2 edition of Functional importance of Munc18-1 and its interaction with syntaxin1 in PC12 cells. found in the catalog.
Functional importance of Munc18-1 and its interaction with syntaxin1 in PC12 cells.
Written in English
Munc18-1 is a 67kDa mammalian orthologue to unc18 in C.elegans , which was first identified as a syntaxin binding protein. Deletion of Munc18-1 results in severely hampered exocytosis in neurons and chromaffin cells. Utilising RNA interference, we have established PC12 cells in which Munc18-1 is stably knocked down by more than 90%. Our secretion assays revealed a 40-70% decrease in Ca-dependent vesicle exocytosis in the Munc18-1 Knockdown (KD) cells. In addition there was also a 40-50% decrease in the proportion of docked vesicles within Munc18-1 KD PC12 cells. Analysis of the localization of syntaxin1 in Munc18-1 KD cells reveals a significant intracellular mislocalization compared to controls. Such syntaxin1 mislocalization was also nearly completely rescued upon reintroduction of Munc18-1 into the Munc18-1 KD PC12 cells. This clearly supports our hypothesis that Munc18-1 acts as a molecular chaperone to syntaxin1 to facilitate the transport of syntaxin1 to the cell membrane in PC12 cells.
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Probing syntaxin 1 association with Munc, in real-time and in living cells, we found that modification of Ser14 modulated the dynamics of this interaction, specifically at the plasma membrane. The SM (Sec1/Munclike) protein Munc and the soluble N -ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc interacts with neuronal SNARE proteins in two .
Structure of the Munc/syntaxin-1 complex (Burkhardt et al., ) highlighting mutations used in this study. Munc is shown colored yellow to green (N to C terminus) and syntaxin-1 is. Munc is known to bind to syntaxin-1 and stabilize it in a closed conformation by which its N-terminal Habc domain is folded back on its SNARE domain thus preventing any interaction with VAMP.
Munc was originally described as an essential docking factor in chromaffin cells. Recent findings showed that Munc has an additional role in the regulation of the cortical F-actin network. Unlike overexpression of either Munc or SNAP in munc single-null cells, overexpression of these proteins in munc/synaptotagmin-1 double-null cells no longer rescued the docking phenotype (Figures 6A, 6C, S1, and S5). This indicates that synaptotagmin-1 is required for syntaxin-1/SNAPdependent docking and also excludes the.
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This Munc mutant loses its ability to bind and chaperone syntaxin-1 and to restore secretion in Munc/-2 double-knockdown PC12 cells (Han et al., ). A second binding mode is mediated by the interaction between the N-terminal peptide (residues 1–20) of syntaxin-1 and the hydrophobic pocket (residues –) in domain-1 of MuncCited by: The most convincing evidence for this interaction is the demonstration of fluorescence resonance energy transfer (FRET) between overexpressed Munc and syntaxin1 in live cells and its reduction by mutations (Liu et al., ).
However, it is not clear whether the alternative binding mode between syntaxin1 and Munc also would give rise to Cited by: Munc18–1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18–1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear.
Here we show that syntaxin 1 levels are reduced by 70% in munc18–1 knockout mice. Pulse Cited by: To determine the effect of the interaction of Munc18‐1 with Syntaxin 1 on its Dyrk1A‐mediated phosphorylation, GST–Syntaxin 1 ( μg), His‐Munc18‐1 ( μg), or both were pre‐incubated on ice for 30 min and then in vitro kinase assay with Dyrk1A was performed as described by: However, the residual syntaxin 1 in munc18–1 knockout mice is still correctly targeted to synap-ses and efﬁciently forms SDS-resistant SNARE complexes, demonstrating that Munc18–1 is not required for syntaxin 1 function as such.
These data demonstrate that the Munc18–1 interaction with syntaxin 1 is physiologically important, butCited by: Munc Is Critical for Plasma Membrane Localization of Syntaxin1 but Not of SNAP in PC12 Cells Article (PDF Available) in Molecular biology of the cell 19(2) March with 79 Reads.
Role of Cortical F-Actin in Regulated Secretion in Chromaffin Cells. In addition to the minimal docking machinery (syntaxin-1, SNAP, synaptotagmin-1, and Munc), the dense cortical network of filamentous actin (F-actin) underneath the plasma membrane was shown to mediate regulatory secretion in chromaffin cells (Aunis and Bader ; Trifaró et al.
