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An intermembrane space protein facilitates completion of mitochondrial division in yeast

Olivia M. Connor, Srujan K. Matta, Jonathan R. Friedman

Preprint posted on 1 April 2023 https://www.biorxiv.org/content/10.1101/2023.03.31.535139v1

Article now published in Journal of Cell Biology at https://doi.org/10.1083/jcb.202303147

Mitochondrial fission: a solo mission for Dnm1 (Drp1 in humans) or does it need an inside accomplice? A new preprint by Connor et al. uncovers a novel mitochondrial intermembrane protein that's crucial for Dnm1-mediated fission!

Selected by Leeba Ann Chacko

Updated 15 August 2023 with a postLight by Leeba Ann Chacko

Congratulations to Olivia M. Connor, Srujan K. Matta, and Jonathan R. Friedman! Their preprint titled “An intermembrane space protein facilitates completion of mitochondrial division in yeast” has been published in the Journal of Cell Biology under the title “Completion of mitochondrial division requires the intermembrane space protein Mdi1/Atg44”.

Overall, there are no major changes between the preprint and the published paper in terms of the core findings and theoretical models. Both versions explore the positioning of Mdi1 relative to Dnm1 at division sites and propose that Mdi1 may bind and locally distort regions of the IMM and/or OMM, allowing Dnm1 to more easily constrict and divide both membranes.

The published version addresses some of the preprint’s limitations by incorporating additional controls and experimental insights. In particular, the authors performed immunofluroscence using the functional internal 2xFLAG-tagged version of Mdi1. This technique enabled them to confirm that the endogenous protein indeed localizes to specific focal points within mitochondria.

The published version also explores the relationship between Dnm1 and Mdi1 more deeply, using simultaneous imaging—an experiment aligned with a question raised in the preLight. This revealed that both proteins must interact with each other for a successful division to take place.

In summary, the transition from preprint to published paper has maintained the core understanding while adding valuable experimental refinements that strengthen the insights into mitochondrial division and the role of Mdi1 in this process.

Please take your time to explore both the preprint and the published version to appreciate the continuity and enhancements of this work.

Question to authors: In your view, what were the most important improvements in your study as a result of peer review?’

Jonathan R. Friedman: Our revised manuscript included a number of added controls and new data such as the positioning of Dnm1 relative to Mdi1 at division sites. However, one of the most critical improvements in the manuscript was in response to a reviewer who asked whether the observations we make with regards to Mdi1 localization could be an artifact of a non-functional tag. We also shared this concern as a major caveat of our conclusions in the original preprint. The reviewer suggested we perform immunofluorescence utilizing our functional internal FLAG-tagged version of Mdi1. While this was a bit of a technical challenge, we were able to demonstrate that the endogenous protein indeed localizes to focal structures within mitochondria. This observation was key for supporting the conclusions we can make from live cell imaging experiments.

Effect of Dnm1 and Mdi1 on mitochondrial fission (created with BioRender.com)

 

Background:

Mitochondria are organelles that have a double membrane and change shape through fission and fusion. Dnm1 (Drp1 in humans) is an outer mitochondrial membrane (OMM) protein that aids mitochondrial fission by constricting and dividing the mitochondria. It is unclear if specialized inner mitochondrial proteins independently aid the fission of the inner mitochondrial membrane (IMM). MTP18 and S-OPA1 are two IMM proteins that impact mitochondrial morphology, however, the precise way they trigger mitochondrial fission remains unclear (Wai & Langer, 2016).

A recent study using electron tomography on mammalian cells suggests that both the OMM and IMM undergo simultaneous constriction via external forces from actin and septins (Mageswaran et al., 2021). Conversely, Spier et al. were able to observe independent IMM fission in human cells by expressing a human codon-optimized version of FtsZ, which is a protein responsible for bacterial division (Spier et al., 2020).

