Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging

Abstract

The mobility of cell surface MHC molecules and their ability to form dynamic associations may be related to the physiological status of the cell and to the potential to bind effector T lymphocytes. To investigate these properties, we have prepared HLA DR specific monoclonal antibodies coupled in a 1:1 mole ratio to the fluorescent phycobiliprotein, R-phycoerythrin (PE). We show that these small particles can be sequentially imaged using a cooled slow-scan charge coupled device camera and hence can be used for single particle tracking experiments. We have applied this technique to investigate the movements of HLA DR molecules on fibroblasts transfected with human DR alpha and DR beta genes. PE-IgG was bound to the transfected fibroblasts and particle tracks were 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 contributions of these three types of motion showed cell to cell variability. Velocities of directed motion were of the order of 2 nm second-1 whilst domain diameters were in the range 200–800 nm. Diffusion coefficients for random diffusion were in the range 1 × 10(−13)-5 × 10(−12) cm2 second-1. The higher mobilities were observed for the lower intensity fluorescent spots, which possibly correspond to images of single particles. Much lower mobility was observed with a cell where the spot intensities were approximately double that of the lower intensity spots. These spots could be images of double particles implying the association of at least two HLA DR alpha beta dimers. These data are relevant to the study of MHC class II cell surface redistribution and antigen presentation in specific immunity.