To test if a functional syntaxin 1 N-peptide is required for the Muncmediated inhibition, a syntaxin 1 construct lacking the N-terminal 24 amino acids (d24) and a syntaxin 1 point mutation (L8A) impairing the interaction of Munc with the.
Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (“closed” vs.
“open”) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. In all cases, Munc rescued defective secretion of Munc/2 DKD cells significantly better than Munc (Fig. 3B). Interestingly, the recovery of the syntaxin-1 level was also higher in Munc rescue than that of Munc (Fig.
3A). Thus, our results confirm that functional specificity exists between the two isoforms in neuronal. using Munc null neurons, chromafﬁn cells and Munc/-2 knockdown PC12 cells, allowing remarkable progress to be made in the structural/functional understanding of Munc In this review, we summarize these recent advances and attempt to propose an updated model of the pleiotropic func-tions of Munc in neuroexocytosis.
Structure and Function Relationship of Syntaxin-1 in Neurotransmitter Release Seungmee Park Doctor of Philosophy Department of Physiology University of Toronto Abstract Regulated exocytosis is a cellular process that requires the calcium-dependent fusion of.
The Munc domain 3a – loop is not required for Sx1a transport to the plasma membrane We have previously shown that Sx1a transport to the cell surface is defective in DKD-PC12 cells (Han et al., ; Malintan et al., ). Most importantly, it was shown that the inability of various Munc mutants to mediate fusion and exocytosis.
The plasticity in syntaxin1 expression is an indication of the strong physiological significance of the functional interaction between Munc and syntaxin1. This establishes the functional comparability between our Munc knockdown PC12 model and the secretory cells from Munc knockout mice.
We attempted to identify the precise. Hence, these data exclude functional interplay between Syb2 (LR) and Munc, suggesting that the functional importance of the LR of Syb2 is related to its interaction with the MUN domain.
Fig. Article Munc and the Syntaxin-1 N Terminus Regulate Open-Closed States in a t-SNARE Complex Highlights d Munc18 stabilizes a syntaxin/SNAP25 complex d Syntaxin is ina closed state when associated withSNAP25 in the presence of Munc18 d The syntaxin N terminus regulates open and closed states in t-SNAREs d The syntaxin/SNAPMunc18 complex is a starting point for.
In addition, phosphorylation of syntaxin‐1 at Ser14, which inhibits the interaction between Munc18‐1 and syntaxin‐1 in neuroblastoma cells, dramatically decreases the neurosecretory response (Rickman and Duncan ). These results suggest that Munc18‐1 binding to syntaxin‐1 N‐terminus plays a critical role in SNARE‐mediated fusion.
(C–G) DKD-PC12 cells were transfected with control vector, Munc wild-type (wt)-emGFP, or MuncCY-emGFP for 48 hr, prior to incubation at 37°C or 32°C for 24 hr. (C) Cells incubated at 37°C were fixed, immunolabeled for the Golgi marker GM, and the nuclei stained using DAPI.
emGFP was imaged under identical conditions for cytosolic labeling (i and ii) or at low laser intensity. In Munc knockdown PC12 cells, syntaxin-1, but not the other target membrane (t)-SNARE, synaptosomal-associated protein (SNAP), is mislocalized and concentrated in the perinuclear region.
Syntaxin-1 mislocalization was rescued upon reexpression of wild-type Munc. In addition to the homotypic protein–protein interactions discussed earlier, more specific SNARE sorting mechanisms may exist, because overexpression of both syntaxin-1 and syntaxin-4 weakens the segregation of domains enriched in these SNAREs in the plasma membrane of PC12 cells (Sieber et al., ).
Specifically the N-terminal regulatory. Munc, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Mund binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear.Although Munc was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear.
In fact, recent studies of Munc mutants have suggested that Munc plays a critical role for docking of .Comparably, biochemical investigations on the interaction of Munc with the core SNARE machinery have provided data that are difﬁcult to reconcile.
Indisputable, however, is that Munc binds tightly to monomer Syntaxin1. Interestingly, this interaction involves two sites on opposing surfaces of.