Molecular form follows function: (un)snaring the SNAREs

doi:10.1016/j.tins.2008.06.003

Trends in Neurosciences

Volume 31, Issue 9, September 2008, Pages 435-443

https://doi.org/10.1016/j.tins.2008.06.003 Get rights and content

Exocytotic release of transmitters is mediated by the ternary SNARE complex. The form of this complex is consistent with its function in the positioning of vesicles to the plasma membrane and their fusion to it. Recent advances in single-molecule techniques, however, bring an additional layer of complexity to this process, implicating that there might be various modes of operation. For example, the binary syntaxin–synaptobrevin 2 complex, in addition to the ternary complex containing SNAP25, might enable vesicular docking. Single-molecule techniques allow direct measurements of the distance/extension, rupture force, spontaneous dissociation times and interaction energy for SNARE protein–protein interactions. These measurements are complementary to results and conclusions drawn from other techniques. Consequently, single-molecule techniques promise tremendous opportunities for in vitro investigations of SNARE proteins to improve our understanding of their role in exocytosis.

Introduction

The view that brain function follows its form dates back to ancient Roman times. The most prominent figure in Roman medicine, the Greek physician Galen (years ∼129–200), tried to deduce brain function from its physical properties. He poked around the cerebrum and the cerebellum with his finger. By comparing the stiffness, he assigned the softer cerebrum to be the receiver of sensation, whereas the harder cerebellum he thought commanded the muscles. Now nearly 19 centuries later his reasoning might appear dubious, but surprisingly he was not too far from the truth. In a similar manner in this review, we explore the relationship between form/structure of the SNARE complex and its function using contemporary tools. Our journey starts with a primer on the SNARE complex form, mainly guided by X-ray crystallography. We then briefly discuss SNARE function as proposed from biochemical, genetic and electrophysiological approaches followed by a comparison with the information gathered by single-molecule techniques, most notably fluorescence resonant energy transfer (FRET) and atomic force microscopy (AFM).

Section snippets

SNARE primer

The soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptor (SNARE) complex [1] is ubiquitously used for membrane fusion ranging from yeast to human [2]. In the brain, the major cell types, neurons and astrocytes, use regulated exocytosis for their intercellular communication [3]. In these cells, the increase in intracellular Ca²⁺ leads to the fusion of secretory organelles to the plasma membrane, causing the release of transmitters, stored within secretory

Single-molecule measurements: a closer look

The main motivation for using a single-molecule approach is to detect heterogeneities in nonequilibrium dynamics, which bulk biochemical approaches would not be able to detect directly. Indeed, the last decade has seen the development of special instrumentation for the manipulation of single molecules and for measuring their mechanical properties and structural characteristics with nm resolution. These techniques include pipette suction [34], magnetic beads [35], optical traps [36], FRET [37]

Concluding remarks

The primary purpose of this review was to briefly relate SNARE complex form and function. The acquisition of a comprehensive knowledge of the role of SNARE proteins in exocytosis requires multifaceted use of traditional biochemical, genetic and electrophysiological approaches combined with relatively recent single-molecule techniques. The emergent picture is that in isolated systems, Sx1–Sb2 can form parallel coiled-coils in the absence of SNAP25 and such a single binary complex can be

Acknowledgements

The authors’ work is supported by a grant from the National Institute of Mental Health (MH 069791) and a grant from the Department of Defense/Defense Microelectronics Activity under Award no. DOD/DMEA-H94003-06-2-0608.

