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A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2022-08-02 , DOI: 10.1016/j.jbc.2022.102336
Andaleb Kholmukhamedov 1 , Li Li 1 , Christopher C Lindsey 2 , Jiangting Hu 1 , Anna-Liisa Nieminen 3 , Kenji Takemoto 1 , Gyda C Beeson 2 , Chad M Beneker 4 , Campbell McInnes 4 , Craig C Beeson 2 , John J Lemasters 5
Affiliation  

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.



中文翻译:

一种新的荧光传感器 mitoferrofluor 表明极化和去极化线粒体中存在可螯合铁

线粒体螯合铁会导致多种损伤过程的严重性,包括缺血/再灌注、氧化应激和药物毒性。然而,缺乏在活细胞中测量该物种的方法。为了测量活细胞中的线粒体螯合铁,我们在这里合成了一种新的荧光指示剂 mitoferrofluor (MFF)。我们设计了阳离子 MFF 以电泳方式在极化线粒体中积累,然后反应基团与线粒体蛋白形成共价加合物,即使在随后的去极化之后也能保留 MFF。我们还在无细胞培养基中显示 Fe 2+(和 Cu 2+),但不是 Fe 3+、Ca 2+, 或其他生物学相关的二价阳离子, 强烈淬灭 MFF 荧光。使用共聚焦显微镜,我们在肝细胞中证明红色 MFF 荧光与线粒体膜电位 (ΔΨ m ) 指示剂罗丹明 123 (Rh123) 的绿色荧光共定位,表明选择性积累到线粒体中。与 Rh123 不同,线粒体在ΔΨm崩溃后保留了 MFF 。此外,通过膜渗透性 Fe 3+向细胞内输送铁/8-羟基喹啉 (FeHQ) 使肝细胞和其他细胞系中的 MFF 荧光淬灭约 80%,这可通过膜渗透性过渡金属螯合剂吡哆醛异烟酰腙显着恢复。我们还显示 FeHQ 淬灭了细胞溶质共加载的钙黄绿素(另一种 Fe 2+指示剂)的荧光,证实 FeHQ 中的 Fe 3+在细胞内还原为 Fe 2+。最后,在添加钙动员毒胡萝卜素后,MFF 荧光没有改变,这表明 MFF 对线粒体 Ca 2+的生理相关增加不敏感。总之,新型传感器试剂MFF荧光是线粒体可螯合Fe 2+的指示剂在具有极化线粒体的正常肝细胞以及经历 ΔΨm 损失的细胞

更新日期:2022-08-02
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