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1.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31740529

RESUMO

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Exocitose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Listeria monocytogenes/patogenicidade , Proteínas Nucleares/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Listeriose/metabolismo , Listeriose/microbiologia , Polimerização , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia
2.
Adv Exp Med Biol ; 1175: 93-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583585

RESUMO

Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.


Assuntos
Astrócitos/citologia , Sistema Nervoso Central/citologia , Exocitose , Vesículas Secretórias/fisiologia , Humanos , Fusão de Membrana
3.
Muscle Nerve ; 60(6): 790-800, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531871

RESUMO

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.


Assuntos
Envelhecimento/patologia , Diafragma/ultraestrutura , Síndromes Miastênicas Congênitas/patologia , Junção Neuromuscular/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Acetilcolina/metabolismo , Envelhecimento/metabolismo , Animais , Diafragma/metabolismo , Diafragma/fisiopatologia , Modelos Animais de Doenças , Endocitose , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose , Técnicas de Silenciamento de Genes , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Placa Motora , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Mecânica Respiratória/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
4.
Curr Opin Plant Biol ; 52: 69-76, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31509792

RESUMO

The exocyst is an evolutionary conserved complex that mediates tethering of post-Golgi vesicles derived from the conventional secretory pathway to the plasma membrane (PM), before SNARE-mediated fusion. Through its tethering function, connecting secretory vesicles to the PM, it mediates spatiotemporal regulation of exocytosis. As an integral element of the secretory machinery, the exocyst has been implicated in a large variety of processes. However, emerging evidence suggests that it may also cater for unconventional secretory pathways, as well as autophagy. The exocyst entertains a multitude of interactions with proteins and membrane phospholipids, reflecting its highly dynamic nature and the complex regulatory processes that hardwire it with cellular signalling networks. However, our molecular understanding of this essential complex remains fragmentary. Here we review recent work focusing on the molecular features that have revealed both commonalities with yeast and animals, as well as unique characteristics of the plant exocyst.


Assuntos
Exocitose , Proteínas de Transporte Vesicular , Animais , Membrana Celular , Proteínas SNARE , Saccharomyces cerevisiae , Via Secretória , Vesículas Secretórias
5.
Int J Nanomedicine ; 14: 6035-6060, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534335

RESUMO

Background: The clearance of nanomaterials (NMs) from the liver is essential for clinical safety, and their hepatic clearance is primarily determined by the co-disposition process of various types of hepatic cells. Studies of this process and the subsequent clearance routes are urgently needed for organic NMs, which are used as drug carriers more commonly than the inorganic ones. Materials and methods: In this study, the co-disposition of chitosan-based nanoparticles (CsNps) by macrophages and hepatocytes at both the cellular and animal levels as well as their subsequent biological elimination were investigated. RAW264.7 and Hepa1-6 cells were used as models of Kupffer cells and hepatocytes, respectively. Results: The cellular studies showed that CsNps released from RAW264.7 cells could enter Hepa1-6 cells through both clathrin- and caveolin-mediated endocytosis. The transport from Kupffer cells to hepatocytes was also studied in mice, and it was observed that most CsNps localized to the hepatocytes after intravenous injection. Following the distribution in hepatocytes, the hepatobiliary-fecal excretion route was shown to be the primary elimination route for CsNps, besides the kidney-urinary excretion route. The elimination of CsNps in mice was a lengthy process, with a half time of about 2 months. Conclusion: The demonstration in this study of the transport of CsNps from macrophages to hepatocytes and the subsequent hepatobiliary-fecal excretion provides basic information for the future development and clinical application of NMs.


Assuntos
Quitosana/farmacologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Nanopartículas/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Portadores de Fármacos/metabolismo , Exocitose , Hepatócitos/efeitos dos fármacos , Cinética , Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Nanopartículas/ultraestrutura , Fótons
6.
Nat Commun ; 10(1): 4125, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511515

RESUMO

Vesicular release from neurons promotes myelin sheath growth on axons. Oligodendrocytes express proteins that allow dendrites to respond to vesicular release at synapses, suggesting that axon-myelin contacts use similar communication mechanisms as synapses to form myelin sheaths. To test this, we used fusion proteins to track synaptic vesicle localization and membrane fusion in zebrafish during developmental myelination and investigated expression and localization of PSD95, a dendritic post-synaptic protein, within oligodendrocytes. Synaptic vesicles accumulate and exocytose at ensheathment sites with variable patterning and most sheaths localize PSD95 with patterning similar to exocytosis site location. Disruption of candidate PDZ-binding transsynaptic adhesion proteins in oligodendrocytes cause variable effects on sheath length and number. One candidate, Cadm1b, localizes to myelin sheaths where both PDZ binding and extracellular adhesion to axons mediate sheath growth. Our work raises the possibility that axon-glial communication contributes to myelin plasticity, providing new targets for mechanistic unraveling of developmental myelination.


Assuntos
Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Sinapses/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/metabolismo , Exocitose , Modelos Biológicos , Proteínas do Tecido Nervoso , Oligodendroglia/citologia , Vesículas Sinápticas/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra
8.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480244

RESUMO

Major depressive disorder is one of the most common neuropsychiatric disorders worldwide. The treatment of choice that shows good efficacy in mood stabilization is based on selective serotonin reuptake inhibitors (SSRIs). Their primary mechanism of action is considered to be the increased synaptic concentration of serotonin through blockade of the serotonin transporter (SERT). In this study, we described an alternative mode of action of fluoxetine (FLX), which is a representative member of the SSRI class of antidepressants. We observed that FLX robustly decreases both glutamatergic and gamma-Aminobutyric acid (GABA)-ergic synaptic release in a SERT-independent manner. Moreover, we showed that this effect may stem from the ability of FLX to change the levels of main components of the SNARE (solubile N-ethylmaleimide-sensitive factor attachment protein receptor) complex. Our data suggest that this downregulation of SNARE fusion machinery involves diminished activity of protein kinase C (PKC) due to FLX-induced blockade of P/Q type of voltage-gated calcium channels (VGCCs). Taken together, by virtue of its inhibition at SERT, fluoxetine increases extracellular serotonin levels; however, at the same time, by reducing SNARE complex function, this antidepressant reduces glutamate and GABA release.


Assuntos
Fluoxetina/farmacologia , Ácido Glutâmico/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismo , Animais , Canais de Cálcio/metabolismo , Exocitose/efeitos dos fármacos , Feminino , Humanos , Modelos Neurológicos , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Proteína Quinase C/metabolismo , Ratos Wistar
9.
Nat Commun ; 10(1): 4306, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541089

RESUMO

The mucus layer is the first line of innate host defense in the gut that protects the epithelium by spatially separating commensal bacteria. MUC2 mucin is produced and stored by goblet cells that is constitutively exocytosed or hyper secreted upon sensing a threat. How coordinated mucus exocytosis maintains homeostasis in the intestinal epithelium and modulates the immunological landscape remains elusive. Here we describe how the vesicle SNARE protein VAMP8 coordinates mucin exocytosis from goblet cells. Vamp8-/- exhibit a mild pro-inflammatory state basally due to an altered mucus layer and increased encounters with microbial antigens. Microbial diversity shifts to a detrimental microbiota with an increase abundance of pathogenic and mucolytic bacteria. To alleviate the heavy microbial burden and inflammatory state basally, Vamp8-/- skews towards tolerance. Despite this, Vamp8-/- is highly susceptible to both chemical and infectious colitis demonstrating the fragility of the intestinal mucosa without proper mucus exocytosis mechanisms.


Assuntos
Colo/metabolismo , Exocitose/fisiologia , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucina-2/metabolismo , Proteínas R-SNARE/metabolismo , Animais , Biodiversidade , Colo/patologia , Citocinas/metabolismo , Células Caliciformes/patologia , Homeostase , Humanos , Intestinos/patologia , Camundongos Knockout , Microbiota , Mucina-2/genética , Muco/metabolismo , Fenótipo , Proteínas R-SNARE/genética , Proteínas SNARE/metabolismo
10.
Nat Commun ; 10(1): 4041, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492901

RESUMO

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma, have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium. Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.


Assuntos
Proteínas de Transporte/metabolismo , Organelas/metabolismo , Fosfolipídeos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Exocitose , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Parasitos/metabolismo , Parasitos/ultraestrutura , Fosfolipídeos/química , Proteínas de Protozoários/genética
11.
Nat Commun ; 10(1): 3532, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387992

RESUMO

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.


Assuntos
Endossomos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Crescimento Neuronal , Transmissão Sináptica , Sinaptotagminas/metabolismo , Animais , Células Cultivadas , Retículo Endoplasmático/metabolismo , Exocitose , Feminino , Complexo de Golgi/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , Cultura Primária de Células , Sinaptotagminas/genética
13.
Nat Commun ; 10(1): 3838, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444335

RESUMO

Developing biomimetic nanoparticles without loss of the integrity of proteins remains a major challenge in cancer chemotherapy. Here, we develop a biocompatible tumor-cell-exocytosed exosome-biomimetic porous silicon nanoparticles (PSiNPs) as drug carrier for targeted cancer chemotherapy. Exosome-sheathed doxorubicin-loaded PSiNPs (DOX@E-PSiNPs), generated by exocytosis of the endocytosed DOX-loaded PSiNPs from tumor cells, exhibit enhanced tumor accumulation, extravasation from blood vessels and penetration into deep tumor parenchyma following intravenous administration. In addition, DOX@E-PSiNPs, regardless of their origin, possess significant cellular uptake and cytotoxicity in both bulk cancer cells and cancer stem cells (CSCs). These properties endow DOX@E-PSiNPs with great in vivo enrichment in total tumor cells and side population cells with features of CSCs, resulting in anticancer activity and CSCs reduction in subcutaneous, orthotopic and metastatic tumor models. These results provide a proof-of-concept for the use of exosome-biomimetic nanoparticles exocytosed from tumor cells as a promising drug carrier for efficient cancer chemotherapy.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Exossomos/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Exocitose , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/química , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Porosidade , Estudo de Prova de Conceito , Silício/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Nat Commun ; 10(1): 3904, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467284

RESUMO

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.


Assuntos
Fusão de Membrana/fisiologia , Proteínas SNARE/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Membrana Celular/metabolismo , Exocitose , Humanos , Insulina , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso , Células PC12 , Ratos
15.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382643

RESUMO

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Lipídeos de Membrana/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/química , Proteínas de Arabidopsis/química , Membrana Celular/química , Membrana Celular/genética , Exocitose/genética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/genética , Tricomas/química , Tricomas/genética , Proteínas de Transporte Vesicular/química
16.
Anal Sci ; 35(8): 835-838, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31281129

RESUMO

Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.


Assuntos
Exocitose , Teste de Complementação Genética , Transportador de Glucose Tipo 4/análise , Luciferases/metabolismo , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética
17.
PLoS Biol ; 17(7): e3000363, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31318874

RESUMO

For many years, double-layer phospholipid membrane vesicles, released by most cells, were not considered to be of biological significance. This stance has dramatically changed with the recognition of extracellular vesicles (EVs) as carriers of biologically active molecules that can traffic to local or distant targets and execute defined biological functions. The dimensionality of the field has expanded with the appreciation of diverse types of EVs and the complexity of vesicle biogenesis, cargo loading, release pathways, targeting mechanisms, and vesicle processing. With the expanded interest in the field and the accelerated rate of publications on EV structure and function in diverse biomedical fields, it has become difficult to distinguish between well-established biological features of EV and the untested hypotheses or speculative assumptions that await experimental proof. With the growing interest despite the limited evidence, we sought in this essay to formulate a set of unsolved mysteries in the field, sort out established data from fascinating hypotheses, and formulate several challenging questions that must be answered for the field to advance.


Assuntos
Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Modelos Biológicos , Animais , Transporte Biológico/fisiologia , Endocitose/fisiologia , Exocitose/fisiologia , Vesículas Extracelulares/classificação , Humanos , Tamanho da Partícula
18.
Blood ; 134(12): 979-982, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31262780

RESUMO

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular hemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von Willebrand factor [VWF]). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this via a proximity proteomics approach employing the peroxidase APEX2 coupled with 2 known WPB-associated proteins: the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells, and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. Whereas these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteômica/métodos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Biotinilação , Células Cultivadas , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Exocitose/fisiologia , Humanos , Enzimas Multifuncionais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via Secretória/fisiologia
19.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1618-1633, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31260699

RESUMO

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys- mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR31-145 from multidrug resistance protein into SNAP-23 Cys- mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys- and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys--MDR31-145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.


Assuntos
Cisteína/química , Exocitose/fisiologia , Mastócitos/metabolismo , Proteínas de Transporte Vesicular/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Linhagem Celular , Cisteína/genética , Hormônio do Crescimento Humano , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Mutação , Engenharia de Proteínas , Ratos , Análise de Sequência de Proteína , Transfecção
20.
PLoS One ; 14(7): e0220007, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31339911

RESUMO

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.


Assuntos
Vesículas Extracelulares/efeitos dos fármacos , Via Secretória , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Exocitose , Vesículas Extracelulares/metabolismo , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Macrolídeos/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores
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