Figure 5
Figure 5. DC-derived f-actin dynamics are important for stabilization of MHC class I molecules at the synapse and for maintenance of its regulatory features. (A) Mature ΔWASP DC/resting NK-cell cocultures were fixed after 1 or 20 minutes of interaction and f-actin was stained with bodipy conjugated phallacidin (green). DAPI was used to stain nuclear DNA (blue). Arrows indicate the synapse. The graph represents the quantification of f-actin staining intensity at the synapse compared with the staining at the opposite side of the same NK cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (B) Mature ΔWASP DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and stained with bodipy conjugated phallacidin to stain f-actin (green) and anti-KIRs antibodies (red). The fluorescence intensities of KIRs and f-actin stainings were plotted along the indicated trajectory, from A to B. The synapse area in cellular conjugates is indicated by boxes. (C) Mature ΔWASP and control DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and treated for TEM analysis. Original magnifications are 4200×, 13 500×, 4200× and 46 000×, from left to right. The graph represents the size of several regions in the synaptic clefts of 5 DC/NK-cell conjugates for each condition. (D) Mature control or ΔWASP DC co-cultures with resting NK cells were fixed after 20 minutes of interaction. F-actin (green), nuclear DNA (blue) and tubulin, perforin, IL-15Rα, or MHC class I (red) were stained. Arrows point to molecule enrichment at the synapse. The number of conjugates with MTOCs or perforin granules adjacent from the synapse were plotted, in the upper graph, as percentages of total number of conjugates analyzed. The lower graph represents the quantification of IL-15Rα and MHC class I staining intensity at the synapse, compared with the staining at the opposite side of the same conjugated cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. Images are representative of 1 experiment for TEM and at least 3 independent experiments for the others. Original magnifications are 100× for all the light microscopy images. Values on graph bars are medians and error bars represent interquartile ranges from the analysis of at least 100 conjugates from at least 3 independent experiments. ↑↑ indicates fold enrichment > 2. *P < .05. Statistics were performed with a null value of 25%, representing random distribution. Scale bars are 10 μm unless specified otherwise.

DC-derived f-actin dynamics are important for stabilization of MHC class I molecules at the synapse and for maintenance of its regulatory features. (A) Mature ΔWASP DC/resting NK-cell cocultures were fixed after 1 or 20 minutes of interaction and f-actin was stained with bodipy conjugated phallacidin (green). DAPI was used to stain nuclear DNA (blue). Arrows indicate the synapse. The graph represents the quantification of f-actin staining intensity at the synapse compared with the staining at the opposite side of the same NK cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (B) Mature ΔWASP DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and stained with bodipy conjugated phallacidin to stain f-actin (green) and anti-KIRs antibodies (red). The fluorescence intensities of KIRs and f-actin stainings were plotted along the indicated trajectory, from A to B. The synapse area in cellular conjugates is indicated by boxes. (C) Mature ΔWASP and control DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and treated for TEM analysis. Original magnifications are 4200×, 13 500×, 4200× and 46 000×, from left to right. The graph represents the size of several regions in the synaptic clefts of 5 DC/NK-cell conjugates for each condition. (D) Mature control or ΔWASP DC co-cultures with resting NK cells were fixed after 20 minutes of interaction. F-actin (green), nuclear DNA (blue) and tubulin, perforin, IL-15Rα, or MHC class I (red) were stained. Arrows point to molecule enrichment at the synapse. The number of conjugates with MTOCs or perforin granules adjacent from the synapse were plotted, in the upper graph, as percentages of total number of conjugates analyzed. The lower graph represents the quantification of IL-15Rα and MHC class I staining intensity at the synapse, compared with the staining at the opposite side of the same conjugated cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. Images are representative of 1 experiment for TEM and at least 3 independent experiments for the others. Original magnifications are 100× for all the light microscopy images. Values on graph bars are medians and error bars represent interquartile ranges from the analysis of at least 100 conjugates from at least 3 independent experiments. ↑↑ indicates fold enrichment > 2. *P < .05. Statistics were performed with a null value of 25%, representing random distribution. Scale bars are 10 μm unless specified otherwise.

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