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. 2013 Mar;24(5):659-67.
doi: 10.1091/mbc.E12-10-0721. Epub 2013 Jan 2.

Fis1, Mff, MiD49, and MiD51 Mediate Drp1 Recruitment in Mitochondrial Fission

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Free PMC article

Fis1, Mff, MiD49, and MiD51 Mediate Drp1 Recruitment in Mitochondrial Fission

Oliver C Losón et al. Mol Biol Cell. .
Free PMC article

Abstract

Several mitochondrial outer membrane proteins-mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)-have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.

Figures

FIGURE 1:
FIGURE 1:
Mitochondrial elongation in Fis1-null, Mff-null, and Fis1/Mff-null MEFs. (A) Mitochondrial morphology in wild-type and mutant cells. Mitochondria were visualized by immunofluorescence against Tom20. Scale bar, 10 μm. Inset scale bar, 5 μm. (B) Scoring of mitochondrial network morphologies for the indicated cell lines. Each cell was scored into one of four morphological categories. ***p < 0.001, **p < 0.005, *p < 0.02. (C) Morphometric analysis. Well-resolved mitochondria (Tom20) in the cell periphery were used for morphometric analysis. Total mitochondrial number and area were measured; the ratio of these two values was plotted as percentage of wild-type. Twenty ROIs from two independent experiments were analyzed. Error bars, SEM. *p < 0.0001. (D) FRAP analysis. For each of the indicated cell lines, the graph shows the fluorescence recovery over 10 s after photobleaching. Fluorescence data were collected every 200 ms postbleach. (E) Endpoint analysis of FRAP data. The fluorescence recovery ± SEM at 10 s postbleaching is shown. For D and E, 20 FRAP trials were averaged. **p < 0.001, *p < 0.01. (F, G) Fis1-null (F) and Mff-null (G) cells were transiently transfected with expression constructs for Fis1, Mff, or an empty control vector. Cells were scored into one of four morphological categories. F, fragmented; <50, <50% of mitochondria are long tubules; >50, > 50% of mitochondria are long tubules; N, net-like. **p < 0.005, *p < 0.05. Statistical testing was performed by combining the F and <50 classes, which represent cells with mostly short mitochondria. (H) Cells from the indicated cell lines were treated with 50 μM CCCP for 1 h, 100 μM etoposide for 5 h, 1% O2 for 24 h, or 0.1% serum for 3 d. Histograms show the percentage of cells with fragmented or mostly short tubular mitochondria. For B, F, G, and H, data are the averages ± SEM from three independent experiments, with 100 cells scored per experiment. All statistical testing was performed with the Student's t test.
FIGURE 2:
FIGURE 2:
Drp1 recruitment and assembly on mitochondria are affected in Fis1-null and Mff-null cells. (A) Drp1 puncta in the indicated cell lines. To improve visualization of mitochondrial Drp1, the cells were briefly treated with 0.001% digitonin before fixation to reduce the level of cytosolic Drp1. Mitochondria were highlighted by immunofluorescence against Tom20. Scale bar (left), 10 μm. Center and right, magnified images of the boxed regions. Scale bar, 5 μm. Right panel, a mask corresponding to the mitochondrial channel was applied to the Drp1 channel to obtain only mitochondrial Drp1 fluorescence. The heat map reflects Drp1 fluorescence intensity (FI). (B) Density of mitochondrial Drp1 puncta. (C) Drp1 fluorescence per puncta. In B and C, the data are normalized to the wild-type control. Error bars, SEM. Twenty-five ROIs were analyzed from 10 to 12 cells for each group. *p < 0.05, **p < 0.005. Statistical testing was performed with the Student's t test. (D) Drp1 recruitment to mitochondria. Cytosol and purified mitochondrial fractions were prepared from the indicated MEF cells and analyzed by Western blotting for Drp1, Tom20 (mitochondrial marker), and β-tubulin (TUBB; cytosolic marker). The mitochondrial lanes were loaded with 30-fold more cell equivalents than with the cytosolic lanes. Both short and long exposures for the Drp1 blot are presented.
FIGURE 3:
FIGURE 3:
Knockdown of MiD49 or MiD51 causes mitochondrial elongation and enhances the Fis1-null, Mff-null, and Fis1/Mff-null phenotypes. (A) Mitochondrial morphology in wild-type MEFs treated with control siRNA, siRNA against MiD49, siRNA against MiD51, or both. Scale bar, 10 μm. Insets are magnified images of the boxed regions. Scale bar, 5 μm. Mitochondria were highlighted by expression of Cox8-DsRed. (B) Western blot of cell lysates containing single and double knockdown of the MiDs. SOD2 is a loading control. (C) Scoring of mitochondrial network morphologies for knockdown experiments in wild-type, Fis1-null, Mff-null, and Fis1/Mff-null cells. Data were obtained from three independent experiments, with 100 cells scored per experiment. Error bars, SEM. C, collapsed; L, long; N, net-like; S, short.
FIGURE 4:
FIGURE 4:
Overexpression of MiD49 or MiD51 causes enhanced phosphorylation of Drp1 on S637. (A) Increased Drp1 S637-PO4 in cells overexpressing MiD49 or MiD51. Top, lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1 and the Myc-tagged MiD. Bottom, Drp1 was immunoprecipitated, and the levels of Drp1 S637-PO4 were detected with a phosphospecific Drp1 antibody. Drp1 was used as a loading control for the immunoprecipitated samples. (B) Drp1 S616-PO4 levels in cells overexpressing MiD49 or MiD51. Lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1, Drp1 S616-PO4, and the Myc-tagged MiD. Actin was used as a loading control. (C) Increased Drp1 S637-PO4 recruitment to mitochondria in cells overexpressing MiD49 or MiD51. Top, cytosol and crude mitochondrial fractions were prepared from the indicated HeLa cells and analyzed by Western blotting for Drp1, superoxide dismutase 2 (SOD2; mitochondrial), and β-tubulin (TUBB; cytosolic). The mitochondrial lanes were loaded with 20-fold more cell equivalents than with the cytosolic lanes. Bottom, the loading of mitochondrial fractions was normalized for the total Drp1 level. The relative levels of Drp1 S616-PO4 and Drp1 S637-PO4 on mitochondria were assessed by Western blotting with phosphospecific antibodies. In A–C, densitometry was performed on the S616-PO4 and Drp1 S637-PO4 blots and was normalized to the total Drp1 in each sample. Values are presented as proportions of wild type. (D) Binding of MiD49 and MiD51 to phospho-mutants of Drp1. 293T cells were cotransfected with Myc-tagged MiD and Drp1 mutants as indicated. Cells were treated with cross-linker and solubilized. Top, expression of Drp1 and Myc-MiD in the lysates. Bottom panel, anti-Myc immunoprecipitates were analyzed for Drp1. Note that this assay only detects transfected Drp1 (compare lanes 2, 3–7, and 10). A, phospho-null mutant; D, phosphomimetic mutant; S, wild-type Drp1.
FIGURE 5:
FIGURE 5:
Mitochondrial elongation and increased Drp1 S637-PO4 caused by MiD overexpression can be reversed by CCCP. (A) Reduction of Drp1 S637-PO4 levels in CCCP treated cells. Left, lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1. Cells were treated with dimethyl sulfoxide (DMSO; indicated by D) or CCCP (indicated by C). Right, Drp1 S637-PO4 was analyzed as in Figure 4A. Both short and long exposures for the Drp1 S637-PO4 blot are presented. (B) Drp1 S616-PO4 levels do not change in CCCP-treated cells. Lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed as in Figure 4B. Cells were treated with DMSO (indicated by D) or CCCP (indicated by C). Densitometry was performed on the S616-PO4 and Drp1 S637-PO4 blots and was normalized to the total Drp1 in each lane. Values are presented as ratios of the CCCP/DMSO values for each group. (C) CCCP reverses mitochondrial elongation in MiD-overexpressing MEFs. Cells were transfected with MiD49-Myc or MiD51-Myc and subsequently treated with vehicle (DMSO) or CCCP. MiD-expressing cells were identified by Myc immunofluorescence, and mitochondria were visualized with Tom20 immunofluorescence. In the DMSO-treated samples, note that MiD-Myc expression causes mitochondrial elongation. In the CCCP-treated samples, note that MiD-Myc–expressing cells have fragmented mitochondria. Scale bar, 10 μm. (D) Scoring of mitochondrial network morphologies for MEFs transfected with MiD49-Myc, MiD51-Myc, or empty vector and treated with DMSO or CCCP. The data are from three independent experiments, each with 100 cells scored. Error bars, SEM. C, collapsed; L, long; N, net-like; S, short.
FIGURE 6:
FIGURE 6:
MiD49 and MiD51 can restore CCCP-induced mitochondrial fragmentation in Fis1/Mff-null cells. (A) Restoration of Drp1 puncta in Fis1/Mff-null cells. Fis1/Mff-null cells were transfected with MiD49-Myc, MiD51-Myc, or empty vector. Cells were analyzed for MiD-Myc expression (anti-Myc), mitochondrial morphology (Cox8-Dendra2), and Drp1. Scale bar, 10 μm. Right, magnified images of the boxed regions. Scale bar, 5 μm. (B) Density of Drp1 puncta on mitochondria. Twenty ROIs were analyzed in 8–10 cells per group. Data are normalized to wild-type cells. Error bars, SEM. *p < 0.001. DM, Fis1/Mff double mutant. (C) Scoring of mitochondrial network morphologies for Fis1/Mff-null cells transfected with MiD49-Myc, MiD51-Myc, or empty vector and treated with DMSO or CCCP. Data are from three independent experiments, each with 100 cells scored. Error bars, SEM. C, collapsed; L, long; N, net-like; S, short. **p = 0.002, *p = 0.02. (D) Representative micrographs of Fis1/Mff-null cells transfected with MiD49-Myc or MiD51-Myc and treated with DMSO or CCCP. MiD-Myc–expressing cells were analyzed for mitochondrial morphology (Tom20). With CCCP treatment, note that the MiD-Myc–transfected cells have fragmented mitochondria, whereas the nontransfected cells are resistant to CCCP-induced fragmentation. Scale bar, 10 μm. Statistical testing was performed with the Student's t test.

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