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Abstract

Protein secretion has been for long believed to be exclusively taking place through the conventional ER/Golgi-dependent secretory pathway. However, in the last few decades, more and more studies pointed at several different routes for protein lacking a signal peptide to get secreted from cells. It is now accepted from the scientific community that protein secretion from cells can also follow unconventional, ER/Golgi-independent pathways. These processes have been collectively termed Unconventional Protein Secretion (UPS). Fibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in hematopoiesis, wound repair, and tumor-induced angiogenesis. It exerts its biological functions in the extracellular space, in an autocrine and paracrine manner. Despite this, it lacks a signal peptide and does not follow the conventional ER/Golgi-dependent secretory pathway. FGF2 has been in fact one of the first protein found to follow a UPS pathway. It directly translocates across the plasma membrane in a complete folded state. FGF2 gets recruited to the plasma membrane through interaction with the α1 subunit of the Na,K-ATPase. Following interaction with Tec kinase, FGF2 gets phosphorylated. Subsequently, FGF2 oligomerizes in a PI(4,5)P2-dependent manner. The resulting oligomer is able to span the membrane, forming a toroidal lipidic pore. At this stage, membrane-proximal heparan sulfate chains capture FGF2 on cell surfaces. Within this study, employing a recently established single molecule TIRF recruitment assay, I found the α1 subunit of the Na,K-ATPase to be the first physical contact of FGF2 at the inner plasma membrane leaflet. I found this interaction to be a prerequisite for the subsequent FGF2 binding to PI(4,5)P2. Two surface cysteine residues on FGF2, in position 77 and 95, have been for long a mystery in our laboratory with regards to their specific role. With a combination of cell-based recruitment and secretion assays, I contributed to elucidate their respective role. I found both residues to be involved in FGF2 recruitment to the inner plasma membrane leaflet, as well as in translocation to cell surfaces. Nevertheless, comparing FGF2 variants carrying either single substitutions or a double substitution of these cysteines, I observed the residue in position 95 to contribute in a stronger manner to both parameters. Combining my results with other findings from our laboratory, cysteine 95 appears to be involved in intermolecular disulfide bridge formation, while cysteine 77 seems to be involved in the interaction with the α1. Glypican-1 has been recently discovered to be the dedicated HSPGs for FGF2 secretion. Under conditions of low FGF2 expression levels, I found Glypican-1 to increase FGF2 secretion from cells in a much higher manner compared to conditions of overexpressed FGF2. With this finding, I contributed to the conclusion that Glypican-1 is the rate-limiting factor for the unconventional secretion of FGF2. Finally, I found many hints pointing at a possible involvement of liquid-ordered domains in the unconventional secretion of FGF2. This was based on my findings on the positive modulation of both cholesterol and sphingomyelin, two important components of liquidordered domains on the plasma membrane, on the unconventional secretion of FGF2.