Fig. 7. Overexpression of the GTPase G2 motif mutant GFP-Mfn1^T109A caused mitochondrial fragmentation. (A) Live COS-7 cells nine hours after transient transfection with GFP-Mfn1^T109A (green) counterstained with Mitotracker (red). Arrow, transfected cell. High magnification view of fragmented mitochondria, showing GFP-Mfn1^T109A around the surface of individual mitochondria stained with mitotracker (Inset). (B) COS-7 cells transfected with GFP-Mfn1^T109A, showing effects of low (arrow) or high (double arrow) levels of GFP-Mfn1^T109A expression. (C) Example from COS-7 cell culture co-transfected with GFP-Mfn1^T109A and wildtype Mfn1. Samples in B and C imaged by fluorescence of the GFP moiety of the GFP-Mfn1^T109A fusion protein. (D) COS-7 cells from culture co-transfected with GFP-Mfn1^T109A (green) and HIS-tagged wildtype Mfn1 (Mfn1-V5/HIS) (red, immunofluorescence using anti-HIS-tag antibodies also labels nuclei). Fine mitochondrial network in a cell expressing GFP-Mfn1^T109A with low levels of Mfn1-V5/HIS (double arrow). Grape-like mitochondrial cluster in a cell co-expressing GFP-Mfn1^T109A and high levels of V5/HIS-tagged Mfn1 (arrow). Lower panels: GFP-Mfn1^T109A-transfected cell with no detectable Mfn1-V5/HIS protein, from the same co-transfection experiment.