Fission, fusion, and respective machineries converge. (A and B) Cartoon description (A) and corresponding FLiP experiment (B) that reveals consecutive tethering, fusion, and then fission all occurring at the same Mfn1 punctum. A mitochondrion expressing GFP-Mfn1 and a matrix marker (mito-mScarlet, magenta) was photobleached (in box 2), which is located on one side of the Mfn1 punctum (Mfn1 in green, white arrow). Images shown are prebleach (t = −14 s), postbleach (t = 0 s), fusion at an Mfn1 punctum (at t = 7 s), prefission (25 s), and then postfission at an Mfn1 punctum (at t = 58 s). (C) A representative image of a U-2 OS cell expressing mCh-Drp1, GFP-Mfn1, and mito-BFP and magnified merged time-lapse images of the inset show the dynamics and colocalization of fission and fusion machineries over time. Note that both GFP-Mfn1 and mCh-Drp1 puncta colabel a fusion event (white arrow) and a neighboring fission event (magenta arrow). (D) Representative image of a HeLa cell expressing mCh-Drp1, GFP-Mfn1, and mito-BFP and magnified merged time-lapse images of the inset show the dynamics and colocalization of fission and fusion machineries over time. Note that both GFP-Mfn1 and mCh-Drp1 puncta co-label a fission event (magenta arrow) and remain labeled through a subsequent fusion event at the same spot (magenta arrow). (E) Table shows the percentage of fission or fusion events that are marked by GFP-Mfn1 and/or mCh-Drp1 puncta in U-2 OS and HeLa. Scale bars for whole cell = 5 µm; insets, 1 µm.