LCs engage in trans-TJ endocytosis via langerin. See Video 4 for more details. (A) Visualization of langerin and MHC II on activated (asterisks) and resting (cell to the right) LCs in relation to TJs as shown by ZO-1 staining. Dashed lines indicate TJs. Langerin accumulated in TJ-docked dendrite tips of activated LCs (arrows). Dendrites of resting LCs (arrowheads) lack langerin accumulation. (B) Biotinylation of LC surface membrane reveals endocytic activity by a TJ-penetrated dendrite (arrow). Dashed lines represent TJs. Dendrites of resting LCs (arrowheads) lack biotin signals. (C) Rotated views of an endocytosing LC dendrite from B. The streak of biotin signal in the dendrite continues within the TJ barrier (arrowheads). (D) Colocalization of biotinylated molecules (green) with langerin (red) in TJ-penetrated dendrites as shown by MHC II (blue) staining. (E) FITC-OVA (green) application demonstrates trans-TJ uptake of Ags by LCs in vivo. FITC-OVA signals colocalized with langerin (red) in TJ-penetrating dendrites (MHC II, blue; arrowheads). Arrows point to accumulation of FITC and langerin signals in perinuclear area of an activated LC. (F) Numbers of FITC-OVA–positive and –negative dendrites. The number of dendrites that docked or did not dock with TJs was counted on resting and activated LCs. (G) GFP-expressing Escherichia coli application demonstrates GFP signal within LC dendrite (arrows). Bars, 10 µm. Data presented in A–E and G is representative of three mice and data in F of three independent experiments.