Role of syntaxin3 an apical polarity protein in poorly polarized keratinocytes: regulation of asymmetric barrier formations in the skin epidermis.

The skin epidermis exhibits an asymmetric structure composed of multilayered keratinocytes and those in the outer layers form two-way physical barriers, cornified cell envelope (CCE), and tight junctions (TJs). While undifferentiated keratinocytes in the basal layer continuously deliver daughter cells outward, which undergo successive differentiation with losing their polarized characteristics, they retain the expression of several polarity proteins. In the present study, we revealed that the t-SNARE protein syntaxin3, a critical element for the formation of the apical compartment in simple epithelial cells, is required to confer the ability to organize the physical barriers on "poorly polarized" keratinocytes in epidermal outer layers. HaCaT keratinocytes with genetic ablation of syntaxin3 readily succumbed to hydrogen peroxide-induced cell death. Additionally, they lost the ability to organize TJ and CCE structures, accompanied by notable downregulation of transglutaminase1 and caspase14 (a cornification regulator) expression. These syntaxin3-knockout cells appeared to restore oxidative stress tolerance and functional TJ formation ability, in response to the inducible re-expression of exogenous syntaxin3. While plausible mechanisms underlying these phenomena remain unclear, syntaxin3, an apical polarity protein in the simple epithelia, has emerged as a potentially crucial element for barrier formation in poorly polarized keratinocytes in polarized epidermal tissue.

Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3f/f(iCre75) and Stx3f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Syntaxin 3 interacts with serotonin transporter and regulates its function.

Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.

Kv2.1 clusters on ß-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

MYO5B, STX3, and STXBP2 mutations reveal a common disease mechanism that unifies a subset of congenital diarrheal disorders: A mutation update.

Microvillus inclusion disease (MVID) is a rare but fatal autosomal recessive congenital diarrheal disorder caused by MYO5B mutations. In 2013, we launched an open-access registry for MVID patients and their MYO5B mutations (www.mvid-central.org). Since then, additional unique MYO5B mutations have been identified in MVID patients, but also in non-MVID patients. Animal models have been generated that formally prove the causality between MYO5B and MVID. Importantly, mutations in two other genes, STXBP2 and STX3, have since been associated with variants of MVID, shedding new light on the pathogenesis of this congenital diarrheal disorder. Here, we review these additional genes and their mutations. Furthermore, we discuss recent data from cell studies that indicate that the three genes are functionally linked and, therefore, may constitute a common disease mechanism that unifies a subset of phenotypically linked congenital diarrheal disorders. We present new data based on patient material to support this. To congregate existing and future information on MVID geno-/phenotypes, we have updated and expanded the MVID registry to include all currently known MVID-associated gene mutations, their demonstrated or predicted functional consequences, and associated clinical information.

Apical Membrane Alterations in Non-intestinal Organs in Microvillus Inclusion Disease.

OBJECTIVES: Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation. METHODS: We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking. RESULTS: Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia. CONCLUSIONS: These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.

Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

Molecular basis for the interaction of the mammalian amino acid transporters B0AT1 and B0AT3 with their ancillary protein collectrin.

Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B(0)AT1/3 fusion constructs, we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B(0)AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.

Loss of syntaxin 3 causes variant microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a disorder of intestinal epithelial differentiation characterized by life-threatening intractable diarrhea. MVID can be diagnosed based on loss of microvilli, microvillus inclusions, and accumulation of subapical vesicles. Most patients with MVID have mutations in myosin Vb that cause defects in recycling of apical vesicles. Whole-exome sequencing of DNA from patients with variant MVID showed homozygous truncating mutations in syntaxin 3 (STX3). STX3 is an apical receptor involved in membrane fusion of apical vesicles in enterocytes. Patient-derived organoid cultures and overexpression of truncated STX3 in Caco-2 cells recapitulated most characteristics of variant MVID. We conclude that loss of STX3 function causes variant MVID.

Effluent syntaxin3 from dying cells affords protection against apoptosis in epidermal keratinocytes.

Ultra-violet B (UVB)-induced oxidative stress crucially perturbs the epidermal homeostasis, and the skin is endowed with protective mechanisms to take action against such damage. Here, we show the possible involvement of t-SNARE protein syntaxin3, a membrane fusion mediator of cytoplasmic vesicles, and which is released from dying keratinocytes, to play a role in this response. UVB irradiation, which generates reactive oxidative stress in cells, was shown to lead to the keratinocyte cell death accompanied by a release of cytoplasmic syntaxin3. We found that such extracellularly sourced syntaxin3 completely blocked the processing of a crucial effector for apoptotic cell death, caspase-3, and thus facilitated the survival of keratinocytes damaged by oxidative stress. These results demonstrate the latent prosurvival function of syntaxin3 and underline the importance of intracellular molecular elements for the maintenance of homeostasis in epidermal keratinocytes.

Increased STX3 transcript and protein levels were associated with poor prognosis in two independent cohorts of esophageal squamous cell carcinoma patients.

BACKGROUND: Some conventional prognostic biomarkers for esophageal squamous cell carcinoma (ESCC) have the disadvantage that they have only been investigated at the level of either mRNA or protein levels or only in individual cohorts. Associations between Syntaxin 3 (STX3) expression and malignancy have been reported in several tumor types but not in ESCC. Here, we investigated the levels of both STX3 mRNA and protein, and its prognostic potential in two independent cohorts of patients with ESCC. METHODS: STX3 mRNA levels were examined in surgical specimens by quantitative PCR in a cohort that included 176 ESCC patients. STX3 protein levels were investigated in surgically resected ESCC tissues by immunohistochemistry using tissue microarrays in a different cohort of 177 ESCC patients. Correlations were analyzed between the expression of STX3 mRNA and protein with clinicopathological factors and long-term prognosis. RESULTS: Quantitative PCR indicated a significant association between high level of STX3 mRNA expression and lymph node involvement, pathological stage, and poor overall survival. The multivariate analysis demonstrated that high STX3 mRNA expression was independently associated with poor overall survival outcomes. Immunohistochemistry revealed that STX3 protein expression in ESCC tissues and high STX3 protein expression were also significantly correlated with unfavorable overall survival. CONCLUSIONS: Overexpression of STX3 mRNA and protein may serve as potential prognostic biomarkers for ESCC patients.

Syntaxin 3 is haplosufficient for long-term photoreceptor survival in the mouse retina.

Biallelic loss-of-function mutations in the syntaxin 3 gene have been linked to a severe retinal dystrophy in humans that presents in early childhood. In mouse models, biallelic inactivation of the syntaxin 3 gene in photoreceptors rapidly leads to their death. What is not known is whether a monoallelic syntaxin 3 loss-of-function mutation might cause photoreceptor loss with advancing age. To address this question, we compared the outer nuclear layer of older adult mice (≈ 20 months of age) that were heterozygous for syntaxin 3 with those of similarly-aged control mice. We found that the photoreceptor layer maintains its thickness in mice that are heterozygous for syntaxin 3 relative to controls and that photoreceptor somatic counts are comparable. In addition, dendritic sprouting of the rod bipolar cell dendrites into the outer nuclear layer, which occurs following the loss of functional rod targets, was similar between genotypes. Thus, syntaxin 3 appears to be haplosufficient for photoreceptor survival, even with advancing age.

Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain.

SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A's JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A's JMD; and (3) palmitoylation deficiency mutations in STX1A's TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca2+-evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A's JMD regulates the palmitoylation of STX1A's TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A's JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.

Advanced Microscopy for Liver and Gut Ultrastructural Pathology in Patients with MVID and PFIC Caused by MYO5B Mutations.

Mutations in the actin motor protein myosinVb (myo5b) cause aberrant apical cargo transport and the congenital enteropathy microvillus inclusion disease (MVID). Recently, missense mutations in myo5b were also associated with progressive familial intrahepatic cholestasis (MYO5B-PFIC). Here, we thoroughly characterized the ultrastructural and immuno-cytochemical phenotype of hepatocytes and duodenal enterocytes from a unique case of an adult MYO5B-PFIC patient who showed constant hepatopathy but only periodic enteric symptoms. Selected data from two other patients supported the findings. Advanced methods such as cryo-fixation, freeze-substitution, immuno-gold labeling, electron tomography and immuno-fluorescence microscopy complemented the standard procedures. Liver biopsies showed mislocalization of Rab11 and bile canalicular membrane proteins. Rab11-positive vesicles clustered around bile canaliculi and resembled subapical clusters of aberrant recycling endosomes in enterocytes from MVID patients. The adult patient studied in detail showed a severe, MVID-specific enterocyte phenotype, despite only a mild clinical intestinal presentation. This included mislocalization of numerous proteins essential for apical cargo transport and morphological alterations. We characterized the heterogeneous population of large catabolic organelles regarding their complex ultrastructure and differential distribution of autophagic and lysosomal marker proteins. Finally, we generated duodenal organoids/enteroids from biopsies that recapitulated all MVID hallmarks, demonstrating the potential of this disease model for personalized medicine.

p.His16Arg of STXBP1 (MUNC18-1) Associated With Syntaxin 3B Causes Autosomal Dominant Congenital Nystagmus.

Congenital nystagmus (CN) is an ocular movement disorder manifested as involuntary conjugated binocular oscillation and usually occurs in early infancy. The pathological mechanism underlying CN is still poorly understood. We mapped a novel genetic locus 9q33.1-q34.2 in a larger Chinese family with autosomal dominant CN and identified a variant (c.47A>G/p.His16Arg) of STXBP1 by exome sequencing, which fully co-segregated with the nystagmus phenotype in this family and was absent in 571 healthy unrelated individuals. The STXBP1 encodes syntaxin binding protein 1 (also known as MUNC18-1), which plays a pivotal role in neurotransmitter release. In unc-18 (nematode homolog of MUNC18-1) null Caenorhabditis elegans, we found that the p.His16Arg exhibits a compromised ability to rescue the locomotion defect and aldicarb sensitivity, indicating a functional defect in neurotransmitter release. In addition, we also found an enhanced binding of the p.His16Arg mutant to syntaxin 3B, which is a homolog of syntaxin 1A and specifically located in retinal ribbon synapses. We hypothesize that the variant p.His16Arg of STXBP1 is likely to affect neurotransmitter release in the retina, which may be the underlying etiology of CN in this family. Our results provide a new perspective on understanding the molecular mechanism of CN.

Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2-Deficient Intestinal Organoids.

Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.

Microvillus Inclusion Disease Variant in an Infant with Intractable Diarrhea.

Microvillus inclusion disease (MVID) is a rare autosomal recessive congenital enteropathy characterized by intractable secretory diarrhea. We report a case of MVID variant with a homozygous gene mutation in syntaxin 3 (STX3). The patient is a male Saudi infant who presented shortly after birth with severe vomiting, metabolic acidosis, and mild diarrhea. Electron microscopy study for small intestinal biopsy was consistent with MVID. MYO5B gene mutation was excluded; subsequently, whole exome sequencing (WES) was performed, which revealed homozygous gene mutation in STX3. Using WES in clinical environment can be a useful tool for diagnosing difficult and rare inherited congenital enteropathies.

Loss of MYO5B in mice recapitulates Microvillus Inclusion Disease and reveals an apical trafficking pathway distinct to neonatal duodenum.

BACKGROUND AND AIMS: Inactivating mutations in MYO5B cause severe neonatal diarrhea in Microvillus Inclusion Disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush border enzymes. METHODS: We developed three mouse models of germline, constitutively intestinal targeted and inducible intestinal targeted deletion of MYO5B. The mice were evaluated for enterocyte cellular morphology. RESULTS: Germline MYO5B KO mice showed early diarrhea and failure to thrive with evident microvillus inclusions and loss of apical transporters in the duodenum. IgG was present within inclusions. Apical transporters were lost and inclusions were present in the duodenum, but were nearly absent in the ileum. VillinCre;MYO5BF/F mice showed similar pathology and morphological changes in duodenal enterocytes. In contrast, when MYO5B KO was induced with tamoxifen treatment at 8 weeks of age, VillinCreERT2;MYO5BF/F mice developed severe diarrhea with loss of duodenal brush border enzymes, but few inclusions were observed in enterocytes. However, if tamoxifen is administered to 2-day-old VillinCreERT2;MYO5BF/F mice, prominent microvillus inclusions were observed. CONCLUSIONS: The microvillus inclusions that develop after MYO5B loss reveal the presence of an unrecognized apical membrane trafficking pathway in neonatal duodenal enterocytes. However, the diarrheal pathology after MYO5B loss is due to deficits in transporter presentation at the apical membrane in duodenal enterocytes.

Human cytomegalovirus miR-US33-5p inhibits viral DNA synthesis and viral replication by down-regulating expression of the host Syntaxin3.

During infection with human cytomegalovirus (HCMV), overexpression of hcmv-miR-US33 can inhibit the lytic viral replication and down-regulate US29 mRNA. However, it remains unknown whether inhibition of viral replication by miR-US33 is mediated by down-regulation of expression of US29 or another host gene. Here, we identified the host gene Syntaxin3 (STX3) to be a direct target of hcmv-miR-US33-5p using Hybrid-PCR and luciferase-reporter assays. It was further demonstrated that the levels of STX3 protein were down-regulated in hcmv-miR-US33-5p-overexpressing cells. Experiments with STX3-specific siRNA, or with an inhibitor of hcmv-miR-US33-5p confirmed that hcmv-miR-US33-5p-mediated inhibition of HCMV DNA synthesis and of viral replication are specifically mediated by down-regulation of STX3 expression.

The dendritic SNARE fusion machinery involved in AMPARs insertion during long-term potentiation.

Sorting endosomes carry α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) from their maturation sites to their final destination at the dendritic plasma membrane through both constitutive and regulated exocytosis. Insertion of functional AMPARs into the postsynaptic membrane is essential for maintaining fast excitatory synaptic transmission and plasticity. Despite this crucial role in neuronal function, the machinery mediating the fusion of AMPAR-containing endosomes in dendrites has been largely understudied in comparison to presynaptic vesicle exocytosis. Increasing evidence suggests that similarly to neurotransmitter release, AMPARs insertion relies on the formation of a SNARE complex (soluble NSF-attachment protein receptor), whose composition in dendrites has just begun to be elucidated. This review analyzes recent findings of the fusion machinery involved in regulated AMPARs insertion and discusses how dendritic exocytosis and AMPARs lateral diffusion may work together to support synaptic plasticity.