Quantitative analysis of clustering on biological membranes: methodology and application to ligand-induced asialoglycoprotein receptor redistribution on rat hepatocytes.
Ligand-induced receptor clustering is the first step in receptor mediated endocytosis of asialoglycoproteins by rat hepatocytes. This well-characterized receptor was used as a model system to set up a general method for the quantitative analysis and the visualization of molecular clustering on surface replicas, using a two-step approach. In the first step, aiming at the quantitation of clustering, gold-labeled asialoglycoprotein receptors on the cell surface were assumed to reflect two populations, one of clustered and one of not-clustered receptors. The experimental distribution of nearest neighbor distances of labeled receptors was adjusted by least square fitting to the sum of two functions, each corresponding to the theoretical nearest neighbor distance distribution of randomly distributed points, corresponding to non-clustered and clustered particle concentrations, respectively. The resolution of the experimental nearest neighbor distribution into these two components yielded an objective estimate of the number of labeled receptors in clusters and of the total surface of clusters. The second step, aiming at the visualization of clusters, rested on the fact that the distance of a point to its neighbors is shorter in a cluster than outside the cluster. Accordingly, each particle was ordered according to the sum of the distances to its nearest neighbors. Clustered particles with the lowest cumulative distances were extracted, in proportion of the extent of clustering determined in the first step, and displayed on the computer screen. This translated the two components into the two corresponding populations of identified particles. This method demonstrated that, if rat hepatocytes were incubated at 4 degrees C with either asialofetuin-gold complexes before fixation, or with asialofetuin prior to fixation and immunogold labeling of the ASGP-R, up to 65% of the receptors became clustered on the dorsal cell surface in areas where receptors could be concentrated up to 20-fold, as compared with randomly distributed molecules. In ultrathin sections, clusters essentially corresponded to clathrin-coated pits. In principle, this method is generic and can be applied to other biological systems, especially when statistical analysis is needed for studying the dynamics of the clustering process.
Van Der Smissen
P
P
Cell Biology Unit, University of Louvain Medical School, Brussels, Belgium.
Courtoy
P J
PJ
Baudhuin
P
P
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D003198:Computer Simulation; D060748:Fetuins; D017979:Gold Colloid; D008024:Ligands; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008954:Models, Biological; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000509:alpha-Fetoproteins
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D060748:Fetuins; D017979:Gold Colloid; D008024:Ligands; D011956:Receptors, Cell Surface; D000509:alpha-Fetoproteins; C017029:asialofetuin