Transient contraction of mitochondria induces depolarization through the inner membrane dynamin OPA1 protein - PubMed (original) (raw)
Transient contraction of mitochondria induces depolarization through the inner membrane dynamin OPA1 protein
Hakjoo Lee et al. J Biol Chem. 2014.
Abstract
Dynamin-related membrane remodeling proteins regulate mitochondrial morphology by mediating fission and fusion. Although mitochondrial morphology is considered an important factor in maintaining mitochondrial function, a direct mechanistic link between mitochondrial morphology and function has not been defined. We report here a previously unrecognized cellular process of transient contraction of the mitochondrial matrix. Importantly, we found that this transient morphological contraction of mitochondria is accompanied by a reversible loss or decrease of inner membrane potential. Fission deficiency greatly amplified this phenomenon, which functionally exhibited an increase of inner membrane proton leak. We found that electron transport activity is necessary for the morphological contraction of mitochondria. Furthermore, we discovered that silencing the inner membrane-associated dynamin optic atrophy 1 (OPA1) in fission deficiency prevented mitochondrial depolarization and decreased proton leak without blocking mitochondrial contraction, indicating that OPA1 is a factor in coupling matrix contraction to mitochondrial depolarization. Our findings show that transient matrix contraction is a novel cellular mechanism regulating mitochondrial activity through the function of the inner membrane dynamin OPA1.
Keywords: Bioenergetics; Drp1; Dynamin; Electron Transfer; Membrane; Membrane Fusion; Mitochondria; Mitochondrial Fission; OPA1; Proton Leak.
Figures
FIGURE 1.
TMRE flickering and transient synchronous nodulation of mitochondrial tubules in DLP1-KO MEFs. A, time-sequence images of DLP1-KO cells show loss and recovery of TMRE fluorescence. Cells with TMRE loss are indicated by arrowheads. Time-lapse images were acquired at 10-s intervals. Frame numbers are given at the bottom. Scale bar, 20 μm. B, three-dimensional surface plots of a fluorescence image time-sequence for quantifying TMRE flickering in DLP1-KO MEFs. Six sequential frames of the TMRE imaging field (1344 × 1024 pixels, x and y axes) containing 221 cells are shown. Typically, 100 sequential frames were processed using the “Stacks-T-functions” of ImageJ. Images were inverted to display the loss of TMRE fluorescence as upward peaks. Peaks in the z axis represent losses/decreases of TMRE fluorescence compared with those in the preceding frame. The peaks in each frame were counted and presented as events/100 frames/100 cells. C, matrix-targeted GFP shows transient contraction of mitochondrial tubules. Synchronous nodulation appeared and disappeared within 20 s. t, time. Scale bar, 10 μm.
FIGURE 2.
Concomitance of mitochondrial contraction and TMRE loss in DLP1-KO MEFs. A, dual-color imaging shows that matrix contraction (GFP) in the filamentous mitochondrial tubules coincides with TMRE loss. Scale bar, 10 μm. B, matrix contraction occurs toward branch points in mitochondrial networks and coincides with depolarization. Scale bar, 10 μm. C, analyses of fluorescence intensity at the contracted nodules. Increases in GFP signals are accompanied by decreases in TMRE fluorescence. D, images from 1-s interval time sequences. TMRE is retained in the contracted nodules for ∼10 s before its disappearance. Scale bar, 10 μm. E, fluorescence intensity analyses at the contracted nodules show that increases of GFP signals precede the loss of TMRE.
FIGURE 3.
Mitochondrial contraction-depolarization coupling in H9c2 cells. A–A″, dual imaging of TMRE and GFP in mitochondria of normal H9c2 cells shows transient contraction accompanied by depolarization. Scale bar, 10 μm. B–B‴, partial mitochondrial depolarization indicated by TMRE dimming in normal H9c2 cells coincides with mitochondrial contraction. Scale bar, 10 μm. C, inhibition of mitochondrial fission in H9c2 cells by expression of DLP1-K38A significantly increased TMRE flickering. D, merged images from TMRE-GFP dual imaging in the DLP1-K38A-expressing H9c2 cell show prominent mitochondrial contractions associated with TMRE loss (arrows). Scale bar, 10 μm.
FIGURE 4.
TMRE flickering requires electron transport chain activity and membrane potential. A, antimycin A treatment diminished TMRE flickering in DLP1-KO MEFs. B, TMRE flickering quantified for every five frames. Antimycin A was added in frame 21. Error bars are S.E. C, examples of fixed snapshots showing mitochondrial morphologies in DLP1-KO MEFs, either with or without antimycin A for 2 min. Nodulated mitochondrial tubules in untreated DLP1-KO MEFs and un-nodulated mitochondria in antimycin A-treated DLP1-KO cells are shown. Scale bar, 10 μm. D, counting cells containing nodulated mitochondria shows that a 2-min treatment with antimycin A and a 5-min treatment with FCCP significantly decreased mitochondrial contraction. Error bars are S.E. E, nutrient starvation (Starv) of DLP1-KO MEFs abolished TMRE flickering. Addition of pyruvate (Pyr) alone did not increase TMRE flickering. Pyruvate/oligomycin (Olm) induced a substantial increase of TMRE flickering. Error bars are S.E. F, membrane potential assessments by TMRE. TMRE fluorescence in completely uncoupled mitochondria after the FCCP treatment was subtracted (F − Fu). Pyruvate/oligomycin increased membrane potential, whereas pyruvate alone had no significant effect. Error bars are S.E.
FIGURE 5.
OPA1 silencing decreases TMRE flickering and proton leak in DLP1-KO MEFs. A, quantification of TMRE flickering at 72 h post-transduction shows that down-regulation of OPA1 (OPA1 KD) substantially decreased TMRE flickering, whereas mitofilin knockdown had no significant effect. Error bars are S.E. **, p < 0.01. B, marked 8-fold decrease in TMRE flickering in a stable clone expressing OPA1 shRNA in DLP1-KO MEFs (OPA1-KD/DLP1-KO). n = 4. Error bars are S.E. C, DLP1-KO MEFs show increased processing of l-OPA1 to s-OPA1. D, cellular oxygen consumption analyses. Leak respiration in the presence of oligomycin (Olm) was higher in DLP1-KO MEFs. Knockdown of OPA1 in DLP1-KO cells normalized the leak respiration. E, DLP1-KO cells showed a substantial increase in the leak ratio compared with wild-type MEFs (WT) due to frequent large scale depolarization during TMRE flickering. Silencing OPA1 in DLP1-KO cells normalized leak ratio. OCR, oxygen consumption rate. n = 6. Error bars are S.E. **, p < 0.01.
FIGURE 6.
OPA1 silencing prevents mitochondrial depolarization without inhibiting mitochondrial contraction. A, silencing OPA1 in DLP1-KO cells prevents TMRE loss during mitochondrial contraction. Scale bar, 5 μm. B, fluorescence intensity analyses show no TMRE loss with increased GFP signal during mitochondrial contraction in OPA1-KD/DLP1-KO cells.
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