Fig. 3: Combination of time-resolved neutron reflectometry and interfacial shear rheology shows the existence of a AP2/PtdIns(4,5)P₂ entropic network at increasing surface coverage.

A Normalized amount of AP2 protein (Γ/Γ∞, with Γ ∞ being the saturation concentration) interacting with a lipid monolayer enriched in PtdIns(4,5)P₂ and TGN38 measured using time-resolved SNR in a restricted Q region and compared with the increase in surface pressure measured (ΔΠ). B Linear dependence between ΔΠ and the fraction coverage (ϕ = Γ/Γ∞). C The suggested transition mechanism from a fluid state to a percolated network state occurs as the coverage of AP2 on a PtdIns(4,5)P₂-enriched membrane (ϕ) rises. D Time dependence of the interfacial shear viscoelastic moduli, divided in the elastic and viscous modulus (G’ and G´´, respectively). Data shows an increase in both G’ and G” after injecting AP2 in the subphase. E Re-plotted G’ and G” with respect to the surface coverage ϕ, revealing two differentiated regimes: a fluid-dominated state below ϕ = 0.73 and a gel-like state above this threshold that can be rationalized as percolated network according to (C). F G´ plotted as a function of the distance to the percolation threshold showing a power law exponent f = 2.3. G Comparative for monolayers enriched with and without different cargoes such as TGN38 and CD4. Straight lines are fits of the experimental data to obtain an average binding time plotted in (H). G”/G’ ratio of the different monolayers studied before and after the injection of AP2 is plotted in (I). J shows the values of G´ and G´´ for monolayers with different composition. The initial surface pressure Π₀ in all the cases has been fixed to 25 ± 1 mN/m and the bulk concentration of the protein to 10 nM.