Fig. 1. GTP and Mfn1-dependent mitochondrial tethering. (A) Schematic drawing of the mitochondrial tethering reaction using isolated mitochondria. (B) Mitochondria (mit, a-e) or salt-washed mitochondria (wash mit, f and g) were prepared from HeLa cells coexpressing mit-GFP and Mfn1-FLAG, or mit-RFP and Mfn1-FLAG. Mitochondria labeled with GFP or RFP were mixed, and incubated with (b-e and g) or without (a and f) GTP at 30°C. The reaction mixtures were analyzed by confocal microscopy. Magnified images of bound mitochondria are shown (c-e). (C) GFP-labeled mitochondria and RFP-labeled mitochondria prepared from the cells expressing Mfn1-FLAG (filled bars), or without Mfn1-FLAG (open bars) were incubated as in B, and the number of bound mitochondria were counted as described in Materials and Methods. (D) Schematic drawing of pull-down assay for the binding reaction between sonicated mitochondrial vesicles. (E) The PNS fractions from HeLa cells expressing Mfn1-HA or Mfn1-FLAG were sonicated, mixed and incubated in the presence or absence of GTP. The reaction mixtures were then treated with or without Triton X-100 and subjected to immunoprecipitation using anti-HA antibody. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG antibody. In a separate experiment, Mfn1^K88T-FLAG-harboring mitochondria were used in lieu of Mfn1-FLAG-harboring mitochondria.