scholarly journals The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor

2012 ◽  
Vol 443 (1) ◽  
pp. 223-229 ◽  
Author(s):  
Ying Lai ◽  
Yeon-Kyun Shin
Keyword(s):  
Charge Asymmetry ◽  
Vesicle Fusion ◽  
Asymmetric Distribution ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Synaptotagmin 1 ◽  
A Charge ◽  
Synaptic Vesicle Fusion ◽  
Acidic Lipids

Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3–10-fold without Ca2+. However, with Ca2+ there was an additional 2–5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50 μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.

scholarly journals The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

1998 ◽  
Vol 9 (6) ◽  
pp. 1235-1252 ◽  
Author(s):  
Owais Saifee ◽  
Liping Wei ◽  
Michael L. Nonet
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Acetylcholinesterase Inhibitor ◽  
Coiled Coil ◽  
Vesicle Fusion ◽  
Loss Of Function ◽  
Hydrophobic Membrane ◽  
Coiled Coil Domain ◽  
Synaptic Vesicle Fusion

We describe the molecular cloning and characterization of theunc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those ofDrosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

scholarly journals Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion

2017 ◽  
Vol 12 (9) ◽  
pp. 2014-2028 ◽  
Author(s):  
Xiaoxia Liu ◽  
Alpay Burak Seven ◽  
Junjie Xu ◽  
Victoria Esser ◽  
Lijing Su ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
In Vitro Reconstitution ◽  
Synaptic Vesicle Fusion

scholarly journals Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas A. Courtney ◽  
Huan Bao ◽  
Joseph S. Briguglio ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Major Focus ◽  
C2 Domains ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

scholarly journals Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

1973 ◽  
Vol 136 (3) ◽  
pp. 611-622 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Supernatant Fraction ◽  
Nuclear Fraction ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Ammonium Sulphate Fractionation ◽  
Stimulation Of

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5α-dihydrotestosterone–protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5α-dihydrotestosterone–receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5α-dihydrotestosterone–receptor complexes than is the ‘nucleoplasmic’ enzyme. Stimulation occurs less readily in the presence of Mn2+and at high ionic strength than in the presence of Mg2+and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5α-dihydrotestosterone–receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

scholarly journals Effects of α-amanitin on the stimulation of prostatic ribonucleic acid polymerase by prostatic steroid–protein receptor complexes

1974 ◽  
Vol 140 (3) ◽  
pp. 565-567 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Selective Inhibitor ◽  
High Ionic Strength ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Stimulation Of

Stimulation of prostatic RNA polymerase in vitro by prostatic 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone)–receptor complexes has been previously reported. By use of the selective inhibitor, α-amanitin, we have shown that both nucleolar and extranucleolar RNA polymerase activities may be stimulated, but stimulation is abolished at high ionic strength.

scholarly journals Morphine Antidependence ofErythroxylum cuneatum(Miq.) Kurz in Neurotransmission ProcessesIn Vitro

2016 ◽  
Vol 2016 ◽  
pp. 1-9
Author(s):  
Noor Azuin Suliman ◽  
Mohamad Aris Mohd Moklas ◽  
Che Norma Mat Taib ◽  
Mohd Ilham Adenan ◽  
Mohamad Taufik Hidayat Baharuldin ◽  
...  
Keyword(s):  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Human Neuroblastoma Cell ◽  
Chronic Morphine ◽  
Human Neuroblastoma ◽  
Opiate Abuse ◽  
Protein Receptor ◽  
Synaptotagmin 1 ◽  
And Control

