<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy"^^ . "Protein secretion has been for long believed to be exclusively taking place through the\r\nconventional ER/Golgi-dependent secretory pathway. However, in the last few decades, more\r\nand more studies pointed at several different routes for protein lacking a signal peptide to\r\nget secreted from cells. It is now accepted from the scientific community that protein secretion\r\nfrom cells can also follow unconventional, ER/Golgi-independent pathways. These processes\r\nhave been collectively termed Unconventional Protein Secretion (UPS).\r\nFibroblast Growth Factor 2 (FGF2) is a cell survival factor involved in hematopoiesis, wound\r\nrepair, and tumor-induced angiogenesis. It exerts its biological functions in the extracellular\r\nspace, in an autocrine and paracrine manner. Despite this, it lacks a signal peptide and does\r\nnot follow the conventional ER/Golgi-dependent secretory pathway. FGF2 has been in fact one\r\nof the first protein found to follow a UPS pathway. It directly translocates across the plasma\r\nmembrane in a complete folded state. FGF2 gets recruited to the plasma membrane through\r\ninteraction with the α1 subunit of the Na,K-ATPase. Following interaction with Tec kinase,\r\nFGF2 gets phosphorylated. Subsequently, FGF2 oligomerizes in a PI(4,5)P2-dependent\r\nmanner. The resulting oligomer is able to span the membrane, forming a toroidal lipidic pore.\r\nAt this stage, membrane-proximal heparan sulfate chains capture FGF2 on cell surfaces.\r\nWithin this study, employing a recently established single molecule TIRF recruitment assay, I\r\nfound the α1 subunit of the Na,K-ATPase to be the first physical contact of FGF2 at the inner\r\nplasma membrane leaflet. I found this interaction to be a prerequisite for the subsequent\r\nFGF2 binding to PI(4,5)P2.\r\nTwo surface cysteine residues on FGF2, in position 77 and 95, have been for long a mystery\r\nin our laboratory with regards to their specific role. With a combination of cell-based\r\nrecruitment and secretion assays, I contributed to elucidate their respective role. I found both\r\nresidues to be involved in FGF2 recruitment to the inner plasma membrane leaflet, as well as\r\nin translocation to cell surfaces. Nevertheless, comparing FGF2 variants carrying either single\r\nsubstitutions or a double substitution of these cysteines, I observed the residue in position\r\n95 to contribute in a stronger manner to both parameters. Combining my results with other\r\nfindings from our laboratory, cysteine 95 appears to be involved in intermolecular disulfide\r\nbridge formation, while cysteine 77 seems to be involved in the interaction with the α1.\r\nGlypican-1 has been recently discovered to be the dedicated HSPGs for FGF2 secretion. Under\r\nconditions of low FGF2 expression levels, I found Glypican-1 to increase FGF2 secretion from\r\ncells in a much higher manner compared to conditions of overexpressed FGF2. With this\r\nfinding, I contributed to the conclusion that Glypican-1 is the rate-limiting factor for the\r\nunconventional secretion of FGF2.\r\nFinally, I found many hints pointing at a possible involvement of liquid-ordered domains in\r\nthe unconventional secretion of FGF2. This was based on my findings on the positive\r\nmodulation of both cholesterol and sphingomyelin, two important components of liquidordered\r\ndomains on the plasma membrane, on the unconventional secretion of FGF2."^^ . "2022" . . . . . . . "Roberto"^^ . "Saleppico"^^ . "Roberto Saleppico"^^ . . . . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (PDF)"^^ . . . "Roberto Saleppico_PhDThesis.pdf"^^ . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (Other)"^^ . . . . . . "small.jpg"^^ . . . "Molecular analysis of the unconventional secretory\r\npathway of FGF2 employing TIRF microscopy (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #32465 \n\nMolecular analysis of the unconventional secretory \npathway of FGF2 employing TIRF microscopy\n\n" . "text/html" . .