Spatio-temporal control of FGF2 recruitment and membrane translocation exerted by phosphoinositide and kinase switches.
Fibroblast Growth Factor 2 (FGF2) is a cell survival factor with a major role in tumor-induced angiogenesis. FGF2 exerts its biological functions in the extracellular space where it activates FGF receptors for both auto- crine and paracrine signaling. Intriguingly, consistent with the lack of a signal peptide for ER/Golgi-dependent protein secretion, FGF2 was shown to be secreted from cells by direct protein translocation across the plasma membrane. In recent years, we have revealed several key aspects of the molecular mechanism and the molecular machinery behind this unconventional pathway of protein secretion. This process involves (i) se- quential interactions of FGF2 with ATP1A1, Tec kinase and PI(4,5)P2 at the inner leaflet of the plasma mem- brane, (ii) Tec-kinase-mediated tyrosine phosphorylation of FGF2, (iii) PI(4,5)P2-dependent formation of mem- brane-spanning FGF2 oligomers as well as (iv) disassembly and extracellular trapping of FGF2 mediated by cell surface heparan sulfate proteoglycans. Several lines of evidence suggest that PI(4,5)P2-induced oligomer- ization of FGF2 triggers the formation of lipidic membrane pores with a toroidal architecture. As part of this arrangement, the headgroups of PI(4,5)P2 and other plasma membrane lipids contribute a hydrophilic surface in the periphery of the pore with the FGF2 oligomer accommodated in its center. Based upon the addition of FGF2 subunits at the cytoplasmic leaflet and removal of FGF2 subunits at the outer leaflet, an assembly/dis- assembly mechanism has been proposed explaining directional transport of FGF2 across the plasma mem- brane. The latter process is driven by cell surface heparan sulfates that outcompete PI(4,5)P2 for the interac- tion with FGF2 at the outer leaflet resulting in accumulation of FGF2 on cell surfaces.
The goal of this project is to study the spatio-temporal coordination of FGF2 recruitment at the inner leaflet and FGF2 membrane translocation to the outer plasma membrane leaflet in living cells. In particular, we will analyze how this process is directed by the three molecular switches involved, Tec kinase, the phosphoinosi- tide PI(4,5)P2 and the redox/oligomerization switch that governs the formation of membrane-inserted FGF2 membrane translocation intermediates. Building on the results from the first funding period, we aim at (i) analyzing the precise functional oligomeric state of FGF2 as a membrane translocation intermediate, (ii) quan- tifying the kinetics of the various steps involved in FGF2 secretion by establishing a synchronized system with optogenetic control and single molecule sensitivity, (iii) revealing the nanoscopic architecture of the FGF2 membrane translocation machinery using super-resolution TIRF microscopy at the single particle level and (iv) identifying so far unknown components of the molecular machinery of FGF2 membrane translocation using trifunctional derivatives of PI(4,5)P2 in photo-crosslinking experiments. Our experimental findings will be com- bined with theoretical approaches including molecular dynamics simulations and mathematical modeling. In this way, we aim at establishing a comprehensive understanding of the molecular mechanism of the uncon- ventional secretory pathway of FGF2, an unusual pathway of protein secretion that trigger FGF2-dependent tumor cell survival and tumor-induced angiogenesis.