Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes.

The dynamics of protein distribution in endocytic membranes are relevant for many cellular processes, such as protein sorting, organelle and membrane microdomain biogenesis, protein-protein interactions, receptor function, and signal transduction. We have developed an assay based on Fluorescence Resonance Energy Microscopy (FRET) and novel mathematical models to differentiate between clustered and random distributions of fluorophore-bound molecules on the basis of the dependence of FRET intensity on donor and acceptor concentrations. The models are tailored to extended clusters, which may be tightly packed, and account for geometric exclusion effects between membrane-bound proteins. Two main criteria are used to show that labeled polymeric IgA-ligand-receptor complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells: 1), energy transfer efficiency (E%) levels are independent of acceptor levels; and 2), with increasing unquenched donor: acceptor ratio, E% decreases. A quantitative analysis of cluster density indicates that a donor-labeled ligand-receptor complex should have 2.5-3 labeled complexes in its immediate neighborhood and that clustering may occur at a limited number of discrete membrane locations and/or require a specific protein that can be saturated. Here, we present a new sensitive FRET-based method to quantify the co-localization and distribution of ligand-receptor complexes in apical endocytic membranes of polarized cells.