Task 2: Membrane dynamics details


(PARTNERS  2, 4, 5, 7)

Mt can fuse together to make larger compartments or divide to make smaller ones, and defects in these processes impact the cell’s capacity to provide energy. Using the yeast mt model, Partner 4 made a major contribution to our understanding of the dynamics of mt membranes fusion by large GTPases of the Dynamin-Related Protein (DRP) family called mitofusins, Mitofusins from adjacent mt auto-oligomerize in trans to mediate mt tethering and fusion of mt outer membranes. By combining an in vitro mt fusion assay with cryoelectron-tomography (CET) analysis, we discovered and characterized the Mitochondrial Docking Complex (Fig. 2), the first example of a macro-molecular structure assembled by mitofusins (Brandt, Elife, 2016). In a new collaboration between Partners 2 and 4, we used molecular dynamics simulations in a lipid bilayer combined with experimental validation to generate the first near full-length model of a mitofusion protein, Fzo1 (Fig. 3) (De Vecchis, Sci Rep, 2017). Partner 4 further demonstrated that the Ubiquitin-Proteasome System (UPS) monitors mt fusion by coordinating an intricate balance between mitofusin turnover and fatty acid desaturation, thus linking DRP-mediated fusion with lipid homeostasis (Cavellini, Nat Commun, 2017), and that the UPS regulates mitophagy in yeast (Belgareh-Touze, Autophagy, 2017). Lastly, CrFzl, the C. reinhardtii ortholog of Fzo1, was shown to localize to the cp to regulate photosynthesis during high light stress by mediating fusion of thylakoid membranes (Findinier et al., submitted). Together, these advances have dramatically improved our knowledge of DRP-mediated fusion and its role in organizing energy transducing membranes in both mt and cp.

In the E. coli model, Partner 5 studied how the interaction between lipids and energy transducing proteins shape bacterial membranes. The E. coli expression host C43(DE3) undergoes massive membrane proliferation upon homologous overproduction of AtpF, the b subunit of the F1Fo ATP synthase. These intracellular membranes (ICMs) are organized in a network similar to that found in cyanobacteria and contain elevated levels of cardiolipids. Using electron and fluorescence microscopy, and flow cytometry we showed that the hexagonal lipid phases observed in a wild type host were replaced by lamellar onion like structures in a strain lacking its three cardiolipid synthase genes. This suggests that contacts between cardiolipids and AtpF play a crucial role in ICM structuration, as observed in mitochondrial inner membranes (Carranza, Biochim Biophys Acta, 2017). Partner 5 developed this concept further by showing that interactions between cardiolipids and the secretion protein SecY stimulates the activity of the proton motive force (PMF)-dependent secretion machinery (Corey, Proc Natl Acad Sci U S A, 2018). We have provided more mechanistic insight on how lipids modulate the function of MPs by using high resolution NMR to show that the G-protein coupled receptor (GPCR) BLT2 inserted in nanodiscs explores four different conformational states. Increasing the sterol content modified the conformational landscape towards the active conformation, demonstrating a conformational coupling between the receptor and the membrane environment that is likely to be fundamental for membrane signaling (Fig. 4) (Casiraghi, Biochemistry, 2018; Chipot, Chem Rev, 2018). Following the 2015 mid-term report recommendation to increase our focus on MP structures, DYNAMO supported the structural study of a membrane-associated virulence factor of the Gram-negative bacterium Haemophilus influenzae called HxuA by Partner 5 using X-ray crystallography. HxuA belongs to the « Two-Partner-Secretion A » (TpsA) class of surface associated virulence factors and binds the heme-binding protein hemopexin (Hpx) from the serum of the host, allowing acquisition of the essential heme molecule. We determined the first TpsA structure and its complex with hemopexin was validated by mutational analysis (Fig. 5) (Zambolin, Nat Commun, 2016).

Partners 5 and 7 have a worldwide recognized expertise in the development of innovative tools in protein chemistry. Partner 5 was a pioneer in the development of amphipols (APols) as an alternative to detergents for the solubilization of MPs and was involved in the first cryo-electron microscopy (cryo-EM) study using the APol A8-35 to trap mt respiratory complexes (Illustrated in the Scientific Background of the Nobel Prize in Chemistry 2017). Since the “revolution in resolution”, up to 50 publications have described the use of APols for high-resolution structure determination of protein complexes by cryo-EM. In a second generation of grafted APols, Partner 5 developed a new labelling method for visualizing the transmembrane domain of MPs using a biotinylated APol and streptavidin (Perry et al. 2018, under revision). We also showed that hexahistidine-grafted APols are useful for MP immobilization (Giusti, Biomacromolecules, 2015). We have found new applications for amphiphilic polymers in the stabilization of soluble proteins, for example amyloid-beta oligomers (Serra-Batiste, Front Mol Biosci, 2018), and to protect antibodies and scFv fragments from aggregation. Indeed, Partner 7 showed that amphiphilic macromolecules effectively decreased the rate of thermally-induced aggregation of antibodies and enhanced refolding yield (Martin, Macromol Biosci, 2017), by enhancing colloidal stability involving both hydrophobic and coulombic interactions (Frka-Petesic, Langmuir, 2016; Martin, Langmuir, 2015). Partner 7 has also tailored diverse systems to perturb lipid membranes on demand. For instance, hydrophobic derivatives of polyacrylic acid adsorbed non-specifically on giant liposomes and on the plasma membrane of mammalian cells (COS, HEK) (Marie, J Membr Biol, 2014) enable non-toxic formation of nanopores upon mild variation of the polymer's hydrophobicity. Temperature responsive polymer chains are alternative triggers for dynamic and reversible control of the adhesion of living cells and clustering of cell receptors (Dalier, ACS Appl Mater Interfaces, 2018; Dalier, Biomacromolecules, 2016).

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