Figure 3.

Mature Kv1.3 channels move to the immunological synapse by lateral diffusion along the plane of the plasma membrane. A. Newly synthesized Kv1.3 channels are not recruited in the immune synapse. T cells were treated with cycloheximide (CHX, 10 uM) and Kv1.3 polarization in the IS compared with that of control cells (CTR, no CHX). Cells were maintained with and without CHX overnight, then exposed to CD3/CD28 beads for 15 min and fixed. Top: photomicrographs of T/bead conjugates in presence (CHX) or absence (CTR) of CHX. Kv1.3 was labeled with EC-Kv1.3 antibody and identified with Alexa Fluor 488 secondary antibody (right panels). Beads are marked by x in the BF images and the IS by arrows in the fluorescent images. Bottom: Average Kv1.3 recruitment in the IS in CTR and CHX-treated cells. The % of cells that display Kv1.3 accumulation in the IS is calculated as described in figure 2D. The data shown are the average responses collected from 3 separate experiments with a total of 49-89 T cell-bead conjugates/experiment. B. Endocytosis and exocytosis are not involved in Kv1.3 movement to the immune synapse. Left: Comparisons were made between cells treated overnight with DIP (which inhibits endocytosis) and its inactive control (scramble DIP) at 50 uM. We measured the percentage of Kv1.3 recruitment in the IS after 15 min exposure to beads as described in A. Right: Comparisons were made between cells maintained at 20°C (a condition that inhibits exocytosis) for 2 hr and throughout the experiment, and cells maintained at 37°C. The percentage Kv1.3 recruitment into the IS was determined after 15 min treatment with CD3/CD28 beads. Top panels show Kv1.3 recruitment in the IS in the different experimental conditions. The corresponding average data are shown below in the left panel for DIP-, scramble DIP-treated cells and control (CTR, non treated) cells and in the right panel for 20°C and 37°C (n=3 separate experiments; 50 to 100 conjugates/experiment).