Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions

¹ University of California Davis
² Lawrence Livermore National Laboratories

^* To whom correspondence should be addressed. E-mail: mllongo{at}ucdavis.edu.

Submitted on October 11, 2005
Revised on November 12, 2005
Accepted on 1 March 2006

	Abstract

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1 and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC), in supported lipid bilayers. Using "slow cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles, GUVs) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area to perimeter ratio (A_D/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion" and find that the microstructures are remarkably similar above a cholesterol mole fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection..

Key Words: GUV, HIV, raft, sphingolipid, supported lipid bilayer

This article has been cited by other articles:

				Z. D. Schultz and I. W. Levin Lipid Microdomain Formation: Characterization by Infrared Spectroscopy and Ultrasonic Velocimetry Biophys. J., April 15, 2008; 94(8): 3104 - 3114. [Abstract] [Full Text] [PDF]

				C. D. Blanchette, W.-C. Lin, C. A. Orme, T. V. Ratto, and M. L. Longo Domain Nucleation Rates and Interfacial Line Tensions in Supported Bilayers of Ternary Mixtures Containing Galactosylceramide Biophys. J., April 1, 2008; 94(7): 2691 - 2697. [Abstract] [Full Text] [PDF]

				W.-C. Lin, C. D. Blanchette, and M. L. Longo Fluid-Phase Chain Unsaturation Controlling Domain Microstructure and Phase in Ternary Lipid Bilayers Containing GalCer and Cholesterol Biophys. J., April 15, 2007; 92(8): 2831 - 2841. [Abstract] [Full Text] [PDF]

				M. C. Howland, A. W. Szmodis, B. Sanii, and A. N. Parikh Characterization of Physical Properties of Supported Phospholipid Membranes Using Imaging Ellipsometry at Optical Wavelengths Biophys. J., February 15, 2007; 92(4): 1306 - 1317. [Abstract] [Full Text] [PDF]

				M. L. Kraft, P. K. Weber, M. L. Longo, I. D. Hutcheon, and S. G. Boxer Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry. Science, September 29, 2006; 313(5795): 1948 - 1951. [Abstract] [Full Text] [PDF]