Fig. 6. Dependence of Mfn1-induced mitochondrial cluster formation on a wild type Mfn1 GTPase domain. (A) Diagram of wildtype and mutant GFP-Mfn1 expression constructs. (B-E) Immunofluorescence images of COS-7 cells showing GFP (green) and counterstained with anti-cytochrome C (red). (B) Cells transiently transfected with wildtype GFP-Mfn1 (arrows), showing typical clustered networks with large mitochondria (red) around the periphery. (C,D) Cells from culture transiently transfected with mutant GFP-Mfn1^K88T showing that the majority of transfected cells have normal appearing mitochondrial arrays (arrowheads) Higher magnification view. Some transfected cells (30%) still had clustered mitochondria (arrow), although they did not usually show the cluster-of-grape morphology. Instead, the GFP and anti-cytochome c commonly colocalized and the clustered mitochondria commonly lacked the large regions stained with anti-cytochome c but not GFP. (E) COS-7 cells transiently transfected with an N-terminal deletion mutant GFP-Mfn1^GTPase lacking the entire GTPase domain. Most transfected cells had clustered mitochondria in which the GFP and anti-cytochome c colocalized and did not show grape-like clusters (arrow).