scholarly journals Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

2011 ◽  
Vol 195 (7) ◽  
pp. 1159-1170 ◽  
Author(s):  
Zhao Wang ◽  
Huisheng Liu ◽  
Yiwen Gu ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Transmission ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Cytoplasmic Domain ◽  
Snare Proteins ◽  
Antagonist Activity ◽  
Vesicle Docking ◽  
Synaptotagmin I ◽  
Target Membrane

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

scholarly journals Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I

2002 ◽  
Vol 158 (2) ◽  
pp. 273-282 ◽  
Author(s):  
Lara K. Mahal ◽  
Sonia M. Sequeira ◽  
Jodi M. Gureasko ◽  
Thomas H. Söllner
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Fusion Reaction ◽  
Snare Proteins ◽  
Calcium Sensor ◽  
Synaptic Vesicle Protein ◽  
Synaptotagmin I ◽  
In Trans ◽  
Liposome Fusion ◽  
Stimulation Of

Ñeurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay. Synaptotagmin I dramatically stimulated membrane fusion by facilitating SNAREpin zippering. This stimulatory effect was topologically restricted to v-SNARE vesicles (containing VAMP 2) and only occurred in trans to t-SNARE vesicles (containing syntaxin 1A and SNAP-25). Interestingly, calcium did not affect the overall fusion reaction. These results indicate that synaptotagmin I can directly accelerate SNARE-mediated membrane fusion and raise the possibility that additional components might be required to ensure tight calcium coupling.

scholarly journals The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Radhika C. Desai ◽  
Bimal Vyas ◽  
Cynthia A. Earles ◽  
J. Troy Littleton ◽  
Judith A. Kowalchyck ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Essential Step ◽  
Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

scholarly journals SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cristina Nogueira ◽  
Patrik Erlmann ◽  
Julien Villeneuve ◽  
António JM Santos ◽  
Emma Martínez-Alonso ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fusion ◽  
Protein Secretion ◽  
Family Members ◽  
Retrograde Transport ◽  
Cytoplasmic Domain ◽  
Snare Complex ◽  
Snare Proteins ◽  
General Protein

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.

Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

1995 ◽  
Vol 14 (10) ◽  
pp. 2317-2325 ◽  
Author(s):  
T. Hayashi ◽  
S. Yamasaki ◽  
S. Nauenburg ◽  
T. Binz ◽  
H. Niemann
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Vesicle Membrane

scholarly journals T202. THE EFFECT OF ANTIPSYCHOTIC DRUGS ON MEMBRANE FUSION: AN IN VITRO STUDY

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S308-S309
Author(s):  
Vladimir Adrien ◽  
Hugo Fumat ◽  
Cédric Tessier ◽  
Philippe Nuss ◽  
David Tareste
Keyword(s):  
Membrane Fusion ◽  
Lipid Composition ◽  
Synaptic Vesicles ◽  
Significant Proportion ◽  
Membrane Dynamics ◽  
Snare Proteins ◽  
Synaptic Membranes ◽  
Synaptic Plasma Membrane

Abstract Background Common clinical use of antipsychotics (AP) drugs shows that their therapeutic mode of action still needs further clarification although it is admitted that the Dopamine receptor D2 (D2R) antagonism plays a significant role. For instance, clozapine (CLOZ) - which is known to be the most effective AP in treating schizophrenic symptoms - has strikingly the lowest D2R antagonism. Non direct receptor-related effects might thus be involved in the activity of AP at the synapse level. AP, as well as neurotransmitters, are mostly lipophilic and insert within membranes. This characteristic is of interest as a significant proportion of schizophrenic patients has specific and abnormal membrane lipid composition. This possible proxy of the disease biotype can participate in the disease’s physiopathology but also be critical for the effect of AP drugs. We hypothesize that AP insertion into lipid membranes also contribute to their therapeutic effect. AP-induced modifications of synaptic membranes biophysics are likely to influence neurotransmission. In this study, we focus on the effect of AP on membrane fusion, a crucial step for the exocytosis of neurotransmitters. Methods Liposomes modelling synaptic vesicles were reconstituted in saline buffer. Two standard ternary and quaternary lipid mixtures have been studied: phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine (PC:PE:PS) [65:25:10] and the synaptic-like PC:PE:PS:sphingomyelin:cholesterol (PC:PE:PS:SM:CHOL) [25:25:10:10:30]. Some liposomes were protein-free and others were functionalized with Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) proteins, which trigger in vivo the fusion of synaptic vesicles with the pre-synaptic plasma membrane. The liposome size was checked by Dynamic Light Scattering. Insertion of AP within the membrane was checked by second derivative spectroscopy. Fusion was measured by Fluorescence Resonance Energy Transfer in the absence or presence of CLOZ or chlorpromazine (CPZ) at various lipid:AP ratios (10:1 to 100000:1). Protein-free liposomes were fused with Polyethylene glycol (PEG) and SNARE liposomes through the action of cognate SNARE proteins residing in their membrane. Results Liposomes of the same lipid composition were of the same size, with no effect of the addition of AP drugs at various concentrations. Molar partition coefficient of AP drugs within the membrane of protein-free liposomes was approximately 70–85%. CPZ or CLOZ inhibited the fusion of PC:PE:PS liposomes by about 20–40%. When liposomes were synaptic-like (PC:PE:PS:SM:CHOL), the inhibition of fusion by AP drugs reached 50%. CLOZ also inhibited SNARE-mediated fusion of PC:PE:PS liposomes by about 30%. This effect on SNARE-mediated fusion was not observed with CPZ. Discussion Altogether, these results, despite preliminary, could help to understand partially a non direct receptor-related effect of antipsychotics. Indeed, these drugs also seem to modify membrane dynamics at the synapse level. This seems to be particularly the case of CLOZ on SNARE-mediated fusion and could explain its specific therapeutic efficiency.

