Identifying Critical Components of Native Ca²⁺‐triggered Membrane Fusion

Abstract

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.

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

“…Settle CV-CV fusion assays revealed no differences between fresh and incubated eggs, indicating that CV were able to form critical inter-membrane attachments necessary for a robust fusion response (Fig. 2b) [8,26,27,31,38,39,72,84,97]. Overlapping activity curves and kinetic responses found for both CV-CV and CV-PM fusion ( Fig.…”

“…8d and 9c) suggests dissociation of PIP from a direct role in fusion (i.e., FFM). As fusion efficiency is linked to integrity of CHOL-rich microdomains [27,39,72], comparable inhibition of fusion kinetics with LY29, Wrt, and neomycin suggests effects on the functional status/stability of such domains. Thus, changes in PIP may affect modulatory components, reorganizing and/or activating efficiency factors in the microdomain-associated PFM.…”

Lipid Dynamics in Exocytosis

“…The ability of microdomains to sequester specific proteins and to exclude others makes them ideally suited to spatially organize cellular pathways. Hence, some cholesterol-enriched microdomains seem to concentrate components of the exocytotic/fusion machineries (Furber et al 2009b(Furber et al , 2010. For instance, a number of recent studies examining the distribution of SNARE proteins in membrane domains in various cell types suggest that SNAREs, like SNAP-25 partially associate with detergent resistant, cholesterol-enriched microdomains (Chamberlain et al 2001;Lang et al 2001).…”

“…Fluorescent analogs that can be used to promote the mechanism and simultaneously label the proteins involved will now be useful to identify the protein and/or lipid targets of these thiol probes. This approach combined with the isolation of cholesterol-enriched regions and 2D gel electrophoresis has resulted in the detection of a limited number of potential protein candidates that are currently being identified using mass spectrometry (Furber et al 2009b(Furber et al , 2010. This integrated functional-molecular analytical approach, assessing protein and lipid contributions, promises to teach us much more about the fundamental mechanisms underlying the Ca 2?…”

How to Make a Stable Exocytotic Fusion Pore, Incompetent of Neurotransmitter and Hormone Release from the Vesicle Lumen?

“…There are several mechanisms by which cholesterol is capable of contributing to membrane fusion [67,68]. First, as a component of lipid rafts, cholesterol can organize essential proteins and lipids at the fusion site [67].…”