdRED system would be valuable. Following the reviewer's suggestion, we performed a live-cell imaging experiment using MERCdRED to monitor dynamic changes in MERCS in response to nutrient starvation and subsequent recovery. The preliminary results were obtained as shown in Reviewer-only Figure 1, which strongly suggests the utility of MERCdRED for detecting reversible MERCS formation. The data will be added in Figure 5 if the reproducibility is confirmed. This new data set highlights the distinct utility of the MERCdRED system in studying MERCS dynamics.

    We acknowledge that the SPLICS system has been widely adopted for studying membrane contact sites. In the revised manuscript, we will include a comparative discussion of MERCdRED, SPLICS, and other existing MERCS reporters, particularly with respect to their capabilities in capturing the reversible nature of these contacts.

    The genetic (PDZD8, FKBP8) and nutritional (starvation) interventions are very helpful to benchmark the system. The description of the methods and data appear to be reproducible and the stastical analyses are acceptable.

    We thank the reviewer for their positive evaluation of our data and analyses.

    Minor comments

    As mentioned above, it would be helpful to reference and compare the current study in the context of reversability, which the current MERCdRED system has the potential to provide beyond the state-of-the-art.

    We thank the reviewer for this helpful suggestion. We will include additional discussion comparing the reversibility of the MERCdRED system with that of existing tools, highlighting the potential advantages of MERCdRED in capturing dynamic and reversible MERCS.

    Reviewer #3 (Significance (Required)):

    Significance

    The major strength of this study is the development of a stable cell line that allows for the study of MERCs, which has the potential to study the reversible nature of these membrane contact sites. It is debatable as a stable cell line rather than a transient transfection offers a major advancement, even if it does make the study of the system more straightforward, especially if the phenomenon of reversibilty is to be explored.I believe that the CLEM study offers a very informative and precise way to benchmark the ddFP system. Defining how MERC formation and separation (once again the reversibility discovery) have impacts in cell physiology beyond the distances altered by starvation would improve the study. Examining the impact on calcium homeostasis, lipid metabolism, and other aspects of biology that are known to be influenced by MERCs would be interesting. As such, there are no new conceptual, mechanistic, or functional advances, simply minor technical advances in the creation of a stable cell line followed by very solid benchmarking experiments. More complex tri-partite interactions, studied elsewhere, which are conceptually very important for cell and organelle biology, have not been attempted here. Similarly, the notion of studied different types of MERCs, which have been proposed to be important for cell biology, has not been explored using this single reporter. The target audience for this study is one that is interested in membrane contact sites and quantitative biology. My expertise is in mitochondrial fluorescence imaging and biology. I am not an expert in CLEM.

    We thank the reviewer for their thoughtful and detailed comments. We would like to respectfully emphasize that the establishment of a clonal cell line has enabled us to uncover a striking and unexpected biological finding-namely, that nutrient starvation leads to a reduction, rather than an increase, in MERCS formation, and that this change is regulated by PDZD8. This observation directly contradicts previous reports and highlights the value of our robust and quantitative system for re-evaluating previously held assumptions.

    We agree that demonstrating the reversibility of MERCS formation using our system would further strengthen the utility and reliability of the MERCdRED platform. To address this, as mentioned above, we performed a live-cell imaging to assess the dynamic reversibility of MERCS formation (Reviewer-only Figure 1) and will add the results in the revised manuscript.

    We agree that investigation of tri-partite interactions is conceptually important for understanding the broader landscape of organelle communication. However, assessing tri-partite organelle contacts is beyond the scope of this study. We recognize that this is one of the key directions for future studies and believe that the MERCdRED platform is a promising tool for exploring such complex interactions.

    Regarding different types of MERCS, we would like to clarify that our study does address this point to some extent. We identified distinct features of MERCS behavior by comparing structures of different sizes-an aspect that, to our knowledge, has not been previously examined. These findings contribute conceptually to our understanding of the dynamic and heterogeneous nature of ER-mitochondria contacts.

    We believe that our methodological development provides important mechanistic insights into MERCS dynamics, as described above. In line with the reviewer's suggestion, we will investigate the physiological impacts of MERCS remodeling in regulating metabolism in response to nutrient starvation. We hope these forthcoming data will further enhance the biological relevance of our findings.

    Taken together, we believe our study provides both a solid technical advance and novel mechanistic insights into MERCS biology, which will be of interest to researchers working on membrane contact sites, organelle dynamics, and cell physiology.

    We will revise the manuscript to more clearly convey the significance and implications of this study.

