Mitochondrial fission requires DRP1 but not dynamins

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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.

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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

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.

Correspondence to Ira Milosevic or Nuno Raimundo.

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The authors declare no competing interests.

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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). eh, Mitochondrial fission occurs independently of DNM1, DNM2 and DNM3. e, TOM20 immunofluorescence of wild-type and dynamin triple-knockout cells (n = 30 cells each). fh, 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). ln, 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. (fh, ln, 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). df, Mitochondrial morphology analysis of cells in c, as in Fig. 1b–d (n = 30 cells each). gi, 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 (gi). Error bars represent s.d. (df) 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.

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

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