Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Matters Arising
  • Published:

Mitochondrial fission requires DRP1 but not dynamins

Nature volume 570, pages E34–E42 (2019)Cite this article

Subjects

The Original Article was published on 31 October 2016

ARISING FROM J. E. Lee Nature https://doi.org/10.1038/nature20555 (2016)

Mitochondrial fission is necessary for the maintenance of the mitochondrial network, and relies on the GTPase dynamin-related protein 1 (DRP1; also known as DNM1L, dynamin-1-like protein)1,2. DRP1 forms helical oligomers that wrap around the mitochondrial outer membrane and scission it3,4. Recently, it was proposed that DRP1 is not sufficient to execute mitochondrial fission, and that another GTPase, dynamin-2 (DNM2, also known as DYN2), was an essential component of the mitochondrial division machinery5. Here we report that mouse fibroblasts lacking all three mammalian dynamin proteins (DNM1, DNM2 and DNM3)6 (dynamin triple-knockout cells), as well as cells with knockdown of DNM2 only, do not display defects in mitochondrial fission or mitochondrial hyperfusion, which were readily detected after knockdown of DRP1, even in the dynamin triple-knockout cells. The same was observed when examining peroxisomal fission (mitochondria and peroxisomes share the fission machinery7). Thus, our data show that DRP1 is essential for mitochondrial and peroxisomal fission, whereas DNM1–DNM3 are dispensable.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Mitochondrial morphology is not affected in the absence of DNM1, DNM2 and DNM3.
Fig. 2: DRP1, but not dynamin proteins, is necessary for induced-mitochondrial fission.

Data availability

Data supporting the findings of this study are available from the authors upon reasonable request.

References

  1. Bleazard, W. et al. The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1, 298–304 (1999).

    Article  CAS  Google Scholar 

  2. Cereghetti, G. M. et al. Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria. Proc. Natl Acad. Sci. USA 105, 15803–15808 (2008).

    Article  ADS  CAS  Google Scholar 

  3. Kalia, R. et al. Structural basis of mitochondrial receptor binding and constriction by DRP1. Nature 558, 401–405 (2018).

    Article  ADS  CAS  Google Scholar 

  4. Ji, W. K., Hatch, A. L., Merrill, R. A., Strack, S. & Higgs, H. N. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites. eLife 4, e11553 (2015).

    Article  Google Scholar 

  5. Lee, J. E., Westrate, L. M., Wu, H., Page, C. & Voeltz, G. K. Multiple dynamin family members collaborate to drive mitochondrial division. Nature 540, 139–143 (2016).

    Article  ADS  CAS  Google Scholar 

  6. Park, R. J. et al. Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors. J. Cell Sci. 126, 5305–5312 (2013).

    Article  CAS  Google Scholar 

  7. Schrader, M., Costello, J. L., Godinho, L. F., Azadi, A. S. & Islinger, M. Proliferation and fission of peroxisomes—an update. Biochim Biophys Acta 1863, 971–983 (2016).

    Article  CAS  Google Scholar 

  8. Sesaki, H. & Jensen, R. E. Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape. J. Cell Biol. 147, 699–706 (1999).

    Article  CAS  Google Scholar 

  9. Otera, H. et al. Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells. J. Cell Biol. 191, 1141–1158 (2010).

    Article  CAS  Google Scholar 

  10. Kashatus, J. A. et al. Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth. Mol. Cell 57, 537–551 (2015).

    Article  CAS  Google Scholar 

  11. Ishihara, N. et al. Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice. Nat. Cell Biol. 11, 958–966 (2009).

    Article  CAS  Google Scholar 

  12. Karbowski, M. et al. Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J. Cell Biol. 164, 493–499 (2004).

    Article  CAS  Google Scholar 

  13. Francy, C. A., Clinton, R. W., Fröhlich, C., Murphy, C. & Mears, J. A. Cryo-EM Studies of Drp1 reveal cardiolipin interactions that activate the helical oligomer. Sci. Rep. 7, 10744 (2017).

