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Laboratory of Cellular and Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

Correspondence to Joshua Zimmerberg: Joshz{at}helix.nih.gov

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.

Abbreviations used in this paper: CF, carboxy fluorescein; COD, cholesterol oxidase; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; MβCD, methyl-β-cyclo dextrin; RBC, red blood cells; RFI, relative fluorescence intensity; TMD, transmembrane domain.

© 2008 Biswas et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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