Vesicle Docking Research Articles

Binding of the exocyst complex to the SNARE regulator Sec1

Vesicles in eukaryotic cells act as intracellular carriers to transport cargo between membrane‐bound organelles and the plasma membrane. Cellular trafficking is a highly regulated process that is critical for maintaining cellular growth, homeostasis, signaling and division. These cellular trafficking events ultimately culminate in membrane fusion, where secretory vesicles release their contents to the extracellular space. However, the molecular and structural underpinnings of trafficking and membrane fusion still remains misunderstood. The process of intracellular membrane fusion relies on several conserved protein families, including SNAREs, Sec1/Munc18 (SM) proteins, multi‐subunit tethering complexes (e.g. exocyst), and Rab GTPases. Each of these regulatory factors plays multiple roles in assisting vesicle docking and SNARE assembly, indicating a trafficking system with overlapping sets of reactions. Exocyst, a hetero‐octameric protein complex, is proposed to tether vesicles to the plasma membrane and promote specific SNARE‐mediated membrane fusion. Previous binding experiments using recombinant proteins revealed interactions between two exocytic SNARE proteins and Sec1 with a single subunit of exocyst, Sec6. Currently, Sec6 is proposed to bind both binary (Sso1:Sec9), and ternary (Sso1:Sec9:Snc2) SNARE complexes during SNARE complex assembly. The timing in which Sec1 binds to the ternary SNARE complex and exocyst to mediate membrane fusion is unknown. Temperature sensitive sec1 yeast cells are defective for cell growth, SNARE complex assembly, and secretion of protein and lipid cargo at the restrictive temperature. This evidence suggests that Sec1 may play both a general and a specific role during membrane trafficking. Recent structural evidence from a mutant “activated” yeast exocyst complex shows increased flexibility and dynamics of two subunits of exocyst, Exo70 and Sec6, which lead to increased binding to SNAREs compared to wild‐type exocyst. Our study aims to further investigate the mechanisms of SNARE assembly and membrane fusion, using biochemical and EM methods to characterize the interactions of yeast exocyst, Sec1 and SNARE proteins. Using purified forms of each of the proteins and complexes, interactions between Sec1, SNARE proteins, and exocyst (wild type and activated mutant) are being analyzed and quantified.

Disruption of Exocytosis in Sympathoadrenal Chromaffin Cells from Mouse Models of Neurodegenerative Diseases.

Synaptic disruption and altered neurotransmitter release occurs in the brains of patients and in murine models of neurodegenerative diseases (NDDs). During the last few years, evidence has accumulated suggesting that the sympathoadrenal axis is also affected as disease progresses. Here, we review a few studies done in adrenal medullary chromaffin cells (CCs), that are considered as the amplifying arm of the sympathetic nervous system; the sudden fast exocytotic release of their catecholamines—stored in noradrenergic and adrenergic cells—plays a fundamental role in the stress fight-or-flight response. Bulk exocytosis and the fine kinetics of single-vesicle exocytotic events have been studied in mouse models carrying a mutation linked to NDDs. For instance, in R6/1 mouse models of Huntington’s disease (HD), mutated huntingtin is overexpressed in CCs; this causes decreased quantal secretion, smaller quantal size and faster kinetics of the exocytotic fusion pore, pore expansion, and closure. This was accompanied by decreased sodium current, decreased acetylcholine-evoked action potentials, and attenuated [Ca2+]c transients with faster Ca2+ clearance. In the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS), CCs exhibited secretory single-vesicle spikes with a slower release rate but higher exocytosis. Finally, in the APP/PS1 mouse model of Alzheimer’s disease (AD), the stabilization, expansion, and closure of the fusion pore was faster, but the secretion was attenuated. Additionally, α-synuclein that is associated with Parkinson’s disease (PD) decreases exocytosis and promotes fusion pore dilation in adrenal CCs. Furthermore, Huntington-associated protein 1 (HAP1) interacts with the huntingtin that, when mutated, causes Huntington’s disease (HD); HAP1 reduces full fusion exocytosis by affecting vesicle docking and controlling fusion pore stabilization. The alterations described here are consistent with the hypothesis that central alterations undergone in various NDDs are also manifested at the peripheral sympathoadrenal axis to impair the stress fight-or-flight response in patients suffering from those diseases. Such alterations may occur: (i) primarily by the expression of mutated disease proteins in CCs; (ii) secondarily to stress adaptation imposed by disease progression and the limitations of patient autonomy.

Oxidation of Reduced Graphene Oxide via Cellular Redox Signaling Modulates Actin-Mediated Neurotransmission.

Neurotransmission is the basis of brain functions, and controllable neurotransmission tuning constitutes an attractive approach for interventions in a wide range of neurologic disorders and for synapse-based therapeutic treatments. Graphene-family nanomaterials (GFNs) offer promising advantages for biomedical applications, particularly in neurology. Our study suggests that reduced graphene oxide (rGO) serves as a neurotransmission modulator and reveals that the cellular oxidation of rGO plays a crucial role in this effect. We found that rGO could be oxidized via cellular reactive oxygen species (ROS), as evidenced by an increased number of oxygen-containing functional groups on the rGO surface. Cellular redox signaling, which involves NADPH oxidases and mitochondria, was initiated and subsequently intensified rGO oxidation. The study further shows that the blockage of synaptic vesicle docking and fusion induced through a disturbance of actin dynamics is the underlying mechanism through which oxidized rGO exerts depressant effects on neurotransmission. Importantly, this depressant effect could be modulated by restricting the cellular ROS levels and stabilizing the actin dynamics. Taken together, our results identify the complicated biological effects of rGO as a controlled neurotransmission modulator and can provide helpful information for the future design of graphene materials for neurobiological applications.

The Structural Basis of Opposing Functions of Alpha-Synuclein in Vesicle Exocytosis

Alpha-synuclein (aSyn), the principal protein forming toxic intraneuronal aggregates in Parkinson's disease, has a poorly defined physiological function related to regulation of neurotransmitter release. The N-terminal ∼100-residue membrane-binding domain of aSyn interconverts between an extended helix and a broken helix conformation (N-terminal Helix-1 and C-terminal Helix-2), with unknown significance. Using a functional assay in RBL-2H3 cells, we find that wild-type aSyn switches from an inhibitor of exocytosis at a low expression level to a potentiator at a higher expression level, with a corresponding increase in redistribution of vesicles from the perinuclear region to plasma membrane-proximal zone. Using solution-state NMR, we find that mutations disproportionately affecting the membrane affinity of Helix-2 in the broken helix conformation of aSyn cause the protein to lose the exocytosis inhibitory function. Conversely, aSyn mutations affecting the membrane affinity globally in the extended helix conformation are unable to redistribute vesicles and continue inhibiting exocytosis at high expression level. Furthermore, uncoupling Helix-2 from Helix-1 by a flexible linker region causes loss of exocytosis inhibitory function at a low expression level. It also shows increased affinity of Helix-1 to isolated vesicles and redistribution of vesicles, but lack of potentiation of exocytosis at a high expression level. We propose a model where aSyn inhibits exocytosis by binding at the interface between docked vesicles and plasma membrane in a broken helix conformation. Conversely, aSyn binding to isolated vesicles in an extended helix conformation disperses vesicle clusters and increases vesicle availability at the plasma membrane. Further progression to docking and promotion of exocytosis requires partial displacement of aSyn from the vesicle surface and is hampered by a high-affinity interaction. These two opposing functions are apparently balanced in normal physiology but may become unbalanced in the context of disease.

Binding of Complexin to t-SNARE Complex is Mediated by SNAP25

Presynaptic membrane fusion is mediated by t-SNAREs, consisting of syntaxin and SNAP25 on the target membrane, and v-SNARE, i.e., synaptobrevin on the vesicle membrane. The formation of four-helical bundles by these three SNARE proteins produce the necessary energy to merge two opposing membranes, and these processes are tightly regulated by a host of effector proteins, such as Munc18, Munc13, synaptotagmin, complexin, etc. Complexin is a highly charged protein that contributes to clamping of calcium-triggered SNARE-mediated membrane fusion. We have shown that complexin can bind to the target SNARE complex in the absence of synaptobrevin. In the presence of both complexin and Munc18, purified vesicles can be docked to target membranes in a clamped, non-fusing state in the absence of calcium. Subsequently, these docked vesicles can be triggered with Ca2+ to fuse with high efficiency, reminiscent of physiological Ca2+ triggered exocytotic fusion in vivo. Here, we report a new biophysical and biochemical characterization of complexin binding to the t-SNARE complex. Our data show that complexin binding to the t-SNARE complex is strong, but has a relatively high off rate. This off-rate is regulated by the 2nd SNARE helix of SNAP25. We propose that this dynamic interplay between complexin and SNAP25 is important for assembling an intermediate SNARE complex which is critical for Ca2+ triggering in presynatic membrane fusion.

Munc13 Recruits SNAP25 to Facilitate SNARE Complex Assembly

Synaptic vesicle exocytosis in neurons requires SNARE complex formation during which VAMP2 from vesicles zippers with t-SNAREs (SNAP25 and Syntaxin1a) on the plasma membrane. Munc13-1 acts as a chaperone for this process by both tethering vesicles to PIP2-microdomains on the plasma membrane containing Syntaxin1a and by activating Syntaxin1a. How membrane-anchored SNAP25 enters this reaction is unknown. Here we report a novel function of Munc13 in the recruitment of SNAP25 to initiate the SNARE priming process. Using biochemical analyses, including quantitative binding studies, we show the MUN domain of Munc13 directly binds the SNAP25 loop region (connecting the two helices). Indeed, replacing the SNAP25 loop with the loop region of closely-related SNAP23 or with a non-specific GGGGS repeat sequence abrogated the Munc13 interaction. Interestingly, the Munc13 mutant NF->AA (1128/1131) that has been shown to be incapable of activating Syntaxin1a, still bound SNAP25 suggesting the binding sites on Munc13 for Syntaxin1a and SNAP25 are different. Furthermore, total internal reflection fluorescence (TIRF) microscopy analysis revealed that Munc13 co-clusters with SNAP25 on a supported lipid bilayer setup and these clusters typically contain 5-6 molecules each of Munc13 and SNAP25. This co-clustering was absent in the GGGGS mutant. Overall, our data suggest Munc13 recruits SNAP25 to the site of vesicle docking via direct MUN-SNAP25 loop interaction and by virtue of increasing its local concentration, it facilitates the initiation of the SNARE complex formation.

Functional Electron Microscopy, “Flash and Freeze,” of Identified Cortical Synapses in Acute Brain Slices

SummaryHow structural and functional properties of synapses relate to each other is a fundamental question in neuroscience. Electrophysiology has elucidated mechanisms of synaptic transmission, and electron microscopy (EM) has provided insight into morphological properties of synapses. Here we describe an enhanced method for functional EM (“flash and freeze”), combining optogenetic stimulation with high-pressure freezing. We demonstrate that the improved method can be applied to intact networks in acute brain slices and organotypic slice cultures from mice. As a proof of concept, we probed vesicle pool changes during synaptic transmission at the hippocampal mossy fiber-CA3 pyramidal neuron synapse. Our findings show overlap of the docked vesicle pool and the functionally defined readily releasable pool and provide evidence of fast endocytosis at this synapse. Functional EM with acute slices and slice cultures has the potential to reveal the structural and functional mechanisms of transmission in intact, genetically perturbed, and disease-affected synapses.

A thermo-responsive, self-assembling biointerface for on demand release of surface-immobilised proteins.

Dedicated chemistries for on-demand capture and release of biomolecules at the solid-liquid interface are required for applications in drug delivery, for the synthesis of switchable surfaces used in analytical devices and for the assembly of next-generation biomaterials with complex architectures and functions. Here we report the engineering of a binary self-assembling polypeptide system for reversible protein capture, immobilisation and controlled thermo-responsive release from a solid surface. The first element of the binary system is a universal protein substrate immobilised on a solid surface. This protein is bio-inspired by the neuronal SNAP25, which is the protein involved in the docking and fusion of synaptic vesicles to the synaptic membrane. The second element is an artificial chimeric protein engineered to include distinct domains from three different proteins: Syntaxin, VAMP and SNAP25. These native proteins constitute the machinery dedicated to vesicle trafficking in eukaryotes. We removed approximately 70% of native protein sequence from these proteins and constructed a protein chimera capable of high affinity interaction and self-assembly with immobilised substrate. The interaction of the two parts of the engineered protein complex is strong but fully-reversible and therefore the chimera can be recombinantly fused as a tag to a protein of interest, to allow spontaneous assembly and stimuli-sensitive release from the surface upon heating at a predetermined temperature. Two thermo-responsive tags are reported: the first presents remarkable thermal stability with melting temperature of the order of 80 °C; the second disassembles at a substantially lower temperature of about 45 °C. The latter is a promising candidate for remote-controlled localised delivery of therapeutic proteins, as physiologically tolerable local increase of temperatures in the 40-45 °C range can be achieved using magnetic fields, infra-red light or focused ultrasound. Importantly, these two novel polypeptides provide a broader blueprint for the engineering of future functional proteins with predictable folding and response to external stimuli.

Influence Blocking by Gadolinium in Calcium Diffusion on Synapse Model: A Monte Carlo Simulation Study.

Background: Gadolinium (Gd3+) is a chemical element belonging to the lanthanide group and commonly used in magnetic resonance imaging (MRI) as a contrast agent. However, recently, gadolinium has been reported deposition in the body after a patient receives multiple injections. Gadolinium is a potent block and competes with calcium diffusion into the presynaptic. There has not been a precise mechanism of gadolinium blocking calcium channel as a channel of calcium diffusion to presynaptic until now.Objective: This study aims to investigate the mechanism of calcium influx model and the effect of neurotransmitter release to the synaptic cleft influenced by the presence of Gd3+. Material and Methods: Monte Carlo Cell simulation was used to analyze simulation and also Blender was used to create and visualize the model for synapse. The synapse modeled by a form resembling the actual synapse base on a spherical shape.Results: The presence of gadolinium around the presynaptic has been disturbing diffusion of calcium influx presynaptic. The result shows that the presence of gadolinium around the presynaptic has caused a decrease in the amount of calcium influx presynaptic. These factors contribute to reducing the establishment of the active membrane, then the amount of synaptic vesicle docking and finally the amount of released neurotransmitter.Conclusion: Gadolinium and calcium compete with each other across of calcium channel. The presence of gadolinium has caused a chain effect for signal transmission at the chemical synapse, reducing the amount of active membrane, synaptic vesicle docking, and releasing neurotransmitter.

Overexpression of an ALS-associated FUS mutation in C. elegans disrupts NMJ morphology and leads to defective neuromuscular transmission.

ABSTRACTThe amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.This article has an associated First Person interview with the first author of the paper.

Synapses: Multitasking Global Players in the Brain

Abstract Synapses are key elements in the communication between neurons in any given network of the normal adult, developmental and pathologically altered brain. Synapses are composed of nearly the same structural subelements: a presynaptic terminal containing mitochondria with an ultrastructurally visible density at the pre- and postsynaptic apposition zone. The presynaptic density is composed of a cocktail of various synaptic proteins involved in the binding, priming and docking of synaptic vesicles inducing synaptic transmission. Individual presynaptic terminals (synaptic boutons) contain a couple of hundred up to thousands of synaptic vesicles. The pre- and postsynaptic densities are separated by a synaptic cleft. The postsynaptic density, also containing various synaptic proteins and more importantly various neurotransmitter receptors and their subunits specifically composed and arranged at individual synaptic complexes, reside at the target structures of the presynaptic boutons that could be somata, dendrites, spines or initial segments of axons. Beside the importance of the network in which synapses are integrated, their individual structural composition critically determines the dynamic properties within a given connection or the computations of the entire network, in particular, the number, size and shape of the active zone, the structural equivalent to a functional neurotransmitter release site, together with the size and organization of the three functionally defined pools of synaptic vesicles, namely the readily releasable, the recycling and the resting pool, are important structural subelements governing the ‘behavior’ of synaptic complexes within a given network such as the cortical column. In the late last century, neuroscientists started to generate quantitative 3D-models of synaptic boutons and their target structures that is one possible way to correlate structure with function, thus allowing reliable predictions about their function. The re-introduction of electron microscopy (EM) as an important tool achieved by modern high-end, high-resolution transmission-EM, focused ion beam scanning-EM, CRYO-EM and EM-tomography have enormously improved our knowledge about the synaptic organization of the brain not only in various animal species, but also allowed new insights in the ‘microcosms’ of the human brain in health and disease.

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10.

MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25- or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in β-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and -3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in β-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion.This article has an associated First Person interview with the first author of the paper.

First report of a VAMP1 associated pre-synaptic congenital myasthenic syndrome from South-East Asia

Biallelic mutations in VAMP1, a member of synaptobrevin family have been reported previously to cause a rare form of pre-synaptic congenital myasthenic syndrome (CMS) in two middle-eastern families. We present a case of pre-synaptic CMS from Southern India with VAMP1 novel mutation identified by clinical exome sequencing (CES).Infomed consent obtained. A 5-year-old girl born to consanguineous parentage and delivered by LSCS for absent labour pains, developed respiratory distress and feeding difficulty postnatally requiring oxygen supplementation. Profound hypotonia, muscle weakness and ophthalmoparesis was observed during neonatal period. After 9 months of ryle’s tube feeding, percutaneous endoscopic gastrostomy (PEG) was performed. Fatigable ptosis, hypophonia and frequent lower respiratory tract infections were noticed from 2 years of age. She did not attain motor mile stones but attained normal mental milestones. Examination revealed myopathic facies, ophthalmoparesis, bifacial and bulbar weakness, contractures at ankle and knee, kyphoscoliosis and areflexia. Her elder sister died in neonatal period following meconium aspiration and respiratory distress. Her maternal cousin, a 12-year-old boy is reported to have fatiguable ocular, limb and bulbar weakness. Creatine kinase level was 366 IU/L. Child did not co-operate for repetitive nerve stimulation test. CES revealed a homozygous frameshift mutation c.66delT (p.Gly23AlafsTer6) in exon 2 of VAMP1 resulting in loss of v-SNARE domain important for vesicle docking at NMJ. Child showed mild improvement in neck, truncal movements and voice after 2 weeks of oral salbutamol and pyridostigmine. This is the first case report of VAMP1 pre-synaptic CMS from south-east Asia.

The role of the exocyst in renal ciliogenesis, cystogenesis, tubulogenesis, and development.

The exocyst is a highly conserved eight-subunit protein complex (EXOC1–8) involved in the targeting and docking of exocytic vesicles translocating from the trans-Golgi network to various sites in renal cells. EXOC5 is a central exocyst component because it connects EXOC6, bound to the vesicles exiting the trans-Golgi network via the small GTPase RAB8, to the rest of the exocyst complex at the plasma membrane. In the kidney, the exocyst complex is involved in primary ciliognesis, cystogenesis, and tubulogenesis. The exocyst, and its regulators, have also been found in urinary extracellular vesicles, and may be centrally involved in urocrine signaling and repair following acute kidney injury. The exocyst is centrally involved in the development of other organs, including the eye, ear, and heart. The exocyst is regulated by many different small GTPases of the RHO, RAL, RAB, and ARF families. The small GTPases, and their guanine nucleotide exchange factors and GTPase-activating proteins, likely give the exocyst specificity of function. The recent development of a floxed Exoc5 mouse line will aid researchers in studying the role of the exocyst in multiple cells and organ types by allowing for tissue-specific knockout, in conjunction with Cre-driver mouse lines.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy

Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy

Phosphorylation of CEP83 by TTBK2 is necessary for cilia initiation.

Primary cilia are microtubule-based organelles that play important roles in development and tissue homeostasis. Tau-tubulin kinase-2 (TTBK2) is genetically linked to spinocerebellar ataxia type 11, and its kinase activity is crucial for ciliogenesis. Although it has been shown that TTBK2 is recruited to the centriole by distal appendage protein CEP164, little is known about TTBK2 substrates associated with its role in ciliogenesis. Here, we perform superresolution microscopy and discover that serum starvation results in TTBK2 redistribution from the periphery toward the root of distal appendages. Our biochemical analyses uncover CEP83 as a bona fide TTBK2 substrate with four phosphorylation sites characterized. We also demonstrate that CEP164-dependent TTBK2 recruitment to distal appendages is required for subsequent CEP83 phosphorylation. Specifically, TTBK2-dependent CEP83 phosphorylation is important for early ciliogenesis steps, including ciliary vesicle docking and CP110 removal. In summary, our results reveal a molecular mechanism of kinase regulation in ciliogenesis and identify CEP83 as a key substrate of TTBK2 during cilia initiation.

Pannexin-1 limits the production of proinflammatory cytokines during necroptosis.

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apoptosis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs endosomal trafficking and biogenesis of small extracellular vesicles as well as the production of proinflammatory cytokines during the early steps of necroptosis; however, the molecular basis continues to be elucidated. Here, we find that MLKL oligomers activate Pannexin-1 (PANX1) channels, concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the small GTPase RAB27A and RAB27B isoforms, which regulate intracellular vesicle trafficking, docking, and fusion with the plasma membrane. Although cells in which PANX1 is silenced or inhibited normally undergo necroptotic death, they display enhanced production of cytokines such as interleukin-8, indicating that PANX1 may tamper with inflammation. These data identify a novel signaling nexus between MLKL, RAB27, and PANX1 and propose ways to interfere with inflammation associated with necroptosis.

366-OR: TCF19 Promotes Functional Beta-Cell Mass and Proliferative Capacity by Regulating DNA Damage Repair Pathways

Transcription factor 19, TCF19, is a novel gene associated with type 1 and type 2 diabetes. We have previously shown that Tcf19 is involved in the regulation of proliferation and apoptosis in the INS1 β-cell line. To determine the role of Tcf19 in diabetes pathogenesis, we generated germline whole body Tcf19 knockout mice (wbTcf19KO). Lean wbTcf19KO mice have slightly increased fasting glucose and decreased in vivo glucose-stimulated insulin secretion. We focused on the pancreatic islet in further analysis of this model due to the high expression of Tcf19 in the mouse islet and our preliminary findings in a β-cell line. RNAseq from wbTcf19KO mouse islets identified significantly decreased expression of genes involved in maintaining β-cell identity, vesicle docking, and Ca2+ sensing (Nkx6.1, Nkx2.2, Pdx1, Sort1, Syt7). Additionally, the proliferative marker, Ki67, was downregulated by 3-fold. Similarly, with immunohistochemistry of pancreatic sections, there was a 20% decrease in Ki67+ β-cells in wbTcf19KO mice. Despite no difference in overall β-cell mass compared to control mice, wbTcf19KO mice have an altered islet size distribution with an increased quantity of small islets. In addition, wbTcf19KO mice fail to increase proliferative genes in the islet, such as cyclinD2 and Ki67, in response to a short term high fat diet (HFD). Islet RNA seq analysis also showed an upregulation of genes involved in the DNA damage pathway, inflammation, and pro apoptotic genes (Ddx60, Gadd45a, Dtx3l, Chop, Bak, Ifit1, Cxcl10). Baseline levels of DNA damage in the wbTcf19KO islets are increased, as measured by γH2AX on western blot. wbTcf19KO mouse islets also have increased DNA damage after in vivo challenge with extended HFD feeding or ex vivo after cytokine exposure. Taken together, we conclude that Tcf19 plays an important role in regulating β-cell DNA damage repair pathways to prevent apoptosis and allow proliferation. Disclosure G.H. Yang: None. J. Han: None. S. Lodh: None. J.T. Blumer: None. D. Fontaine: None. D.B. Davis: None.

Analysis, Simulation, and Optimization of Stochastic Vesicle Dynamics in Synaptic Transmission

AbstractSynaptic transmission is the mechanism of information transfer from one neuron to another (or from a neuron to a muscle or to an endocrine cell). An important step in this physiological process is the stochastic release of neurotransmitter from vesicles that fuse with the presynaptic membrane and spill their contents into the synaptic cleft. We are concerned here with the formulation, analysis, and simulation of a mathematical model that describes the stochastic docking, undocking, and release of synaptic vesicles and their effect on synaptic signal transmission. The focus of this paper is on the parameter p0, the probability of release for each docked vesicle when an action potential arrives. We study the influence of this parameter on the statistics of the release process and on the theoretical capability of the model synapse in reconstructing various desired outputs based on the timing and amount of neurotransmitter release. This theoretical capability is assessed by formulating and solving an optimal filtering problem. Methods for parameter identification are proposed and applied to simulated data. © 2019 Wiley Periodicals, Inc.