Opiate abuse has been studied to cause adaptive changes observed in the presynaptic release and the mediated-synaptic plasticity proteins. The involvement of neuronal SNARE proteins reveals the role of the neurotransmitter release in expressing the opioid actions. The present study was designed to determine the effect of the alkaloid extract ofErythroxylum cuneatum(E. cuneatum) against chronic morphine and the influences ofE. cuneatumon neurotransmission processes observedin vitro. The human neuroblastoma cell line, SK-N-SH, was treated with the morphine, methadone, orE. cuneatum. The cell lysates were collected and tested forα-synuclein, calmodulin, vesicle-associated membrane protein 2 (VAMP 2), and synaptotagmin 1. The extract ofE. cuneatumwas observed to upregulate the decreased expression of dependence proteins, namely,α-synuclein and calmodulin. The effects were comparable to methadone and control. The expressions of VAMP 2 and synaptotagmin 1 were normalised by the plant and methadone. The extract ofE. cuneatumwas postulated to treat dependence symptoms after chronic morphine and improve the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein involved in synaptic vesicle after.

scholarly journals Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Ying Lai ◽  
Jiajie Diao ◽  
Daniel J Cipriano ◽  
Yunxiang Zhang ◽  
Richard A Pfuetzner ◽  
...  
Keyword(s):  
Lipid Content ◽  
Point Contact ◽  
Vesicle Fusion ◽  
Neuronal Cultures ◽  
Spontaneous Release ◽  
Terminal Domain ◽  
Synaptotagmin 1 ◽  
Free Membrane ◽  
Synaptic Vesicle Fusion ◽  
Single Vesicle

Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (<xref ref-type="bibr" rid="bib5">Diao et al., 2012</xref>). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.

scholarly journals Alpha-Synuclein and Calpains Disrupt SNARE-Mediated Synaptic Vesicle Fusion During Manganese Exposure in SH-SY5Y Cells

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 258 ◽  
Author(s):  
Can Wang ◽  
Zhuo Ma ◽  
Dong-Ying Yan ◽  
Chang Liu ◽  
Yu Deng ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Alpha Synuclein ◽  
Convincing Evidence ◽  
Snare Complex ◽  
Attachment Protein ◽  
Over Expression ◽  
Synaptic Vesicle Fusion

Synaptic vesicle fusion is mediated by an assembly of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs), composed of syntaxin 1, soluble NSF-attachment protein (SNAP)-25, and synaptobrevin-2/VAMP-2. Previous studies have suggested that over-exposure to manganese (Mn) could disrupt synaptic vesicle fusion by influencing SNARE complex formation, both in vitro and in vivo. However, the mechanisms underlying this effect remain unclear. Here we employed calpeptin, an inhibitor of calpains, along with a lentivirus vector containing alpha-synuclein (α-Syn) shRNA, to examine whether specific SNAP-25 cleavage and the over-expression of α-Syn disturbed the formation of the SNARE complex in SH-SY5Y cells. After cells were treated with Mn for 24 h, fragments of SNAP-25-N-terminal protein began to appear; however, this effect was reduced in the group of cells which were pre-treated with calpeptin. FM1-43-labeled synaptic vesicle fusion decreased with Mn treatment, which was consistent with the formation of SNARE complexes. The interaction of VAMP-2 and α-Syn increased significantly in normal cells in response to 100 μM Mn treatment, but decreased in LV-α-Syn shRNA cells treated with 100 μM Mn; similar results were observed in terms of the formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusion. Our data suggested that Mn treatment could increase [Ca2+]i, leading to abnormally excessive calpains activity, which disrupted the SNARE complex by cleaving SNAP-25. Our data also provided convincing evidence that Mn could induce the over-expression of α-Syn; when combined with VAMP-2, α-Syn prevented VAMP-2 from joining the SNARE complex cycle.

scholarly journals Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

2021 ◽  
Author(s):  
Manindra Bera ◽  
Sathish Ramakrishnan ◽  
Jeff Coleman ◽  
Shyam S Krishnakumar ◽  
James E Rothman
Keyword(s):  
Specific Interaction ◽  
Vesicle Fusion ◽  
Assembly Process ◽  
Inhibitory Function ◽  
Physiological Relevance ◽  
Vesicular Release ◽  
Molecular Determinants ◽  
Synaptotagmin 1 ◽  
Single Vesicle

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

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