INDIVIDUAL RESEARCHERMargarida Barroso , Ph.D.
Education1991 - Ph.D. from University of Lisbon, Lisbon, Portugal
How proteins find their rightful place in cells and how that process goes awry in diseases is a fundamental question in biomedical research. Many diseases, such as cystic fibrosis and hypercholesterolemia, are due to defects in protein sorting and transport. The mechanism of protein transport has been extensively analyzed and a general model of the process has been elucidated. However, how cells are able to recognize and sort out membrane proteins is still poorly understood. We are particularly interested in understanding how membrane-bound receptors are distributed and sorted out throughout intracellular transport pathways (Figure 1). We propose that the clustering of receptors is an integral part of the membrane protein sorting process. To test this hypothesis, Fluorescence Resonance Energy Transfer (FRET) was used to characterize the distribution of receptors in endocytic membranes and assay the molecular basis of receptor clustering (Figure 1-2). Different membrane-bound receptors such as polymeric IgA-receptor[1-2], which plays an important role in pathogen neutralization and immune response, transferrin-receptor, which delivers iron into the cells, and low-density lipoprotein-receptor, which is involved in cholesterol homeostasis, have been analyzed using FRET assays combined with mathematical modeling[1-3] (Figure 1-2). FRET is measured between receptor-ligand complexes, in which ligands are labeled with two types of fluorophores ("donor" and "acceptor"), in endocytic membranes of polarized cells using Laser Scanning Confocal Microscopy (Figure 2). The dependence of energy transfer efficiency (E%) on donor and acceptor concentrations is used to distinguish a clustered arrangement from a random distribution of labeled receptor-ligand complexes, and the E% dependence on other variables will provide further insights into the clustering mechanisms[1-3] (Figure 3).
- Wallrabe H, Elangovan M, Burchard A, Periasamy A and Barroso M. Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. Biophys J 85: 559-571, 2003.
- Wallrabe H, Stanley M, Periasamy A and Barroso M. One- and two-photon FRET microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes. J Biomed Opt 8: 339-346, 2003
- Elangovan M, Wallrabe H, Chen Y, Day RN, Barroso M and Periasamy A. Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods 29: 58-73, 2003.
- Wallrabe H and Barroso M. FRET reveals the organization of different receptor-ligand complexes (polymeric IgA-R and Transferrin-R) in endocytic membranes of polarized MDCK cells. Proc. SPIE, Vol. 5323 (in press), Microscopy in the Biomedical Sciences IV; A. Periasamy (Ed.), 2004.