    Article  ADS  Google Scholar 

  14. Kamerkar, S. C., Kraus, F., Sharpe, A. J., Pucadyil, T. J. & Ryan, M. T. Dynamin-related protein 1 has membrane constricting and severing abilities sufficient for mitochondrial and peroxisomal fission. Nat. Commun. 9, 5239 (2018).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This work was funded by ERG StG 337327 MitoPexLysoNETWORK (N.R.), DFG E. Noether MI-1702/1(I.M.), Schram Stiftung (I.M.) and DFG SFB1190-P02 (I.M., N.R.), ASPPA Association and Fondazione Cariparo (T.B.F.). We thank P. de Camilli for the dynamin triple-knockout cells.

Author information

Author notes

  1. These authors contributed equally: Tiago Branco Fonseca, Ángela Sánchez-Guerrero

Authors and Affiliations

  1. Institute of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany

    Tiago Branco Fonseca, Ángela Sánchez-Guerrero & Nuno Raimundo

  2. European Neuroscience Institute Göttingen, A Joint Initiative of the University Medical Center Göttingen and the Max-Planck-Society, Göttingen, Germany

    Tiago Branco Fonseca, Ángela Sánchez-Guerrero & Ira Milosevic

  3. Department of Biology, University of Padova, Padova, Italy

    Tiago Branco Fonseca

Authors
  1. Tiago Branco Fonseca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ángela Sánchez-Guerrero
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ira Milosevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nuno Raimundo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.R. and I.M. designed and supervised the study. T.B.F., A.S.-G., I.M. and N.R. performed the experiments, analysed data and prepared the figures. N.R. wrote the manuscript which was then edited by all authors.

Corresponding authors

Correspondence to Ira Milosevic or Nuno Raimundo.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Schematic representation of the experimental approach.

a, b, Cre-loxP recombination system (a) and protocol to obtain dynamin triple-knockout and DRP1-knockdown cells (b). c, d, Representative immunoblot (c) and quantification (d) of expression levels of DNM1–DNM3 (n = 3). e–h, Mitochondrial fission occurs independently of DNM1, DNM2 and DNM3. e, TOM20 immunofluorescence of wild-type and dynamin triple-knockout cells (n = 30 cells each). f–h, Respective mitochondrial morphology analysis of the cells in e, as in Fig. 1b–d (n = 30 cells each). i, j, Immunoblot (i) and quantification (j) of DNM2 in scrambled and DNM2-knockdown fibroblasts (n = 3). α-tubulin was used as a loading control. k, TOM20 immunofluorescence of fibroblasts transfected with scrambled and DNM2 DsiRNA (n = 30 cells each). l–n, Respective mitochondrial morphology analysis of cells in k (n = 30 cells each). o, p, Immunoblot and quantification analyses of mitochondrial fission (o) and fusion (p) proteins of DNM2 in fibroblasts transfected with scrambled and DNM2 DsiRNA (n = 3). q, r, Immunoblot (q) and quantification (r) of DNM2 in scrambled and DNM2-knockdown HeLa cells (n = 3). s, TOM20 immunofluorescence of HeLa cells transfected with scrambled and DNM2 DsiRNA (n = 25 cells each). t, Respective mitochondrial area of cells in s (n = 25 cells each). u, Representative time-lapse of a mitochondrial fission event in scrambled and DNM2-knockdown HeLa cells expressing Mito-GFP (n = 15 cells each). Error bars represent s.d. (f–h, l–n, t) and s.e.m. (d, j, o, p, r). *P < 0.05, ***P < 0.001, one-tailed Student’s t-test. Scale bars, 10 µm (e, k, s) and 2 µm (u).

