SINGLE-PARTICLE TRACKING OF CELL-SURFACE HLA-DR MOLECULES USING R-PHYCOERYTHRIN LABELED MONOCLONAL-ANTIBODIES AND FLUORESCENCE DIGITAL IMAGING

The mobility of cell surface MHC molecules and their ability to form d ynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investiga te these properties, we have prepared HLA DR specific monoclonal antib odies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE), We show that these small particles can be seque ntially imaged using a cooled slow-scan charge coupled device camera a nd hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecul es on fibroblasts transfected with human DR alpha and DR beta genes, P E-IgG was bound to the transfected fibroblasts and particle tracks wer e obtained by sequential imaging over a period of typically 30 minutes , Analysis of particle tracks revealed the presence of directed motion and domain-limited diffusion in addition to random diffusion. The con tributions of these three types of motion showed cell to cell variabil ity, Velocities of directed motion were of the order of 2 nm second(-1 ) whilst domain diameters were in the range 200-800 nm, Diffusion coef ficients for random diffusion were in the range 1x10(-13)-5x10(-12) cm (2) second(-1). The higher mobilities were observed for the lower inte nsity fluorescent spots, which possibly correspond to images of single particles, Much lower mobility was observed with a cell where the spo t intensities were approximately double that of the lower intensity sp ots. These spots could be images of double particles implying the asso ciation of at least two HLA DR alpha beta dimers, These data are relev ant to the study of MHC class II cell surface redistribution and antig en presentation in specific immunity.