MiP2005: Session 5 - Young Investigator Presentation

Mitochondrial Physiology Network 10.9: 57-59 (2005) - download pdf

 

Role of mitochondrial network organization in the regulation of energy production in living human cells: a multi-approach study.

Giovanni Benard1, P Parrone2, B Faustin1, C Rocher1, C Lales1, D Pierron1, JC Martinou2, T Letellier1, R Rossignol1

1U 688 - INSERM, Physiopathologie Mitochondriale, Université Victor Segalen Bordeaux 2, Bordeaux, France ; 2Dept. Cell Biology, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva, Switzerland. - benard.giovanni@tiscali.fr 

Recent advances in cell biology have helped to create a picture of mitochondrial organization whereby the organelle exists as a single dynamic network (mt-network), either forming a reticulum or a fragmented collection of vesicles (Figure 1). Several observations have revealed that its tridimensional organization is variable in human living cells, both under normal or pathological situations. However, little is known about the determinants and the bioenergetic consequences of these changes. Also, the fuctionning of mitochondria as a network, even heterogeneous, needs to be demonstrated. Such analysis is complex since mitochondria participate in a multiplicity of cellular functions such as energetics, ROS production, calcium signaling or apoptosis. Changes in mt-network organization can be observed in association with different physiological situations including cell cycle, cytoskeletal trafficking, energy status, pathology or apoptosis (see Table 1). These changes can be mediated by fission and fusion proteins, but their regulation is poorly understood. The sole contribution of physicochemical transitions in mitochondrial lipid bi-layers also remains to be investigated. Here we present our work on the relationships between the energy status of the cell and mitochondrial network organization. Using fluorescence microscopy and ratiometric GFP biosensors we observed, in living human cells, specific and opposite transformations of mitochondrial overall structure (as well as internal organization) in response to in situ activation or inhibition of mitochondrial energy production using different effectors such as glucose deprivation, or treatment with rotenone, KCN, antimycin A, and amyloid peptide. For comparison, we looked at the mt-network features in a variety of cells taken from patients with a mitochondrial disease. We present also novel results we obtained on a human cell line where DRP1, a protein involved in fission of the mt-network was stably knocked out using the tet-induced siRNA technology. In these cells, we observed important changes in mitochondrial membrane fluidity and mt-network organization, associated with the uncoupling of  oxidative phosphorylation and the impairement of cell proliferation in absence of glucose. Taken together, our observations point out toward a strong implication of mt-network organization in the regulation of OXPHOS, under normal or pathological situations.

 

Figure 1: Three main configurations of the mitochondrial network in living HeLa cells stained with a matrix targeted GFP.

 

Table 1: Changes in mt-network organization under various physiological, pathological or experimental situations.

 

TRIGGER

METHODOLOGY<o:p></o:p>

MT-NETWORK<o:p></o:p>

CELLULAR IMPLICATIONS<o:p></o:p>

REF. <o:p></o:p>

ENERGY

STATUS<o:p></o:p>

Change in the type of cellular energy substrate: oxidative versus glycolytic <o:p></o:p>

Ratiometric GFPs, pH and redox sensinsitive, TEM and confocal analysis<o:p></o:p>

In oxidative mode: thin tubular, branched. In glycolytic mode: larger tubules, peri-nuclear<o:p></o:p>

Adaptation of mt-network to the carbon source.<o:p></o:p>

[1]<o:p></o:p>

[2]<o:p></o:p>

CELL

CYCLE<o:p></o:p>

Synchronisation of human cells in culture (143B)<o:p></o:p>

Mitotracker labelling <o:p></o:p>

Fragmentation before S phase, reticular again in G1<o:p></o:p>

Mt-network could be a check point of cell cycle<o:p></o:p>

[3]<o:p></o:p>

Analysis in budding yeast<o:p></o:p>

Destabilization of actin cables or the mitochore <o:p></o:p>

Three classes of mitochondrial motility<o:p></o:p>

Mitochondrial movement requires actin cables<o:p></o:p>

[4]<o:p></o:p>

<o:p> </o:p>

Induction of apoptosis by staurosporin, and silencing of fusion or fssion protein<o:p></o:p>