In yeast, it is thought that the OMM proteins Dnm1 and Fis1 are the sole players involved in mitochondrial division. Mdm33 is a yeast IMM protein involved in mitochondrial division, but its absence does not cause the same morphological defects observed for other fission proteins. It remains elusive whether an independent IMM machinery exists to aid IMM fission. For the first time, Connor et. al. have identified a yeast-specific inter-membrane space (IMS) protein called Mitochondrial Division IMS 1 (Mdi1) that is needed for mitochondrial fission. The authors of this preprint hypothesize that Mdi1 possibly assists Dnm1 by distorting the inner membrane during mitochondrial division and show that Dnm1 requires the harmonious action of the IMS protein to divide the organelle effectively.

Key results:

Deleting Mdi1 results in hyperfused mitochondria, resembling the phenotype observed in Δdnm1 or Δfis1 cells. Furthermore, the hyperfused mitochondrial morphology persists in Δmdi1 cells despite the loss of function of the IMM fusion protein, fzo1.

Mitochondrial DNA (mtDNA) is typically preserved in hyperfused mitochondria but is lost in fragmented mitochondria. As a result, cells with fragmented mitochondria (non-functional fzo1) exhibit growth defects when grown on non-fermentable media, whereas those with normal (WT) or hyperfused mitochondria (Δdnm1) do not. Deleting Dnm1 from cells lacking fzo1 function rescues the growth defect phenotype. Intriguingly, this is not the case when Mdi1 is deleted.

Next, the authors investigate the sub-mitochondrial localization of Mdi1 by using a split GFP system, where an IMS protein is tagged with GFP1-10 and Mdi1 with a complementary GFP11 tag. The researchers observe discrete GFP puncta on the mitochondria, confirming the localization of Mdi1 to the IMS. Additionally, Mdi1 puncta are located near the site of mitochondrial fission.

Mid1 and Dnm1 can individually localize to mitochondrial constriction sites, but neither can independently trigger fission. Dnm1 requires Mid1 for fission, as demonstrated by the inability of Dnm1 to mediate fission in the absence of Mid1. Interestingly, mitochondrial constriction induced by the mitochondrial uncoupling agent CCCP persists without Dnm1, Fis1, or Mid1.

Mdi1 is conserved across several fungal species, and the authors find that its function is also conserved in a related yeast, Schizosaccharomyces Pombe. The authors identify a predicted amphipathic α-helix in Mdi1 that, when altered, affects mitochondrial morphology. This preprint shows that efficient mitochondrial division requires the coordinated action of Mdi1 and Dnm1.

What I liked about this preprint:

I have worked on Dnm1 in the past and the loss of this protein has a characteristic and beautiful (in my opinion) net-like morphology. The moment I saw the title of this preprint I was immediately intrigued because, for the first time, I came across a piece of work that explores the possibility of an internal mitochondrial protein, altering mitochondrial morphology the way Dnm1 does!

Questions for the authors:

  1. It was observed in S.pombe cells that altering the mitochondrial morphology affects the presence and structure of mtDNA (Dong et al., 2022). The authors also found that Δfzo1 cells grew slower than Δdnm1Δfzo1 cells in non-fermentable media. It is intriguing that Δmdi1 fzo1-1 cells did not grow in ethanol/glycerol media at 37°C despite the mitochondrial morphology staying mostly interconnected. Could it be possible that the mtDNA in these cells is affected similar to what is observed for Δfzo1 S.pombe cells?
  2. In Fig. S1A and B, it is not clear to me what the difference between constriction and hyper-constriction is. Is there a way to quantitatively measure the difference in the sizes of constriction sites?
  3. In Fig. 2D and E, you examine the percentage of Mdi1 foci present in the vicinity of a division site. Would it be beneficial to quantify the total number of division events compared to the number of Mdi1 foci present in the mitochondria i.e. detectable Mdi1 enrichment but no fission event?
  4. Would it be possible to observe Dnm1 and Mdi1 dynamics simultaneously in the future?

