Song, Lin Ye; (1993) Mechanisms of membrane fusion. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis is concerned with studies on two aspects of membrane fusion. The first aspect is the mechanism of membrane fusion in electrically-induced cell fusion. The second aspect is the mechanism of membrane fusion induced by fusogenic viral peptides. Evidence was found from both fluorescence microscopy and freeze-fracture electron microscopy for the occurrence of hemi-fusion in the electrofusion of human erythrocytes. The conditions that favour hemi-fusion as opposed to complete fusion were characterised, and the possibility that hemi-fusion might precede complete electrically-induced cell fusion are discussed. The procoagulant activity of human erythrocytes, which provides a measure of the translocation of phosphatidylserine from the inner to the outer monolayer of the plasma membrane, has been compared with the percentage cell fusion in experiments on erythrocyte fusion induced by electrical breakdown pulses under differing experimental conditions. It seems possible that a localised, surface exposure of phosphatidylserine may contribute to the "long-lived fusogenic state". Divalent cations in the pulsing medium may interact with phosphatidylserine molecules, translocated from the inner to the outer monolayer of the erythrocyte plasma membrane by breakdown pulses, to stabilise the pulsed erythrocyte membrane against haemolysis, and to assist the formation of pearl chains of pulsed cells. It was found that the entry of sugar molecules, via electropores in the plasma membrane, facilitated the rounding-up of electrically fused erythrocytes into giant cells, while impermeable molecules e.g. poly (ethylene glycol) or dextran inhibited this process. The secondary structures and orientations of the fusion peptides (corresponding to the N-terminus of HA2 protein of strains A/PR/8/34 and X31 influenza viruses (HA and WT peptide, respectively), a peptide (G1E peptide) with a substitution of glutamic acid for the glycine residue at the N-terminal of the WT peptide, and the fusion peptide corresponding to the N-terminus of gp41 protein of ARV2 strain HIV virus were investigated in egg PC phospholipid bilayers with polarized, attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR). Both dried and hydrated samples were studied. The relationships between the observed secondary structures and orientation of these fusion peptides and how they may induce membrane fusion are discussed.

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