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Biophys. J. BioFAST: First Published October 13, 2006. doi:10.1529/biophysj.106.088815
© 2006 by the Biophysical Society.

A more recent version of this article appeared on January 1, 2007.
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Angel M. Davey
Ronn P. Walvick
Yuexin Liu
Ahmed A. Heikal
Erin D. Sheets
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CELL BIOPHYSICS

Membrane order and molecular dynamics associated with IgE receptor crosslinking in mast cells

Angel M. Davey 1, Ronn P. Walvick 1, Yuexin Liu 1, Ahmed A. Heikal 1 and Erin D. Sheets 2*

1 Pennsylvania State University
2 Penn State Univ, Dept Chem

* To whom correspondence should be addressed. E-mail: eds11{at}psu.edu.

Submitted on May 13, 2006
Revised on July 3, 2006
Accepted on 20 September 2006


  Abstract
Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy (FLIM) and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (Fc{varepsilon}RI) is extensively crosslinked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids [the lipid analog diI-C18 or glycosphingolipids]) and lipid-anchored proteins co-redistribute with crosslinked IgE-Fc{varepsilon}RI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C18 and Alexa 488-labeled IgE-Fc{varepsilon}RI in the domains where these molecules co-localize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.

Key Words: IgE receptor, fluorescence anisotropy, fluorescence lifetime imaging, lipid domains, two-photon FLIM




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 J. Biol. Chem.Home page
A. M. Davey, K. M. Krise, E. D. Sheets, and A. A. Heikal
Molecular Perspective of Antigen-mediated Mast Cell Signaling
J. Biol. Chem., March 14, 2008; 283(11): 7117 - 7127.
[Abstract] [Full Text] [PDF]


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Copyright © 2006 by the Biophysical Society.