Adaptor proteins MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and are specific for mitochondrial fission

Abstract

Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.

Keywords: Cell Biology; Confocal Microscopy; Drp1; Fission; Mammal; MiD49; MiD51; Mitochondria; Morphology; Peroxisomes.

FIGURE 1.

Induction of MiD49 and MiD51 expression causes mitochondrial elongation by Drp1 inactivation and mitofusin-dependent elongation. A, MiD49-induced wild-type, Mfn1^−/−, Mfn2^−/−, and Mfn-DKO MEFs were treated with 4-OHT for 0–48 h to induce MiD49 expression. Following induction of MiD49, whole cell lysates were subjected to SDS-PAGE and analyzed by Western blotting with antibodies against Mfn1, Mfn2, human MiD49, and β-actin. B, following induction of MiD49 for either 0 or 24 h with 4-OHT (4HT), cells were stained with MitoTracker Red (red) and Hoechst (blue). The dashed boxes in the left panels are enlarged to enhance visibility in the right panels (Inset). Scale bars = 20 μm. C, Mfn1^−/−, Mfn2^−/−, and Mfn-DKO cells were treated with or without 4-OHT to induce MiD49 protein expression before being subjected to assessment of mitochondrial phenotypes (mean ± S.E. (n = 3), 100 cells counted per experiment). D, Mfn2^−/− MEFs were transfected with plasmids encoding GFP-Drp1^K38A, MiD49-GFP, MiD51-GFP, mito-GFP, or GFP-Drp1. Cells were stained with MitoTracker Red (red) and Hoechst (blue). Scale bars = 20 μm. E, cells from D were blind-counted for mitochondrial morphology (mean ± S.E. (n = 3), 100 cells counted per experiment).

FIGURE 2.

MiD49- and MiD51-induced mitochondrial elongation is Mfn1/2-dependent. A, schematic depicting a potential model of MiD49/51 involvement in mitochondrial fusion. Mitochondrial mixing following fusion of a cell expressing mito-GFP with another cell expressing mito-DsRed can be detected by the presence of colocalization (upper panel, yellow). In the absence of both Mfn1 and Mfn2, fusion cannot occur in mitochondria due to the lack of tethering (middle panel). This method was used to test whether MiD49/51 overexpression could act in a manner similar to Mfn1 or Mfn2 in mitochondrial tethering (lower panel). B, PEG fusion assay of MEFs expressing mito-EGFP or mito-DsRed. Cell hybrids were formed from the fusion of wild-type to wild-type (WT × WT) cells, Mfn-DKO to Mfn1^−/− or Mfn2^−/− MEFs, and MiD49- or MiD51-induced Mfn-DKO cells to WT, Mfn1^−/−, or Mfn2^−/− MEFs. Cells were induced to express MiD49 or MiD51 for 24 h prior to cell fusion. The dashed boxes in the left panels are magnified in the right panels (Inset). Scale bars = 20 μm. g, green; r, red. C, colocalization of mito-GFP and mito-DsRed following PEG-mediated cell fusion was analyzed by line scans measuring relative fluorescence intensity. The degree of mitochondrial fusion is shown as Pearson correlation units (r; mean ± S.E., n = >10 cells/condition).

FIGURE 3.

Elongated peroxisomes are formed following induction of MiD51 expression. A, HeLa cells expressing GFP-Fis1, GFP-Mff, or MiD51-GFP were immunostained with antibodies against PEX14 to visualize peroxisomes (red). Cells were visualized by confocal microscopy. The dashed boxes in the left panels are enlarged to enhance visibility in the Inset panels. Arrows identify peroxisomes showing clear colocalization with GFP-Fis1 and GFP-Mff but not MiD51-GFP. Scale bars = 10 μm. B, wild-type and MiD51-induced MEFs were treated with 4-OHT for 0–72 h. Confocal images of cells immunostained with antibodies against PEX14 were analyzed, and average peroxisome length was determined (mean ± S.E. (n = 3), >15 images/experiment/condition) and normalized to uninduced control cells. C, MEFs before (−) and 72 h after (+) 4-OHT treatment to induce MiD51 expression were immunostained with antibodies against cytochrome c (mitochondria; red) and PEX14 (peroxisomes; green). Cells were analyzed by confocal microscopy. The dashed boxes in the merge panels are enlarged in the inset panels. Scale bars = 20 μm. D, peroxisomes in wild-type and MiD51-induced MEFs were analyzed by electron microscopy. Peroxisomes were visualized by staining for catalase. The dashed boxes in the upper panels are magnified in the lower panels (Inset). Asterisks indicate mitochondria, and arrows indicate peroxisomes. Scale bars = 2 μm. E, the average peroxisome number in wild-type and MiD51-induced MEFs before and after 4-OHT treatment was determined (mean ± S.E. (n = 3), >15 cells/experiment/condition).

FIGURE 4.

Induction of MiD51 expression sequesters mitochondrial Drp1 and blocks peroxisomal fission. A, MiD51-induced MEFs were treated with or without 4-OHT for 72 h. Cells were then fixed and immunostained for peroxisomes and Drp1 (Drp1 (green, g) and PEX14 (red, r)). The dashed boxes in the left panels are magnified in the right panels (Inset). B, the intensity of Drp1 fluorescence at peroxisomes was quantified and normalized to the wild-type control (mean ± S.E. (n = 3), > 10 cells/experiment/condition). C, MiD51-induced MEFs were treated with or without 4-OHT for 72 h. Cells were then fixed and immunostained for Drp1 (green) and mitochondria (Tom20; red). D, the amount of cytosolic Drp1 relative to the total was quantified (mean ± S.E. (n = 3), > 9 cells/experiment/condition). E, MiD51-induced MEFs were transfected with plasmids encoding GFP-Drp1 (green) and then treated with or without 4-OHT. Cells were stained with MitoTracker Deep Red to visualize mitochondria and immunostained for peroxisomes (PEX14; red). Scale bars = 20 μm.

FIGURE 5.

MiD51^ΔTM can recruit Drp1 to mitochondria, lysosomes, and peroxisomes. A, schematic of MiD51^ΔTM-GFP translocation to subcellular organelles via heterodimerization. The FKBP domain at the N terminus of MiD51 is stimulated to interact with the membrane-localized FRB domain upon treatment with the rapamycin analog A/C Heterodimerizer (A/C Het). B, in the presence of A/C Heterodimerizer, cytosolic FKBP-MiD51^ΔTM-GFP was directed to the mitochondrial (FRB-mito; upper panels), lysosomal (lyso-FRB; middle panels), and peroxisomal (perox-FRB; lower panels) outer membranes. Cells were immunostained for Drp1 (red) and mitochondria (Tom20; gray/blue). The dashed boxes in the Merge panels are magnified in the Inset panels, with GFP and Drp1 (red) shown. C, GFP-Fis1^ΔTM-FKBP and GFP-Mff^ΔTM-FKBP in the presence of lyso-FRB were directed to lysosomes following treatment with A/C Heterodimerizer. Cells were immunostained for Drp1 (red) and Tom20 (gray/blue). D, HeLa cells expressing GFP-FKBP, GFP-Fis1 ^ΔTM-FKBP, GFP-Mff ^ΔTM-FKBP, or FKBP-MiD51^ΔTM-GFP together with lyso-FRB were treated with A/C Heterodimerizer and immunostained for Drp1 (red). The dashed boxes in the left panels are magnified in the right panels (Inset). Scale bars = 20 μm. E, line scans of images from D were analyzed for colocalization of Drp1 and GFP as described by Pearson correlation units (r; mean ± S.E. of three independent experiments, n > 55 measurements, two measurements/cell).