scholarly journals The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

2001 ◽  
Vol 154 (6) ◽  
pp. 1117-1124 ◽  
Author(s):  
Cynthia A. Earles ◽  
Jihong Bai ◽  
Ping Wang ◽  
Edwin R. Chapman
Keyword(s):  
Membrane Fusion ◽  
Real Time ◽  
Pc12 Cells ◽  
Binding Sites ◽  
C2 Domain ◽  
C2 Domains ◽  
Synaptotagmin I ◽  
Target Membrane ◽  
Complete Disruption

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.

scholarly journals Superfluous Role of Mammalian Septins 3 and 5 in Neuronal Development and Synaptic Transmission

2008 ◽  
Vol 28 (23) ◽  
pp. 7012-7029 ◽  
Author(s):  
Christopher W. Tsang ◽  
Michael Fedchyshyn ◽  
John Harrison ◽  
Hong Xie ◽  
Jing Xue ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Synapse Formation ◽  
Vesicle Traffic ◽  
Axon Development ◽  
Central Synapses ◽  
Synaptic Vesicle Fusion

ABSTRACT The septin family of GTPases, first identified for their roles in cell division, are also expressed in postmitotic tissues. SEPT3 (G-septin) and SEPT5 (CDCrel-1) are highly expressed in neurons, enriched in presynaptic terminals, and associated with synaptic vesicles. These characteristics suggest that SEPT3 or SEPT5 might be important for synapse formation, maturation, or synaptic vesicle traffic. Since Sept5 −/− mice do not show any overt neurological phenotypes, we generated Sept3 −/− and Sept3 −/− Sept5 −/− mice and found that SEPT3 and SEPT5 are not essential for development, fertility, or viability. Changes in the expression of septins were noted in the absence of SEPT3, SEPT5, and both septins. SEPT5 association with other septins in brain tissue was unaffected by the removal of SEPT3. No abnormalities were observed in the gross morphology and synapses of the hippocampus. Similarly, axon development and synapse formation were unaffected in vitro. In cultured hippocampal neurons, the size of the recycling synaptic vesicle pool was unaltered in the absence of SEPT3. Furthermore, synaptic transmission at two different central synapses was not significantly affected in Sept3 −/− Sept5 −/− mice. These results indicate that SEPT3 and SEPT5 are dispensable for neuronal development as well as for synaptic vesicle fusion and recycling.

scholarly journals Ring-like oligomers of Synaptotagmins and related C2 domain proteins

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Maria N Zanetti ◽  
Oscar D Bello ◽  
Jing Wang ◽  
Jeff Coleman ◽  
Yiying Cai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Structural Aspect ◽  
C2 Domain ◽  
Early Step ◽  
Vesicle Docking ◽  
Ring Assembly ◽  
Synaptotagmin 1

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx.

A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion

Cell ◽  
1993 ◽  
Vol 75 (3) ◽  
pp. 409-418 ◽  
Author(s):  
Thomas Söllner ◽  
Mark K. Bennett ◽  
Sidney W. Whiteheart ◽  
Richard H. Scheller ◽  
James E. Rothman
Keyword(s):  
Synaptic Vesicle ◽  
Protein Assembly ◽  
Vesicle Docking

Molecular mechanisms of synaptic vesicle docking and membrane fusion

1994 ◽  
Vol 6 (3) ◽  
pp. 167-176 ◽  
Author(s):  
Beverly Wendland ◽  
Richard H. Scheller
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Molecular Mechanisms ◽  
Vesicle Docking
Sign in / Sign up

Export Citation Format

Share Document