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Fig. 5 | BMC Biology

Fig. 5

From: MIEF1/2 orchestrate mitochondrial dynamics through direct engagement with both the fission and fusion machineries

Fig. 5

Drp1 binding-deficient mutants of MIEFs interact with pro-fusion proteins and promote mitochondrial fusion. a Schematic representation of full-length MIEF1 and MIEF2, as well as their Drp1 binding-deficient mutants fused to V5-tag at the C terminus. b Drp1 binding-deficient mutants of MIEFs still interact with Mfn1 and Mfn2. 293T cells were transfected with empty vector, MIEF1-V5, MIEF1Δ160-169-V5, MIEF2-V5, or MIEF2Δ151-160-V5. Cell lysates were used for co-IP with anti-V5 beads, followed by Western blotting with indicated antibodies. c, d Representative confocal images of mitochondrial fusion from the PEG-based fusion assay in WT 293T polykaryons transfected with empty vector, MIEF1Δ160-169 or MIEF2Δ151-160. Cells with stable expression of mitoRFP or mitoGFP were co-cultured, and transfected with empty vector (control), MIEF1Δ160-169-V5 or MIEF2Δ151-160-V5 as indicated, then subjected to the PEG cell fusion assay. The mitochondrial fusion is indicated by co-localization of mitoRFP and mitoGFP (i.e., yellow mitochondria) (c). The extent of mitochondrial fusion in individual hybrid cells was analyzed via the Pearson’s correlation coefficient (PCC, mean ± SEM) in three independent experiments for each condition and data are summarized in (d) (n represents the total number of cells analyzed for each condition). e, f The mito-PAGFP-based mitochondrial fusion assay confirmed that Drp1 binding-deficient mutants MIEF1Δ160-169 or MIEF2Δ151-160 still enhanced mitochondrial fusion. n represents the number of cells analyzed. ***P < 0.001; **P < 0.01; *P < 0.05 (f)

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