Exploring the structure function relationship of membrane-pore-forming FGF2 oligomers - a single molecule approach

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Fibroblast Growth Factor 2 (FGF2) is a strong mitogen promoting angiogenesis in health and disease. Under pathological conditions it is functioning as a major activator of tumor-induced angiogenesis and also acts as a survival factor of tumor cells mediated by an autocrine signaling loop suppressing apoptosis. Despite its defined extracellular functions, FGF2 lacks a signal peptide and, therefore, is exported from cells by an unconventional, ER/Golgi-independent mechanism of protein secretion. Understanding the molecular mechanism by which FGF2 is secreted from tumor cells may pave the way to develop a new class of anti-angiogenic inhibitors with a high potential in cancer therapy.FGF2 secretion from cells was found not to involve intracellular vesicle intermediates but rather is mediated by direct translocation across the plasma membrane. This process is initiated by the recruitment of FGF2 at the inner leaflet of the plasma membrane mediated by the phosphoinositide PI(4,5)P2. This interaction causes FGF2 to oligomerize and, stimulated by phosphorylation of FGF2 at a specific surface tyrosine residue, results in membrane insertion of a FGF2 oligomer concomitant with the formation of a toroidal membrane pore. This structure has been interpreted as an intermediate in FGF2 membrane translocation. In a final step, cell surface heparan sulfate proteoglycans trap FGF2 molecules at the outer leaflet resulting in translocation of FGF2 molecules into the extracellular space.The goal of this research proposal is to follow an interdisciplinary approach at the interface between cell biology, biochemistry and biophysics. It aims at the molecular analysis of the structure function relationship of FGF2 oligomers to obtain insight into how a lipidic membrane pore can be formed induced by PI(4,5)P2-dependent FGF2 oligomerization. Specifically, we plan to follow a single molecule approach to be able to identify and analyze functional FGF2 oligomers forming membrane pores both in vitro and in a novel cell-based system. These studies aim at i.) investigating the number of FGF2 molecules engaged in pore-forming FGF2 oligomers and the size of the membrane pore associated with FGF2 oligomers, ii.) determining the distance between individual FGF2 molecules within pore-forming oligomers, iii.) analyzing their dynamics and the lifetime of membrane pores and iv.) revealing a potential role for lipidic nano-domains surrounding membrane pores formed by FGF2 oligomers. Finally, we aim at validating the results from reconstitution experiments in a novel cell-based system. These studies thus have a strong potential to reveal important parameters of the functional architecture of membrane-inserted FGF2 oligomers and, therefore, from these studies, we expect fundamental advances in our understanding of the functional role of these structures as intermediates of the unconventional secretory pathway of FGF2.