Journal: Journal of cell science

Article Title: Dissociation of β2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.

doi: 10.1242/jcs.259498

Figure Lengend Snippet: Fig. 1. MHC I HC–HC association requires dissociation of β2m but no disulfide bonds. (A) Schematic of MHC class I states at the plasma membrane. Dissociation of peptide from the HC–β2m–peptide trimer results in an ‘empty’ HC–β2m heterodimer. Dissociation of β2m then produces free HCs (FHCs), which can form FHC associations (HC–HC dimer and oligomer shown). Other forms such as disulfide-linked dimers are known depending on the allotype (see the text). (B) Schematic representation of the two-hybrid antibody micropattern assay. Cells expressing a class I HC GFP fusion (green) and an N-terminally HA-tagged class I HC (gray) are seeded onto glass slides that are printed with micrometer-sized patterns of fluorescently labeled anti-HA antibodies. Dissociation of β2m generates FHCs of both constructs, which diffuse freely in the plasma membrane and eventually associate with each other to form HC–HC dimers (center) or oligomers (not shown). The HC–HC associations, which contain both HA-tagged and GFP-fused FHCs, localize in the pattern elements and are visible as pattern- shaped GFP fluorescence on the plasma membrane. (C) Representative fluorescence micrograph showing one single STF1 cell expressing both HA-tagged and GFP-fused H-2Kb in phase contrast (left), the purple anti-HA antibody pattern on the glass slide (middle), and green Kb–GFP colocalizing with the antibody pattern (right). The arrows point to the fluorophore observed in the respective panel and emphasize the plane of the image. Scale bar: 20 μm. (D) Interaction (arrow) occurs between HA-tagged Kb and Db–GFP FHCs (37°C) but not between HC–β2m heterodimers (25°C). Scale bars: 20 μm. (E) HC–HC interaction does not involve intracellular disulfide bond formation, since the FHCs (37°C) of HA–Kb and Kb(C332S)–GFP, which lacks the cytosolic cysteine, interact in the micropattern assay (arrow). Scale bars: 20 μm. (F) HA-B*27:05 (B27), but not Kb, forms covalent dimers. HA–Kb (Kb in the label) and B27 molecules were immunoprecipitated from the lysate of transduced STF1 cells with an anti-HA monoclonal antibody, separated by reducing (+ DTT) or nonreducing (– DTT) SDS- PAGE, and monomers (heavy chain) and covalent homodimers as indicated were detected by western blotting with an anti-HA antiserum. * denotes a background band. (G) There is no interaction of Kb–GFP with F pocket-stabilized HA–Kb(Y84C/A139C), a disulfide-stabilized Kb variant with increased β2m affinity. Scale bars: 20 μm. Images in C–G are representative of at least three independent experiments with the exception of F, which was performed twice.

Article Snippet: MHC molecules were visualized on the membranes with polyclonal rabbit anti-HA antibody as primary antibody (1:1000, ab9110, Abcam, Cambridge, UK) and alkaline phosphatase-conjugated anti-rabbitIgG serum from goat as secondary antibody (1706518, Biorad, Munich, Germany).

Techniques: Clinical Proteomics, Membrane, Expressing, Labeling, Construct, Fluorescence, Immunoprecipitation, SDS Page, Western Blot, Variant Assay