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1.
Proc Natl Acad Sci U S A ; 121(2): e2309161121, 2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38170748

RESUMEN

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.


Asunto(s)
Células Cromafines , Proteínas SNARE , Animales , Ratones , Membrana Celular/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Fusión de Membrana/fisiología , Proteínas SNARE/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(42): e2409636121, 2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-39374398

RESUMEN

The Ca2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al., bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion.


Asunto(s)
Calcio , Neurotransmisores , Proteínas SNARE , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Calcio/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Neurotransmisores/metabolismo , Sintaxina 1/metabolismo , Sintaxina 1/genética , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteína 25 Asociada a Sinaptosomas/genética , Ratas , Unión Proteica , Transmisión Sináptica
3.
Proc Natl Acad Sci U S A ; 121(34): e2409341121, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39145939

RESUMEN

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and ß2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.


Asunto(s)
Complejo 2 de Proteína Adaptadora , Chaperonas Moleculares , Complejo 2 de Proteína Adaptadora/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Unión Proteica , Endocitosis/fisiología , Transporte de Proteínas
4.
Proc Natl Acad Sci U S A ; 120(39): e2307899120, 2023 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-37733740

RESUMEN

The human blood-brain barrier (BBB) comprises a single layer of brain microvascular endothelial cells (HBMECs) protecting the brain from bloodborne pathogens. Meningitis is among the most serious diseases, but the mechanisms by which major meningitis-causing bacterial pathogens cross the BBB to reach the brain remain poorly understood. We found that Streptococcus pneumoniae, group B Streptococcus, and neonatal meningitis Escherichia coli commonly exploit a unique vesicle fusion mechanism to hitchhike on transferrin receptor (TfR) transcytosis to cross the BBB and illustrated the details of this process in human BBB model in vitro and mouse model. Toll-like receptor signals emanating from bacteria-containing vesicles (BCVs) trigger K33-linked polyubiquitination at Lys168 and Lys181 of the innate immune regulator TRAF3 and then activate the formation of a protein complex containing the guanine nucleotide exchange factor RCC2, the small GTPase RalA and exocyst subcomplex I (SC I) on BCVs. The distinct function of SEC6 in SC I, interacting directly with RalA on BCVs and the SNARE protein SNAP23 on TfR vesicles, tethers these two vesicles and initiates the fusion. Our results reveal that innate immunity triggers a unique modification of TRAF3 and the formation of the HBMEC-specific protein complex on BCVs to authenticate the precise recognition and selection of TfR vesicles to fuse with and facilitate bacterial penetration of the BBB.


Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Humanos , Animales , Ratones , Recién Nacido , Factor 3 Asociado a Receptor de TNF , Transcitosis , Bacterias , Receptores de Transferrina
5.
Plant J ; 116(6): 1633-1651, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37659090

RESUMEN

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.


Asunto(s)
Arabidopsis , Proteínas SNARE , Membrana Celular/metabolismo , Fusión de Membrana , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Tirosina/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo
6.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33468652

RESUMEN

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.


Asunto(s)
Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sinaptotagmina I/metabolismo , Animales , Calcio/metabolismo , Regulación de la Expresión Génica , Liposomas/química , Liposomas/metabolismo , Fusión de Membrana , Proteínas Munc18/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neurotransmisores/genética , Neurotransmisores/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Ratas , Transmisión Sináptica , Vesículas Sinápticas/química , Proteína 25 Asociada a Sinaptosomas/genética , Sinaptotagmina I/genética , Sintaxina 1/genética , Sintaxina 1/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/metabolismo
7.
Int J Mol Sci ; 25(12)2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38928373

RESUMEN

Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but have smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and wavy, with a complex venation pattern. The mutant leaves also develop necrotic patches and undergo premature senescence. RNA-seq revealed transcriptome changes likely leading to reduced cell wall integrity and an increase in the unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Mutación , Hojas de la Planta , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Pared Celular/genética , Fenotipo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo
8.
J Neurosci ; 42(12): 2385-2403, 2022 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-35063999

RESUMEN

Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca2+ entry and neurotransmitter release. However, Ca2+ requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca2+ entry may occur through small numbers or even single open Ca2+ channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca2+ entry, using cell-attached patch clamp and lattice light-sheet microscopy (LLSM), resolving small channel numbers evoking Ca2+ entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations of 0-6 event sizes between individual APs and stochastic variation of precise locations of Ca2+ entry within the AZ. Further, total Ca2+ channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca2+ entry via as few as one channel may trigger neurotransmitter release.SIGNIFICANCE STATEMENT Presynaptic Ca2+ entry through voltage-gated calcium channels (VGCCs) causes neurotransmitter release. To understand neurotransmission, its modulation, and plasticity, we must quantify Ca2+ entry and its relationship to vesicle fusion. This requires direct recordings from active zones (AZs), previously possible only at calyceal terminals containing many AZs, where few channels open following action potentials (APs; Sheng et al., 2012), and even single channel openings may trigger release (Stanley, 1991, 1993). However, recording from more conventional terminals with single AZs commonly found centrally has thus far been impossible. We addressed this by cell-attached recordings from acutely dissociated single lamprey giant axon AZs, and by lattice light sheet microscopy of presynaptic Ca2+ entry. We demonstrate nanodomains of presynaptic VGCCs coupling with primed vesicles with 1:1 stoichiometry.


Asunto(s)
Calcio , Terminales Presinápticos , Animales , Femenino , Lampreas , Masculino , Neurotransmisores , Terminales Presinápticos/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas
9.
J Neurosci ; 42(6): 980-1000, 2022 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-34949691

RESUMEN

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts via Gßγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gßγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors, which alter presynaptic Ca2+ In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENT Two G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gßγ, but one (a GABAB receptor) inhibits Ca2+ entry whereas another (a 5-HT1B receptor) competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, whereas 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration.


Asunto(s)
Terminales Presinápticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transmisión Sináptica/fisiología , Animales , Femenino , Hipocampo/fisiología , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley
10.
Int J Mol Sci ; 24(9)2023 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-37175944

RESUMEN

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.


Asunto(s)
Colorantes Fluorescentes , Liposomas Unilamelares , Liposomas Unilamelares/química , Fosfatidilcolinas , Fusión de Membrana
11.
Traffic ; 21(10): 636-646, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32851733

RESUMEN

Exocytosis is a vesicle fusion process driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulin-stimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR-Cas9 genome editing to delete the two tomosyn-encoding genes in adipocytes. We observed that both basal and insulin-stimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α-SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin-stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn-arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.


Asunto(s)
Exocitosis , Transportador de Glucosa de Tipo 4/metabolismo , Insulina , Proteínas del Tejido Nervioso/fisiología , Proteínas R-SNARE/fisiología , Membrana Celular/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucosa de Tipo 4/genética , Humanos , Insulina/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Proteínas R-SNARE/genética
12.
Am J Hum Genet ; 104(4): 721-730, 2019 04 04.
Artículo en Inglés | MEDLINE | ID: mdl-30929742

RESUMEN

VAMP2 encodes the vesicular SNARE protein VAMP2 (also called synaptobrevin-2). Together with its partners syntaxin-1A and synaptosomal-associated protein 25 (SNAP25), VAMP2 mediates fusion of synaptic vesicles to release neurotransmitters. VAMP2 is essential for vesicular exocytosis and activity-dependent neurotransmitter release. Here, we report five heterozygous de novo mutations in VAMP2 in unrelated individuals presenting with a neurodevelopmental disorder characterized by axial hypotonia (which had been present since birth), intellectual disability, and autistic features. In total, we identified two single-amino-acid deletions and three non-synonymous variants affecting conserved residues within the C terminus of the VAMP2 SNARE motif. Affected individuals carrying de novo non-synonymous variants involving the C-terminal region presented a more severe phenotype with additional neurological features, including central visual impairment, hyperkinetic movement disorder, and epilepsy or electroencephalography abnormalities. Reconstituted fusion involving a lipid-mixing assay indicated impairment in vesicle fusion as one of the possible associated disease mechanisms. The genetic synaptopathy caused by VAMP2 de novo mutations highlights the key roles of this gene in human brain development and function.


Asunto(s)
Discapacidad Intelectual/genética , Hipotonía Muscular/genética , Trastornos del Neurodesarrollo/genética , Neuronas/metabolismo , Sinapsis/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/genética , Adolescente , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Epilepsia/metabolismo , Exocitosis , Femenino , Heterocigoto , Humanos , Lípidos/química , Imagen por Resonancia Magnética , Masculino , Fusión de Membrana , Trastornos del Movimiento/genética , Mutación , Trastornos del Neurodesarrollo/metabolismo , Neurotransmisores/metabolismo , Fenotipo , Dominios Proteicos , Proteínas R-SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/fisiología
13.
Biochem Biophys Res Commun ; 618: 113-118, 2022 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-35717905

RESUMEN

Lipid giant vesicles represent a versatile minimal model system to study the physicochemical basis of lipid membrane fusion. Membrane fusion processes are also of interest in synthetic cell research, where cell-mimicking behavior often requires dynamically interacting compartments. For these applications, triggered fusion compatible with transcription-translation systems is key in achieving complexity. Recently, a photosensitive surfactant, azobenzene trimethylammonium bromide (AzoTAB), has been reported to induce membrane fusion by a photoinduced conformational change. Using imaging flow cytometer (IFC) and confocal microscopy we quantitatively investigated photoinduced AzoTAB-mediated fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine vesicles. The IFC analysis result showed that the fusion rate could reach about 40% following AzoTAB addition and UV irradiation in optimized conditions. We confirmed the compatibility between AzoTAB-induced vesicle fusion and a synthetic cell-free protein translation system using green fluorescent protein as reporter. With the techniques presented, cell-sized vesicle fusion can be quantitatively analyzed and optimized, paving the way to controllable synthetic cells with fundamental biological functions like the ability to express proteins from encapsulated plasmids.


Asunto(s)
Bromuros , Fusión de Membrana , Compuestos Azo , Biosíntesis de Proteínas , Compuestos de Amonio Cuaternario
14.
Nano Lett ; 21(18): 7479-7485, 2021 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-34491760

RESUMEN

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.


Asunto(s)
Fenómenos Biológicos , Transferencia Resonante de Energía de Fluorescencia , Fusión de Membrana , Microscopía , Proteínas SNARE
15.
Int J Mol Sci ; 23(3)2022 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-35163009

RESUMEN

The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma.


Asunto(s)
Exocitosis/efectos de los fármacos , Células Neuroendocrinas/metabolismo , Esfingosina/metabolismo , Animales , Transporte Biológico , Humanos , Fusión de Membrana , Proteínas SNARE/metabolismo , Esfingosina/farmacología
16.
EMBO J ; 36(6): 816-829, 2017 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-28137749

RESUMEN

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin-1 adopts a closed conformation when bound to Munc18-1, preventing binding to synaptobrevin-2 and SNAP-25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13-1 catalyzes the transition from the Munc18-1/syntaxin-1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin-1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18-1/syntaxin-1/MUN complex, in which syntaxin-1 still adopts a closed conformation tightly bound to Munc18-1, whereas the syntaxin-1 linker region changes its conformation, similar to that of the LE mutant of syntaxin-1 when bound to Munc18-1. We suggest that the conformational change of the syntaxin-1 linker region induced by Munc13-1 initiates ternary SNARE complex formation in the neuronal system.


Asunto(s)
Exocitosis , Proteínas Munc18/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Sinapsis/fisiología , Animales , Células Cultivadas , Humanos , Modelos Biológicos , Conformación Proteica , Proteínas Qa-SNARE/química , Ratas
17.
Bioessays ; 41(8): e1900056, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31264240

RESUMEN

The exocyst is a conserved octameric complex that physically tethers a vesicle to the plasma membrane, prior to membrane fusion. It is important not only for secretion and membrane delivery but also, in mammalian cells, for cytokinesis, ciliogenesis, autophagy, tumorigenesis, and host defense. The combination of genome editing and advanced light microscopy of exocyst subunits in living cells has recently shown the complex to be much more dynamic than previously appreciated, and exposed how little we still know about its function and regulation.


Asunto(s)
Membrana Celular/metabolismo , Exocitosis/fisiología , Fusión de Membrana/fisiología , Proteínas de Transporte Vesicular/metabolismo , Animales , Autofagia , Carcinogénesis , Compartimento Celular , Citoplasma/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Proteínas SNARE/metabolismo
18.
Proc Natl Acad Sci U S A ; 115(36): E8421-E8429, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30127032

RESUMEN

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.


Asunto(s)
Exocitosis/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Proteínas Munc18 , Péptidos , Proteínas SNARE , Animales , Células COS , Chlorocebus aethiops , Cinética , Ratones , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Péptidos/química , Péptidos/farmacología , Ratas , Proteínas SNARE/química , Proteínas SNARE/metabolismo
19.
Sensors (Basel) ; 21(9)2021 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-34068538

RESUMEN

The paper describes the architecture of a Spiking Neural Network (SNN) for time waveform analyses using edge computing. The network model was based on the principles of preprocessing signals in the diencephalon and using tonic spiking and inhibition-induced spiking models typical for the thalamus area. The research focused on a significant reduction of the complexity of the SNN algorithm by eliminating most synaptic connections and ensuring zero dispersion of weight values concerning connections between neuron layers. The paper describes a network mapping and learning algorithm, in which the number of variables in the learning process is linearly dependent on the size of the patterns. The works included testing the stability of the accuracy parameter for various network sizes. The described approach used the ability of spiking neurons to process currents of less than 100 pA, typical of amperometric techniques. An example of a practical application is an analysis of vesicle fusion signals using an amperometric system based on Carbon NanoTube (CNT) sensors. The paper concludes with a discussion of the costs of implementing the network as a semiconductor structure.

20.
Plant J ; 99(4): 703-716, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31009161

RESUMEN

The trans-Golgi network (TGN) is a major site for sorting of cargo to either the vacuole or apoplast. The TGN-localized coiled-coil protein TNO1 is a putative tethering factor that interacts with the TGN t-SNARE SYP41 and is required for correct localization of the SYP61 t-SNARE. An Arabidopsis thaliana tno1 mutant is hypersensitive to salt stress and partially mislocalizes vacuolar proteins to the apoplast, indicating a role in vacuolar trafficking. Here, we show that overexpression of SYP41 or SYP61 significantly increases SYP41-SYP61 complex formation in a tno1 mutant, and rescues the salt sensitivity and defective vacuolar trafficking of the tno1 mutant. The TGN is disrupted and vesicle budding from Golgi cisternae is reduced in the tno1 mutant, and these defects are also rescued by overexpression of SYP41 or SYP61. Our results suggest that the trafficking and Golgi morphology defects caused by loss of TNO1 can be rescued by increasing SYP41-SYP61 t-SNARE complex formation, implicating TNO1 as a tethering factor mediating efficient vesicle fusion at the TGN.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Red trans-Golgi/metabolismo , Red trans-Golgi/fisiología , Aparato de Golgi/metabolismo , Unión Proteica/fisiología , Transporte de Proteínas/fisiología
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