Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2

FGF2 is a cell survival factor involved in tumor-induced angiogenesis that is secreted through an unconventional secretory pathway based upon direct protein translocation across the plasma membrane. Here, we demonstrate that both PI(4,5)P2-dependent FGF2 recruitment at the inner plasma membrane leaflet and FGF2 membrane translocation into the extracellular space are positively modulated by cholesterol in living cells. We further revealed cholesterol to enhance FGF2 binding to PI(4,5)P2-containing lipid bilayers. Based on extensive atomistic molecular dynamics (MD) simulations and membrane tension experiments, we proposed cholesterol to modulate FGF2 binding to PI(4,5)P2 by (i) increasing head group visibility of PI(4,5)P2 on the membrane surface, (ii) increasing avidity by cholesterol-induced clustering of PI(4,5)P2 molecules triggering FGF2 oligomerization, and (iii) increasing membrane tension facilitating the formation of lipidic membrane pores. Our findings have general implications for phosphoinositide-dependent protein recruitment to membranes and explain the highly selective targeting of FGF2 toward the plasma membrane, the subcellular site of FGF2 membrane translocation during unconventional secretion of FGF2

Cholesterol promotes clustering of PI(4,5)P-2 driving unconventional secretion of FGF2

Peer reviewe

Nanodomain organization of the FGF2 secretory machinery

Fibroblast growth factor 2 (FGF2) is a potent mitogen involved in angiogenesis and tumor cell survival. Although a secretory protein involved in autocrine and paracrine signaling, FGF2 does not contain a signal peptide and bypasses the classical ER-Golgi-dependent route of protein secretion. In fact, FGF2 is secreted unconventionally (type I UPS) via direct translocation across the plasma membrane by self-sustained pores. FGF2 is recruited to the plasma membrane via interaction with the ⍺1 subunit of the Na,K-ATPase. Thereafter FGF2 interacts with Tec kinase, which phosphorylates FGF2. FGF2 binds to the phosphoinositide PI(4,5)P2, which drives its dimerization and oligomerization into membrane-spanning complexes. FGF2 is then captured on the cell surface through binding to heparan sulfate proteoglycans (HSPGs). The GPI-anchored HSPG glypican-1 (GPC1) was identified in our laboratory via a genome-wide BioID screen. In the here presented thesis, GPC1 CRISPR-Cas9 knockout (KO) cell lines were shown to secrete significantly less FGF2 in cell surface biotinylation assays. GPC1 reintroduction and stable overexpression did not only restore secretion to wild-type levels but even further increased secretion. FGF2 endocytosis was not affected by GPC1 KO or overexpression. As I could correlate GPC1 levels to FGF2 secretion in TIRF microscopy, GPC1 can be considered a rate-limiting factor for FGF2 secretion. GPC5, another endogenously expressed glypican in HeLa cells, did not reduce FGF2 secretion when knocked out. Also, GPC5 could not compensate for loss of GPC1 in the here demonstrated data. GPC6 knockout in U2OS cells only led to a modest decrease in FGF2 secretion, yet further decreased FGF2 secretion in GPC1 KO cells in my experiments. Organization of FGF2 and the components needed for secretion into nanodomains would facilitate fast FGF2 secretion from cells. FGF2, ⍺1 and GPC1 were indeed shown to be in proximity to each other in the here conducted proximity ligation assays. Also, I found all three components in detergent resistant membrane (DRM) fractions, whereby GPC1 was the determining factor for FGF2 DRM localization. Super resolution STED experiments showed a homogenous distribution of ⍺1 and GPC1 throughout the membrane. Interestingly, I found FGF2 and PI(4,5)P2 in areas enriched in cholesterol detected via EGFP-Gram1b G187L transfection, which is supported by recent findings demonstrating that cholesterol promotes FGF2 secretion. Caveolins, cholesterol-binding proteins that assemble into detergent resistant membrane fractions, were analyzed regarding their effect on FGF2 secretion. In my experiments caveolin-1 (Cav1) and -2 (Cav2) affected FGF2 secretion in HeLa S3 and U2OS. In HeLa cells, caveolin-2 KO cells showed reduced FGF2 secretion in biotinylation experiments. Intriguingly, caveolin-1 failed to localize into DRM fractions in this context and also ⍺1 and FGF2 levels were reduced in liquid ordered detergent resistant fractions. Cav2 KO in U2OS on the other hand, did not impact FGF2 secretion in my hands, although TIRF data demonstrated Cav1 to be involved in FGF2 secretion. Caveolin function in FGF2 nanodomain organization remained unresolved. Pulldown experiments I conducted using trifunctional lipid probes demonstrated an interaction between PI(3,4,5)P3 and the ⍺1 subunit of the Na,K-ATPase, further supporting the hypothesis of specialized membrane domains involved in FGF2 membrane translocation into the extracellular space

Glypican-1 drives unconventional secretion of fibroblast growth factor 2

Fibroblast growth factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse subclasses consisting of syndecans, perlecans, glypicans, and others, Glypican-1 (GPC1) is the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression