Cholesterol facilitates the native mechanism of Ca2+-triggered membrane fusion

Abstract

The process of regulated exocytosis is defined by the Ca2+-triggered fusion of two apposed membranes, enabling the release of vesicular contents. This fusion step involves a number of energetically complex steps and requires both protein and lipid membrane components. The role of cholesterol has been investigated using isolated release-ready native cortical secretory vesicles to analyze the Ca2+-triggered fusion step of exocytosis. Cholesterol is a major component of vesicle membranes and we show here that selective removal from membranes, selective sequestering within membranes, or enzymatic modification causes a significant inhibition of the extent, Ca2+ sensitivity and kinetics of fusion. Depending upon the amount incorporated, addition of exogenous cholesterol to cholesterol-depleted membranes consistently recovers the extent, but not the Ca2+ sensitivity or kinetics of fusion. Membrane components of comparable negative curvature selectively recover the ability to fuse, but are unable to recover the kinetics and Ca2+ sensitivity of vesicle fusion. This indicates at least two specific positive roles for cholesterol in the process of membrane fusion: as a local membrane organizer contributing to the efficiency of fusion, and, by virtue of its intrinsic negative curvature, as a specific molecule working in concert with protein factors to facilitate the minimal molecular machinery for fast Ca2+-triggered fusion.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…CSC and CV preparations, treatments, and fusion assays CSC/CV were isolated from fresh and 20-h incubated eggs using standard protocols [27,30,31]. Acute treatments and fusion assays were carried out in baseline intracellular media (BIM-210 mM potassium glutamate, 500 mM glycine, 10 mM NaCl, 10 mM 1,4-piperazinediethanesulfonic acid (PIPES), 1 mM MgCl 2 , 0.05 mM CaCl 2 , 1 mM ethyleneglycoltetraacetic acid (EGTA); pH 6.7) with the addition of protease inhibitors and 2.5 mM adenosine triphosphate (ATP) [30].…”

“…CV, isolated from fresh eggs, were treated with cinnamycin and neomycin for 30 min (25°C) followed by fusion assays. For fusion recovery, CV suspensions (OD 405 1.0), isolated from Smst-/Atst-incubated eggs, were treated with 2 mM CHOL-loaded hpβcd or 200 μM αT for 30 min (25°C), centrifuged, and suspended in fresh BIM for fusion assays; CHOL-loaded hpβcd was prepared as described [27,42,70]. αT was delivered in hexadecane (<0.1% solvent, final) [26,27].…”

“…For fusion recovery, CV suspensions (OD 405 1.0), isolated from Smst-/Atst-incubated eggs, were treated with 2 mM CHOL-loaded hpβcd or 200 μM αT for 30 min (25°C), centrifuged, and suspended in fresh BIM for fusion assays; CHOL-loaded hpβcd was prepared as described [27,42,70]. αT was delivered in hexadecane (<0.1% solvent, final) [26,27]. CV/CSC end-point and kinetic fusion assays and CV 'settle' fusion assays to assess inter-membrane attachment were as described [27,31,41,87,98].…”

“…αT was delivered in hexadecane (<0.1% solvent, final) [26,27]. CV/CSC end-point and kinetic fusion assays and CV 'settle' fusion assays to assess inter-membrane attachment were as described [27,31,41,87,98]. Briefly, suspensions of free-floating CV were aliquoted into multiwell plates and low speed centrifugation was used to bring the CV gently into contact at the bottom of the wells.…”

“…This integrated approach capitalizes on the metabolic activity of unfertilized sea urchin eggs to effect endogenous alterations of molecular components involved in docking, priming, and Ca 2+ -triggered fusion; CHOL, PE, and PIP levels were altered via inhibition of synthesis rather than by physically disrupting membrane structure with reagents that remove these lipids from the membrane [26,27]. This approach thus firmly establishes critical but also differing roles for CHOL and PE in membrane fusion and indicates modulatory roles for PIP-these are unlikely to act directly in membrane merger but specific species likely underlie the final priming steps in the docked state; depriming from this releaseready state does not, however, result in undocking of vesicles.…”