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

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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 ... continued below

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Blanchette, C D; Lin, W; Ratto, T V & Longo, M L March 3, 2006.

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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.

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PDF-file: 24 pages; size: 0.2 Mbytes

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  • Journal Name: Biophysical Journal, vol. 90, N/A, March 24, 2006, pp. 4466-4478

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  • Report No.: UCRL-JRNL-219567
  • Grant Number: W-7405-ENG-48
  • Office of Scientific & Technical Information Report Number: 899110
  • Archival Resource Key: ark:/67531/metadc880449

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Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

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  • March 3, 2006

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  • Sept. 22, 2016, 2:13 a.m.

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  • Nov. 28, 2016, 6:22 p.m.

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Blanchette, C D; Lin, W; Ratto, T V & Longo, M L. Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions, article, March 3, 2006; Livermore, California. (digital.library.unt.edu/ark:/67531/metadc880449/: accessed November 17, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.