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1.  Munc13-4 Is an Effector of Rab27a and Controls Secretion of Lysosomes in Hematopoietic Cells 
Molecular Biology of the Cell  2005;16(2):731-741.
Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Δ608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.
doi:10.1091/mbc.E04-10-0923
PMCID: PMC545907  PMID: 15548590
2.  Munc18c Depletion Selectively Impairs the Sustained Phase of Insulin Release 
Diabetes  2009;58(5):1165-1174.
OBJECTIVE
The Sec1/Munc18 protein Munc18c has been implicated in Syntaxin 4–mediated exocytosis events, although its purpose in exocytosis has remained elusive. Given that Syntaxin 4 functions in the second phase of glucose-stimulated insulin secretion (GSIS), we hypothesized that Munc18c would also be required and sought insight into the possible mechanism(s) using the islet β-cell as a model system.
RESEARCH DESIGN AND METHODS
Perifusion analyses of isolated Munc18c- (−/+) or Munc18c-depleted (RNAi) mouse islets were used to assess biphasic secretion. Protein interaction studies used subcellular fractions and detergent lysates prepared from MIN6 β-cells to determine the mechanistic role of Munc18c in Syntaxin 4 activation and docking/fusion of vesicle-associated membrane protein (VAMP)2-containing insulin granules. Electron microscopy was used to gauge changes in granule localization.
RESULTS
Munc18c (−/+) islets secreted ∼60% less insulin selectively during second-phase GSIS; RNAi-mediated Munc18c depletion functionally recapitulated this in wild-type and Munc18c (−/+) islets in a gene dosage-dependent manner. Munc18c depletion ablated the glucose-stimulated VAMP2–Syntaxin 4 association as well as Syntaxin 4 activation, correlating with the deficit in insulin release. Remarkably, Munc18c depletion resulted in aberrant granule localization to the plasma membrane in response to glucose stimulation, consistent with its selective effect on the second phase of secretion.
CONCLUSIONS
Collectively, these studies demonstrate an essential positive role for Munc18c in second-phase GSIS and suggest novel roles for Munc18c in granule localization to the plasma membrane as well as in triggering Syntaxin 4 accessibility to VAMP2 at a step preceding vesicle docking/fusion.
doi:10.2337/db08-1059
PMCID: PMC2671048  PMID: 19188424
3.  Munc13-4*rab27 complex tethers secretory lysosomes at the plasma membrane 
Natural Killer (NK) cells and Cytotoxic T lymphocytes (CTL) are critical for the immune response against virus infections or transformed cells. They kill target cells via polarized exocytosis of lytic proteins from secretory lysosomes (SL). Rab27a and munc13-4 interact directly and are required for target cell killing. How they cooperate in the intricate degranulation process is not known. We identified critical residues in munc13-4 for rab27 interaction and tested binding mutants in several complementation assays. In a rat mast cell line we replaced endogenous munc13-4 with ectopically expressed munc13-4 constructs. Unlike wild type munc13-4, binding mutants fail to rescue β-hexosaminidase secretion. In accord, expression of binding mutants in CTL of Familial Hemophagocytic Lymphohistiocytosis type 3 patients, does not rescue CD107 appearance on the plasma membrane. Total Internal Reflection Fluorescence (TIRF) imaging shows that munc13-4*rab27a restricts motility of SL in the subapical cytoplasm. We propose that rab27*munc13-4 tethers SL to the plasma membrane, a requirement for formation of a cognate SNARE complex for fusion.
PMCID: PMC3291317  PMID: 22482013
cytotoxic T cell; Degranulation; mast cell; munc13-4; rab27; secretory lysosomes; tethering
4.  Regulated Mucin Secretion from Airway Epithelial Cells 
Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 106 Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.
doi:10.3389/fendo.2013.00129
PMCID: PMC3776272  PMID: 24065956
secretion; exocytosis; mucin; mucus; MARCKS; Munc18; Munc13; synaptotagmin
5.  Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding 
PLoS Computational Biology  2011;7(3):e1001097.
The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.
