Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate
Figure 2
FGF-2 NP in the pericellular matrix are clustered.
TEM of plasma membrane sheets reveals clustering and the heterogeneous spatial distribution of FGF2-NP at high resolution. Five hundred and fifty pM (A) or 2.8 nM (B) of FGF2-NP were added to living cells before washing, plasma membrane sheet preparation, and fixation. No labelling was observed when using 2.8 nM of control nanoparticles (non-specific binding control with NP-TrisNiNTA, not conjugated to FGF2) (C). (D) 2.8 nM FGF2-NP in the presence of 50 µg/mL heparin-derived dodecasaccharide (DP12). In this condition, FGF-2 binding to the HS of the pericellular matrix was abolished, but not the interaction with the FGFR. The little labelling that was observed corresponded to complexes of FGF2-NP with FGFR and DP12. Scale bar, 200 nm. Representative images. (E) Average number of nanoparticles per µm2 (mean +/− SD) for 550 pM (purple), 2.8 nM FGF2-NP (green). Labelling was strongly reduced in the presence of 50 µg/mL of DP12 (grey) for both concentrations of FGF2-NP. Little labelling was also observed when 550 pM of FGF2-NP was added to the cell with an excess of unlabelled FGF2 protein (50 µM) in the absence (white) or in the presence (stripped) of DP12. The number of photomicrographs of 1.578 µm per 1.578 µm or 1.578 µm per 2.1 µm analysed were 69 for 550 pM FGF2-NP, 65 for 550 pM FGF2-NP in the presence of DP12, 63 for 550 pM FGF2-NP in the presence of unlabelled FGF2, 57 for 550 pM FGF2-NP in the presence of DP12 and unlabelled FGF2, and 27 for 2.8 nM FGF2-NP and 116 for 2.8 nM FGF2-NP in the presence of DP12. Non-parametric Kolmogorov-Smirnov performed on the data gave the following p values: 550 pM FGF2-NP against 550 pM FGF2-NP with DP12, p = 0; 550 pM FGF2-NP against 550 pM FGF2-NP with excess FGF2, p = 0; 550 pM FGF2-NP against 550 pM FGF2-NP with DP12 and excess FGF2, p = 0; 550 pM FGF2-NP against 2.8 nM FGF2-NP, p = 2.12883E−9 all 2.8 nM FGF2-NP against 2.8 nM FGF2-NP with DP12, p = 2.22045E−16. (F) FGF2-NP clustering at 550 pM (purple) and 2.8 nM (green) was characterised by K-function analysis (Materials and Methods). 24 and 27 photomicrographs of 1.578 µm per 1.578 µm were analysed, respectively. Values of L(r)-r above the 99% confidence interval (CI) (black) indicate significant clustering within the defined x-axis radius values (r). Clustering of FGF2-NP was observed at 550 pM and its extent increased with FGF2-NP concentration.