Description of the PhD thesis project
How cells maintain strong differences in lipid composition among their compartments despite constant intracellular membrane exchanges by transport carriers remains mysterious. Thus, there is a 5-fold enrichment in sphingomyelin (SM) in the plasma membrane (PM) as compared to the trans-Golgi network (TGN) where SM is synthesized. SM enrichment in transport carriers cannot be explained by a bending energy minimization-based mechanism of lipid sorting, since SM form stiff membranes. We had hypothesized that protein-assisted lipid sorting could explain SM enrichment in transport carriers.
Indeed, we recently found that caveolin-depleted cells have lower SM levels in their PM. The membrane proteins Caveolins traffic from TGN to PM, which suggests that they could enrich SM at the PM, by assisting their inclusion in transport vesicles at the TGN. Caveolins are the structural components of small PM invaginations, called caveolae, rich in SM and cholesterol, that are involved in lipid homeostasis, mechanosensing and cellular signaling. How caveolins assist in SM recruitment is unclear, since caveolins are inserted like hairpins in the inner leaflet of the PM, whereas SM is in the outer leaflet.
The PhD objective is to uncover the physical mechanism of SM sorting by caveolin in asymmetric membranes. The student will develop novel in vitro reconstituted systems based on Giant Unilamellar Vesicles (GUV), and purified caveolin. S/he will study how caveolin influences SM sorting in curved membranes using quantitative fluorescence microscopy and the nanotube pulling assay with optical tweezers we established.
Caveolin reconstitution in GUV is already operational but so far only in membranes with symmetric leaflets. A challenge will be to prepare GUVs with asymmetric leaflets, mimicking the PM and to reconstitute caveolin in the opposite leaflet of SM-cholesterol. The new tools and physical models of this project will unravel protein-assisted lipid transport by caveolin and beyond.
International, interdisciplinary & intersectoral aspects of the project
The thesis will be co-supervised by P. Bassereau (Biophysicist) and C. Lamaze (Cell biologist). The project has already started with a joint post-doc (J. Podkalicka), biochemist and biologist working on the in vivo aspects, who will directly interact with the PhD.
For reconstitution, the Bassereau group collaborates closely with D. Lévy and M. Dezzi, structural biologists that are experts in membrane proteins. We collaborate with A. Kenworthy (Virginia Univ.), who studies caveolin structure and C. Burd (Yale Univ.) who develops SM markers.
A scientist of the L’Oréal company will participate to the thesis committee and the student will have the possibility to interact with the L’Oréal advanced microscopy platform. Finally, the thesis will have the "Doctor Europaeus" label.
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