    1. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer #2

    Major points:

    In Figure 1E (and the rest of the manuscript), the meaning of the label "MERCdRED on Mito" is unclear. A portion of the MERCdRED signal does not co-localize with mitochondria. The authors should clearly define what "MERCdRED on Mito" represents which appears to be the intensity of the MERCdRED signal within the mitochondrial mask. How about the global MERCdRED signal intensity? When the authors knocked-out PDZD8, did the global fluorescence intensity of the MERCdRED signal decrease?

    As the reviewer pointed out, some red signals appear outside of mitochondria in MERCdRED cells, which are presumably due to autofluorescence. While the global red channel fluorescence intensity also decreased upon PDZD8 conditional knockout (cKO), as shown in Reviewer-only Figure 2A, the reduction was less pronounced than the decrease observed when only the red signals on mitochondria were measured (Reviewer-only Figure 2B). We consider the mitochondrial red signals to represent MERCdRED signals, and we agree that the label "MERCdRED on Mito" may be misleading. To improve clarity, we revised the figure labels as follows: "MERCdRED" was changed to "Red channel," and "MERCdRED on Mito" was changed to "MERCdRED (Red signals on Mito)."

    1. While the authors demonstrate that MERCdRED can quantify a reduction in MERCs (e.g., in PDZD8 knockout conditions), it would be valuable to assess its sensitivity to increases in MERCs as well. For example, previous work from the authors (Nakamura et al., 2025) showed that FKBP8 overexpression leads to an increase in MERCs.

    We thank the reviewer for suggesting this valuable experiment. To assess whether the dynamic range of MERCdRED covers increased MERCS formation, we overexpressed PDZD8 in MERCdRED cells. Notably, PDZD8 overexpression resulted in a significant increase in MERCdRED signal intensity, demonstrating that the system is indeed capable of detecting enhanced MERCS formation. These new data were added in the revised manuscript as new Figure 3D-E.

    Minor points:

    1. Please revise the sentence "First, signals from MERCdRED fluorescence overlapped with the mitochondrial marker Tomm20-iRFP were detected by confocal microscopy in living cells."

    We revised this sentence to "First, fluorescence from MERCdRED and the mitochondrial marker Tomm20-iRFP wasdetected by confocal microscopy in living cells."

    1. Description of analyses that authors prefer not to carry out

    Reviewer #2

    Major points:

    1. The authors claim that their construct enables balanced expression of the RA and GB moieties of the reporter. This should be substantiated by showing protein expression levels via Western blot analysis.

    We thank the reviewer for pointing this out. In our system, Tomm20-GB and RA-Sec61β are expressed from a single plasmid using a self-cleaving P2A peptide sequence, which ensures that the two proteins are produced in equimolar amounts upon translation. Therefore, their expression levels are expected to be approximately equal. Given that comparing the expression levels of these two proteins by Western blotting would require extensive work, including obtaining reconstituted proteins to normalize band intensities, but remains inconclusive due to the semi-quantitative nature of the method, we have decided not to pursue this approach.

    Minor points:

    1. In Figure 2, the ER structures are not segmented in the EM images. It would enhance the manuscript to show the three-dimensional spatial relationship between mitochondria and the ER, rather than only highlighting the regions identified as contacts.

    We agree that visualizing the entire ER structure would enhance the reader's understanding of the three-dimensional spatial relationship between mitochondria and the ER. However, complete segmentation of the ER in EM images is extremely labor-intensive. Given the scope and focus of this study, we have decided not to include full ER segmentation in this manuscript.

  2. Review Commons

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

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors develop a new system to study mitochondrial-ER contact sites in living mouse embryonic fibroblast cells and explore the impact that nutritent starvation has on these contact sites in real time. By stably expressing a bicistroic reporter construct of a dimerization-dependent fluorescent protein that will generate a signal once the two moities, one anchored in the ER by Sec61b, and the other anchored in the outer membrane of mitochondria, via TOM20, comme in close apposition. This cell model is validated using sofisticated CLEM experiments and via the ablation of known regulators of MERCs, such as PDZD8 and FKBP8.

    Major comments

    The authors claim to have developed a new for the study of MERCs. They indeed have benchmarked this system using very sophisticated CLEM approaches and through the ablation of known regulators of MERCs, all of which is very carefully performed and convincing. They argue that the generation of a stable cell line via lentiviral delivery is an improvement over the transient transfection approaches that have been applied in the past (see cited references), which I would generally agree. However, they have not contrasted or compared their system to the widly-used SLPICs system from the Tito Cali group (Vallese, F. et al. An expanded palette of improved SPLICS reporters detects multiple organelle contacts in vitro and in vivo. Nat. Commun. 11, 6069 (2020)) which measures bi and tri-partite interactions with other membrane contacts, including mitochondria and ER at two specific distances, which in my opinion has been more extensivley used to study cell and tissue physiology. They accurately point out that the reversability of this and other systems is challenging and it would be important to define highlight whether the current system allows the study of reversible MERCs. It does not appear as though the reversability of MERCs has been explored in this study. The genetic (PDZD8, FKBP8) and nutritional (starvation) interventions are very helpful to benchmark the system. The description of the methods and data appear to be reproducible and the stastical analyses are acceptable.

