Figure 6.

Impaired CD3 ubiquitylation enhances IS formation and increases ERK phosphorylation in DP thymocytes. (A–C) DP thymocytes from CD3^WT or CD3^KR Rg mice were purified by FACS and added to a synthetic planar lipid bilayer containing unlabelled His-tagged ICAM-1 and fluorescently labelled streptavidin conjugated to biotinylated anti-TCRβ antibodies. Interactions were fixed following 15 min stimulation, visualized using spinning disk confocal microscopy and analysed using Slidebook software. (A) The area of individual DP T-cell synapses obtained in two independent experiments. (B, C) The morphology of individual synapses were classified according to the representative images (B) and shown graphically (C—Scale bar=5 mm). (D, E) Thymocytes from CD3^WT or CD3^KR Rg mice were isolated, rested in 0.5% FCS RPMI for 1 h at 37°C, and then activated by crosslinking using a CD3ɛ mAb in the presence (+U0126) or absence (+Veh.) of the MEK inhibitor U0126. Thymocytes were then fixed at the indicated time, permeablized, and stained with CD4, CD8, and pERK Abs. (D) CD4+CD8+ DP thymocytes were gated and pERK expression measured. (E) Representative flow cytometry dot plots are shown 10 min after stimulation. Statistical significance was determined using an unpaired t test in Prism software.