Figure 1. Single-molecule localization... | Rockefeller University Press

Figure 1.

$Figure 1. Single-molecule localization and tracking of DY647IFN binding to endogenous cell surface IFNAR. (a) Ligand-induced assembly of a dynamic ternary complex. The effective ligand binding affinity to the cell surface receptor depends on the dynamic equilibrium between the binary and ternary complex. (b) Live-cell IFNα2 binding assay by single-molecule imaging on HeLa. (b, left) A fluorescence image showing individual DY647IFNα2-wt bound to the cell surface receptor. (b, right) Trajectories of IFNα2-wt molecules from the boxed region. The boundaries of the cell are indicated by a yellow dotted line. (c) Density of DY647IFNα2-wt molecules localized on the surface of individual HeLa cells imaged in the presence of 2 nM DY647IFNα2-wt. For comparison, the density of DY647IFNα2-wt molecules on HeLa cells blocked with unlabeled IFNα2-wt is shown in addition to IFNAR2-deficient U5A cells. Data distribution of the second and third quartile (box), median (line), mean (closed square), and whiskers (1.5× interquartile range) is shown. (d) Normalized bleaching of DY647IFNα2-wt (>150 particles at t = 0) bound to endogenous receptors on HeLa at standard conditions and 10-fold increased laser power. Representative curves are shown for at least five experiments. (e) Single-step bleaching of labeled IFNs depicted as a 3D kymograph. Bleaching events are indicated by green arrows. (f) Single-step bleaching events of three individually labeled IFNs (representative curves for >100 bleached particles). (g) Diffusion properties of cell-bound DY647IFNα2-wt presented as the step length distribution for a time lapse of 160 ms (5 frames, black curve), which was obtained by fitting the step length histogram by considering three components corresponding to an immobile as well as a slow and a fast mobile fraction (Fig. S1). (h) Diffusion properties of cell-bound DY647IFNα2-dn and fit according to a two-component model. (i) Comparison of the step-length histogram for DY647IFNα2-wt and DY647IFNα2-dn. The data shown in g–i are pooled from at least two independent experiments, each with >650 analyzed trajectories (≥15 steps) per IFN mutant. (j) Changes in mobility of a model transmembrane protein dimerized by a monoclonal antibody. The data shown are pooled from eight independent experiments with >400 analyzed trajectories for each experiment (≥15 steps).$

Single-molecule localization and tracking of ^DY647IFN binding to endogenous cell surface IFNAR. (a) Ligand-induced assembly of a dynamic ternary complex. The effective ligand binding affinity to the cell surface receptor depends on the dynamic equilibrium between the binary and ternary complex. (b) Live-cell IFNα2 binding assay by single-molecule imaging on HeLa. (b, left) A fluorescence image showing individual ^DY647IFNα2-wt bound to the cell surface receptor. (b, right) Trajectories of IFNα2-wt molecules from the boxed region. The boundaries of the cell are indicated by a yellow dotted line. (c) Density of ^DY647IFNα2-wt molecules localized on the surface of individual HeLa cells imaged in the presence of 2 nM ^DY647IFNα2-wt. For comparison, the density of ^DY647IFNα2-wt molecules on HeLa cells blocked with unlabeled IFNα2-wt is shown in addition to IFNAR2-deficient U5A cells. Data distribution of the second and third quartile (box), median (line), mean (closed square), and whiskers (1.5× interquartile range) is shown. (d) Normalized bleaching of ^DY647IFNα2-wt (>150 particles at t = 0) bound to endogenous receptors on HeLa at standard conditions and 10-fold increased laser power. Representative curves are shown for at least five experiments. (e) Single-step bleaching of labeled IFNs depicted as a 3D kymograph. Bleaching events are indicated by green arrows. (f) Single-step bleaching events of three individually labeled IFNs (representative curves for >100 bleached particles). (g) Diffusion properties of cell-bound ^DY647IFNα2-wt presented as the step length distribution for a time lapse of 160 ms (5 frames, black curve), which was obtained by fitting the step length histogram by considering three components corresponding to an immobile as well as a slow and a fast mobile fraction (Fig. S1). (h) Diffusion properties of cell-bound ^DY647IFNα2-dn and fit according to a two-component model. (i) Comparison of the step-length histogram for ^DY647IFNα2-wt and ^DY647IFNα2-dn. The data shown in g–i are pooled from at least two independent experiments, each with >650 analyzed trajectories (≥15 steps) per IFN mutant. (j) Changes in mobility of a model transmembrane protein dimerized by a monoclonal antibody. The data shown are pooled from eight independent experiments with >400 analyzed trajectories for each experiment (≥15 steps).

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