•Upon exocytosis vesicular cholesterol transfers to the plasmalemma.•Cholesterol enrichment decreases, depletion increases the fusion pore conductance.•A model of cholesterol-dependent force ...preventing fusion pore widening is developed.•Disease-related increase in vesicle cholesterol reduces the fusion pore conductance.
In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.
Display omitted Top: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the plasmalemma regulates the fusion pore.
Abstract
It is believed that in regulated exocytosis the vesicle membrane fuses with the plasma membrane in response to a physiological stimulus. However, in the absence of stimulation, repetitive ...transient fusion events are also observed, reflecting a stable state. The mechanisms by which the initial fusion pore attains stability are poorly understood. We modelled energetic stability of the fusion pore by taking into account the anisotropic, intrinsic shape of the membrane constituents and their in-plane ordering in the local curvature of the membrane. We used cell-attached membrane capacitance techniques to monitor the appearance and conductance of single fusion pore events in cultured rat lactotrophs. The results revealed a bell-shaped distribution of the fusion pore conductance with a modal value of 25 pS. The experimentally observed increase of the fusion pore stability with decreasing fusion pore radius agrees well with the theoretical predictions. Moreover, the results revealed a correlation between the amplitude of transient capacitance increases and the fusion pore conductance, indicating that larger vesicles may attain a stable fusion pore with larger fusion pore diameters.
Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is ...lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N‐terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20‐amino‐acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane‐inserted peptide from α‐helix to β‐structure. The β‐structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA‐expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature‐promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.
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