Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission

Abstract

Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

Keywords: Apoptosis; Dynamin-related protein 1; Fission; GTPase; Mitochondria.

Fig. 1.

Characterization of gene-edited MEF cell lines. (A) Gene-edited cell lines were analyzed for steady-state protein levels using antibodies as indicated. The asterisk denotes a non-specific band. (B) Gene-edited MEFs stained with anti-Tom20 (green) and anti-Drp1 (red) antibodies and imaged with confocal microscopy. The insets show a magnified view of the boxed region. Scale bars: 20 μm. (C) Pearson correlation (r) analysis showing the linear correlation where Drp1 colocalizes with Tom20-stained mitochondria. Data are mean±s.e.m. n=15. (D) FRAP analysis. For each indicated cell line, the percentage recovery was measured over 60 s following photobleaching. (E) Endpoint analysis of FRAP. Fluorescence recovery at 60 s postbleach from E. Data in D and E are mean±s.e.m., n=5. *P<0.05, **P<0.005, ***P<0.0005; n.s, non-significant (Student's t-test).

Fig. 2.

Peroxosimal defects and rescue of knockout MEFs. (A) WT, ΔMff and ΔMiD49/51/Mff/Fis1 MEFs were stained with Hoechst 33258 (blue) and immunostained with antibodies against Pex14 (green) and cytochrome c (red) and imaged using confocal microscopy. Scale bars: 20 μm. (B) Peroxisome length was quantified from images in A and Fig. S2A and expressed as a fold change (mean±s.e.m.; n≥30,) over WT. *P<0.05 (Student's t-test). (C) ΔMiD49/51/Mff/Fis1 MEFs were transfected with either MiD49–GFP, MiD51–GFP, GFP–Fis1 or GFP–Mff. Cells were stained with Hoechst 33258 (blue) and immunostained for Tom20 (top panels) or cytochrome c (bottom panel) (red) and either Drp1 or Pex14 (gray) as indicated and imaged with confocal microscopy. Scale bars: 20 μm. Insets show a magnified view of the boxed region; in C, line-scans (15 µm) from the position indicated in the image are also shown to determine colocalization of GFP-tagged adaptors (green) with Drp1 (red).

Fig. 3.

Loss of MiD49 and MiD51 in MEFs confers resistance to cell death. (A) Gene-edited MEFs treated with 20 μM CCCP for 2 h, stained with antibodies against cytochrome c and imaged using confocal microscopy. Scale bars: 20 μm. (B) Cells from A were counted and mitochondrial morphology scored as indicated. Data are mean±s.e.m., n=3. (C) Cells were treated with 20 µM etoposide and 20 µM QVD for 8 h (+Etop.), immunostained for cytochrome c (green) and Tom20 (red) and imaged using confocal microscopy. Diffuse cytochrome c staining is observed in etoposide-treated WT and single-adaptor-knockout cells. Scale bars: 20 μm. (D) Gene-edited cells were treated with 20 μM etoposide for 16 h, stained with annexin-V–FITC and propidium iodide (PI) and analyzed by performing FACS. Data are mean±s.e.m., n=4. *P<0.05, **P<0.005, ***P<0.0005 (Student's t-test). (E) Gene-edited cells were treated with 20 μM etoposide for 16 h, then the heavy membrane and supernatant fractions were isolated and analyzed by western blotting with antibodies as indicated. (F) The percentage of cytochrome c released into the supernatant fraction upon etoposide treatment. Data are mean±s.e.m., n=4.

Fig. 4.

BioID proximity-labeling analysis of MiD51, Mff and Fis1. (A) BirA*-tagged MiD51 and MiD51^ΔPEYFP (schematics at top) were expressed in ΔMiD49/51 MEFs, stained with Hoechst 33258 (blue) and immunostained using antibodies against the HA tag (green) and Tom20 (red). (B) BioID proximity labeling and enrichment of proteins in BirA*–MiD51 relative to BirA*–MiD51^ΔPEYFP. A Student's t-test was performed on the log-transformed LFQ intensities from three biological replicates of MiD51 and MiD51^ΔPEYFP enrichments, and plotted against the difference between the mean log-transformed intensities. The thresholds for enriched were set at P≤0.05 and t-test difference at >1.5 (log₁₀ LFQ intensities). (C) BirA*–Mff and BirA*–Mff^2RM (schematics at top) were expressed in ΔMff MEF cells, stained with Hoechst 33258 (blue) and immunostained using anti-Flag (green) and anti-Pex14 (red) antibodies. (D) As for B, comparing relative enrichment between Mff and Mff^2RM. (E) BirA*-tagged Fis1 (schematic at top) was expressed in ΔFis1 MEF cells, stained with Hoechst 33258 (blue) and immunostained for Flag (green) and Tom20 (red). (F) As for B, comparing relative enrichment between Fis1 and Fis1^TMD. The insets show a magnified view of the boxed region. Scale bars: 20 μm.

Fig. 5.

MiD51 and Mff have opposing effects on Drp1 GTPase activity. Drp1 GTPase activity stimulated by Ni-liposomes in the presence or absence of the indicated Drp1 adaptor variants was plotted by (A) linear regression, and the mean k_cat values were calculated (B). (C) Drp1 GTPase activity stimulated by Ni-liposomes with the incorporation of Mff at 4.5 min as indicated. (D) Drp1 GTPase activity stimulated by Ni-liposomes with indicated adaptors added at 4.5 min and where indicated, 20 µM ADP (hatched lines) added at 8.5 min. Data mean±s.d. for n≥3.