Author Summary
Protein phosphorylation plays a significant regulatory role in multi-component systems engaged in signal transduction or coordination of cellular processes, by activating or deactivating proteins. The potential of phosphorylation to induce substantial conformational changes in proteins, thereby changing their affinity to target proteins, has already been shown but the dynamics of the process is not fully elucidated. In the present study, we investigated, by molecular dynamics simulations, the dynamic conformational changes in munc18a, a protein that is crucial for neurotransmitter release and interacts tightly with the SNARE syntaxin-1. We further investigated the conformational changes that occur in munc18a when it is phosphorylated, reducing its affinity to syntaxin-1a. The results of the simulations suggest that there is a conformational flexibility of the syntaxin-unbounded munc18a that allows changes in the shape of the syntaxin-1a binding cavity. In silico insertion of phosphomimetic mutations into munc18a led to a reduction in the flexibility and closure of the syntaxin-binding site. We suggest that the reduced affinity of phosphorylated munc18a to syntaxin-1a stems from the difficulty of syntaxin-1a to bind to the munc18a closed-cavity conformation, induced by the PKC phosphorylation of munc18a.
doi:10.1371/journal.pcbi.1001097
PMCID: PMC3048386  PMID: 21390273
6.  Advances in Mucous Cell Metaplasia 
Mucous cell metaplasia is induced in response to harmful insults and provides front-line protection to clear the airway of toxic substances and cellular debris. In chronic airway diseases mucous metaplasia persists and results in airway obstruction and contributes significantly to morbidity and mortality. Mucus hypersecretion involves increased expression of mucin genes, and increased mucin production and release. The past decade has seen significant advances in our understanding of the molecular mechanisms by which these events occur. Inflammation stimulates epidermal growth factor receptor activation and IL-13 to induce both Clara and ciliated cells to transition into goblet cells through the coordinated actions of FoxA2, TTF-1, SPDEF, and GABAAR. Ultimately, these steps lead to up-regulation of MUC5AC expression, and increased mucin in goblet cell granules that fuse to the plasma membrane through actions of MARCKS, SNAREs, and Munc proteins. Blockade of mucus in exacerbations of asthma and chronic obstructive pulmonary disease may affect morbidity. Development of new therapies to target mucus production and secretion are now possible given the advances in our understanding of molecular mechanisms of mucous metaplasia. We now have a greater incentive to focus on inhibition of mucus as a therapy for chronic airway diseases.
doi:10.1165/rcmb.2009-0151TR
PMCID: PMC2830403  PMID: 19520914
mucus; goblet cell; airway epithelium; asthma; COPD
7.  Munc18-2 and Syntaxin 3 control distinct essential steps in mast cell degranulation1 
Mast cell degranulation requires N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)6 and mammalian unc18 (Munc18) fusion accessory proteins for membrane fusion. However, it is still unknown how their interaction supports fusion. Here we found that siRNA-mediated silencing of the isoform Munc18-2 in mast cells inhibits cytoplasmic secretory granule (SG) release but not CCL2 chemokine secretion. Silencing of its SNARE binding partner Syntaxin 3 (STX3) also markedly inhibited degranulation, while combined knock-down produced an additive inhibitory effect. Strikingly, while Munc18-2 silencing impaired SG translocation, silencing of STX3 inhibited fusion demonstrating unique roles of each protein. Immunogold studies showed that both Munc18-2 and STX3 are located on the granule surface, but also within the granule matrix and in small nocodazole-sensitive clusters of the cytoskeletal meshwork surrounding SG. After stimulation clusters containing both effectors were detected at fusion sites. In resting cells, Munc18-2, but not STX3, interacted with tubulin. This interaction was sensitive to nocodazole treatment and decreased after stimulation. Our results indicate that Munc18-2 dynamically couples the membrane fusion machinery to the microtubule cytoskeleton and demonstrate that Munc18-2 and STX3 perform distinct, but complementary, functions to support, respectively, SG translocation and membrane fusion in mast cells.
doi:10.4049/jimmunol.1301277
PMCID: PMC3905451  PMID: 24323579
8.  Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner 
The Journal of Cell Biology  2014;204(5):759-775.
PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.
Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.
doi:10.1083/jcb.201308026
PMCID: PMC3941046  PMID: 24590174
9.  The presynaptic Munc13-1 binds alcohol and modulates alcohol self-administration in Drosophila 
Journal of neurochemistry  2013;126(6):715-726.
Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and involved in presynaptic plasticity in brain. Ethanol, butanol and octanol quenched the intrinsic fluorescence of the C1 domain of Munc13-1 with EC50s of 52 mM, 26 mM and 0.7 mM, respectively. Photoactive azialcohols photolabeled Munc13-1 C1 exclusively at Glu-582, which was identified by mass spectrometry. Mutation of Glu-582 to alanine, leucine and histidine reduced the alcohol binding two- to five-fold. Circular dichroism studies suggested that binding of alcohol increased the stability of the wild type Munc13-1 compared with the mutants. If Munc13-1 plays some role in the neural effects of alcohol in vivo, changes in the activity of this protein should produce differences in the behavioral responses to ethanol. We tested this prediction with a loss-of-function mutation in the conserved Dunc-13 in Drosophila melanogaster. The Dunc-13P84200/+ heterozygotes have 50% wild type levels of Dunc-13 mRNA and display a very robust increase in ethanol self-administration. This phenotype is reversed by the expression of the rat Munc13-1 protein within the Drosophila nervous system. The present studies indicate that Munc13-1 C1 has binding site(s) for alcohols and Munc13-1 activity is sufficient to restore normal self-administration to Drosophila mutants deficient in Dunc-13 activity.