References:

  1. Dong, F., Zhu, M., Zheng, F., & Fu, C. (2022). Mitochondrial fusion and fission are required for proper mitochondrial function and cell proliferation in fission yeast. The FEBS Journal, 289(1), 262–278. https://doi.org/10.1111/FEBS.16138
  2. Mageswaran, S. K., Grotjahn, D. A., Zeng, X., Barad, B. A., Medina, M., Hoang, M. H., Dobro, M. J., Chang, Y.-W., Xu, M., Yang, W. Y., & Jensen, G. J. (2021). Nanoscale details of mitochondrial fission revealed by cryo-electron tomography. BioRxiv, 2021.12.13.472487. https://doi.org/10.1101/2021.12.13.472487
  3. Spier, A., Sachse, M., Tham, N. T., Matondo, M., Cossart, P., & Stavru, F. (2020). Bacterial FtsZ induces mitochondrial fission in human cells. BioRxiv, 2020.01.24.917146. https://doi.org/10.1101/2020.01.24.917146
  4. Wai, T., & Langer, T. (2016). Mitochondrial Dynamics and Metabolic Regulation. Trends in Endocrinology & Metabolism, 27(2), 105–117. https://doi.org/10.1016/J.TEM.2015.12.001

Tags: dnm1, fission, mdi1, mitochondria, yeast

Posted on: 5 May 2023 , updated on: 16 August 2023

doi: https://doi.org/10.1242/prelights.34475

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Author's response

Jonathan R. Friedman shared

1) It was observed in pombe cells that altering the mitochondrial morphology affects the presence and structure of mtDNA (Dong et al., 2022). The authors also found that Δfzo1 cells grew the slower than Δdnm1Δfzo1 cells in non-fermentable media. It is intriguing that Δmdi1 fzo1-1 cells did not grow in elhanol/glycerol media at 37C despite the mitochondrial morphology staying mostly interconnected. Could it be possible that the mtDNA in these cells are affected similar to what is observed in Δfzo1 in S.pombe cells?

We agree it is interesting that ∆mdi1 fzo1-1 and ∆dnm1 fzo1-1 cells do not have identical growth rates in respiratory media at 37°C. We think this could be an indication that mitochondrial division proceeds to some small degree in the absence of Mdi1, and that this ultimately leads to loss of mtDNA due to the inability to fuse. However, since Mdi1 localizes inside mitochondria, we want to explore the possibility that Mdi1 plays a role, potentially through interacting partners, in coupling mitochondrial DNA to sites of division. Another possibility is that the differential growth is related to mitophagy, which, according to recent work by Fukuda et al., requires Mdi1 but not Dnm1.

2) It Fig. S1A and B, it is not clear to me what the difference between constriction and hyper-constriction is. Is there a way to quantitatively measure the difference in the sizes of constriction sites?

A limitation of our study is that mitochondrial morphology is observed by fluorescence microscopy, which is an indirect measure of membrane continuity. We refer to division sites that are induced by CCCP treatment as hyper-constrictions because they lead to thin threads of interconnected mitochondrial signal. However, unfortunately we are not able to resolve or quantitatively assess the diameter of mitochondrial constriction sites with this approach.

3) In Fig. 2D and E, you examine the percentage of Mdi1 foci present in the vicinity of a division site. Would it be beneficial to quantify the total number of division events compared to the number of Mdi1 foci present in the mitochondria i.e detectable Mdi1 enrichment but no fission event.

This is an interesting point and something we tried to clarify in the revision. As in the case of Dnm1, there are many more Mdi1 foci at any given time than there are division events. Because photobleaching issues prevented any long-term imaging of Mdi1, we are not sure if all of these foci will eventually mark a mitochondrial division event. However, one hypothesis is that some of these sites represent inactive pools of Mdi1.

4) Would it be possible to observe Dnm1 and Mdi1 dynamics simultaneously in the future?

In our revision, we attempted this experiment. While we were not able to take long enough movies to determine which protein localizes to the division site first, we could clearly see that the majority of Dnm1-marked division sites were labelled with Mdi1. Interestingly, we also observed that about half of Dnm1 sites were co-localized with Mdi1, and conversely, half of Mdi1 sites appeared to be Dnm1 positive. Our current model is that the two proteins have to find each other in order for a productive division to occur, but whether this is stochastic or regulated in some way will be the focus of future work.

 

 

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