Date of Award
1993
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Scanning fluorescence correlation spectroscopy (S-FOS) is introduced as a technique to measure the distribution of receptors on cell surfaces. The receptors are made observable by the binding of a specific ligand that is fluorescently labeled. This technique is based on fluctuation analysis through direct calculation of correlation functions. It uses a confocal microscope configuration to obtain fluorescence records as a function of laser beam position on the surface of cells. The fluorescence records present fluctuations that have a characteristic persistence length equal to the laser beam width. The calculated correlation function decays as the laser beam intensity profile which is Gaussian in nature. The zero-lag value of the properly normalised correlation function is a direct measure of the surface density of randomly distributed molecules at the cell surface.;Receptor distributions are inferred from S-FCS experiments in which the labeled-ligand concentration is varied. Experiments performed on mouse fibroblasts with succinyl concanavalin A as receptor-ligand, determined that the succinyl concanavalin A binds with high affinity to numerous monomeric receptors and binds with low affinity to few large receptor clusters. The respective surface densities of these receptor species were measured to be 25 and 2 per square micrometer.;An investigation of the distribution of the epidermal growth factor (EGF) receptors on the surface of A431 cells was carried out to examine whether or not these receptors were distributed uniformly at the cell surface. S-FCS experiments were performed using two monoclonal antibodies directed against the receptor, IgG 29.1 which binds to the carbohydrate portion of the receptor and IgG 528 that binds to its protein core. Both antibodies gave similar results. We conclude that EGF receptors are distributed in domains at the surface of A431 cells. The surface density of these domains is about 8 per square micrometer where each domain contains about 130 receptors. This result implies that the mode of lateral diffusion of the EGF receptor is one of rapid exchange between domains.
Recommended Citation
St-Pierre, Paul, "Submicron Correlations Of Receptor-positions On Cell Surfaces" (1993). Digitized Theses. 2207.
https://ir.lib.uwo.ca/digitizedtheses/2207