Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells.
Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.
Ishihara
Naotada
N
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 812-8582, Fukuoka, Japan.
Jofuku
Akihiro
A
Eura
Yuka
Y
Mihara
Katsuyoshi
K
D000818:Animals; D002455:Cell Division; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008561:Membrane Fusion; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D049411:Utrophin
D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D049411:Utrophin; D049452:Green Fluorescent Proteins; C107312:Fzo protein, Drosophila; D020558:GTP Phosphohydrolases