(A) Tomographic slice of in vitro attachment reactions with mitochondria isolated from fzo1△ cells; scale bar 100 nm. (B) Levels of Ugo1 and Mgm1 in whole-cell extracts prepared from wildtype or Fzo1-overexpressing cells. Ugo1 levels did not vary, which is consistent with an imbalance with mitofusins upon Fzo1 overexpression. Note that the ratio between long and short forms of Mgm1 was slightly shifted toward the short form in cells overexpressing Fzo1. This may contribute to the changes in cristae morphology upon Fzo1 overexpression as seen in the electron micrographs shown in (D). (C) Mitochondrial morphology in representative WT and FZO1 o.e. cells. (D) Electron micrographs from Figure 7D at higher resolution. Fusion intermediates (fused outer membranes, separated inner membranes) and attached intermediates (attached outer membranes) are indicated by red and green arrowheads, respectively. (E-F) in vitro mitochondrial attachment upon Fzo1 overexpression. (E) Method summary: Mitochondria isolated from cells expressing either mito-GFP or mito-mCherry were mixed in equal amounts and processed for in vitro attachment reactions before analysis by fluorescence microscopy. Attached mitochondria (red-red; green-green; green-red) were counted in reactions stopped before centrifugation (t −10), after centrifugation (t 0) or after 10 min incubation on ice (t +10). (F) Ratios of attached mitochondria from wild-type (WT, blue) or Fzo1 overexpressing (FZO1 o.e., red) cells at t −10, t 0 and t +10. Ratios were normalized to the WT attachment at t 0. Mitochondria from Fzo1-overexpressing mitochondria were attached twice as frequently than mitochondria from wild-type cells at all time points, both after and before centrifugation (t −10). With Fzo1 overexpression, centrifugation stimulated in vitro attachment by a factor of two, compared to wild-type (t 0). In contrast, incubation on ice (t +10) had a weak effect on attachment in both wild-type and Fzo1 overexpressing conditions (compare t +10 with t 0), consistent with the requirement of this step for the transition from tethering to docking rather than for de novo attachment in vitro.