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First published online 13 February 2007
doi: 10.1242/jcs.03381

Journal of Cell Science 120, 838-848 (2007)
Published by The Company of Biologists 2007
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Mitochondrial bioenergetics and structural network organization

Giovanni Benard1, Nadège Bellance1, Dominic James2, Philippe Parrone2, Helder Fernandez3, Thierry Letellier1 and Rodrigue Rossignol1,*

1 Institut National de la Santé et de la Recherche Médicale (INSERM), U688 Physiopathologie Mitochondriale, Universite Victor Segalen-Bordeaux 2, 146 rue Leo-Saignat, F-33076 Bordeaux cedex, France
2 Department of Cell Biology, University of Geneva, 30, Quai Ansermet 1211 Geneva 4, Switzerland
3 Laboratoire de Biologie Moleculaire et de Sequencage, Institut de Biochimie et Genetique Cellulaires, UMR Universite Victor Segalen Bordeaux 2-CNRS 5095, box 64, 146 rue Leo Saignat, F-33076 Bordeaux Cedex, France


Figure 1
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  		Fig. 1. Silencing of DRP1 in HeLa cells results in alterations of the mt-network morphology. (A-C) mt-network organization was observed in (A) wild-type HeLa cells, and (B) non-induced and (C) induced HeLa cells carrying the tet-inducible siRNA vector. Notice the budding of mt-network around the nucleus and the long, poorly ramified, tubules in C. Bars, 10 µm. (D) Western blots performed with 10 µg of cell-lysate proteins obtained from these cells.

 

Figure 2
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  		Fig. 2. Mitochondrial energetics is altered in HeLa cells where DRP1 levels were decreased. (A) Cellular endogenous respiration (black bars) was for wild-type (WT), not-induced (NI) or induced (I) HeLa cells cultured in galactose medium. The rate of respiration under non-phosphorylating conditions (gray bars) was obtained in cells incubated with oligomycin and atractyloside. (B) The rate of mitochondrial ATP synthesis was measured by bioluminometry for permeabilized cells with 10 mM pyruvate-malate and 2 mM ADP as substrates for wild-type ({triangleup}), not-induced ({diamondsuit}) or induced ({blacksquare}) HeLa cells, and wild-type HeLa cells incubated with oligomycin and atractyloside ({circ}). Results in A and B are given as the mean ± s.d., n=3; *P<0.05, statistically significant change compared with the NI cells.

 

Figure 3
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  		Fig. 3. Mitochondrial membrane fluidity is increased in HeLa cells with decreased DRP1 levels. (A) Mitochondrial respiratory rate measured in the presence of ascorbate-TMPD in wild-type (WT), not-induced (NI) or induced (I) HeLa cells. (B) Isolated activity of complex IV measured by spectrophotometry in the same cells. (C) The total content in cytochrome c determined by recording difference absorbance spectra. (D) DPH anisotropy measured in isolated mitochondria by fluorometry. The data indicate an increase in organelle membrane fluidity when DRP1 was silenced by RNAi. Results are given as the mean ± s.d., n=3; *P<0.05, statistically significant change compared with the NI cells.

 

Figure 4
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  		Fig. 4. Effect of rotenone treatment on MRC5 fibroblasts. (A) Cell viability in cells treated with different rotenone concentrations for 4 hours. (B) Rotenone titration curve of mitochondrial complex-I activity ({triangleup}), cellular endogenous respiration ({diamondsuit}), and cell proliferation ({circ}), expressed as percent of untreated control. (C) Inhibition of cell respiration and proliferation in galactose medium, expressed as percent of untreated control. Notice the threshold effect. (D) Morphological changes of the mt-network in cells treated with increasing rotenone concentrations. i, 0 ng/ml; ii, 3 ng/ml; iii, 6 ng/ml. Results are given as the mean ± s.d., n=3; *P<0.05, statistically significant change compared with the not induced.

 

Figure 5
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  		Fig. 5. Effect of rotenone treatment on mitochondrial {Psi}{Delta}, cytosolic ROS and matrix redox potential. (A) Determination of {Psi}{Delta} in MRC5 cells treated with increasing concentrations of rotenone, measured with the JC1 probe and expressed as the ratio of red to green fluorescence. (B) Determination of the cytosolic ROS concentration by fluorescence intensity of the H2-DCFDA probe, expressed as the percentage of control value. Results are given as the mean ± s.d., n=3. (C) Measurement of the redox potential of the mitochondrial matrix in MRC5 cells treated with increasing rotenone concentrations, measured with redox-GFP and expressed as the percentage of the fluorescence intensity (emission ratio of 511 nm, excitation set at 400 nm and 480 nm) measured in control cells (not shown). *P<0.05, statistically significant change compared with the control.

 

Figure 6
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  		Fig. 6. Mitochondrial flux-force-structure diagram. (A-C) The energy state was modulated in living cells grown in a galactose medium (a), by three different means, which include: OXPHOS uncoupling with CCCP (b,c), respiratory chain inhibition with rotenone (d-f) and inhibition of mitochondrial ATP synthesis with oligomycin (g,h). All chemicals were added to the culture medium and incubated at 37°C. Rotenone was used for 4 hours at 2.4, 3.4 (d), 6.9 (e), 9.6 and 12 ng/ml (f). CCCP was used for 30 minutes at 5 and 10 µM (b), and 15 and 20 µM (c). Oligomycin was used at 0.1 and 0.2 ng/ml (g), and 0.5 ng/ml (h) for 3 hours. Staurosporin was used at 1 µM for 6 hours (k). Results for cells grown in glucose medium are also shown (a'). (A) Diagram showing the variation of mitochondrial respiration as a function of {Delta}{Psi}, expressed as a percentage of the control value and measured on MRC5 cells grown in galactose. (B) ROS steady-state level measured in the cytosol (black bars) or the mitochondrion (gray bars). Letters refer to the experimental conditions described in A. (C) mt-network organization observed in the above described situations labeled a-k. Also shown is the mt-network organization of cells taken from patients with (i) a severe complex-I defect and (j) multiple respiratory-chain deficiency. These cells lines are also positioned on the diagram shown in A, according to their bioenergetic coordinates. Fragmentation of the mt-network was also observed in MRC5 cells treated for 6 hours with staurosporin 1 µM (k). In addition, we discovered the details of mitochondrial ultrastructure in cells treated with rotenone (1) and CCCP (2), compared with the control (3).

 

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