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Syntaxin-1A (stx1a) repression causes a neurodevelopmental disorder phenotype, low latent inhibition (LI) behavior, by disrupting 5-hydroxytryptaminergic (5-HTergic) systems. Herein, we discovered that lysine acetyltransferase (KAT) 3B increases stx1a neuronal transcription and TTK21, a KAT3 activator, induces stx1a transcription and 5-HT release in vitro. Furthermore, glucose-derived CSP-TTK21 could restore decreased stx1a expression, 5-HTergic systems in the brain, and low LI in stx1a (+/−) mice by crossing the blood-brain barrier, whereas the KAT3 inhibitor suppresses stx1a expression, 5-HTergic systems, and LI behaviors in wild-type mice. Finally, in wild-type and stx1a (−/−) mice treated with IKK inhibitors and CSP-TTK21, respectively, we show that KAT3 activator-induced LI improvement is a direct consequence of KAT3B-stx1a pathway, not a side effect. In conclusion, KAT3B can positively regulate stx1a transcription in neurons, and increasing neuronal stx1a expression and 5-HTergic systems by a KAT3 activator consequently improves the low LI behavior in the stx1a ablation mouse model.
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Citation Excerpt :
For example, each neuronal vesicle contains ∼70 Sb2 molecules [39], while astrocytic vesicles contain ∼20 Sb2 molecules [40]. While the nanoanatomy of a single lactotroph vesicle is unknown, we can make an assumption that Sb2 would be relatively abundant, so that Sb2 molecules would be present throughout vesicle membrane and not solely located at the fusion site where 1–2 molecules are needed [7]. Those Sb2 molecules present on the diametrically opposite side of the vesicle docking/hemifusion site we probed here using Sx1A-functionalized tip to record single Sx1A-Sb2 mechanical interactions.
Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex formation, which includes an assembly of the intermediate cis binary complex between Sx1A and SNAP25 on the plasma membrane to engage Sb2 on the vesicle. However, whether a trans binary Sx1A-Sb2 complex alone could mediate vesicle docking in a cellular environment remains unclear. To address this issue, we used atomic force microscopy (AFM) in the force spectroscopy mode combined with fluorescence imaging. Using AFM tips functionalized with the full Sx1A cytosolic domain, we probed native Sb2 studding the membrane of secretory vesicles docked at the plasma membrane patches, referred to as “inside-out lawns”, identified based on fluorescence stains and prepared from primary culture of lactotrophs. We recorded single molecule Sx1A-Sb2 mechanical interactions and obtained measurements of force (∼183 pN) and extension (∼21.6 nm) necessary to take apart Sx1A-Sb2 binding interactions formed at tip-vesicle contact. Measured interactive force between a single pair of Sx1A-Sb2 molecules is sufficient to hold a single secretory vesicle docked at the plasma membrane within distances up to that of the measured extension. This finding further advances a notion that native vesicle docking can be mediated by a single trans binary Sx1A-Sb2 complex in the absence of SNAP25.
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Sensory nerve endings respond to various stimuli and subsequently transmit afferent informations to central nervous system, but their responsibility has been suggested to be modulated by glutamate. In the present study, we examined the immunohistochemical localization of vesicular glutamate transporter 1 (vGLUT1) and vGLUT2 in baroreceptor nerve endings immunoreactive for P2X2 and P2X3 purinoceptors in the rat carotid sinus by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3-immunoreactive flat leaf-like axon terminals were immunoreactive to vGLUT2, but not to vGLUT1. Among members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, immunoreactivities for synaptosomal-associated protein, 25 kDa, Syntaxin1, and vesicle-associated membrane protein 2 (VAMP2) were localized in P2X2- and P2X3-immunoreactive axon terminals. Punctate immunoreactive products for VAMP2 and vGLUT2 were co-localized in axon terminals. These results suggest that vGLUT2 is localized in P2X3-immunoreactive baroreceptor terminals in the carotid sinus, and these terminals may release glutamate by exocytosis in order to modulate baroreceptor function in the carotid sinus.
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Interacting partners of PRRSV GP5 and M were identified using a porcine macrophage cDNA library yeast two-hybrid screen. Subsequently, the interactions between PRRSV GP5/M and the cellular protein Snapin were mapped using truncated versions of the GP5 and M proteins in a yeast two-hybrid assay to localize the interactions. The Snapin gene from the African green monkey kidney cell line MARC-145, which is permissive to PRRSV, was cloned and sequenced, and compared to porcine Snapin. Cellular Snapin expression was reduced in PRRSV-infected cells via Snapin-specific siRNA targeting.
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Trends in Neurosciences

ReviewMolecular form follows function: (un)snaring the SNAREs

Introduction

Section snippets

SNARE primer

Single-molecule measurements: a closer look

Concluding remarks

Acknowledgements

Cell

J. Biol. Chem.

Biophys. J.

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Biophys. J.

Biophys. J.

Methods Cell Biol.

J. Biol. Chem.

Cell

Biophys. J.

Biophys. J.

Structure

Structure

Biophys. J.

Biophys. J.

SNAP receptors implicated in vesicle targeting and fusion

Nature

SNAREs – engines for membrane fusion

Nat. Rev. Mol. Cell Biol.

Vesicular release of glutamate mediates bidirectional signaling between astrocytes and neurons

J. Neurochem.

Synaptotagmin IV regulates glial glutamate release

Proc. Natl. Acad. Sci. U. S. A.

Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4

Nat. Struct. Mol. Biol.

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution

Nature

A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants

Science

Review
Molecular form follows function: (un)snaring the SNAREs

Structural basis for the evolutionary inactivation of Ca²⁺ binding to synaptotagmin 4