synj with ptdins 4 5 p 2  (Echelon Biosciences)

Journal: bioRxiv

Article Title: Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

doi: 10.1101/2024.04.19.590255

Figure Lengend Snippet: Simultaneous colocalization of AP2 and PtdIns(4,5)P 2 clusters in Langmuir monolayers at the air water interface were reported by fluorescence images of TGN-PtdIns(4,5)P 2 monolayer at the air/buffer interface labelled with 1 mol % Bodipy-TMR-PtdIns(4,5)P 2 (A) interacting with AP2-labelled with Alexa Fluor 488 (B). Transferred lipid monolayers immersed in buffer were examined by fluid-phase, tapping-mode AFM in absence (C) and presence (D) of AP2. (E) shows the thickness histograms of samples shown in (C) and (D) with straight lines being the corresponding Gaussian distributions, respectively. (F) Time-resolved AFM images shows AP2 yielded an increase of thickness and FWHM after injection until a plateau is reached when AP2 saturates the monolayer. Data points are represented as mean ± S.D. (n = 10).

Article Snippet: Lipid monolayers were doped with 0.1 mol % BODIPY-TMR PtdIns(4,5)P 2 and observed using an inverted bright-field microscope (Nikon Eclipse) with an extra-long working distance (WD 3.7–2.7 mm, NA 0.60) objective of 40× magnification.

Techniques: Fluorescence, Injection

Journal: bioRxiv

Article Title: Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

doi: 10.1101/2024.04.19.590255

Figure Lengend Snippet: (A) Increase in Surface Pressure (ΔΠ) as a function of time for different concentration of AP2 on lipid monolayers enriched in 10 wt. % PtdIns(4,5)P 2 and TGN38 (2wt. %). The initial surface pressure Π 0 in all the cases has been fixed to 25 ± 1 mN/m similar to the lateral pressure in a plasma membrane leaflet. (B) Normalized surface pressure plotted against to the time elapsed after a lag period and fitted by a stretched exponential (straight lines) following . (C) Concentration dependence of the average relaxation ratio and a linear fit (straight lines). (D) Maximal increase in surface pressure ΔΠ ∞ for lipid monolayers at different composition at Π 0 =25 ± 1 mN/m and a fixed concentration of AP2 of 500 nM. (E) Tensiometry binding analysis for AP2 recruited to a monolayer enriched in PtdIns(4,5)P 2 and TGN38 or CD4 at different values of Π 0 . The increment in pressure, ΔΠ, is proportional to the amount of protein binding to the lipid monolayer at the air/water interface. Straight lines are fits to the experimental data obtained through the Hill-Langmuir relation . (F) The increment in pressure, ΔΠ as a function of AP2 concentration for CD4 and TGN38 monolayers after subtracting PtdIns(4,5)P 2 contribution. ( G ) Uppermost surface pressure increments due to the interaction of AP2 with a monolayer at Π 0 = 25 ± 1 mN/m, at different concentration of PtdIns(4,5)P 2 . ( H ) Dissociation constants for PtdIns(4,5)P 2 monolayers enriched in CD4 and TGn38 at different initial surface pressure. Bar diagrams in D, G, H are represented as mean ± S.D. (n = 5).

Article Snippet: Lipid monolayers were doped with 0.1 mol % BODIPY-TMR PtdIns(4,5)P 2 and observed using an inverted bright-field microscope (Nikon Eclipse) with an extra-long working distance (WD 3.7–2.7 mm, NA 0.60) objective of 40× magnification.

Techniques: Concentration Assay, Membrane, Binding Assay, Protein Binding

Journal: bioRxiv

Article Title: Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

doi: 10.1101/2024.04.19.590255

Figure Lengend Snippet: (A) Normalized amount of AP2 protein (Γ/G∞, with Γ∞ being the saturation concentration) interacting with a lipid monolayer enriched in PtdIns(4,5)P 2 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/G∞). (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)P 2 -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 Π 0 in all the cases has been fixed to 25 ± 1 mN/m and the bulk concentration of the protein to 10 nM.

Article Snippet: Lipid monolayers were doped with 0.1 mol % BODIPY-TMR PtdIns(4,5)P 2 and observed using an inverted bright-field microscope (Nikon Eclipse) with an extra-long working distance (WD 3.7–2.7 mm, NA 0.60) objective of 40× magnification.

Techniques: Concentration Assay, Membrane, Shear, Binding Assay, Injection

Journal: bioRxiv

Article Title: Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

doi: 10.1101/2024.04.19.590255

Figure Lengend Snippet: (A) Calculated volume fraction profiles corresponding to the preferred orientation of open conformation AP2 bound to PtdIns(4,5)P 2 -enriched monolayers in absence and presence of TGN38 or CD4 peptides. (B) Surface coverage and (C) Violin plots showing the distance to the interface were calculated by fitting the theoretical and experimental SLD values for each orientation of the protein in open conformation (PDB: 2xa7). The black vertical line indicates the interquartile range, the box plot, shows the ends of the first and third quartiles and the central dot the median value (n=256).

Article Snippet: Lipid monolayers were doped with 0.1 mol % BODIPY-TMR PtdIns(4,5)P 2 and observed using an inverted bright-field microscope (Nikon Eclipse) with an extra-long working distance (WD 3.7–2.7 mm, NA 0.60) objective of 40× magnification.

Techniques: