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Fig. 2. Segments R1, R3, R6 and R7, and GTPase activity of Mfn2^TM are necessary for mitochondrial fragmentation in COS7 cells. (A) Schematic diagram of the Mfn2 deletion and point mutants used to analyze mitochondrial fragmentation, and percentage of transfected cells with fragmented mitochondria. All constructs are epitope-tagged with the 3 xFLAG sequence present at the N terminus. Boxed C, predicted coiled-coil domain; gray, GTPase domain; black, predicted transmembrane domains. The G2 Mfn2-T130A mutation is equivalent to a mutation in the Ras GTPase that abolishes effector interactions. (B) COS7 cells were transiently transfected with expression vector for FLAG-Mfn2^TM (a,b) and FLAG-Mfn2^R4/R5/TM (c,d). After 16 hours, the cells were labeled with MitoTracker to visualize mitochondria (b,d), fixed, and stained with anti-FLAG antibody followed by Alexa Fluor 488-conjugated secondary antibody (a,c). The star indicates untransfected cell with normal reticular mitochondria. Bar, 20 µm.

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