MIEF1/2 function as adaptors to recruit Drp1 to mitochondria and regulate the association of Drp1 with Mff

Mitochondrial dynamics is a fundamental cellular process and recruitment of Drp1 to mitochondria is an essential step in mitochondrial fission. Mff and MIEF1/2 (MiD51/49) serve as key receptors for recruitment of Drp1 to mitochondria in mammals. However, if and how these receptors work together in mitochondrial fission is poorly understood. Here we show that MIEFs interact with both Drp1 and Mff on the mitochondrial surface and serve as adaptors linking Drp1 and Mff together in a trimeric Drp1-MIEF-Mff complex. Thus, MIEFs can regulate the interaction between Drp1 and Mff, and also Mff-induced Drp1 accumulation on mitochondria. It is shown that loss of endogenous MIEFs severely impairs these processes. Additionally, in cells depleted of endogenous MIEF1/2, high levels of exogenous MIEFs sequester Drp1 on the mitochondrial surface, resulting in mitochondrial elongation, whereas low-to-moderate levels of MIEFs promote mitochondrial fission, leading to mitochondrial fragmentation. In sum, the data suggest that MIEFs and Mff work coordinately in Drp1-mediated mitochondrial fission and that the level of MIEF1/2 relative to Mff sets the balance between mitochondrial fission and fusion.


Supplemental Figures
. Simultaneous knockdown of MIEF1/2 reduces the amount of Drp1 on mitochondria, leading to mitochondrial elongation.
(A) Confocal images of mitochondrial morphology and Drp1 distribution in 293T cells treated with MIEF1 and MIEF2 siRNAs in different combinations as indicated, and stained with MitoTracker (red) and anti-Drp1 antibody (green). Nuclei were stained with DAPI (blue). Insets represent high magnification views of the boxed areas. Scale bar, 10 µm.
(B) Percentages (mean ± SEM) of cells with indicated mitochondrial morphologies in 293T cells treated with control siRNA, or MIEF1 and MIEF2 siRNAs in combinations as indicated.
(C) Quantitative co-localization of endogenous Drp1 (green) with mitochondria (MitoTracker Red) was analyzed using the Pearson's correlation coefficient (PCC) (mean ± SEM) of at least 5 images (each containing 5-10 cells). The analysis is based on two independent experiments. (B) Sequence alignments around the edited gRNA targeting sites of wild-type (WT) and mismatches (blue color) in MIEF1-and MIEF2-single knockout and MIEF1/2-double knockout clonal cell lines. The data were obtained from PCR cloning followed by sequencing.   (E) Western blot analysis of MIEF1/2 DKO cells compared to wild-type control.

Figure S5. Distinct effects of introduced mouse MIEFs or mouse Mff respectively on mitochondrial morphology and Drp1 distribution in cells depleted of endogenous MIEFs or Mff (A, B)
Lower cellular levels of exogenous mMIEF1 and mMIEF2 induced a mitochondrial fission phenotype in cells depleted of endogenous MIEF1 or MIEF2 respectively by siRNA, whereas higher levels of exogenous mMIEFs leads to a mitochondrial fusion phenotype. Confocal images (left panels) of 293T cells treated with either MIEF1 or MIEF2 siRNA, followed by introduction of either mouse MIEF1-Myc (mMIEF1) or mouse MIEF2-Myc (mMIEF2), and stained with MitoTracker (red), anti-Drp1 (green) and anti-Myc (blue) antibodies. Cells marked in (A) by (a) to (c) represent: empty vector-transfected cell (a); low-level of mMIEF1-Myc (b); high-level of mMIEF1-Myc (c). Cells marked in (B) by (a) to (d) represent: empty vector-transfected cell (a); low-level of introduced mMIEF2-Myc (b); high-level of introduced mMIEF2-Myc (c and d). Insets represent high magnification views of the boxed areas. Expression levels of mMIEF1-and mMIEF2-Myc in transfected 293T cells exhibiting a mitochondrial fission or a fusion phenotype were measured by immunofluorescence intensity (mean ± SD) (right panels) using the ImageJ in at least 10 cells for each condition.

Cell cultures and transfection
The 293T cells were cultured in Dulbecco's modified Eagle's medium (HyClone) with 10% fetal bovine serum. Transient transfection of plasmids was performed using Lipofectamine TM 2000 transfection reagent (Invitrogen) according to the manufacturer´s protocol.

Establishment of 293T cell lines with stable expression of MIEF2-V5
The 293T cells were transfected with the MIEF2-V5 plasmid (pcDNA3.1V5/His) with neomycin (G418) resistance. From the following day, the transfected cells were cultured for two weeks in regular growth medium containing G418 (2 mg/ml) for selection of stably transfected colonies. Cell lines with stable expression of MIEF2-V5 were validated by immunoblotting and immunofluorescence microscopy.

Expression Constructs
All expression plasmids used in this study, except the GFP-Mff∆50 construct, have been described in previous studies and are listed here in Supplemental Table S1. Empty vectors of pcDNA3.1V5/His and pAcGFP-C In-Fusion® Ready Vector (Clontech) were used as controls.

RNA interference (RNAi) for gene silencing
Information on specific siRNAs used in this study is listed in the Supplemental Table S2 and as control we used a scrambled Stealth RNAi™ siRNA Negative Control Kit (12935-100) with similar GC content recommended by Invitrogen.

Western blotting
Protein extracts were separated by electrophoresis using NuPAGE® Novex Bis-Tris Gel (Invitrogen) and transferred to PVDF membranes with Transfer Pack (Bio-Rad). After blocking with 10% nonfat dry milk in PBS, membranes were incubated with primary antibodies followed by the peroxidase-conjugated secondary antibody (GE Healthcare), and immunocomplexes were detected with the Pierce ECL Western Blotting Substrate (Thermo Scientific). Intensity of the bands on Western blots was measured using ImageJ.

Co-immunoprecipitation (co-IP)
Co-IP experiments were carried out as described (Hajek et al., 2007;Liu et al., 2013;Zhao et al., 2011). Briefly, cultured 293T cells were washed with PBS buffer, proteins were in vivo cross-linked by incubating the cells in PBS buffer containing 1% formaldehyde (FA) and the cells were scraped off with a rubber scraper and suspended in lysis buffer (PBS containing 1% NP-40 and protease inhibitor cocktail complete EDTA-free) (Roche Diagnostics). The cell suspensions were sonicated and centrifuged to remove insoluble debris. For the co-IP of exogenous proteins, the resulting supernatants were incubated with anti-V5 agarose, anti-Myc agarose, anti-Flag agarose (Novus Biologicals) or anti-GFP mAb-Agarose beads (MBL); For the co-IP of endogenous proteins, 2 µg of antibody against targeting protein was incubated with Dynabeads® protein G for 1 h. After washing with lysis buffer twice, the antibody-conjugated beads were added to the resulting supernatants. Thereafter the beads were washed with PBS containing 1% NP-40 followed by PBS. The immunocomplexes captured on the agarose beads conjugated with antibody were dissolved in SDS-sample buffer and subjected to immunoblotting.