doi:10.1111/jnc.12315
PMCID: PMC3766433  PMID: 23692447
Munc13-1; presynaptic; protein kinase C; alcohol; diazirine; circular dichroism; photolabeling; mass spectrometry; fluorescence; homology model; alcohol self-administration; Drosophila
10.  Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells 
Molecular Biology of the Cell  2009;20(23):4962-4975.
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.
doi:10.1091/mbc.E09-08-0712
PMCID: PMC2785739  PMID: 19812250
11.  Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion 
The Journal of Cell Biology  2012;197(2):301-312.
Munc13-4 is a Ca2+-dependent membrane- and SNARE-binding protein that promotes membrane fusion.
Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis.
doi:10.1083/jcb.201109132
PMCID: PMC3328385  PMID: 22508512
12.  Munc18b Is a Major Mediator of Insulin Exocytosis in Rat Pancreatic β-Cells 
Diabetes  2013;62(7):2416-2428.
Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic β-cells to exocytose insulin becomes compromised in diabetes. β-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted β-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6–8 SGs) not normally (2–3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of β-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.
doi:10.2337/db12-1380
PMCID: PMC3712044  PMID: 23423569
13.  WNK1 Is a Novel Regulator of Munc18c-Syntaxin 4 Complex Formation in Soluble NSF Attachment Protein Receptor (SNARE)-mediated Vesicle Exocytosis* 
The Journal of biological chemistry  2007;282(45):32613-32622.
Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159–491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.
doi:10.1074/jbc.M706591200
PMCID: PMC2423411  PMID: 17848561
14.  Milligram Quantities of Homogeneous Recombinant Full-Length Mouse Munc18c from Escherichia coli Cultures 
PLoS ONE  2013;8(12):e83499.
Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1–2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.
doi:10.1371/journal.pone.0083499
PMCID: PMC3877047  PMID: 24391775
15.  Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding 
Background
Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1.
Results
Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα.
Conclusion
Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.
doi:10.1186/1750-1326-2-23
PMCID: PMC2211485  PMID: 18067682
16.  Mucus hypersecretion in asthma: causes and effects 
Purpose of review
Airway mucus plugging has long been recognized as a principal cause of death in asthma. However, molecular mechanisms of mucin overproduction and secretion have not been understood until recently. These mechanisms are reviewed together with ongoing investigations relating them to lung pathophysiology.
Recent findings
Of the five secreted gel-forming mucins in mammals, only MUC5AC and MUC5B are produced in significant quantities in intrapulmonary airways. MUC5B is the principal gel-forming mucin at baseline in small airways of humans and mice, and therefore likely performs most homeostatic clearance functions. MUC5AC is the principal gel-forming mucin upregulated in airway inflammation and is under negative control by forkhead box a2 and positive control by hypoxia inducible factor-1. Mucin secretion is regulated separately from production, principally by extracellular triphosphate nucleotides that bind P2Y2 receptors on the lumenal surface of airway secretory cells, generating intracellular second messengers that activate the exocytic proteins, Munc13-2 and synaptotagmin-2.
Summary
Markedly upregulated production of MUC5AC together with stimulated secretion leads to airflow obstruction in asthma. As MUC5B appears to mediate homeostatic functions, it may be possible to selectively inhibit MUC5AC production without impairing airway function. The precise roles of mucin hypersecretion in asthma symptoms such as dyspnea and cough and in physiologic phenomena such as airway hyperresponsiveness remain to be defined.
doi:10.1097/MCP.0b013e32831da8d3
PMCID: PMC2709596  PMID: 19077699
airway; asthma; mucin; mucous; mucus
17.  Munc18-1 Is Critical for Plasma Membrane Localization of Syntaxin1 but Not of SNAP-25 in PC12 Cells 
Molecular Biology of the Cell  2008;19(2):722-734.
Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.
doi:10.1091/mbc.E07-07-0662
PMCID: PMC2230596  PMID: 18077557
18.  Munc18 and Munc13 regulate early neurite outgrowth 
Biology of the Cell  2010;102(Pt 8):479-488.
Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth.
Results. We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1–4 (day in vitro 1–4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14–23).
