Dataset Information

Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores.

ABSTRACT: Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered. From the lipid release times we computed pore openness, the fraction of time the pore is open, which increased dramatically with cholesterol. For most lipid compositions tested, SNARE-mediated and nonspecifically nucleated pores had similar openness, suggesting that pore flickering was controlled by lipid bilayer properties. However, with physiological cholesterol levels, SNAREs substantially increased the fraction of fully open pores and fusion was so accelerated that there was insufficient time to recruit t-SNAREs to the fusion site, consistent with t-SNAREs being preclustered by cholesterol into functional docking and fusion platforms. Our results suggest that cholesterol opens pores directly by reducing the fusion-pore bending energy, and indirectly by concentrating several SNAREs into individual fusion events.

SUBMITTER: Stratton BS

PROVIDER: S-EPMC4833774 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores.

Stratton Benjamin S BS Warner Jason M JM Wu Zhenyong Z Nikolaus Joerg J Wei George G Wagnon Emma E Baddeley David D Karatekin Erdem E O'Shaughnessy Ben B

Biophysical journal 20160401 7

Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small ...[more]

PMID: 27074679

Similar Datasets

Project description:Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.

Dataset Information

Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores.

Publications

Cholesterol Increases the Openness of SNARE-Mediated Flickering Fusion Pores.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets