One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes.

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

[1]  D. Hoekstra,et al.  The sub-apical compartment: a novel sorting center? , 2022 .

[2]  K. Mostov,et al.  Definition of Distinct Compartments in Polarized Madin–Darby Canine Kidney (MDCK) Cells for Membrane‐Volume Sorting, Polarized Sorting and Apical Recycling , 2000, Traffic.

[3]  T M Jovin,et al.  Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  L. Brand,et al.  Resonance energy transfer: methods and applications. , 1994, Analytical biochemistry.

[5]  Ammasi Periasamy,et al.  Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations , 2003, The Journal of cell biology.

[6]  Tamar Schlick,et al.  Engineering teams up with computer-simulation and visualization tools to probe biomolecular mechanisms. , 2003, Biophysical journal.

[7]  Dino A. De Angelis.  Why FRET over genomics , 1999 .

[8]  E. Sztul,et al.  Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome , 1994, The Journal of cell biology.

[9]  H Szmacinski,et al.  Fluorescence lifetime imaging. , 1992, Analytical biochemistry.

[10]  B. Herman,et al.  Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. , 1998, Biophysical journal.

[11]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[12]  Horst Wallrabe,et al.  Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. , 2003, Methods.

[13]  C. Futter,et al.  Sorting Mechanisms Regulating Membrane Protein Traffic in the Apical Transcytotic Pathway of Polarized MDCK Cells , 1998, The Journal of cell biology.

[14]  P. Bastiaens,et al.  Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. , 1999, Trends in cell biology.

[15]  A. Kenworthy,et al.  Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer , 1998, The Journal of cell biology.

[16]  I. Mellman,et al.  Differential microtubule requirements for transcytosis in MDCK cells. , 1990, The EMBO journal.

[17]  S. V. van IJzendoorn,et al.  The subapical compartment: a novel sorting centre? , 1999, Trends in cell biology.

[18]  Y. Liu,et al.  Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. , 2001, Biophysical journal.

[19]  A. Kenworthy,et al.  High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. , 2000, Molecular biology of the cell.

[20]  K. Hahn,et al.  Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. , 2000, Methods in enzymology.

[21]  T M Jovin,et al.  FRET microscopy demonstrates molecular association of non‐specific lipid transfer protein (nsL‐TP) with fatty acid oxidation enzymes in peroxisomes , 1998, The EMBO journal.

[22]  A. Periasamy.  Methods in Cellular Imaging , 2001, Methods in Physiology.

[23]  Horst Wallrabe,et al.  Confocal FRET microscopy to measure clustering of ligand-receptor complexes in endocytic membranes. , 2003, Biophysical journal.