Project title
Unconventional protein secretion: Functional analysis of the plasma membrane nanodomain organisation driving FGF2 membrane translocation into the extracellular space

Summary
Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that belongs to a subgroup of extracellular proteins lacking N-terminal signal peptides. Whereas this phenomenon was already recognized in the early 1990s, detailed insights into the molecular mechanisms underlying alternative pathways of protein secretion from eukaryotic cells were obtained only recently. Today, we know about a number of alternative secretory mechanisms, collectively termed unconventional protein secretion (UPS). FGF2 belongs to a subgroup of cargo proteins secreted by direct translocation across the plasma membrane. This feature has been classified as type I UPS and is shared with other unconventionally secreted proteins, such as HIV-Tat and Tau. FGF2 translocation across the membrane is initiated through sequential interactions with the Na,K-ATPase, Tec kinase, and phosphoinositide PI(4,5)P2 at the inner plasma membrane leaflet. Whereas the first two are auxiliary factors of this pathway, the interaction of FGF2 with PI(4,5)P2 triggers the core mechanism of FGF2 membrane translocation. It is based on a lipidic membrane pore that is formed by PI(4,5)P2-induced oligomerization of FGF2. Membrane-inserted FGF2 oligomers are recognized as translocation intermediates that are resolved at the outer plasma membrane leaflet by glypican-1, a heparan sulfate proteoglycan that captures and disassembles FGF2 oligomers on cell surfaces. Recent findings suggest the molecular machinery mediating FGF2 membrane translocation to be highly organized in liquid-ordered plasma membrane nanodomains. The goal of this project will be to obtain detailed insights into the nanodomain organization of this process, providing a molecular understanding of the spatial and temporal coordination of FGF2 secretion into the extracellular space.

References
Lolicato F, Saleppico R, Griffo A, Meyer A, Scollo F, Pokrandt B, Müller HM, Ewers H, Hähl H, Fleury JB, Seemann R, Hof M, Brügger B, Jacobs K, Vattulainen I, Nickel W (2022) Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2. J Cell Biol e202106123 

Sparn C, Meyer, A, Saleppico R, and Nickel W (2022) Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells. 
Trends Biochem Sci 47:699-709 

Sparn C, Dimou E, Meyer A, Saleppico R, Wegehingel S, Gerstner M, Klaus S, Ewers H, Nickel W (2022) Glypican-1 drives unconventional secretion of Fibroblast Growth Factor 2. eLife e75545 

Lolicato F and Nickel W (2022) A role for liquid-ordered plasma membrane nanodomains coordinating the unconventional secretory pathway of Fibroblast Groth Factor 2. 
Front Cell Dev Biol 10:864257 

Dimou E, Cosentino K, Platonova E, Ros U, Sadeghi M, Kashyap P, Katsinelos T, Wegehingel S, Noé F, García-Sáez AJ, Ewers H, Nickel W (2019) Single event visualization of unconventional secretion of FGF2. J Cell Biol 218:683-699 

Dimou E and Nickel W (2018) Unconventional mechanisms of eukaryotic protein secretion. 
Curr Biol 28:R406-R410 

Steringer JP, Lange S, Čujová S, Šachl R, Poojari C, Lolicato F, Beutel O, Müller HM, Unger S, Coskun Ü, Honigmann A, Vattulainen I, Hof M, Freund C, Nickel W (2017) Key steps in unconventional secretion of fibroblast growth factor 2 reconstituted with purified components. eLife e28985 

Methods that will be used
- Super-resolution STED and MinFlux microscopy 
- Single molecule TIRF microscopy 
- Biochemical and biophysical reconstitution experiments 
- Molecular dynamics simulations

Cooperation partners
Dr. Holger Lorenz, ZMBH Imaging Facility, Heidelberg University 
Prof. Helge Ewers, FU Berlin 
Prof. Ilpo Vattulainen, Helsinki University 
Prof. Martin Hof, Prague

Funding SFB/TRR 186; E13/60%

Personal qualifications
We are looking for highly motivated students with a master degree in biochemistry, biophysics, chemistry or molecular biology.

Keywords
advanced super-resolution microscopy, computational biology, molecular dynamics simulations, tumor cell survival, FGF2, fibroblast growth factor 2, protein secretion, membrane trafficking, protein translocation across membranes, membrane nanodomains, liquid-ordered membrane domains

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