Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion

Abstract

Fibroblast growth factor 2 (FGF2) is a critical mitogen with a central role in specific steps of tumor-induced angiogenesis. It is known to be secreted by unconventional means bypassing the endoplasmic reticulum/Golgi-dependent secretory pathway. However, the mechanism of FGF2 membrane translocation into the extracellular space has remained elusive. Here, we show that phosphatidylinositol 4,5-bisphosphate-dependent membrane recruitment causes FGF2 to oligomerize, which in turn triggers the formation of a lipidic membrane pore with a putative toroidal structure. This process is strongly up-regulated by tyrosine phosphorylation of FGF2. Our findings explain key requirements of FGF2 secretion from living cells and suggest a novel self-sustained mechanism of protein translocation across membranes with a lipidic membrane pore being a transient translocation intermediate.

FIGURE 1.

Biochemical analysis of PI(4,5)P₂-dependent FGF2 oligomerization on membrane surfaces. Biochemical analysis employing liposomes with a plasma membrane-like lipid composition were supplemented with either PI(4,5)P₂ or a Ni-NTA lipid as indicated. Following binding to liposomes of the FGF2 variant forms indicated, bound proteins were separated from free proteins by flotation in density gradients. Proteins were separated by SDS-PAGE under reducing conditions and analyzed by Western blotting using anti-FGF2 antibodies.

FIGURE 2.

Analysis of PI(4,5)P₂-dependent FGF2 oligomerization at membrane surfaces using scanning fluorescence cross-correlation spectroscopy. GUVs were prepared with a plasma membrane-like lipid composition containing either PI(4,5)P₂ or a Ni-NTA lipid as indicated. His-tagged variants of FGF2-WT and FGF2-Y82pCMF were labeled with Atto488 and Atto655 dyes resulting in four independent preparations of fluorescent proteins. Mixtures of proteins with the two different dyes were added to the two types of GUVs resulting in four experimental conditions. Based on the FCCS analysis, we obtained auto-correlation curves for the Atto488- (green) and Atto655 (orange)-labeled FGF2 proteins, whose amplitude is inversely proportional to the concentration of the corresponding molecules. The two-color analysis yields the cross-correlation curve (blue), which provides information about the concentration of molecules forming a complex. The results of the fitting of the auto- and cross-correlation curves can then be combined to calculate the percentage of molecules forming a complex in the sample as well as diffusion constants. For further details see “Experimental Procedures.” A–D, representative examples of auto- (green and orange) and cross-correlation curves (blue) derived from two-focus scanning FCCS measurements of individual GUVs under the conditions indicated are shown. Solid lines correspond to fitted curves and dashed lines to raw data. E, percentage of FGF2 molecules found in complexes following correction for channel cross-talk and degree of labeling. Standard deviations are shown. To assess statistical significance, an unpaired two-tailed t test was performed using GraphPad Prism 5.0c (ns = not significant; *, p value ≤0.05; **, p value ≤0.01; ***, p value ≤0.001). F, diffusion coefficient of FGF2 molecules in individual GUVs. Standard deviations are shown. To assess statistical significance an unpaired two-tailed t test was performed using GraphPad Prism 5.0c (ns = not significant; *, p value ≤0.05; **, p value ≤0.01; ***, p value ≤0.001).

FIGURE 3.

FGF2 membrane activity depends on PI(4,5)P₂-mediated membrane recruitment and tyrosine phosphorylation. Carboxyfluorescein was sequestered in liposomes containing a plasma membrane-like lipid composition supplemented with either PI(4,5)P₂ (A and C) or a Ni-NTA lipid (B). Liposomes were incubated with FGF2-WT, FGF2-Y82pCMF, or FGF2-PIP-Mut as indicated. Membrane activity was measured by fluorescence dequenching as described under “Experimental Procedures.” The results shown are representative of four independent preparations of liposomes.

FIGURE 4.

Membrane activity of FGF2-Y82pCMF measured by confocal microscopy using giant unilamellar vesicles. A, confocal images of GUVs with a plasma membrane-like lipid composition either lacking PI(4,5)P₂ (panels a and d), containing PI(4,5)P₂ (panels b and e), or containing the Ni-NTA lipid (panels c and f). All three types of GUVs contained rhodamine B-labeled PE to visualize their membranes (red). Following formation of GUVs, a small fluorescent tracer molecule (Alexa 488, green) was added to monitor a potential membrane activity of added proteins measured by penetration of the lumen of GUVs. GUVs were incubated either in the presence of buffer (panels a–c) or FGF2-Y82pCMF (panels d–f). Following 90 min of incubation, confocal images were recorded (bar, 10 μm). B, quantitative analysis of the experiments shown in A (FGF2-Y82pCMF, panels d–f). GUVs were classified according to their luminal fluorescence compared with the surrounding fluorescence. Cyan bars represent GUVs lacking PI(4,5)P₂. Red bars represent GUVs containing PI(4,5)P₂. Blue bars represent GUVs containing the Ni-NTA lipid. For all conditions, data were derived from at least four independent experiments each of which involved the analysis of ≥300 GUVs per experimental condition. C, quantitative comparison and statistical analysis of membrane activity exerted by FGF2-Y82pCMF using FGF2-WT and FGF2-PIP-Mut as controls. Membrane activity is given as the percentage of GUVs with a luminal fluorescence of ≥50% compared with the surrounding fluorescence. For each experimental condition, the buffer control was subtracted. Standard deviations are shown. To assess statistical significance, an unpaired two-tailed t test was performed using GraphPad Prism 5.0c (ns, not significant; *, p value ≤0.05; **, p value ≤0.01).

FIGURE 5.

FGF2-Y82pCMF generates pores in GUVs containing PI(4,5)P₂ with a defined size cutoff. GUVs with a plasma membrane-like lipid composition containing PI(4,5)P₂ were incubated in the presence of FGF2-Y82pCMF and either Alexa488 (∼1 kDa, A), Alexa488-labeled dextran (∼10 kDa; B), or Alexa488 labeled cytochrome c (∼12 kDa, C). Confocal imaging was conducted as described in the legend to Fig. 4. Luminal penetration of the fluorescent tracers indicated was quantified as described in the legend to Fig. 4B and under “Experimental Procedures” using a threshold of ≥50% of luminal fluorescence compared with the surroundings (D).

FIGURE 6.

PI(4,5)P₂-dependent oligomerization of FGF2-Y82pCMF causes the formation of a toroidal membrane pore as measured by transbilayer diffusion of a membrane lipid. Transbilayer redistribution of pyrene-labeled sphingomyelin in large unilamellar liposomes was analyzed in the presence of either FGF2-Y82pCMF, the wild-type form of FGF2, a PI(4,5)P₂-binding mutant of FGF2, or buffer as indicated. Liposomes contained a plasma membrane (PM)-like lipid composition supplemented with either 2 mol % PI(4,5)P₂ or 2 mol % of the Ni-NTA lipid. Where indicated, liposomes were further supplemented with 5 mol % diacylglycerol (DAG). Following liposome formation, pyrene-labeled sphingomyelin was inserted into the outer leaflet resulting in asymmetrically labeled membranes. Addition of protein was carried out at t = 0 min. The time-dependent decrease of the I_E/I_M ratio indicates diffusion of pyrene-labeled sphingomyelin from the outer to the inner leaflet of liposomes. For further details, see “Experimental Procedures.” A, quantitative comparison of transbilayer diffusion of pyrene-labeled sphingomyelin in the presence of either 1 μm FGF2-Y82pCMF, 1 μm FGF2-WT, or 1 μm FGF2-PIP-Mut using liposomes with a plasma membrane-like lipid composition containing PI(4,5)P₂. B, protein concentration-dependent transbilayer diffusion of pyrene-labeled sphingomyelin caused by FGF2-Y82pCMF using liposomes with a plasma membrane-like lipid composition containing PI(4,5)P₂. C, dependence of transbilayer diffusion of pyrene-labeled sphingomyelin on PI(4,5)P₂-mediated recruitment of FGF2-Y82pCMF (1 μm) and inhibition by diacylglycerol.

FIGURE 7.

Structural model of lipidic membrane pores triggered by PI(4,5)P₂-dependent oligomerization of FGF2. Based on the detection of hexamers (Fig. 1) and the triangular crystal structure of FGF2, we propose a hexameric ring of FGF2 molecules in the center of a lipidic membrane pore. In the context of the reconstitution experiments of this study, this structure would allow for membrane passage of small fluorescent tracers and transbilayer diffusion of membrane lipids. The latter suggests a toroidal organization of membrane lipids at the rim of the membrane pore. In the context of a cell membrane, so far unidentified factors involved in FGF2 secretion may prevent both transbilayer diffusion of membrane lipids and membrane passage of small molecules. Additionally, FGF2 induced membrane pores may represent extremely short lived structures that are rapidly resolved by heparan sulfate proteoglycans at the extracellular side of the membrane. Therefore, they are unlikely to pose a substantial problem to cellular viability. P, tyrosine phosphorylation of FGF2.