Hemagglutinin Spatial Distribution Shifts in Response to Cholesterol in the Influenza Viral Envelope

AbstractInfluenza virus delivers its genome to the host cytoplasm via a process of membrane fusion mediated by the viral hemagglutinin protein. Optimal fusion likely requires multiple hemagglutinin trimers, so the spatial distribution of hemagglutinin on the viral envelope may influence fusion mechanism. We have previously shown that moderate depletion of cholesterol from the influenza viral envelope accelerates fusion kinetics even though it decreases fusion efficiency, both in a reversible manner. Here, we use electron cryo-microscopy to measure how the hemagglutinin lateral density in the viral envelope changes with cholesterol extraction. We extract this information by measuring the radial distribution function of electron density in >4000 viral images per sample, assigning hemagglutinin density by comparing images with and without anti-HA Fab bound. On average, hemagglutinin trimers move closer together: we estimate that the typical trimer-trimer spacing reduces from 94 to 84 Å when ∼90% of cholesterol is removed from the viral membrane. Upon restoration of viral envelope cholesterol, this spacing once again expands. This finding can qualitatively explain the observed changes to fusion kinetics: contemporary models from single-virus microscopy are that fusion requires the engagement of several hemagglutinin trimers in close proximity. If removing cholesterol increases the lateral density of hemagglutinin, this should result in an increase in the rate of fusion

Rapid Fusion of Synaptic Vesicles with Reconstituted Target SNARE Membranes

Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of ∼20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism.status: publishe

Fast SNARE Mediated Synaptic Vesicle Fusion in Supported Membranes

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SNARE Mediated Vesicle Fusion in Supported Membranes: Comparing Synaptic Vesicles with Reconstituted Vesicles

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