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Files in this Data Supplement:
Fig. S1. (A) Distribution of full-width half maximum (FWHM) diameters observed for DC-SIGN clusters on MX-DCSIGN (n=848). (B) Simulated FWHM diameters calculated for objects of various true diameters, as described in Materials and Methods, together with best-fit line to the empirical function defining the relationship between true diameter and FWHM diameter.
Fig. S2. (A) Fixed immature dendritic cell immunostained for DC-SIGN corrected for DiI bleed-through. Green arrows mark sites of apparent DC-SIGN enrichment near the leading edge. (B) The same cell stained for membrane content using DiI and corrected for DC-SIGN-stain bleed-through. Images were taken at ×60 in epifluorescence mode. (C) Ratio of DC-SIGN and DiI images scaled by 100. Green arrows show peripheral DC-SIGN-rich sites from A that persist after normalization for membrane content. (D) To measure overall distribution of DC-SIGN−DiI signal with respect to the leading edge, the edge was defined using the DiI image and radii were drawn at regularly spaced intervals from a single point in the central perinuclear zone to the edge. (E) Mean DC-SIGN−DiI ratio (×100) signal along these radii as a function of distance from the edge. Error bars are one standard deviation, and these data are representative of three cells analyzed with similar results.
Fig. S3. Monte Carlo simulations were used to test non-randomness of trajectories. Head-to-tail distances were measured for each simulated trajectory (inset). A 95% confidence limit (red line) for random trajectory head-to-tail distances was established based on the histogram of values returned from many Monte Carlo simulations (main chart). The head-to-tail distances observed in experimental trajectories were compared with the 95% confidence limit derived from simulated trajectories with the same distribution of step sizes and a uniform random distribution of directionality.
Movie 1. Rotating 3D projection of deconvoluted DC-SIGN staining on a fixed, immunostained Raji-DCSIGN cell, as described in Fig. 3.
Movie 2. Rotating 3D projection of deconvoluted DC-SIGN staining on a fixed, immunostained immature dendritic cell, as described in Fig. 3.
Movie 3. Endocytosis of F(ab)-labeled DC-SIGN cluster (red) in a dendritic cell. The cell was bathed in FITC-BSA, and fluorescein emission was detected in a cytoplasmic confocal section to detect endocytosis events in real time. This movie represents a detail from Fig. 5 and a surface DC-SIGN cluster that is endocytosed is highlighted by the blue box.
Movie 4. DC-SIGN on the lamella of an immature DC briefly stained with Alexa-Fluor-568-conjugated anti-DC-SIGN F(ab). This movie was recorded at 30 Hz and is played back at real-time speed. Marked are the following: the approximate cell boundary (yellow line), stationary DC-SIGN (red arrows), DC-SIGN that undergoes rearward transport (green arrow).
Movie 5. Rapid motion of non-internalized DC-SIGN clusters labeled by F(ab) (red) on a dendritic cell. These rapidly moving surface-localized DC-SIGN clusters are denoted by white and cyan boxes. Note that internalization events are detectable in other regions of the image series.
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