Dynamic re-organisation of Munc18-1 and syntaxin-1A nanodomains on the plasma membrane of neurosecretory cells during exocytosis

2019 ◽  
Author(s):  
Ravi Kiran Kasula
Keyword(s):  
Plasma Membrane ◽  
Neurosecretory Cells ◽  
Syntaxin 1A

scholarly journals Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1

1999 ◽  
Vol 112 (12) ◽  
pp. 1865-1877 ◽  
Author(s):  
J. Rowe ◽  
N. Corradi ◽  
M.L. Malosio ◽  
E. Taverna ◽  
P. Halban ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Transport ◽  
Golgi Complex ◽  
Protein Transport ◽  
Neurosecretory Cells ◽  
Chromogranin B ◽  
Syntaxin 1A ◽  
Endogenous Expression ◽  
Intracellular Membrane Transport

The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.

Mechanisms of CFTR regulation by syntaxin 1A and PKA

2002 ◽  
Vol 115 (4) ◽  
pp. 783-791 ◽  
Author(s):  
Steven Y. Chang ◽  
Anke Di ◽  
Anjaparavanda P. Naren ◽  
H. Clive Palfrey ◽  
Kevin L. Kirk ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Membrane Trafficking ◽  
Anion Channel ◽  
Cell Types ◽  
Snare Proteins ◽  
Snare Protein ◽  
Membrane Turnover ◽  
Open Probability ◽  
Syntaxin 1A

Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl- currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues:(a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or syntaxin 1A association.

Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting

2001 ◽  
Vol 357 (3) ◽  
pp. 625-634 ◽  
Author(s):  
Philip WASHBOURNE ◽  
Victor CANSINO ◽  
James R. MATHEWS ◽  
Margaret GRAHAM ◽  
Robert D. BURGOYNE ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Snare Complex ◽  
Cysteine Residues ◽  
Cell Secretion ◽  
Transport Pathway ◽  
Syntaxin 1A ◽  
Golgi Transport ◽  
Membrane Target

The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7S and 20S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.

scholarly journals Nanobodies reveal an extra-synaptic population of SNAP-25 and Syntaxin 1A in hippocampal neurons

2018 ◽  
Author(s):  
Manuel Maidorn ◽  
Aurélien Olichon ◽  
Silvio O. Rizzoli ◽  
Felipe Opazo
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Hippocampal Neurons ◽  
Cellular Localization ◽  
Super Resolution ◽  
Syntaxin 1A ◽  
Resolution Imaging ◽  
Single Domain Antibodies ◽  
The Subject ◽  
Synaptic Vesicle Fusion

AbstractSynaptic vesicle fusion (exocytosis) is a precisely regulated process that entails the formation of SNARE complexes between the vesicle protein synaptobrevin 2 (VAMP2) and the plasma membrane proteins Syntaxin 1 and SNAP-25. The sub-cellular localization of the latter two molecules remains unclear, although they have been the subject of many recent investigations. To address this, we generated two novel camelid single domain antibodies (nanobodies) specifically binding to SNAP-25 and Syntaxin 1A. These probes penetrated more easily into samples and detected their targets more efficiently than conventional antibodies in crowded regions. When investigated by super-resolution imaging, the nanobodies revealed substantial extra-synaptic populations for both SNAP-25 and Syntaxin 1A, which were poorly detected by antibodies. Moreover, extra-synaptic Syntaxin 1A molecules were recruited to synapses during stimulation, suggesting that these are physiologically-active molecules. We conclude that nanobodies are able to reveal qualitatively and quantitatively different organization patterns, when compared to conventional antibodies.

scholarly journals Novel isoform of syntaxin 1 is expressed in mammalian cells

1997 ◽  
Vol 321 (1) ◽  
pp. 151-156 ◽  
Author(s):  
Mittur N. JAGADISH ◽  
Judy T. TELLAM ◽  
S. Lance MACAULAY ◽  
Keith H. GOUGH ◽  
David E. JAMES ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cdna Clone ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Terminal Region ◽  
Receptor Protein ◽  
Fat Cell ◽  
Syntaxin 1A ◽  
Truncated Protein

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, α-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and -18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immunoprecipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.

scholarly journals The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a Complex

2006 ◽  
Vol 17 (5) ◽  
pp. 2101-2112 ◽  
Author(s):  
Takashi Tsuboi ◽  
Mitsunori Fukuda
Keyword(s):  
Plasma Membrane ◽  
Specific Interaction ◽  
Inhibitory Effect ◽  
Neuroendocrine Cells ◽  
Dense Core ◽  
Dense Core Vesicle ◽  
Dense Core Vesicles ◽  
Syntaxin 1A ◽  
Vesicle Exocytosis ◽  
Linker Domain

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl–induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1·syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.

scholarly journals The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

2016 ◽  
Vol 214 (7) ◽  
pp. 847-858 ◽  
Author(s):  
Ravikiran Kasula ◽  
Ye Jin Chai ◽  
Adekunle T. Bademosi ◽  
Callista B. Harper ◽  
Rachel S. Gormal ◽  
...  
Keyword(s):  
Single Molecule ◽  
Botulinum Neurotoxin ◽  
Secretory Vesicle ◽  
Neurosecretory Cells ◽  
Snare Complex ◽  
Secretory Vesicles ◽  
Deficient Mutant ◽  
Syntaxin 1A ◽  
Single Molecule Analysis ◽  
Snare Complex Formation

Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming.

scholarly journals The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE-interactions

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jasmin Mertins ◽  
Jérôme Finke ◽  
Ricarda Antonia Sies ◽  
Kerstin Rink ◽  
Jan Hasenauer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Secretory Pathway ◽  
Snare Proteins ◽  
Strong Interactions ◽  
Dependent Manner ◽  
Syntaxin 1A ◽  
Membrane Area ◽  
Cellular Processes ◽  
Domain Interactions

SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE-domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin-clusters in a SNARE-domain dependent manner. Our data suggests that SNARE-domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose this mechanisms preserves active syntaxin 1A-SNAP25 complexes at the plasma membrane.

scholarly journals Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells

2009 ◽  
Vol 20 (23) ◽  
pp. 4962-4975 ◽  
Author(s):  
Liping Han ◽  
Tiandan Jiang ◽  
Gayoung A. Han ◽  
Nancy T. Malintan ◽  
Li Xie ◽  
...  
Keyword(s):  
Binding Mode ◽  
Neurosecretory Cells ◽  
Attachment Protein ◽  
Syntaxin 1A ◽  
Stimulatory Action ◽  
Regulated Secretion ◽  
Closed Conformation ◽  
Protein Receptor ◽  
N Terminus ◽  
Sensitive Factor

Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the “closed” conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

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