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

Energy Transfer Efficiency based on One- and Two-Photon FRET

We are investigating membrane-based sorting processes in polarized epithelial MDCK cells, which most likely involves membrane microdomains. We have postulated that proteins contained in these microdomains, cluster, and to prove this, we have internalized differently fluorophore labeled pIgA-R ligands in MDCK cells, stably transfected with polymeric IgA receptors (pIgA-R), from opposite plasma membranes. Our previous work showed that these receptor-ligand complexes colocalize in the apical recycling endosome (ARE), underneath the apical plasma membrane. Quantitative one-photon confocal and 2-photon (2-P) FRET microscopy allowed us to calculate energy transfer efficiency (E%). Unquenched donor levels where established based on a novel algorithm, which corrects the FRET contamination of acceptor bleed-through and donor crosstalk. Using different emission filters also confirmed the veracity of the algorithm. 2-P FRET allows the selection of a specific donor wavelength, which does not precipitate acceptor bleed-through, a clear advantage over 1-P confocal microscopy. Results show that E% is independent of acceptor levels, an indication of a clustered distribution, as in random distribution E% rises with increasing acceptor levels. However, E% decreases with increasing donor and donor:acceptor ratio levels, which we have termed donor geometric exclusion , where some donors in a cluster block others from interacting with an acceptor. We submit that this is a second indicator for a clustered pattern, because in a random, dispersed situation donors are not likely to be in close proximity to have such an effect. We have developed a model explaining this phenomenon