Thilo Jakob, Mark C. Udey Journal of Investigative Dermatology

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Characterization of E-Cadherin-Containing Junctions Involving Skin-Derived Dendritic Cells Thilo Jakob, Mark C. Udey Journal of Investigative Dermatology Volume 112, Issue 1, Pages 102-108 (January 1999) DOI: 10.1046/j.1523-1747.1999.00475.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 E-cadherin and catenins in FSDDC aggregates. (A) Coprecipitation of E-cadherin and catenins. Soluble fractions of triton lysates of FSDDC-A were immunoprecipitated under nondenaturing conditions using anti-E-cadherin MoAb (lanes 2, 4, 6, 8, 10) or contol MoAb (lanes 1, 3, 5, 7, 9). Precipitates were separated on denaturing gels under reducing conditions, transferred, and blotted with MoAb directed against E-cadherin (lanes 1, 2), β-catenin (lanes 3, 4), γ-catenin/plakoglobin (lanes 5, 6), p120CAS (lanes 7, 8), and α-catenin (lanes 9, 10), and visualized using enhanced chemiluminescence (see Materials and Methods). (B) E-cadherin and β-catenin associated with the cytoskeleton. Triton-insoluble fractions of FSDDC-A lysates were solubilized in SDS buffer, separated, transferred, and blotted with anti-E-cadherin (lane 1) and anti-β-catenin (lane 2) MoAb. Journal of Investigative Dermatology 1999 112, 102-108DOI: (10.1046/j.1523-1747.1999.00475.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Confocal micrographs of FSDDC aggregates (A, B) and FSDDC/KC cocultures (C–F). (A, B) Horizontal (x–y plane; top) and vertical (x–z plane; bottom) sections through dome-shaped FSDDC aggregates demonstrate colocalization (yellow) of E-cadherin and catenins in areas of cell–cell contact. FSDDC aggregates were seeded on glass cover slips and allowed to adhere for 4 h at 37°C. After PFA fixation and permeabilization, cell aggregates were stained for E-cadherin (green) and β-catenin (red) (A) or E-cadherin (green) and γ-catenin/plakoglobin (red) (B). (C–F) Focal accumulations of E-cadherin in areas of contact between FSDDC and KC. FSDDC aggregates were dissociated into single cells and seeded onto monolayers of primary KC (see Materials and Methods). After 18 h, cocultures were washed, fixed, permeabilized, and stained for E-cadherin (green) and MHC class II (red) (C, E); E-cadherin (green), F-actin (red), and MHC class II (blue) (D); or E-cadherin (green) and β-catenin (red) (F). Colocalization of MHC class II and F-actin is displayed in pink (D), and colocalization of E-cadherin and β-catenin in yellow (F). Arrowheads designate mature DC (see text for explanation of nomenclature). Scale bars: (A, B) 15 μm; (C, D) 20 μm; (E) 15 μm; (F) 10 μm. Journal of Investigative Dermatology 1999 112, 102-108DOI: (10.1046/j.1523-1747.1999.00475.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ultrastructure of junctions in FSDDC. FSDDC aggregates were harvested, fixed, and embedded for electron microscopy. (A) Field showing cells within FSDDC aggregates and areas of close membrane apposition (box B, C, D) (scale bar: 1 μm). (B–E) Enlargements showing characteristic appearance of representative junctions (scale bar: 0.1 μm). Part (E) includes a junction identified in the same sample, but a different section from that depicted in (A). Journal of Investigative Dermatology 1999 112, 102-108DOI: (10.1046/j.1523-1747.1999.00475.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 β-Catenin, a cytoplasmic protein associated with adherens junctions in epithelia, is localized to cell junctions (arrows) in FSDDC. FSDDC aggregates were harvested, fixed, and embedded for post-embedding immunoelectron microscopy (see Materials and Methods). (A, B) Immunogold detection of structures reactive with anti-β-catenin MoAb. (C) Isotype control. Scale bar: 0.1 μm. Journal of Investigative Dermatology 1999 112, 102-108DOI: (10.1046/j.1523-1747.1999.00475.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Adherens junctions-like structures in areas of contact between FSDDC and keratinocytes. FSDDC aggregates were dissociated into single cells and seeded onto monolayers of primary KC (see Materials and Methods). After 18 h of coculture, cells were fixed and embedded for electron microscopy. (A, B) Two fields from independent experiments showing representative junctions (arrow) in areas of cell–cell contact between FSDDC (DC) and tonofilament (arrowhead)-containing keratinocytes (KC). Scale bar: 0.2 μm. Journal of Investigative Dermatology 1999 112, 102-108DOI: (10.1046/j.1523-1747.1999.00475.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions