2018
2018
Master 2
Mitochondrial Cristae and their role in ATPsynthase functioning
Encadrant : Frédéric Joubert

SummarizeThe membranes in living cells are considered as “active membranes” due to different non-equilibrium reactions occurring in their vicinity. In return, changes of membrane mechanical properties or of their morphology can modulate important biological functions. Mitochondria represent a paradigm of this intrinsic relationship between membrane properties and organelle function. They are key organelles in eukaryotic cells for energy production and apoptosis. A hallmark of the inner mitochondrial membrane (IMM) is the presence of dynamic nano-invaginations called “cristae” (20-50 nm diameter) containing the respiratory complexes responsible for ATP production. Our objective is to demonstrate how such membrane shape and molecular organization operate to influence energy production. Our main hypothesis is that high membrane curvature regions such as cristae facilitate specific phospholipid and oligomeric protein recruitment, allowing the creation of a confined membrane environment that optimizes respiratory chain function and ATPsynthase efficiency.

 

State of the art: Mitochondria are dynamic organelles that constantly adapt their form1, cristae shape and density to energy demand2-3. Relative integrity of cristae morphology is considered as a relevant indicator of the functional state of mitochondria, since cristae contain the F1Fo ATPsynthase that uses the energy of the proton electrochemical gradient created by respiratory complexes to synthesize ATP. Specific alterations of cristae are associated with mitochondrion dysfunctions yielding organ or systemic metabolic consequences and are observed in many pathological situations2,4,5, leading to the assumption thatenergy production is influenced by IMM and cristae shape. A few studies attempted to theoretically explain the role of cristae shape6however the mechanism by which it influences ATP production still remains to be fully and experimentally established.

Currently, we are working on three main hypotheses: 1) the role of the formation of F1Fo ATPsynthase dimers that could be recruited in cristae and optimized mitochondrial function. 2) the role of membrane phospholipid (PL) composition of cristae, especially  the enrichment in cardiolipin (CL) that could activate the membrane environment required for the correct functioning of enzymes and respiratory chain coupling in cristae, and triggers functional plasticity of cristae. 3) the role of confinement that could allow a coupling between enzyme function, proton diffusion and membrane morphology.   

 

Methodology: The internship project will be performed using biophysical approaches and in vitro system reconstitution. We will use Giant Unilamellar Vesicles (GUV) containing ATPsynthase and specific phospholipids. We recently succeeded in incorporating a functional monomeric E. coli ATPsynthase into GUV7, and demonstrated that activation of ATPsynthase modified surrounding membrane mechanical properties. Now we would like to further demonstrate how PL/dimer recruitment in high curvature region helps to improve ATPsynthase function. For this, we will use monomeric and dimeric forms of yeast ATPsynthase. So far, reconstitution of dimeric forms has never been published and is now mastered by our team. These GUV will be used to work at four different levels: 

1) P. Bassereau at Institut Curie has recently demonstrated that CL was enriched in nanometric invaginations (submitted). We will investigate whether ATPsynthase dimers are also specifically recruited in high membrane curvature regions. Using proteo-GUV containing the two ATPsynthase forms with GFP constructs, we will generate membrane invaginations of different diameters either by osmotic shock (deflation8), or by pulling nanotubes at a controlled diameter using optical tweezers (in collaboration with P. Bassereau9,10). The role of PL composition in this process will be addressed by modifying PL composition of GUV in a substantial and controlled manner. 

2) Liposomes will be prepared using lipids mimicking in vivo mitochondrial cristae composition and with lipid extracts from control or pathological mice. Mechanical membrane properties (flickering experiments, in collaboration with I. Lopez Montero in Madrid), proton permeability, bilayer fluidity, lipid packing (membrane zeta potential, Laurdan) will be measured.

3) Finally, we will quantify ATPsynthase activity in the previous reconstituted systems, and evidence how it is impacted by the specific PL/environment or the nano-membrane structuration. Electrochemical gradient required for ATPsynthase functioning will be created by the specific K+ transporter valinomycin7 or by co-incorporating BacterioRhodospin (BR) with the ATPsynthase. Enzyme function will be deduced using fluorescent dyes (pH, membrane potential) or luciferase assay. A specific approach based on the monitoring of fluorescence emitted by nanotubes will be developed to specifically study ATPsynthase function inside the nanotubes.

 

References:

 

[1] Pernas and Scorrano, Annu Rev Physiol78:505-31 (2016); [2] Vafai and Mootha, Nature491:374-83 (2012); [3] Mannella Biochim Biophys Acta1763:542-8 (2006); [4] Acehan et al., Lab Invest87(1):40-8 (2007); [5] Siegmund et al. iScience6, 83-91 (2018) [6] Song et al. Phys Rev E Stat Nonlin Soft Matter Phys88(6):062723 (2013); [7] Almendro-Vedia et al. PNAS114(43):11291-11296 (2017); [8] Steinkuhler et al. Sci Rep2018 8(1):11838 (2018); [9] Sorre et al. PNAS106(14):5622-6 (2009);  [10] Aimon et al.Dev Cell28(2):212-8 (2014);

 

Publications reliés

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase - Proceedings of the National Academy of Sciences of the United States of America
V.G. Almendro-Vedia , P. Natale , M. Mell , S. Bonneau , F. Monroy , F. Joubert , I. López-Montero
  URL Full text PDF Bibtex doi:10.1073/pnas.1701207114

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