Conclusion. These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.
doi:10.1042/BC20100036
PMCID: PMC2895782  PMID: 20497124
development; growth cone; Munc18; neurite outgrowth; organotypic cultures; vesicle release; AA, arachidonic acid; Arp2/3 complex, actin-related protein 2/3 complex; CDC42, cell division cycle 42; dGBSS, dissection Gey's balanced salt solution; DIV, day in vitro; E18, embryonic day 18; EGFP, enhanced green fluorescent protein; HBSS, Hanks balanced salt solution; M13, Munc13-1 and Munc13-2; M18, Munc18-1; MAP2, microtubuleassociated protein 2; NA, numerical aperture; PFA, paraformaldehyde; PLA, phospholipase A2; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor; WT, wild-type
19.  Munc18-1 domain-1 controls vesicle docking and secretion by interacting with syntaxin-1 and chaperoning it to the plasma membrane 
Molecular Biology of the Cell  2011;22(21):4134-4149.
Striking correlations exist between the abilities of domain-1 cleft mutants of Munc18-1 to bind and chaperone syntaxin-1 and their ability to restore vesicle docking and secretion.
Munc18-1 plays pleiotropic roles in neurosecretion by acting as 1) a molecular chaperone of syntaxin-1, 2) a mediator of dense-core vesicle docking, and 3) a priming factor for soluble N-ethylmaleimide–sensitive factor attachment protein receptor–mediated membrane fusion. However, how these functions are executed and whether they are correlated remains unclear. Here we analyzed the role of the domain-1 cleft of Munc18-1 by measuring the abilities of various mutants (D34N, D34N/M38V, K46E, E59K, K46E/E59K, K63E, and E66A) to bind and chaperone syntaxin-1 and to restore the docking and secretion of dense-core vesicles in Munc18-1/-2 double-knockdown cells. We identified striking correlations between the abilities of these mutants to bind and chaperone syntaxin-1 with their ability to restore vesicle docking and secretion. These results suggest that the domain-1 cleft of Munc18-1 is essential for binding to syntaxin-1 and thereby critical for its chaperoning, docking, and secretory functions. Our results demonstrate that the effect of the alleged priming mutants (E59K, D34N/M38V) on exocytosis can largely be explained by their reduced syntaxin-1–chaperoning functions. Finally, our data suggest that the intracellular expression and distribution of syntaxin-1 determines the level of dense-core vesicle docking.
doi:10.1091/mbc.E11-02-0135
PMCID: PMC3204074  PMID: 21900502
20.  Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release 
eLife  2014;3:e01715.
Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP.
DOI: http://dx.doi.org/10.7554/eLife.01715.001
eLife digest
Brain function depends on the rapid transfer of information from one brain cell to the next at junctions known as synapses. Small packages called vesicles play an important role in this process. The arrival of an electrical action potential at the nerve terminal of the first cell causes some vesicles in the cell to fuse with the cell membrane, and this leads to the neurotransmitters inside the vesicles being released into the synapse. The neurotransmitters then bind to receptors on the second cell, which leads to an electrical signal in the second cell. A protein called Munc18-1 has a central role in the fusion of the vesicle at the cell membrane.
The strength of a synapse—that is, how easily the first brain cell can impact the electrical behaviour of the second—can change, and this ‘synaptic plasticity’ is thought to underlie learning and memory. Long-term changes in synaptic strength require additional receptors to be inserted into the membrane of the second cell. However, synapses can also be temporarily strengthened: the arrival of a burst of action potentials—a tetanus—causes some synapses to increase the amount of neurotransmitter they release in response to any subsequent, single, action potential.
This temporary increase in synaptic strength, which is known as post-tetanic potentiation, requires an enzyme called protein kinase C; the role of this enzyme is to phosphorylate specific target proteins (i.e., to add phosphate groups to them). Now, Genç et al. have genetically modified a mouse synapse in vivo and shown that protein kinase C brings about post-tetanic potentiation by phosphorylating Munc18-1. Furthermore, pharmacological experiments show that proteins called phosphatases, which de-phosphorylate proteins, normally terminate the post-tetanic potentiation after about one minute. Taken together, the study identifies a target protein which is phosphorylated by protein kinase C during post-tetanic potentiation. The study also suggests that in addition to its fundamental role in vesicle fusion, the phosphorylation state of Munc18-1 can change the probability of vesicle fusion in a more subtle way, thereby contributing to synaptic plasticity.
DOI: http://dx.doi.org/10.7554/eLife.01715.002
doi:10.7554/eLife.01715
PMCID: PMC3919271  PMID: 24520164
synaptic plasticity; transmitter release; protein kinase C; protein phosphatase; calyx of Held; synaptic transmission; mouse; rat
21.  Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents 
Journal of Clinical Investigation  2000;105(3):377-386.
