A post-docking role for synaptobrevin in synaptic vesicle fusion

doi:10.1016/0896-6273(94)90443-X

Neuron

Volume 12, Issue 6, June 1994, Pages 1269-1279

https://doi.org/10.1016/0896-6273(94)90443-X Get rights and content

Abstract

We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%–68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca²⁺ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

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Cited by (209)

Fast, Ca<sup>2+</sup>-dependent exocytosis at nerve terminals: Shortcomings of SNARE-based models
2014, Progress in Neurobiology
Citation Excerpt :
Thus, additional work will be needed to clarify the importance of syntaxin 1a for the function of vertebrate synapses. Among the seminal studies of SNARE function at the synapse, Hunt et al. (1994) monitored the impact on evoked transmitter release of presynaptic introduction of clostridial neurotoxins known to target synaptobrevin. The toxins caused a slow blockade of evoked transmitter release at the squid giant synapse that was largely complete after 2–3 h (similar results were seen at Aplysia synapses; Schiavo et al., 1992).
Investigations over the last two decades have made major inroads in clarifying the cellular and molecular events that underlie the fast, synchronous release of neurotransmitter at nerve endings. Thus, appreciable progress has been made in establishing the structural features and biophysical properties of the calcium (Ca²⁺) channels that mediate the entry into nerve endings of the Ca²⁺ ions that trigger neurotransmitter release. It is now clear that presynaptic Ca²⁺ channels are regulated at many levels and the interplay of these regulatory mechanisms is just beginning to be understood. At the same time, many lines of research have converged on the conclusion that members of the synaptotagmin family serve as the primary Ca²⁺ sensors for the action potential-dependent release of neurotransmitter. This identification of synaptotagmins as the proteins which bind Ca²⁺ and initiate the exocytotic fusion of synaptic vesicles with the plasma membrane has spurred widespread efforts to reveal molecular details of synaptotagmin's action. Currently, most models propose that synaptotagmin interfaces directly or indirectly with SNARE (soluble, N-ethylmaleimide sensitive factor attachment receptors) proteins to trigger membrane fusion. However, in spite of intensive efforts, the field has not achieved consensus on the mechanism by which synaptotagmins act. Concurrently, the precise sequence of steps underlying SNARE-dependent membrane fusion remains controversial. This review considers the pros and cons of the different models of SNARE-mediated membrane fusion and concludes by discussing a novel proposal in which synaptotagmins might directly elicit membrane fusion without the intervention of SNARE proteins in this final fusion step.
Release of Neurotransmitters
2014, From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience: Third Edition
This chapter summarizes classical knowledge and recent advances in the release of neurotransmitters, emphasizing evidence supporting present concepts, rather than a recitation of dogma. Topics covered include:
- •
  Structural organization of chemical synapses
- •
  Quantal nature of transmission; identification of the synaptic vesicle as the physical quantum
- •
  Vesicle membrane and transmitter cycles
- •
  Biophysics of excitation-secretion coupling; secretory trigger properties
- •
  Calcium microdomains in triggering exocytosis
- •
  Mobilizing vesicles to docking sites and priming vesicles for release
- •
  Presynaptic protein identification by vesicle purification and genetics
- •
  SNARE proteins: assembly, disassembly, and vesicle fusion
- •
  Synaptotagmin: calcium sensor and secretory trigger
- •
  Docking and priming proteins
- •
  Molecular mechanisms of endocytosis and vesicle refilling
- •
  Quantal analysis at neuromuscular junctions and central synapses
- •
  Poisson and binomial models; meaning of statistical parameters
- •
  Limitations of the standard Katz model
- •
  Single and multivesicular release at active zones
- •
  Spontaneous vs. evoked release
- •
  Estimating and manipulating statistical parameters
- •
  Quantal analysis of release modulation
Docking and fusion of synaptic vesicles in cell-free model system of exocytosis
2008, Neurochemistry International
The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.
The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.
Molecular form follows function: (un)snaring the SNAREs
2008, Trends in Neurosciences
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.
A 20-nm step toward the cell membrane preceding exocytosis may correspond to docking of tethered granules
2008, Biophysical Journal
In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse ∼3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.
Coats, Tethers, Rabs, and SNAREs Work Together to Mediate the Intracellular Destination of a Transport Vesicle
2007, Developmental Cell
Citation Excerpt :
Thus, the mere presence of a SNARE cannot be the sole determinant of the direction in which a vesicle is traveling. Furthermore, the interactions of SNAREs are promiscuous (Tsui and Banfield, 2000; von Mollard et al., 1997), and the disruption of SNARE complex formation does not block vesicle tethering (Broadie et al., 1995; Hunt et al., 1994). These findings indicate that SNAREs do not mediate the first point of contact between a vesicle and its target.
Tethering factors have been shown to interact with Rabs and SNAREs and, more recently, with coat proteins. Coat proteins are required for cargo selection and membrane deformation to bud a transport vesicle from a donor compartment. It was once thought that a vesicle must uncoat before it recognizes its target membrane. However, recent findings have revealed a role for the coat in directing a vesicle to its correct intracellular destination. In this review we will discuss the literature that links coat proteins to vesicle targeting events.

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^#: The first two authors contributed equally to this paper.

^∗∗: Present address: Max Delbrück Center for Molecular Medicine Berlin, Federal Republic of Germany.

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Neuron

ArticleA post-docking role for synaptobrevin in synaptic vesicle fusion

Abstract

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Brain Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Neuron

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

Brain Res.

FEBS Lett.

Cell

Cell

Neuron

Neuron

Cell

Neuron

Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse

J. Neurosci.

Calcium action in synaptic transmitter release

Annu. Rev. Neurosci.

The calcium signal for transmitter secretion from presynaptic nerve terminals

Ann. NY Acad. Sci.

Synaptobrevin: an integral membrane protein of 18,000 daltons present in small synaptic vesicles of rat brain

EMBO J.

The molecular machinery for secretion is conserved from yeast to neurons

Synaptic vesicle membrane proteins interact to form a multimeic complex

J. Cell Biol.

Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis

Nature

Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the PAS superfamily

Mol. Cell. Biol.

Article
A post-docking role for synaptobrevin in synaptic vesicle fusion