Membrane Remodeling During Viral Infection, Parasite Inv

Child Health And Human Development

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Abstract

This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. There are two components: 1. Influenza Ha Does Not Partition Into Rafts With Ideal Mixing: Although lipid-dependent protein clustering in biomembranes mediates numerous functions, there is little consensus among membrane models on cluster organization or size. Here, we use influenza viral envelope protein hemagglutinin (HA0) to test the hypothesis that clustering results from proteins partitioning into preexisting, fluid-ordered ?raft? domains, wherein they have a random distribution. Japan HA0 expressed in fibroblasts was visualized by electron microscopy using immunogold labeling and probed by fluorescence resonance energy transfer (FRET). Labeled HA coincided with electron-dense, often noncircular membrane patches. Poisson and K-test (Rip-ley, B.D. 1977. J. R. Stat. Soc. Ser. B.39:172?192) analyses reveal clustering on accessible length scales (20?900 nm). Membrane treatments with methyl-cyclodextrin and glycosphingolipid synthesis inhibitors did not abolish clusters but did alter their pattern, especially at the shortest lengths, as was corroborated by changes in FRET efficiency. The magnitude and density dependence of the measured FRET efficiency also indicated a nonrandom distribution on molecular length scales (6?7 nm). This work rules out the tested hypothesis for HA over the accessible length scales, yet shows clearly how its spatial distribution depends on lipid composition. 2. Membrane Transformation During Malaria Parasite Release from Human Red Blood Cells: Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites, fusion of erythrocyte and parasitophorus vacuolar membranes (PVM), and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts, membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature which contrariwise promote membrane rupture, and without protease inhibitors parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results together with the modulation of release by osmotic stress suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.