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Fig. 4. Loss of mitochondrial membrane potential induced by overexpression of the fusion protein lacking the GTPase domain and transmembrane domain of Mfn2, in COS7 cells. (A) Schematic diagram of the Mfn2 deletion mutants used and percentage of transfected cells with loss of mitochondrial membrane potential. All constructs are epitope-tagged with the 3 xFLAG sequence. Yellow, predicted coiled-coil domains; gray, GTPase domain; black, predicted transmembrane domains. (B) COS7 cells transiently transfected with expression vector for FLAG-Mfn2^GTPase/TM (a,b) and FLAG-Mfn2^R2/R6 (c,d). After 16 hours of transfection, the cells were stained by the membrane potential-sensitive dye MitoTracker Red (b,d), fixed, and then immunostained with anti-FLAG antibody followed by Alexa Fluor 488-conjugated secondary antibody (a,c). An arrow and an arrowhead indicate transfected cells expressing FLAG-Mfn2^GTPase/TM and FLAG-Mfn2^R2/R6, respectively; stars indicates normal mitochondria with a reticular network. (C) COS7 cells transiently transfected with an expression vector for FLAG-Mfn2^GTPase/TM (a-d). After 16 hours of transfection, the cells were stained with MitoTracker as the membrane potential-sensitive dye (b), fixed, and stained with anti-Mfn2-C antibody (a) and anti-Hsp60 antibody as mitochondrial marker (c) followed by Alexa Fluor 350-conjugated secondary antibody and Alexa Fluor 488-conjugated secondary antibody, respectively. A merged image is shown in panel d. (D) Mfn2^GTPase/TM-induced loss of mitochondrial membrane potential without loss of mitochondrial DNA. COS7 cells expressing Mfn2^GTPase/TM were stained with MitoTracker, with Hoechst 33342 as DNA dye (b), and anti-Hsp60 antibody as mitochondrial marker (k) followed by Alexa Fluor 488-conjugated secondary antibody. Merged images are shown in a and d; (b-d) higher magnifications of the boxed area in a. Bar, 20 µm.

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