QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 February 2008
doi: 10.1242/jcs.022418

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Neumann, A. K. Articles by Jacobson, K. Search for Related Content PubMed PubMed Citation Articles by Neumann, A. K. Articles by Jacobson, K.

Distribution and lateral mobility of DC-SIGN on immature dendritic cells–implications for pathogen uptake

¹ Department of Cell and Developmental Biology
² Lineberger Comprehensive Cancer Center
³ Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (91K): [in this window] [in a new window]   Fig. 1. (A-C) DC-SIGN staining (DCN46 mAb) in fixed cells shown for (A) fibronectin-adhered Raji-DCSIGN cells, (B) MX-DCSIGN cells and (C) MDDCs. (D-E) DC-SIGN staining in live cells using Alexa-Fluor-568-conjugated anti-DC-SIGN F(ab) shown for (D) Raji-DC-SIGN cells, (E) MX-DCSIGN cells, (F) MDDCs. All images were focused on the dorsal surface. Scale bars, 10 µm. (G) Distribution of DC-SIGN cluster sizes on MX-DCSIGN cells (n=804).

View larger version (19K): [in this window] [in a new window]   Fig. 2. DC-SIGN cluster photobleaching and recovery. (A) Montage showing the bleaching of a representative DC-SIGN cluster on an MX-DCSIGN cell stained with anti-DC-SIGN F(ab). The bleached region is indicated with a yellow box and bleaching occurred between the third and fourth frames. (B) The complete intensity profile in time of the bleached cluster. (C) A montage showing the bleaching of several DC-SIGN clusters on a dendritic cell. (D) Bleaching and recovery phase for a representative cluster on this dendritic cell. (E) Distribution of mobile fractions (Mf) as measured from the FRAP curves of all DC-SIGN clusters analyzed in both MX-DCSIGN cells and dendritic cells (n=7 in each cell type, error bars denote ± s.d.). Red and blue bars show the mean Mf for all trials per cell type. Scale bars, 1 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 3. (A) Raji-DCSIGN cells visualized by total internal reflectance (TIR) illumination of the ventral membrane after fixation and staining for DC-SIGN (DCN46 mAb). (B) Control staining of fixed Raji-DCSIGN cells with the lipophilic dye, DiI under TIR illumination. (C) Addition of 580 kDa FITC-dextran to the medium to demonstrate accessibility of the ventral membrane surface of adhered Raji-DCSIGN cells. Data shown are mean intensities of ventral membrane regions from TIR images before and after addition of the reagent. Ventral ROIs were defined as the largest box that could fit within the cell outline as determined from a DIC image. (D) Control stainings of fixed Raji-DCSIGN cells for membrane-associated proteins, CD46 and CD59. These images were collected in TIRF mode to show only material within 150 nm of the glass substrate. (E) A fixed MDDC stained for DC-SIGN (DCN46 mAb) and visualized by TIR microscopy. (F) 3D projection images to show the dorsolateral membrane distribution of DC-SIGN on fixed Raji-DCSIGN cells as obtained from a deconvoluted z-stack (step=200 nm) of epifluorescence images. (G) DC-SIGN imaged on the dorsal lamellar membrane of a fixed MDDC in a single epifluorescence image focused 2 µm above the coverslip with edge-region detail expanded as indicated. (H) A fixed MDDC imaged for morphology in DIC and for DC-SIGN distribution by deconvolution of a z-stack of epifluorescence images (step=200 nm). Arrows denote active leading edge membrane regions enriched in DC-SIGN. 3D projections are shown as views from 90° (xy plane) and 0° (from the left side of the cell, yz plane). Scale bars, 10 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 4. (A) Colocalization of internalized FITC-BSA and Alexa-Fluor-568 anti-DC-SIGN F(ab) in a MDDC (scale bar, 10 µm). Representative details of edge (red frame), medial zone (yellow frame), and perinuclear zone regions (blue frame) show non-internalized and internalized DC-SIGN. Three regions in each category (shown by red, yellow and blue boxes) were chosen for quantitative colocalization analysis. (B) Proportion of colocalization of DC-SIGN and internalized FITC-BSA per region (t-test *P<0.001; **P<0.05). (C) Histogram of the ratio of internalized clusters versus surface clusters as a function of distance from the edge of the cell. This analysis is representative of four independent experiments.

View larger version (78K): [in this window] [in a new window]   Fig. 5. (A) Schematic representation of the live cell internalization experiment. In dendritic cells stained with anti-DC-SIGN F(ab) (red) and bathed in FITC-BSA as an endocytosis marker (green), a confocal slice containing the medial lamellar membrane was observed. Endocytosis results in a colocalized (yellow) signal. (B) DIC image of the cell under observation. (C) A single frame showing the leading edge of a dendritic cell (yellow line) and a region of interest containing a DC-SIGN cluster that exhibits internalization. (D) A montage of selected frames limited to the region of interest defined in (C) showing the internalization of a DC-SIGN cluster. The arrow shows a surface DC-SIGN cluster before internalization is evident. The arrowhead in a subsequent frame shows the appearance of internalization activity conincident with the surface DC-SIGN cluster.

View larger version (10K): [in this window] [in a new window]   Fig. 6. (A) Representative trajectories of DC-SIGN undergoing directed (red) or non-directed (green) motion relative to the cell edge (gray line). The red arrow denotes overall direction of DC-SIGN displacement for the directed motion trajectory. All trajectories were obtained from live MDDCs stained with anti-DC-SIGN F(ab). Scale bar, 1 µm. (B) Mean square displacement versus time for DC-SIGN trajectories exhibiting directed motion (n=4).

View larger version (89K): [in this window] [in a new window]   Fig. 7. Dendritic cells were stained and observed as described in Fig. 5 to distinguish surface (red) and internalized (yellow) DC-SIGN. (A) DIC image of the dendritic cell under observation. (B) A single frame (from supplementary material Movie 5) showing the leading edge of the dendritic cell (yellow line), internalized DC-SIGN (blue boxes), and mobile surface DC-SIGN clusters (white and cyan arrows). (C) Trajectories followed by the mobile surface DC-SIGN clusters emphasized in (B) with trajectory start points indicated by green arrows. (D) Montage of selected frames showing the rapid linear mobility of these spots and their continued lack of colocalization with endocytosis marker (FITC-BSA). Scale bars, 1 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Two hypothetical models of DC-SIGN cluster rearward motion. (A) The constitutive transport model. DC-SIGN clusters (red border) constitutively leave the leading edge and cycle back (green border) to zones of internalization. Pathogen would be passively transported on structures undergoing this cycling. (B) The triggered transport model. DC-SIGN clusters are caught at the leading edge (red border) and must be liberated (orange border) prior to rearward transport (green border). In this model, pathogen binding would influence the probability of rearward transport of a cluster by affecting its release at the leading edge.