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Proceedings Paper

Comparing different FRET techniques to measure clustering of receptor-ligand complexes in endocytic membranes
Author(s): Horst Wallrabe; Ammasi Periasamy; Almut Burchard; Margarida Barroso
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Paper Abstract

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.'

Paper Details

Date Published: 10 July 2003
PDF: 9 pages
Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.485606
Show Author Affiliations
Horst Wallrabe, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)
Almut Burchard, Univ. of Virginia (United States)
Margarida Barroso, Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 4963:
Multiphoton Microscopy in the Biomedical Sciences III
Ammasi Periasamy; Peter T. C. So, Editor(s)

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