Changes in tight junction morphology after anti-CD3 treatment. (A) Immunofluorescent localization of the tight junction proteins claudin-1, claudin-4, claudin-5, and JAM-A in the small intestinal epithelium of wild-type mice demonstrates the localization of these proteins to the tight junction before (left panels) and 3 hours after (right panels) anti-CD3 treatment. Claudin-1, claudin-4, and claudin-5 distributions were not affected by anti-CD3 treatment. However, JAM-A demonstrated limited redistribution to intracellular sites after anti-CD3 treatment. (B) ZO-1 distribution appeared unchanged in transverse sections, but when viewed in en face sections, ZO-1 adopted a thinner, more sinuous distribution in anti-CD3–treated tissue. (C) Occludin was localized to the tight junction in control tissue, but after anti-CD3 treatment, occludin was internalized into intracellular vesicles (arrows), visible in both transverse and en face sections. Scale bars in A–C, 5 μm. (D) Detergent-insoluble subcellular fractions of jejunal epithelial cells were isolated on isopycnic sucrose gradients (black line). Protein concentration was determined for each fraction of gradients prepared from control (blue line) and anti-CD3–treated (red line) mice. A protein peak associated with the low-density, detergent-insoluble membrane fraction was present (bracket). The arrow designates the insoluble high-density pellet (fraction 24). These fractions were assessed by immunoblot. (E) Immunoblots for occludin in lysate (diluted), pellet, and low-density, detergent-insoluble membrane fractions showed loss of occludin from the detergent-insoluble membrane fraction after anti-CD3 treatment.