Abstract
The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery. The human protein, hFis1, participates in mitochondrial fission by recruiting the Drp1 into the mitochondria. Using short hairpin RNA, we reduced the expression levels of hFis1 in mammalian cells. Cells lacking hFis1 showed sustained elongation of mitochondria and underwent significant cellular morphological changes, including enlargement, flattening, and increased cellular granularity. In these cells, staining for acidic senescence-associated beta-galactosidase activity was elevated, and the rate of cell proliferation was greatly reduced, indicating that cells lacking hFis1 undergo senescence-associated phenotypic changes. Reintroduction of the hFis1 gene into hFis1-depleted cells restored mitochondrial fragmentation and suppressed senescence-associated beta-galactosidase activity. Moreover, depletion of both hFis1 and OPA1, a critical component of mitochondrial fusion, resulted in extensive mitochondrial fragmentation and markedly rescued cells from senescence-associated phenotypic changes. Intriguingly, sustained elongation of mitochondria was associated with decreased mitochondrial membrane potential, increased reactive oxygen species production, and DNA damage. The data indicate that sustained mitochondrial elongation induces senescence-associated phenotypic changes that can be neutralized by mitochondrial fragmentation. Thus, one of the key functions of mitochondrial fission might be prevention of the sustained extensive mitochondrial elongation that triggers cellular senescence.
Highlights
The number and morphology of mitochondria within a cell are precisely regulated by the mitochondrial fission and fusion machinery
Our results reveal that hFis1 knockdown leads to sustained mitochondrial elongation and senescence-associated phenotypic changes, and further show that these changes could be suppressed by reconstitution of mitochondrial fission, suggesting that mitochondrial fission opposing mitochondrial fusion is required for normal cell growth in mammalian cells
Collec- knockdown by itself did not affect endogenous cellular levels of tively, these findings indicate that the sustained extensive elon- mitochondrial fission and fusion molecules, gation of mitochondria accompanies the senescence-associ- including Drp1 (Fig. 3D), we conclude that hFis1 depletion did ated morphological changes
Summary
Mitofusin; JC-1, 5,50,6,60-tetrachloro1,10,3, 30-tetrathylbenzimidazole carbocyanide iodide; ⌬m, mitochondrial membrane potential; TGase 2, transglutaminase 2; ROS, reactive oxygen species; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RNAi, RNA interference; shRNA, short hairpin RNA; XTT, sodium 3Ј-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate. Mfn-null or OPA1-RNAi cells show severe defects in mitochondrial fusion as well as cellular dysfunctions, including poor cell growth and decreased cellular respiration (26). Defects in mitochondrial fusion cause cellular dysfunctions that relate to different human diseases. It is unclear how defects in mitochondrial fission, which result in the formation of elongated net-like structures, affect cellular function. We have focused on clarifying the cellular functions of mitochondrial fission by efficiently blocking the fission event using hFis RNAi. Our results reveal that hFis knockdown leads to sustained mitochondrial elongation and senescence-associated phenotypic changes, and further show that these changes could be suppressed by reconstitution of mitochondrial fission, suggesting that mitochondrial fission opposing mitochondrial fusion is required for normal cell growth in mammalian cells. We report and discuss a novel cellular function of mitochondrial fission, a defense role against stressinduced cellular senescence
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