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

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 (AD/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),more » 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.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
899110
Report Number(s):
UCRL-JRNL-219567
TRN: US200706%%495
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biophysical Journal, vol. 90, N/A, March 24, 2006, pp. 4466-4478
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; ADHESION; AGGLUTININS; AIDS VIRUS; ATOMIC FORCE MICROSCOPY; BACTERIA; CELL MEMBRANES; CHOLESTEROL; GALACTOSE; LIPIDS; MEMBRANES; MICA; MICROSTRUCTURE; PROTEINS; SOLUBILITY; SUBSTRATES; SURFACE AREA; VIRUSES

Citation Formats

Blanchette, C D, Lin, W, Ratto, T V, and Longo, M L. Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions. United States: N. p., 2006. Web. doi:10.1529/biophysj.105.072744.
Blanchette, C D, Lin, W, Ratto, T V, & Longo, M L. Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions. United States. doi:10.1529/biophysj.105.072744.
Blanchette, C D, Lin, W, Ratto, T V, and Longo, M L. Fri . "Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions". United States. doi:10.1529/biophysj.105.072744. https://www.osti.gov/servlets/purl/899110.
@article{osti_899110,
title = {Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions},
author = {Blanchette, C D and Lin, W and Ratto, T V and Longo, M L},
abstractNote = {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 (AD/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.},
doi = {10.1529/biophysj.105.072744},
journal = {Biophysical Journal, vol. 90, N/A, March 24, 2006, pp. 4466-4478},
number = ,
volume = ,
place = {United States},
year = {Fri Mar 03 00:00:00 EST 2006},
month = {Fri Mar 03 00:00:00 EST 2006}
}