RNAi OPA1<o:p></o:p>

Confocal analysis<o:p></o:p>

Fragmented, vesicular, cytochrome c release<o:p></o:p>

Loss of Y, higher sensitivity to apoptosis <o:p></o:p>

[5]<o:p></o:p>

- RNAi DRP1( fission)<o:p></o:p>

- RNAi Fis1 (fission)<o:p></o:p>

Elongated<o:p></o:p>

Resistance to apoptosis (staurosporin or actimomycin D)<o:p></o:p>

[6]<o:p></o:p>

PATHOLOGY<o:p></o:p>

Oxphos diseases<o:p></o:p>

Monoclonal antibodies <o:p></o:p>

immunofluorescence<o:p></o:p>

Abnormal: peripheric, ragged,  fragmentation<o:p></o:p>

Alteration of mt-network profile in Oxphos deficient cells<o:p></o:p>

[7]<o:p></o:p>

OXPHOS

ALTERATIONS<o:p></o:p>

Rotenone treatment of Helas and MRC5s<o:p></o:p>

ratiometric GFP pH and redox sensitive<o:p></o:p>

Vesicular with "donuts" <o:p></o:p>

Increased redox potential, decrease in respiration.<o:p></o:p>

Present study<o:p></o:p>

CCCP treatment of Helas and MRC5s<o:p></o:p>

Immuno fluorescence<o:p></o:p>

Fragmented<o:p></o:p>

Yis necessary for fusion<o:p></o:p>

[8]<o:p></o:p>

Treament with 10µM ß-amyloids on MRC5s<o:p></o:p>

GFP ratiometric pH and redox sensitive<o:p></o:p>

Fragmented<o:p></o:p>

Alteration of mt-network profile in Oxphos deficient cells<o:p></o:p>

Present study<o:p></o:p>

Misarrangment of F1F0-ATPsynthase (Yeast)<o:p></o:p>

Cross linking of ATP synthase<o:p></o:p>

Fragmented<o:p></o:p>

Abnormal cell division and mt-DNAtransmission to buds<o:p></o:p>

[9]<o:p></o:p>

<o:p> </o:p>

MONITORING

OF

mt -

FUSION

OR FISSION

PROTEINS<o:p></o:p>

Repression of fusion protein mfm2<o:p></o:p>

Immuno fluorescence<o:p></o:p>

Anti-sense ARNm<o:p></o:p>

Fragmented and clustering<o:p></o:p>

reduced glucose oxidation, Yand cell  respiration<o:p></o:p>

[10]<o:p></o:p>

Over expression of fusion protein mfm1<o:p></o:p>

Immuno fluorescence<o:p></o:p>

Fluorescence microscopy<o:p></o:p>

Super connectivity<o:p></o:p>

Mixing of whole mitochondria content<o:p></o:p>

[11]<o:p></o:p>

Over expression of fusion protein mfn2<o:p></o:p>

Immuno fluorescence<o:p></o:p>

Clustering around the nucleus<o:p></o:p>

Mfn2 GTPase regulates or mediates mitochondrial fusion<o:p></o:p>

[12]<o:p></o:p>

Silencing of fusion proteins mfm 1and 2<o:p></o:p>

RNAi  Mfn1 and 2<o:p></o:p>

cell fusion and GFP <o:p></o:p>

Unmixing of mitochondrial populations from two cells<o:p></o:p>

Unmixing of whole mitochondria content <o:p></o:p>

[8]<o:p></o:p>

Overexpression of the fission protein Drp 1<o:p></o:p>

Overexpression <o:p></o:p>

Fragmented<o:p></o:p>

Alteration of calcium signaling <o:p></o:p>

<o:p> </o:p>

[13]<o:p></o:p>

 

1.    Rossignol R et al. (2004) Cancer Res 64: 985-993.

2.    Jakobset al. (2003) J Cell Sci 116: 2005-2014.

3.    Margineantu D et al. (2002) Mitochondrion 1: 425-435.

4.    Fehrenbacher KL et al. (2004) Curr Biol 14: 1996-2004

5.    Olichon A et al. (2003) J Biol Chem 278: 7743-7746.

6.    Lee YJ et al. (2004) Mol Biol Cell 15: 5001-5011.

7.    Hanson B et al. (2001) Mitochondrion 1: 237-248.

8.    Ishihara N et al. (2003) Biochem Biophys Res Commun 301: 891-898.

9.    Gavin PD et al. (2004) J Cell Sci 117: 2333-2343.

10.   Bach D et al. (2003) J Biol Chem 278: 17190-17197.

11.   Legros F et al (2002) Mol Biol Cell 13: 4343-4354.

12.   Santel A, Fuller MT (2001) J Cell Sci 114: 867-874.

13.   Szabadkai G et al. (2004) Mol Cell 16: 59-68.


to topPrint page

 
 

© Mitochondrial Physiology