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

Matthew A Churchward,Jens R Coorssen,Tatiana Rogasevskaia,Jason Bau,Jana HöFgen

doi:10.1242/jcs.02601

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.

Highlights

Exocytosis is an essential cellular process with numerous distinct stages
The kinetics of fusion showed a comparable dose-dependent inhibition following treatment with m␤cd (Fig. 1B); the initial rate decreased to 8.9±0.9%/second following treatment with 6 mM m␤cd (n=2)
Inhibition was not a result of cortical vesicles (CVs) lysis or the undocking of CVs from the plasma membrane (PM) during treatment (Fig. 2), as cortices treated with m␤cd were morphologically identical to parallel, untreated cortices, except that intact, unfused vesicles remained even after a Ca2+ challenge (Fig. 2A)

Summary

Introduction

Exocytosis is an essential cellular process with numerous distinct stages. In many systems, the defining step of regulated exocytosis is the Ca2+-triggered fusion of two apposed membranes. The mechanism of Ca2+-triggered membrane fusion has been described by both proximity and proteinaceous fusion pore models. Both models seek to account for a mechanism that can overcome the substantial energy barriers obviating the close apposition of two bilayers and the subsequent molecular reorganization required for the merger and coalescence of these membranes (Rand and Parsegian, 1989). Proximity models consider mechanisms that bring two bilayers into close apposition, causing bilayer reorganization and rapid formation of a lipidic fusion pore (Rand and Parsegian, 1989; Kuzmin et al, 2001; Kozlovsky et al, 2002). Positive curvature is the tendency to form convex, micelle-like structures, whereas negative curvature implies formation of concave surfaces at the lipid-water interface (Luzzati and Husson, 1962)

Methods

Results

Discussion

Conclusion