Comparing different FRET techniques to measure clustering of receptor-ligand complexes in endocytic membranes

摘要

Here, we have investigated the molecular mechanisms underlying the dynamics of protein distribution within membranes using Fluorescence Resonance Energy Transfer Microscopy (FRET). We have developed a one-photon (1-P) and two-photon (2-P) FRET assay to differentiate between the clustered and random distribution of membrane-bound fluorophore-labeled receptor-ligand complexes. Our results demonstrate that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized MDCK cells, since energy transfer efficiency (E%) levels are independent from acceptor fluorescence, a standard parameter to confirm clustered distribution. We also describe a second parameter: E% decreases with increasing unquenched donor fluorescence and unquenched donor : acceptor ratios, a phenomenon which we ascribe to some donors preventing others from interacting with an acceptor. We call this effect 'donor geometric exclusion'. Going beyond the determination of clustered vs. random distribution of protein complexes, mathematical models have been developed, tailored to large, tightly packed molecular clusters, estimating their local densities with an adjustable parameter 's'.