    Minor comments

    As mentioned above, it would be helpful to reference and compare the current study in the context of reversability, which the current MERCdRED system has the potential to provide beyond the state-of-the-art.

    Significance

    The major strength of this study is the development of a stable cell line that allows for the study of MERCs, which has the potential to study the reversible nature of these membrane contact sites. It is debatable as a stable cell line rather than a transient transfection offers a major advancement, even if it does make the study of the system more straightforward, especially if the phenomenon of reversibilty is to be explored. I believe that the CLEM study offers a very informative and precise way to benchmark the ddFP system. Defining how MERC formation and separation (once again the reversibility discovery) have impacts in cell physiology beyond the distances altered by starvation would improve the study. Examining the impact on calcium homeostasis, lipid metabolism, and other aspects of biology that are known to be influenced by MERCs would be interesting. As such, there are no new conceptual, mechanistic, or functional advances, simply minor technical advances in the creation of a stable cell line followed by very solid benchmarking experiments. More complex tri-partite interactions, studied elsewhere, which are conceptually very important for cell and organelle biology, have not been attempted here. Similarly, the notion of studied different types of MERCs, which have been proposed to be important for cell biology, has not been explored using this single reporter. The target audience for this study is one that is interested in membrane contact sites and quantitative biology.

    My expertise is in mitochondrial fluorescence imaging and biology. I am not an expert in CLEM.

  3. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript entitled "Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8" by Saeko Aoyama-Ishiwatari et al., describes a novel methodology for visualizing contacts between mitochondria and the endoplasmic reticulum (MERCs) by fluorescence microscopy. Inter-organelle contacts, defined as membrane proximities below ~30 nm, fall below the diffraction limit of conventional light microscopy. The method developed by Hirabayashi's laboratory leverages dimerization-dependent fluorescence complementation to create a reporter capable of both visualizing and quantifying ER-mitochondria contacts (MERCs).

    Major points:

    1. The authors claim that their construct enables balanced expression of the RA and GB moieties of the reporter. This should be substantiated by showing protein expression levels via Western blot analysis.
    2. In Figure 1E (and the rest of the manuscript), the meaning of the label "MERCdRED on Mito" is unclear. A portion of the MERCdRED signal does not co-localize with mitochondria. The authors should clearly define what "MERCdRED on Mito" represents which appears to be the intensity of the MERCdRED signal within the mitochondrial mask. How about the global MERCdRED signal intensity? When the authors knocked-out PDZD8, did the global fluorescence intensity of the MERCdRED signal decrease?
    3. While the authors demonstrate that MERCdRED can quantify a reduction in MERCs (e.g., in PDZD8 knockout conditions), it would be valuable to assess its sensitivity to increases in MERCs as well. For example, previous work from the authors (Nakamura et al., 2025) showed that FKBP8 overexpression leads to an increase in MERCs.

    Minor points:

    1. Please revise the sentence "First, signals from MERCdRED fluorescence overlapped with the mitochondrial marker Tomm20-iRFP were detected by confocal microscopy in living cells."
    2. In Figure 2, the ER structures are not segmented in the EM images. It would enhance the manuscript to show the three-dimensional spatial relationship between mitochondria and the ER, rather than only highlighting the regions identified as contacts.

    Significance

    This timely study provides a valuable and innovative approach to overcoming a longstanding technical limitation in the field, enabling dynamic analysis of ER-mitochondria contacts.

    Expertise: cell biology, membrane contact sites, lipid transfer proteins

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors successfully established a stable cell line expressing MERCdRED, a dimerization-dependent fluorescent protein (ddFP)-based sensor for monitoring mitochondrial-ER contact sites (MERCS). Through light and electron microscopy analyses, they demonstrated that MERCS formation is regulated by nutrient availability and requires PDZD8. While the work is technically sound and well-presented, the biological implications of nutrient-dependent MERCS regulation remain underexplored.

    Major Concerns:

    Although the manuscript is methodologically robust and suitable for a Methods-type article, its biological significance is limited. The findings primarily serve as proof-of-concept for the MERCdRED tool, without substantially advancing our understanding of MERCS regulation.

    Significance

    To enhance the impact of the study, the authors could use this sensor to investigate novel biological questions-such as the molecular pathways linking nutrient sensing to MERCS dynamics-or explore downstream activities of nutrient-dependent MERCS formation. Deeper mechanistic insights would significantly strengthen the work's contribution to the field.

  5. Version published to 10.1101/2025.04.17.649323 on bioRxiv