The CFTR Cl– channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl– currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl– currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II–activated Cl– currents in these cells.
PMCID: PMC377449  PMID: 10675364
22.  Aberrant Morphology and Residual Transmitter Release at the Munc13-Deficient Mouse Neuromuscular Synapse†  
Molecular and Cellular Biology  2005;25(14):5973-5984.
In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and γ-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.
doi:10.1128/MCB.25.14.5973-5984.2005
PMCID: PMC1168806  PMID: 15988013
23.  Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites* 
The Journal of Biological Chemistry  2012;288(7):5102-5113.
Background: The molecular architecture of the secretory machinery is undefined.
Results: Munc18 moves between membrane depots distinct from vesicle docking sites.
Conclusion: Munc18 is not a docking factor, and the membrane environment is likely to determine fusion likelihood.
Significance: It is now possible to test directly previous models of the molecular mechanisms of secretion.
Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.
doi:10.1074/jbc.M112.407585
PMCID: PMC3576115  PMID: 23223447
Membrane Biophysics; Membrane Function; Membrane Proteins; Membrane Trafficking; Vesicles
24.  Munc13-1 Translocates to the Plasma Membrane in a Doc2B- and Calcium-Dependent Manner 
Munc13-1 is a presynaptic protein activated by calcium, calmodulin, and diacylglycerols (DAG) that is known to enhance vesicle priming. Doc2B is another presynaptic protein that translocates to the plasma membrane (PM) upon elevation of internal calcium concentration ([Ca2+]i) to the submicromolar range, and increases both spontaneous and asynchronous release in a calcium-dependent manner. We speculated that Doc2B also recruits Munc13-1 to the PM since these two proteins have been shown to interact physiologically and this interaction is enhanced by Ca2+. However, this calcium-dependent co-translocation has never actually been shown. To examine this possibility, we expressed both proteins tagged to fluorescent proteins in PC12 cells and stimulated the cells to investigate the recruitment hypothesis using imaging techniques. We found that Munc13-1 does indeed translocate to the PM upon elevation in [Ca2+]i, but only when co-expressed with Doc2B. Interestingly, Munc13-1 co-translocates at a slower rate than Doc2B. Moreover, while Doc2B dislocates from the PM as soon as the [Ca2+]i returns to basal levels, Munc13-1 dislocates at a slower rate and a fraction of it accumulates on the PM. This accumulation is more pronounced under subsequent stimulations, suggesting that Munc13-1 accumulation builds up as some other factors accumulate at the PM. Munc13-1 co-translocation and accumulation was reduced when its mutant Munc13-1H567K, which is unable to bind DAG, was co-expressed with Doc2B, suggesting that Munc13-1 accumulation depends on DAG levels. These results suggest that Doc2B enables recruitment of Munc13-1 to the PM in a [Ca2+]i-dependent manner and offers another possible Munc13-1-regulatory mechanism that is both calcium- and Doc2B-dependent.
doi:10.3389/fendo.2013.00119
PMCID: PMC3775473  PMID: 24062723
Munc13; Doc2B; calcium; translocation; phorbol ester
25.  A Mechanism of Munc18b-Syntaxin 3-SANP25 complex assembly in regulated epithelial secretion 
FEBS letters  2007;581(22):4318-4324.
Syntaxin and Munc18 are essential for regulated exocytosis in all eukaryotes. It was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by CDK5 phosphorylation, indicating that structural change disrupts the syntaxin-Munc18 interaction. Here we show that this phosphorylation promotes the assembly of Munc18b-Syntaxin 3-SNAP25 tripartite complex and membrane fusion machinery SNARE. Using siRNAs to screen for genes required for regulated epithelial secretion, we identified the requirements of CDK5 and Munc18b in cAMP-dependent gastric acid secretion. Biochemical characterization revealed that Munc18b bears a syntaxin 3-selective binding site located at its most C-terminal 53 amino acids. Significantly, the phosphorylation of Thr572 by CDK5 attenuates Munc18b-Syntaxin 3 interaction and promotes formation of Munc18b-Syntaxin 3-SNAP25 tripartite complex, leading to an assembly of functional Munc18b-Syntaxin 3-SNAP25-VAMP2 membrane fusion machinery. Thus, our studies suggest a novel regulatory mechanism in which phosphorylation of Munc18b operates vesicle docking and fusion in regulated exocytosis.
doi:10.1016/j.febslet.2007.07.083
PMCID: PMC3690314  PMID: 17716669
SNARE; syntaxin 3; Munc18b; CDK5; exocytosis

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