Extended Data Fig. 2 DRP1, but not dynamin proteins, is necessary for induced-mitochondrial fission.

a, Immunoblots of DNM1, DNM2 and DNM3 (Dyn pan) and DRP1 in scrambled and DRP1-knockdown cells in both wild-type and dynamin triple-knockout cells. b, Relative quantification of data in a (n = 3). c, TOM20 immunofluorescence of wild-type and dynamin triple-knockout cells transfected with scrambled or DRP1 DsiRNA (n = 30 cells each). d–f, Mitochondrial morphology analysis of cells in c, as in Fig. 1b–d (n = 30 cells each). g–i, Representative time-lapse frames of mitochondrial fission events in wild-type mouse embryonic fibroblasts (MEFs) co-transfected with DNM2–mRFP and mito-GFP (n = 21 cells) (g), or with DRP1–GFP, DNM2–mCherry and mito-BFP (n = 15 cells) (h) and of HeLa cells expressing DRP1–GFP, DNM2–mCherry and mito-BFP (n = 12 cells) (i). Scale bars, 10 μm (c) and 2 µm (g–i). Error bars represent s.d. (d–f) and s.e.m. (b). **P < 0.01, ***P < 0.001, one-tailed Student’s t-test.

Extended Data Fig. 3 Peroxisomes do not require dynamin proteins for fission.

a, Schematic model of peroxisomal fission induction by PEX11β and DRP1 overexpression (adapted from ref. 7). PEX11β oligomerizes at the peroxisome division site, promoting peroxisomal membrane elongation, enrichment of fission factors such as FIS1 and MFF, and recruitment of DRP1 at the division site for the scission event. b, PMP70 immunofluorescence of wild-type mock mouse embryonic fibroblasts and wild-type fibroblasts transfected with PEX11β and DRP1 plasmids (n = 30 cells each). c, d, Respective quantification of peroxisomal morphology by number of peroxisomes per ROI (c) and peroxisomal area per ROI (d) for the cells in b (n = 30 cells each). e, PMP70 immunofluorescence of wild-type and dynamin triple-knockout cells co-transfected with PEX11β and DRP1 (n = 30 cells each). f, g, Respective peroxisomal morphology of cells in e, as in c and d (n = 30 cells each). h, Representative time-lapse frames of peroxisomal fission events in wild-type and dynamin triple-knockout cells, co-transfected with the peroxisomal marker Ub-RFP-SKL and DRP1–GFP (n = 21 cells). Scale bars, 10 μm (b, e) and 2 μm (h). Error bars represent s.d. *P < 0.05, one-tailed Student’s t-test.

Supplementary information

Supplementary Information

This file contains Supplementary Methods and uncropped blots from Fig. 1 and Extended Data Fig. 1 and 2.

Reporting Summary

Supplementary Video 1

Mitochondrial fission event (arrow) in dynamin-TKO fibroblasts expressing Mito-GFP for 21h. 63x objective, imaged at 37 °C every 4s, video rate: 10f/s, scale bar 2µm.

Supplementary Video 2

Mitochondrial fission event (arrow) in HeLa cells with dynamin-2 KD expressing Mito-GFP for 22h. 63x objective, imaged at 37 °C every 4s, video rate: 10f/s, scale bar 2µm.

Supplementary Video 3

Dynamin-TKO (left) and dynamin-TKO+Drp1KD (right) fibroblasts expressing Mito-GFP for 22h, treated with CCCP. 63x objective imaged at 37 °C every 4s, video rate: 10f/s, scale bar 10µm.

Supplementary Video 4

Mitochondrial fission event (arrow) in WT fibroblasts expressing Mito-GFP and dynamin-2-mRFP for 22h. 63x objective imaged at 37 °C every 2s, video rate: 10f/s, scale bar 10µm.

Supplementary Video 5

Mitochondrial fission event (arrow) in WT fibroblasts expressing Mito-BFP, Drp1-GFP and dynamin-2-mRFP for 24h. 63x objective imaged at 37 °C every 2s, video rate: 5f/s, scale bar 10µm.

Supplementary Video 6

Mitochondrial fission event (arrow) in HeLa cells expressing Mito-BFP, Drp1-GFP and dynamin-2-mRFP for 24h. 63x objective imaged at 37 °C every 2s, video rate: 5f/s, scale bar 10µm.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fonseca, T.B., Sánchez-Guerrero, Á., Milosevic, I. et al. Mitochondrial fission requires DRP1 but not dynamins. Nature 570, E34–E42 (2019). https://doi.org/10.1038/s41586-019-1296-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-019-1296-y

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing