Detection of dimers of dimers of human leukocyte antigen (HLA)-DR on the surface of living cells by single-particle fluorescence imaging.
The technique of single-particle fluorescence imaging was used to investigate the oligomeric state of MHC class II molecules on the surface of living cells. Cells transfected with human leukocyte antigen (HLA)-DR A and B genes were labeled at saturation with a univalent probe consisting of Fab coupled to R-phycoerythrin. Analysis of the intensities of fluorescent spots on the cell surface revealed the presence of single and double particles consistent with the simultaneous presence of HLA-DR heterodimers and dimers of dimers. The proportion of double particles was lower at 37 degrees C than at 22 degrees C, suggesting that the heterodimers and dimers of dimers exist in a temperature-dependent equilibrium. These results are discussed in the context of a possible role for HLA-DR dimers of dimers in T cell receptor-MHC interactions. The technique is validated by demonstrating that fluorescence imaging can distinguish between dimers and tetramers of human erythrocyte spectrin deposited from solution onto a solid substrate. The methodology will have broad applicability to investigation of the oligomeric state of immunological and other membrane-bound receptors in living cells.
Cherry
R J
RJ
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom. cherr@essex.ac.uk
Wilson
K M
KM
Triantafilou
K
K
O'Toole
P
P
Morrison
I E
IE
Smith
P R
PR
Fernández
N
N
D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D019281:Dimerization; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D059805:HLA-DR alpha-Chains; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007962:Leukocytes / Q000276:immunology; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection
D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D007140:Immunoglobulin Fab Fragments; D046911:Macromolecular Substances; D011994:Recombinant Proteins