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1.
Nat Commun ; 11(1): 5516, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139696

RESUMO

Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a 'closed' conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 mutant phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibit enhanced spontaneous and evoked exocytosis compared to WT animals. Unexpectedly, the open syntaxin KI partially suppresses exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit and unc-31/CAPS, in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravates the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Exocitose/genética , Lipossomos/metabolismo , Transmissão Sináptica/genética , Sintaxina 1/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Técnicas de Introdução de Genes , Mutação , Neurotransmissores/metabolismo , Sintaxina 1/genética
3.
Mol Cancer Ther ; 19(9): 1930-1942, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32737155

RESUMO

Although intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been the gold standard for nonsurgical management of non-muscle-invasive bladder cancer, a considerable number of patients exhibit resistance to the adjuvant treatment with unexplained mechanisms. This study aimed to investigate whether and how androgen receptor (AR) signals modulate BCG cytotoxicity in bladder cancer. AR knockdown or overexpression in bladder cancer lines resulted in induction or reduction, respectively, in intracellular BCG quantity and its cytotoxic activity. Microarray screening identified Rab27b, a small GTPase known to mediate bacterial exocytosis, which was upregulated in BCG-resistant cells and downregulated in AR-shRNA cells. Knockdown of Rab27b, or its effector SYTL3, or overexpression of Rab27b also induced or reduced, respectively, BCG quantity and cytotoxicity. In addition, treatment with GW4869, which was previously shown to inhibit Rab27b-dependent secretion, induced them and reduced Rab27b expression in bladder cancer cells. Meanwhile, AR expression was upregulated in BCG-resistant lines, compared with respective controls. In a mouse orthotopic xenograft model, Rab27b/SYTL3 knockdown or GW4869 treatment enhanced the amount of BCG within tumors and its suppressive effect on tumor growth. Moreover, in non-muscle-invasive bladder cancer specimens from patients subsequently undergoing BCG therapy, positivity of AR/Rab27b expression was associated with significantly higher risks of tumor recurrence. AR activation thus correlates with resistance to BCG treatment, presumably via upregulating Rab27b expression. Mechanistically, it is suggested that BCG elimination from urothelial cells is induced by Rab27b/SYTL3-mediated exocytosis. Accordingly, Rab27b inactivation, potentially via antiandrogenic drugs and/or exocytosis inhibition are anticipated to sensitize the efficacy of BCG therapy, especially in patients with BCG-refractory AR/Rab27b-positive bladder cancer.


Assuntos
Vacina BCG/uso terapêutico , Exocitose/efeitos dos fármacos , Imunoterapia/métodos , Receptores Androgênicos/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas rab de Ligação ao GTP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Vacina BCG/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais
4.
Neuron ; 107(4): 667-683.e9, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32616470

RESUMO

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.


Assuntos
Canais de Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Exocitose/fisiologia , Camundongos Knockout , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo
5.
Curr Opin Neurobiol ; 63: 198-209, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32663762

RESUMO

The synaptotagmin family of molecules is known for regulating calcium-dependent membrane fusion events. Mice and humans express 17 synaptotagmin isoforms, where most studies have focused on isoforms 1, 2, and 7, which are involved in synaptic vesicle exocytosis. Recent work has highlighted how brain function relies on additional isoforms, with roles in postsynaptic receptor endocytosis, vesicle trafficking, membrane repair, synaptic plasticity, and protection against neurodegeneration, for example, in addition to the traditional concept of synaptotagmin-mediated neurotransmitter release - in neurons as well as glia, and at different timepoints. In fact, it is not uncommon for the same isoform to feature several splice isoforms, form homo- and heterodimers, and function in different subcellular locations and cell types. This review aims to highlight the diversity of synaptotagmins, offers a concise summary of key findings on all isoforms, and discusses different ways of grouping these.


Assuntos
Proteínas do Tecido Nervoso , Sinaptotagmina I , Animais , Cálcio/metabolismo , Exocitose , Humanos , Fusão de Membrana , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinaptotagminas/genética
6.
PLoS One ; 15(7): e0235864, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645101

RESUMO

In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are species- or cell-type-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.


Assuntos
Receptores de Peptídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Exocitose , Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Peptídeos/análise , Receptores de Peptídeos/genética , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genética
7.
Arterioscler Thromb Vasc Biol ; 40(9): 2054-2069, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640907

RESUMO

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.


Assuntos
Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Catepsinas/biossíntese , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catepsinas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Exocitose , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout para ApoE , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , Via Secretória , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Clin Sci (Lond) ; 134(12): 1449-1456, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32556178

RESUMO

Hepatitis C virus (HCV) infection and chronic hepatitis C (CHC) are associated with a measurable risk of insulin resistance (IR)/impaired glucose tolerance (IGT)/diabetes mellitus (DM). While loss of hepatic endocrine function contributes to liver cirrhosis in diabetic patients, onset and progression of IR/IGT to diabetes and exacerbation of incident hyperglycemia are ostensibly linked with chronic HCV infection. In this regard, the study by Chen J et al. appearing in Clinical Science (2020) (134(5) https://doi.org/10.1042/CS20190900) attempts to understand the mechanisms underlying the savaging effects of chronic HCV infection on insulin-producing pancreatic ß-cells and hence diabetic onset. The study investigated the role of mitogen-activated protein kinase (MAPK) p38δ-protein kinase D (PKD)-golgi complex axis in impacting insulin exocytosis. It was inferred that an insulin secretory defect of pancreatic ß-cells, owing to disrupted insulin exocytosis, to an extent explains ß-cell dysfunction in HCV-infected or CHC milieu. HCV infection negatively regulates first-phase and second-phase insulin secretion by impinging on PKD-dependent insulin secretory granule fission at trans-golgi network and insulin secretory vesicle membrane fusion events. This commentary highlights the study in question, that deciphered the contribution of p38δ MAPK-PKD-golgi complex axis to ß-cell dysfunction in CHC milieu. This pivotal axis proffers a formidable therapeutic opportunity for alleviation of double burden of glucose abnormalities/DM and CHC.


Assuntos
Exocitose , Complexo de Golgi/metabolismo , Hepatite C Crônica/metabolismo , Hepatite C Crônica/patologia , Insulina/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Animais , Humanos , Camundongos Endogâmicos C57BL , Modelos Biológicos
9.
PLoS One ; 15(6): e0235116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32569321

RESUMO

Here, we examine known GTPase regulators of vesicle trafficking events to assess whether they affect endothelial cell (EC) lumen and tube formation. We identify novel roles for the small GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA, RalB and caveolin-1 in co-regulating membrane trafficking events that control EC lumen and tube formation. siRNA suppression of individual GTPases such as Rab3A, Rab8A, and RalB markedly inhibit tubulogenesis, while greater blockade is observed with combinations of siRNAs such as Rab3A and Rab3B, Rab8A and Rab11A, and RalA and RalB. These combinations of siRNAs also disrupt very early events in lumen formation including the formation of intracellular vacuoles. In contrast, knockdown of the endocytosis regulator, Rab5A, fails to inhibit EC tube formation. Confocal microscopy and real-time videos reveal that caveolin-1 strongly labels intracellular vacuoles and localizes to the EC apical surface as they fuse to form the luminal membrane. In contrast, Cdc42 and Rab11A localize to a perinuclear, subapical region where intracellular vacuoles accumulate and fuse during lumen formation. Our new data demonstrates that EC tubulogenesis is coordinated by a series of small GTPases to control polarized membrane trafficking events to generate, deliver, and fuse caveolin-1-labeled vacuoles to create the apical membrane surface.


Assuntos
Caveolina 1/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Colágeno/metabolismo , Exocitose , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
10.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G74-G86, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32538138

RESUMO

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.


Assuntos
Células Acinares/metabolismo , Movimento Celular/fisiologia , Transporte Proteico/fisiologia , Via Secretória/fisiologia , Amilases/metabolismo , Animais , Células Cultivadas , Exocitose/fisiologia , Glândula Parótida/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(25): 14493-14502, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513685

RESUMO

A high rate of synaptic vesicle (SV) release is required at cerebellar mossy fiber terminals for rapid information processing. As the number of release sites is limited, fast SV reloading is necessary to achieve sustained release. However, rapid reloading has not been observed directly. Here, we visualize SV movements near presynaptic membrane using total internal reflection fluorescence (TIRF) microscopy. Upon stimulation, SVs appeared in the TIRF-field and became tethered to the presynaptic membrane with unexpectedly rapid time course, almost as fast as SVs disappeared due to release. However, such stimulus-induced tethering was abolished by inhibiting exocytosis, suggesting that the tethering is tightly coupled to preceding exocytosis. The newly tethered vesicles became fusion competent not immediately but only 300 ms to 400 ms after tethering. Together with model simulations, we propose that rapid tethering leads to an immediate filling of vacated spaces and release sites within <100 nm of the active zone by SVs, which serve as precursors of readily releasable vesicles, thereby shortening delays during sustained activity.


Assuntos
Cerebelo/fisiologia , Modelos Neurológicos , Fibras Nervosas/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cerebelo/citologia , Exocitose/fisiologia , Feminino , Microscopia Intravital , Masculino , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Ratos , Wisteria
12.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32554695

RESUMO

Accessory genes occurring between the S and E genes of coronaviruses have been studied quite intensively during the last decades. In porcine epidemic diarrhea virus (PEDV), the only gene at this location, ORF3, encodes a 224-residue membrane protein shown to exhibit ion channel activity and to enhance virus production. However, little is known about its intracellular trafficking or about its function during PEDV infection. In this study, two recombinant PEDVs were rescued by targeted RNA recombination, one carrying the full-length ORF3 gene and one from which the gene had been deleted entirely. These viruses as well as a PEDV encoding a naturally truncated ORF3 protein were employed to study the ORF3 protein's subcellular trafficking. In addition, ORF3 expression vectors were constructed to study the protein's independent transport. Our results show that the ORF3 protein uses the exocytic pathway to move to and accumulate in the Golgi area of the cell similarly in infected and transfected cells. Like the S protein, but unlike the other structural proteins M and N, the ORF3 protein was additionally observed at the surface of PEDV-infected cells. In addition, the C-terminally truncated ORF3 protein entered the exocytic pathway but it was unable to leave the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartment (ERGIC). Consistently, a YxxØ motif essential for ER exit was identified in the C-terminal domain. Finally, despite the use of sensitive antibodies and assays no ORF3 protein could be detected in highly purified PEDV particles, indicating that the protein is not a structural virion component.IMPORTANCE Coronaviruses typically express several accessory proteins. They vary in number and nature, and only one is conserved among most of the coronaviruses, pointing at an important biological function for this protein. PEDV is peculiar in that it expresses just this one accessory protein, termed the ORF3 protein. While its analogs in other coronaviruses have been studied to different extents, and these studies have indicated that they share an ion channel property, little is still known about the features and functions of the PEDV ORF3 protein except for its association with virulence. In this investigation, we studied the intracellular trafficking of the ORF3 protein both in infected cells and when expressed independently. In addition, we analyzed the effects of mutations in five sorting motifs in its C-terminal domain and investigated whether the protein, found to follow the same exocytic route by which the viral structural membrane proteins travel, is also incorporated into virions.


Assuntos
Infecções por Coronavirus/veterinária , Exocitose , Interações Hospedeiro-Patógeno , Fases de Leitura Aberta , Vírus da Diarreia Epidêmica Suína/genética , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Engenharia Genética , Redes e Vias Metabólicas , Plasmídeos/genética , Transporte Proteico , Proteômica , Suínos , Proteínas Virais/química , Proteínas Virais/genética
13.
Curr Opin Neurobiol ; 63: 95-103, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32403081

RESUMO

In a presynaptic nerve terminal, the active zone is composed of sophisticated protein machinery that enables secretion on a submillisecond time scale and precisely targets it toward postsynaptic receptors. The past two decades have provided deep insight into the roles of active zone proteins in exocytosis, but we are only beginning to understand how a neuron assembles active zone protein complexes into effective molecular machines. In this review, we outline the fundamental processes that are necessary for active zone assembly and discuss recent advances in understanding assembly mechanisms that arise from genetic, morphological and biochemical studies. We further outline the challenges ahead for understanding this important problem.


Assuntos
Terminações Pré-Sinápticas , Sinapses , Exocitose , Neurônios , Proteínas
14.
Science ; 368(6493): 897-901, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32381591

RESUMO

Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.


Assuntos
Citotoxicidade Imunológica , Granzimas/metabolismo , Complexos Multiproteicos/metabolismo , Perforina/metabolismo , Linfócitos T Citotóxicos/metabolismo , Trombospondina 1/metabolismo , Sistemas CRISPR-Cas , Exocitose , Edição de Genes , Humanos , Células K562 , Trombospondina 1/genética , Tomografia por Raios X
15.
Proc Natl Acad Sci U S A ; 117(24): 13468-13479, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467162

RESUMO

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.


Assuntos
Exocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/metabolismo , Humanos , Lipossomos/metabolismo , Fusão de Membrana , Células PC12 , Domínios de Homologia à Plecstrina , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
17.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276321

RESUMO

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.


Assuntos
Autofagia , Exocitose , Vesículas Extracelulares/fisiologia , Lisossomos/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endossomos/fisiologia , Exossomos/fisiologia , Humanos
18.
Nat Commun ; 11(1): 1896, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312960

RESUMO

Glucagon is released from pancreatic α-cells to activate pathways that raise blood glucose. Its secretion is regulated by α-cell-intrinsic glucose sensing and paracrine control through insulin and somatostatin. To understand the inadequately high glucagon levels that contribute to hyperglycemia in type-2 diabetes (T2D), we analyzed granule behavior, exocytosis and membrane excitability in α-cells of 68 non-diabetic and 21 T2D human donors. We report that exocytosis is moderately reduced in α-cells of T2D donors, without changes in voltage-dependent ion currents or granule trafficking. Dispersed α-cells have a non-physiological V-shaped dose response to glucose, with maximal exocytosis at hyperglycemia. Within intact islets, hyperglycemia instead inhibits α-cell exocytosis, but not in T2D or when paracrine inhibition by insulin or somatostatin is blocked. Surface expression of somatostatin-receptor-2 is reduced in T2D, suggesting a mechanism for the observed somatostatin resistance. Thus, elevated glucagon in human T2D may reflect α-cell insensitivity to paracrine inhibition at hyperglycemia.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Exocitose/fisiologia , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Células Secretoras de Glucagon/patologia , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Insulina/metabolismo , Imagem Óptica , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo
19.
J Neurosci ; 40(21): 4103-4115, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32327530

RESUMO

Volatile anesthetics are widely used for surgery, but neuronal mechanisms of anesthesia remain unidentified. At the calyx of Held in brainstem slices from rats of either sex, isoflurane at clinical doses attenuated EPSCs by decreasing the release probability and the number of readily releasable vesicles. In presynaptic recordings of Ca2+ currents and exocytic capacitance changes, isoflurane attenuated exocytosis by inhibiting Ca2+ currents evoked by a short presynaptic depolarization, whereas it inhibited exocytosis evoked by a prolonged depolarization via directly blocking exocytic machinery downstream of Ca2+ influx. Since the length of presynaptic depolarization can simulate the frequency of synaptic inputs, isoflurane anesthesia is likely mediated by distinct dual mechanisms, depending on input frequencies. In simultaneous presynaptic and postsynaptic action potential recordings, isoflurane impaired the fidelity of repetitive spike transmission, more strongly at higher frequencies. Furthermore, in the cerebrum of adult mice, isoflurane inhibited monosynaptic corticocortical spike transmission, preferentially at a higher frequency. We conclude that dual presynaptic mechanisms operate for the anesthetic action of isoflurane, of which direct inhibition of exocytic machinery plays a low-pass filtering role in spike transmission at central excitatory synapses.SIGNIFICANCE STATEMENT Synaptic mechanisms of general anesthesia remain unidentified. In rat brainstem slices, isoflurane inhibits excitatory transmitter release by blocking presynaptic Ca2+ channels and exocytic machinery, with the latter mechanism predominating in its inhibitory effect on high-frequency transmission. Both in slice and in vivo, isoflurane preferentially inhibits spike transmission induced by high-frequency presynaptic inputs. This low-pass filtering action of isoflurane likely plays a significant role in general anesthesia.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Tronco Encefálico/efeitos dos fármacos , Isoflurano/administração & dosagem , Neurônios/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Feminino , Masculino , Camundongos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
20.
J Nanobiotechnology ; 18(1): 64, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-32334582

RESUMO

BACKGROUND: The primary strategy to repair peripheral nerve injuries is to bridge the lesions by promoting axon regeneration. Thus, the ability to direct and manipulate neuronal cell axon regeneration has been one of the top priorities in the field of neuroscience. A recent innovative approach for remotely guiding neuronal regeneration is to incorporate magnetic nanoparticles (MNPs) into cells and transfer the resulting MNP-loaded cells into a magnetically sensitive environment to respond to an external magnetic field. To realize this intention, the synthesis and preparation of ideal MNPs is an important challenge to overcome. RESULTS: In this study, we designed and prepared novel fluorescent-magnetic bifunctional Fe3O4·Rhodamine 6G@polydopamine superparticles (FMSPs) as neural regeneration therapeutics. With the help of their excellent biocompatibility and ability to interact with neural cells, our in-house fabricated FMSPs can be endocytosed into cells, transported along the axons, and then aggregated in the growth cones. As a result, the mechanical forces generated by FMSPs can promote the growth and elongation of axons and stimulate gene expression associated with neuron growth under external magnetic fields. CONCLUSIONS: Our work demonstrates that FMSPs can be used as a novel stimulator to promote noninvasive neural regeneration through cell magnetic actuation.


Assuntos
Axônios/fisiologia , Óxido Ferroso-Férrico/química , Indóis/química , Nanopartículas de Magnetita/química , Polímeros/química , Rodaminas/química , Animais , Axônios/efeitos dos fármacos , Caderinas/genética , Caderinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Exocitose , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Nanopartículas de Magnetita/toxicidade , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Regeneração Nervosa/efeitos dos fármacos , Ácido Oleico/química , Células PC12 , Polímeros/farmacologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Ratos
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