Data supplements
Files in this Data Supplement:
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- Supplemental text (PDF) - Equations S1-S22 for data fitting and calculations.
Article Figures & Data
Figures
- Figure 1.
Overall integrin conformational states, the equilibria linking them and the strategy to quantify equilibria. (A) The three overall states in an integrin conformational ensemble (Luo et al., 2007) and their thermodynamic parameters. (B) Equations used in this study. (C) Specificities of conformation-specific Fabs.
- Figure 2.
Integrin α4β1 preparations. (A) SDS 7.5% PAGE of α4β1 headpiece with high-mannose N-glycans (lane 1), unclasped ectodomain with high-mannose N-glycans (lane 2), and unclasped ectodomain with complex N-glycans (lane 3). (B) Domain compositions.
- Figure 3.
Intrinsic affinities, ensemble affinities and conformational equilibria of α4β1 headpiece and ectodomain fragments. (A–C) Affinities for FITC-LDVP of α4β1 headpiece with high-mannose N-glycans (A), unclasped ectodomain with high-mannose N-glycans (B) and unclasped ectodomain with complex N-glycans (C) were measured using FP in presence or absence of the indicated Fabs. Errors in plots or inset tables are SD from nonlinear least square fits with triplicates, except values with asterisks are mean and SD from at least three experiments on different days. (D and E) Affinity measurements and calculated thermodynamic parameters for α4β1 (D) and comparison to published (Li et al., 2017) α5β1 measurements (E). Errors are as described for A–C, except values with “#,” which represent mean ± difference from the mean of measurements with distinct Fabs stabilizing the same conformation.
- Figure 4.
Intrinsic and ensemble affinities of α4β1 on the surface of three cell types and deduced conformational equilibria. (A–C) Affinities for FITC-LDVP of intact α4β1 on Jurkat (A), Thp1 (B), and KA4 cells (C) in the presence of the indicated Fabs and cell concentrations. (D) Tabulation of results from A–C. Errors in each panel and inset table are as described in the Fig. 3 legend. (E) Specific fluorescence intensity of cells stained with 25 nM Alexa647-conjugated mAb.
- Figure 5.
Affinities of α4β1 on cell surface for different ligands. (A–C) Basal ensemble affinity of α4β1 for LDVP and VCAM D1D2 on Jurkat (A), KA4 (B), and Thp1 (C) cells determined by binding of 5nM Alexa647-9EG7 Fab. (D) Intrinsic affinity of α4β1 EO conformation for VCAM D1D2, Fn IIICS, and MAdCAM D1D2 measured on Jurkat cells by inhibition of binding of 1nM FITC-LDVP in presence of EO-stabilizing Fabs 9EG7&HUTS4. (E) Intrinsic affinity of α4β1 EO conformation in presence of Fabs 9EG7&HUTS4 measured by saturation binding of Alexa488-VCAM D1D2 using fluorescence flow cytometry. (F) Tabulation of results. Errors in each panel and inset table are as described in Fig. 3 legend.
- Figure 6.
Intrinsic and ensemble affinities of α5β1 on the surface of Thp1 and KA4 cells and deduced conformational equilibria for comparison with α4β1. (A and C) Affinity of α5β1 on Thp1 (A) and KA4 (C) cells for Fn39-10 by enhancement of 5 nM Alexa647-9EG7 Fab binding. (B and D) Affinity of α5β1 on Thp1 (B) and KA4 (D) cells for Alexa488-Fn39-10 in presence of indicated Fabs. (E) Tabulation of results. Data for α5β1 on Jurkat and K562 cell surfaces are published (Li et al., 2017) and shown for comparison. Errors in each panel are as described in Fig. 3 legends.
- Figure 7.
Lack of correlation between affinity of cell surface integrin α4β1 for soluble ligand and cellular adhesiveness. (A and B) Cell adhesion to VCAM-Fc under basal conditions (A) or in presence of 8 µM 9EG7 Fab (B). (C and D) Ensemble affinity of α4β1 on HPB-ALL (C) and U937 (D) cells for FITC-LDVP under basal conditions and in presence of 8 µM 9EG7 Fab to stabilize extension. (E) Comparison of ensemble affinity for FITC-LDVP on five cell types to adhesiveness measured as VCAM-Fc EC50 values under basal or extension-stabilizing conditions. (F) Jurkat or U937 cells were mixed with LDVP or closure-stabilizing Fab mAb13 and assayed for adhesion to 5 nM VCAM-Fc bound to protein A–coated plates as described in Results. Lines in A, B, and F show fits to dose–response curves. Errors in each panel are as described in Fig. 3 legend, except values with “+,” which represent mean and deviation from the mean from two experiments on different days.
- Figure 8.
Regulation of integrin activation by intracellular and extracellular signaling. (A and B) ΔG of each integrin conformational state for integrin headpiece or ectodomain preparations or intact cell surface integrins measured here for α4β1 (A) and here and previously for α5β1 (B; Li et al., 2017). (C) Energy landscape comparisons for intact α5β1 and α4β1 on Jurkat, Thp1, and KA4 cells. (D) Schematic illustration of integrin activation regulated by intracellular protein binding to integrin cytoplasmic tails and application of cytoskeletal force. Ovals represent inhibitors and stars represent adaptors. F represents tensile force exerted across ligand–integrin–adaptor complexes by the cytoskeleton and resisted by immobilized ligand. Distances in the force-bearing pathway between the ligand-binding site and the C terminus of the β-tail domain known from structures and molecular dynamics are shown with arrows (x1, x2, and x3). (E–G) Population of integrin states that mediate cell adhesion in absence (E) or presence of force (F and G). Colors in the key encode the population of integrin states that can mediate cell adhesion (i.e., the sum of adaptor and ligand bound states over all three integrin conformational states). >99% of such adaptor- and ligand-bound integrins are in the EO conformation (Li and Springer, 2017). Ligands are used at concentrations equal to
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August 6, 2018
Volume 217, No. 8