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1.
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
2.
Infect Immun ; 87(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31262980

RESUMO

Pneumonia due to Gram-negative bacteria is associated with high mortality. Acinetobacter baumannii is a Gram-negative bacterium that is associated with hospital-acquired and ventilator-associated pneumonia. Bacteria have been described to release outer membrane vesicles (OMVs) that are capable of mediating systemic inflammation. The mechanism by which A. baumannii OMVs mediate inflammation is not fully defined. We sought to investigate the roles that Toll-like receptors (TLRs) play in A. baumannii OMV-mediated pulmonary inflammation. We isolated OMVs from A. baumannii cultures and intranasally introduced the OMVs into mice. Intranasal introduction of A. baumannii OMVs mediated pulmonary inflammation, which is associated with neutrophil recruitment and weight loss. In addition, A. baumannii OMVs increased the release of several chemokines and cytokines in the mouse lungs. The proinflammatory responses were partially inhibited in TLR2- and TLR4-deficient mice compared to those of wild-type mice. This study highlights the important roles of TLRs in A. baumannii OMV-induced pulmonary inflammation in vivo.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/fisiologia , Pneumonia/microbiologia , Vesículas Secretórias/fisiologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Infecções por Acinetobacter/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos
3.
Brain Res ; 1702: 46-53, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29577889

RESUMO

The evolution of peptidergic signaling systems in the central nervous system serves a distinct and crucial role in brain processes and function. The diversity of physiological peptides and the complexity of their regulation and secretion from the dense core vesicles (DCV) throughout the brain is a topic greatly in need of investigation, though recent years have shed light on cellular and molecular mechanisms that are summarized in this review. Here, we focus on the convergence of peptidergic systems onto the Locus Coeruleus (LC), the sole provider of norepinephrine (NE) to the cortex and hippocampus, via large DCV. As the LC-NE system is one of the first regions of the brain to undergo degeneration in Alzheimer's Disease (AD), and markers of DCV have consistently been demonstrated to have biomarker potential for AD progression, here we summarize the current literature linking the LC-NE system with DCV dysregulation and Aß peptides. We also include neuroanatomical data suggesting that the building blocks of senile plaques, Aß monomers, may be localized to DCV of the LC and noradrenergic axon terminals of the prefrontal cortex. Finally, we explore the putative consequences of chronic stress on Aß production and the role that DCV may play in LC degeneration. Clinical data of immunological markers of DCV in AD patients are discussed.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Locus Cerúleo/fisiologia , Vesículas Secretórias/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Humanos , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Norepinefrina/metabolismo , Norepinefrina/fisiologia , Vesículas Secretórias/patologia
4.
Ann Am Thorac Soc ; 15(Suppl 3): S159-S163, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30431338

RESUMO

The respiratory system is protected from inhaled particles and microbes by the mucociliary system. This system differs between animal species, where pigs and humans have numerous submucosal glands. The polymer-forming mucin, MUC5B, is packed in a highly organized way in granules of the mucus-secreting cells in the glands. Upon secretion, the packed MUC5B is flushed out by a chloride- and bicarbonate-rich fluid from the cystic fibrosis transmembrane conductance regulator-expressing serosal cells located at the most distal part of the gland. The bicarbonate raises the pH and removes calcium from the N terminus of MUC5B, allowing the mucin to be pulled out into a linear polymer. Thousands of such polymers gather in bundles in the submucosal gland duct, and these bundles appear at the opening of the glands. They are moved by the beating cilia, and sweep over the airway surface and are patchily coated with the MUC5AC mucin from the surface goblet cells. The movement of these bundles is controlled by the MUC5AC mucin attachment/detachment to the goblet cells. Thus, higher animals with submucosal glands and large diameters of the proximal airways are efficiently cleaned by the thick mucus bundles sweeping the airway surface and moving particles and bacteria toward the larynx.


Assuntos
Pneumopatias/etiologia , Mucinas/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Pneumopatias/diagnóstico , Pneumopatias/terapia , Depuração Mucociliar/fisiologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/fisiopatologia , Vesículas Secretórias/fisiologia , Suínos
5.
Ann Am Thorac Soc ; 15(Suppl 3): S164-S170, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30431339

RESUMO

Exocytosis of secreted mucins is the final step in their intracellular processing, resulting in their release into the airway lumen to interact with water and ions to form mucus. Mucins are secreted at a low baseline rate and a high stimulated rate, and both rates are regulated by second messengers acting on components of the exocytic machinery. The principal physiologic function of the low baseline rate is to support steady-state mucociliary clearance of inhaled particles and pathogens that enter the airways during normal breathing. Even in the setting of mucin hyperproduction, baseline secretion generally does not induce mucus occlusion. The principal physiologic function of the high stimulated rate of secretion from both submucosal glands and surface goblet cells in proximal airways appears to be to sweep away larger particles, whereas in distal airways it appears to act in concert with mucin hyperproduction to induce mucus occlusion to trap migrating helminths. Pathophysiologically, stimulated mucin secretion in the setting of mucin hyperproduction from allergic or other types of airway inflammation in the absence of helminth infection causes airflow obstruction and infection. Molecular components of the mucin exocytic machinery are increasingly being identified, and surprisingly, many components are not shared between baseline and stimulated machines. The physiologic significance of the presence of two distinct molecular machines is not yet known, such as whether these interact selectively with secretory granules of different sizes or contents. A full understanding of the mechanism and regulation of airway mucin secretion will provide further insight into pathophysiologic processes and may identify therapeutic strategies to alleviate obstructive airway diseases.


Assuntos
Exocitose/fisiologia , Pneumopatias/etiologia , Mucinas/metabolismo , Depuração Mucociliar/fisiologia , Mucosa Respiratória/fisiologia , Humanos , Pneumopatias/diagnóstico , Pneumopatias/terapia , Muco/metabolismo , Vesículas Secretórias/fisiologia
6.
Ann Am Thorac Soc ; 15(Suppl 3): S154-S158, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30431345

RESUMO

Mucociliary clearance is a crucial component of innate defense of the lung. In respiratory diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, mucus with abnormal properties contributes to obstruction of the airways. The failure in function of mucus in airway clearance and pathogen protection leads to chronic infection and risk of death. Polymeric mucins (MUC5AC and MUC5B) provide the structural framework of the airway mucus gel. The intracellular synthesis and assembly of these enormous, polymeric O-linked glycoproteins is a complex, multistage process involving intra- and intermolecular disulfide bond formation and extensive addition of O-glycan chains. The fully formed polymers are packaged in a highly organized and condensed form within secretory granules inside specialized secretory cells, and after the appropriate stimulus, mucins are released and expand to form mucus. This short article brings together the current knowledge on the different steps in the production of mucin polymers and the molecular mechanisms that condense them into a packaged form in secretory granules. It is by unraveling the molecular mechanisms that control intracellular mucin supramolecular structure that we might gain new insight into what determines mucus gel properties in health and disease.


Assuntos
Pneumopatias/etiologia , Mucinas/metabolismo , Humanos , Pneumopatias/diagnóstico , Pneumopatias/terapia , Depuração Mucociliar , Vesículas Secretórias/fisiologia
7.
Biochem Soc Trans ; 46(5): 1021-1027, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30154095

RESUMO

As part of their life cycle, Gram-negative bacteria produce and release microvesicles (outer membrane vesicles, OMVs) consisting of spherical protrusions of the outer membrane that encapsulate periplasmic contents. OMVs produced by commensal bacteria in the gastrointestinal (GI) tract of animals are dispersed within the gut lumen with their cargo and enzymes being distributed across and throughout the GI tract. Their ultimate destination and fate is unclear although they can interact with and cross the intestinal epithelium using different entry pathways and access underlying immune cells in the lamina propria. OMVs have also been found in the bloodstream from which they can access various tissues and possibly the brain. The nanosize and non-replicative status of OMVs together with their resistance to enzyme degradation and low pH, alongside their ability to interact with the host, make them ideal candidates for delivering biologics to mucosal sites, such as the GI and the respiratory tract. In this mini-review, we discuss the fate of OMVs produced in the GI tract of animals with a focus on vesicles released by Bacteroides species and the use of OMVs as vaccine delivery vehicles and other potential applications.


Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Microbioma Gastrointestinal , Bactérias Gram-Negativas/fisiologia , Vesículas Secretórias/fisiologia , Animais , Bacteroides , Encéfalo/microbiologia , Células Epiteliais/microbiologia , Trato Gastrointestinal/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Sistema Imunitário , Mucosa Intestinal/microbiologia
8.
J Physiol ; 596(16): 3759-3773, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29873393

RESUMO

KEY POINTS: Despite their immense physiological and pathophysiological importance, we know very little about the biology of dense core vesicle (DCV) trafficking in the intact mammalian brain. DCVs are transported at similar average speeds in the anaesthetized and awake mouse brain compared to neurons in culture, yet maximal speed and pausing fraction of transport were higher. Microtubule plus (+)-end extension imaging visualized microtubular growth at 0.12 µm/s and revealed that DCVs were transported faster in the anterograde direction. DCV transport slowed down upon presynaptic bouton approach, possibly promoting synaptic localization and cargo release. Our work provides a basis to extrapolate DCV transport properties determined in cultured neurons to the intact mouse brain and reveals novel features such as slowing upon bouton approach and brain state-dependent trafficking directionality. ABSTRACT: Neuronal dense core vesicles (DCVs) transport many cargo molecules like neuropeptides and neurotrophins to their release sites in dendrites or axons. The transport properties of DCVs in axons of the intact mammalian brain are unknown. We used viral expression of a DCV cargo reporter (NPY-Venus/Cherry) in the thalamus and two-photon in vivo imaging to visualize axonal DCV trafficking in thalamocortical projections of anaesthetized and awake mice. We found an average speed of 1 µm/s, maximal speeds of up to 5 µm/s and a pausing fraction of ∼11%. Directionality of transport differed between anaesthetized and awake mice. In vivo microtubule +-end extension imaging using MACF18-GFP revealed microtubular growth at 0.12 µm/s and provided positive identification of antero- and retrograde axonal transport. Consistent with previous reports, anterograde transport was faster (∼2.1 µm/s) than retrograde transport (∼1.4 µm/s). In summary, DCVs are transported with faster maximal speeds and lower pausing fraction in vivo compared to previous results obtained in vitro. Finally, we found that DCVs slowed down upon presynaptic bouton approach. We propose that this mechanism promotes synaptic localization and cargo release.


Assuntos
Anestesia , Transporte Axonal , Axônios/fisiologia , Vesículas Secretórias/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Vigília , Animais , Axônios/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/fisiologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Vesículas Secretórias/efeitos dos fármacos , Córtex Somatossensorial/citologia , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/fisiologia , Sinapses/efeitos dos fármacos , Tálamo/citologia , Tálamo/efeitos dos fármacos , Tálamo/fisiologia
9.
Cell Calcium ; 73: 53-54, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880197

RESUMO

The focus of this special issue (SI) ¼Membrane Merger in Conventional and Unconventional Vesicle Secretion« is regulated exocytosis, a universally conserved mechanism, consisting of a merger between the vesicle and the plasma membranes. Although this process evolved with eukaryotic organisms some three billion years ago (Spang et al., 2015), the understanding of physiology and patobiology of this process, especially at elementary vesicle level, remains unclear. Exocytotic fusion consists of several stages, starting by vesicle delivery to the plasma membrane, initially establishing a very narrow and stable fusion pore, that can reversibly open and close several times before it can fully widen. This allows vesicle cargo to be completely discharged from the vesicle lumen and permits vesicle-membrane resident proteins including channels, transporters, receptors and other signalling molecules, to be incorporated into the plasma membrane. The contributions in this SI bring new insights on the complexity of vesicle-based secretion, including discussion that vesicle anatomy appears to modulate exocytotic fusion pore properties and that the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins (SNARE-proteins), not only facilitate pre- and post-fusion stages of exocytosis, but also serve in vesicle navigation within the cytoplasm.


Assuntos
Membrana Celular/fisiologia , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Proteínas SNARE/fisiologia , Animais , Humanos , Saccharomyces cerevisiae/metabolismo , Vesículas Secretórias/fisiologia
10.
Cell ; 173(4): 934-945.e12, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29606354

RESUMO

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.


Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Fusão de Membrana/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Membrana Celular/química , Células Cromafins/citologia , Células Cromafins/metabolismo , Dinaminas/metabolismo , Estimulação Elétrica , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Masculino , Microscopia Confocal , Modelos Biológicos , Técnicas de Patch-Clamp , Vesículas Secretórias/fisiologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-29496823

RESUMO

The delivery of intracellular material within cells is crucial for maintaining normal function. Myosins transport a wide variety of cargo, ranging from vesicles to ribonuclear protein particles (RNPs), in plants, fungi, and metazoa. The properties of a given myosin transporter are adapted to move on different actin filament tracks, either on the disordered actin networks at the cell cortex or along highly organized actin bundles to distribute their cargo in a localized manner or move it across long distances in the cell. Transport is controlled by selective recruitment of the myosin to its cargo that also plays a role in activation of the motor.


Assuntos
Miosinas/metabolismo , Animais , Transporte Biológico , Corrente Citoplasmática , Humanos , Organelas/fisiologia , Plantas/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Vesículas Secretórias/fisiologia , Vesículas Transportadoras/fisiologia
12.
Curr Biol ; 28(5): 697-710.e13, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29478853

RESUMO

In the endocytic pathway of animals, two related complexes, called CORVET (class C core vacuole/endosome transport) and HOPS (homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease, including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes, but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent, while CORVET-specific subunits have proliferated. VPS8 (vacuolar protein sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup, including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, Δvps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst-sorting receptor Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating that target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.


Assuntos
Endossomos/fisiologia , Proteínas de Protozoários/metabolismo , Vesículas Secretórias/fisiologia , Tetrahymena thermophila/fisiologia , Transporte Biológico/fisiologia , Tetrahymena thermophila/genética
13.
Curr Biol ; 28(1): 146-153.e5, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29290560

RESUMO

Spatial control of exocytosis underlies polarized cell morphogenesis. In rod-shaped fission yeast, exocytic vesicles are conveyed along the actin cytoskeleton by myosin V motors toward growing cell ends [1, 2], the major sites for exocytosis. However, actomyosin-based vesicle delivery is dispensable for polarized secretion and cylindrical cell shape of fission yeast [3]. Thus, additional mechanisms should function in the spatial confinement of exocytosis. Here we report a novel role of endoplasmic reticulum (ER)-plasma membrane (PM) contacts in restricting exocytic sites for polarized fission yeast morphogenesis. We show that fission yeast cells deficient in both ER-PM contacts and actomyosin-based secretory vesicle transport display aberrant globular cell shape due to delocalized exocytosis. By artificially manipulating the strength and extent of ER-PM contacts in wild-type and mutant cells that exhibit induced ectopic exocytosis, we demonstrate that exocytosis and ER-PM contact formation are spatially incompatible. Furthermore, extensive ER-PM junctions at the non-growing lateral cell cortex prevent the PM from exocytic vesicle tethering and hence attenuate growth potential at cell sides. We thus propose that ER-PM contacts function as a new morphogenetic module by limiting exocytosis to growing cell tips in fission yeast. A similar mechanism could apply to other cell types with prominent ER-PM contacts.


Assuntos
Ciclo Celular/fisiologia , Membrana Celular/fisiologia , Polaridade Celular/fisiologia , Retículo Endoplasmático/fisiologia , Exocitose , Schizosaccharomyces/fisiologia , Actomiosina/fisiologia , Morfogênese , Vesículas Secretórias/fisiologia
14.
Gastroenterology ; 154(6): 1805-1821.e5, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29360461

RESUMO

BACKGROUND & AIMS: Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS: We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-µm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS: Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS: In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.


Assuntos
Autofagia/genética , Exocitose/genética , Pâncreas/citologia , Pancreatite/genética , Sintaxina 1/metabolismo , Células Acinares/metabolismo , Animais , Membrana Celular/metabolismo , Ceruletídeo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirurgia , Pancreatite/induzido quimicamente , Vesículas Secretórias/fisiologia , Tripsinogênio/metabolismo
15.
J Neurochem ; 144(3): 241-254, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29178418

RESUMO

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense-core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)-pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT-PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B-/- neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B-/- neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY-pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH-sensitive NPY-pHluorin fluorescence, were all normal in CgA/B-/- neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B-/- neurons. In contrast, CgA/B-/- chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live-cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B-/- neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.


Assuntos
Cromogranina A/fisiologia , Cromogranina B/fisiologia , Exocitose , Neurônios/fisiologia , Biogênese de Organelas , Vesículas Secretórias/fisiologia , Animais , Cromogranina A/genética , Cromogranina B/genética , Feminino , Hipocampo/fisiologia , Hipocampo/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/ultraestrutura , Cultura Primária de Células , Vesículas Secretórias/ultraestrutura , Sinapses/ultraestrutura
16.
Artigo em Inglês | MEDLINE | ID: mdl-28264817

RESUMO

One requirement for establishing polarity within a cell is the asymmetric trafficking of intracellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated trafficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establishment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front-back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.


Assuntos
Polaridade Celular , Exocitose , Animais , Transporte Biológico , Movimento Celular , Citoesqueleto/fisiologia , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Vesículas Secretórias/fisiologia
17.
Mol Biol Cell ; 28(24): 3542-3553, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28904207

RESUMO

Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, we studied the functional relationships of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. Here we show that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). Our results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNA interference had no influence on anterograde DCV runs, and detailed colocalization analysis by superresolution microscopy revealed that Unc-104 and Khc coassociate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. We suggest a mechanism in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, and then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.


Assuntos
Transporte Axonal/fisiologia , Cinesina/metabolismo , Neuropeptídeos/metabolismo , Animais , Axônios/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Cinesina/genética , Neurônios/metabolismo , Neuropeptídeos/fisiologia , Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/fisiologia , Vesículas Secretórias/metabolismo , Vesículas Secretórias/fisiologia
18.
J Biol Chem ; 292(37): 15240-15253, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28765280

RESUMO

Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential phylogenetically conserved octameric protein complex. Exocyst biogenesis is regulated by several processes, but the mechanisms by which the exocyst is degraded are unknown. Here, to unravel the components of the exocyst degradation pathway, we screened for extragenic suppressors of a temperature-sensitive fission yeast strain mutated in the exocyst subunit Sec3 (sec3-913). One of the suppressing DNAs encoded a truncated dominant-negative variant of the 26S proteasome subunit, Rpt2, indicating that exocyst degradation is controlled by the ubiquitin-proteasome system. The temperature-dependent growth defect of the sec3-913 strain was gene dosage-dependent and suppressed by blocking the proteasome, Hsp70-type molecular chaperones, the Pib1 E3 ubiquitin-protein ligase, and the deubiquitylating enzyme Ubp3. Moreover, defects in cell septation, exocytosis, and endocytosis in sec3 mutant strains were similarly alleviated by mutation of components in this pathway. We also found that, particularly under stress conditions, wild-type Sec3 degradation is regulated by Pib1 and the 26S proteasome. In conclusion, our results suggest that a cytosolic protein quality control pathway monitors folding and proteasome-dependent turnover of an exocyst subunit and, thereby, controls exocytosis in fission yeast.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Modelos Biológicos , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Vesículas Secretórias/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/genética , Endocitose/efeitos dos fármacos , Endopeptidases/genética , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Deleção de Genes , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/genética , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/ultraestrutura , Estresse Fisiológico/efeitos dos fármacos , Temperatura , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/genética
19.
Biochim Biophys Acta Gen Subj ; 1861(9): 2293-2303, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28669852

RESUMO

BACKGROUND: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosis. METHODS: Thus we here employed high-resolution cell-attached capacitance measurements and super-resolution structured illumination microscopy to directly examine single vesicle interactions with the plasmalemma in cultured rat astrocytes treated with distinct pharmacological modulators of dynamin activity. Fluorescent dextrans and the lipophilic plasmalemmal marker DiD were utilized to monitor uptake and distribution of vesicles in the peri-plasmalemmal space and in the cell cytosol. RESULTS: Dynamin inhibition with Dynole™-34-2 and Dyngo™-4a prevented vesicle internalization into the cytosol and decreased fusion pore conductance of vesicles that remained attached to the plasmalemma via a narrow fusion pore that lapsed into a state of repetitive opening and closing - flickering. In contrast, the dynamin activator Ryngo™-1-23 promoted vesicle internalization and favored fusion pore closure by prolonging closed and shortening open fusion pore dwell times. Immunocytochemical staining revealed dextran uptake into dynamin-positive vesicles and increased dextran uptake into Syt4- and VAMP2-positive vesicles after dynamin inhibition, indicating prolonged retention of these vesicles at the plasmalemma. CONCLUSIONS: Our results have provided direct evidence for a role of dynamin in regulation of fusion pore geometry and kinetics of endo- and exocytotic vesicles, indicating that both share a common dynamin-regulated structural intermediate, the fusion pore.


Assuntos
Dinaminas/fisiologia , Endocitose , Exocitose , Fusão de Membrana , Vesículas Secretórias/fisiologia , Animais , Células Cultivadas , Dextranos/farmacocinética , Dinaminas/antagonistas & inibidores , Capacitância Elétrica , Feminino , Ratos , Ratos Wistar
20.
Am J Physiol Gastrointest Liver Physiol ; 313(3): G203-G211, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28642299

RESUMO

Patients with alcohol-related cirrhosis (ALD) are prone to infection. Circulating neutrophils in ALD are dysfunctional and predict development of sepsis, organ dysfunction, and survival. Neutrophil granules are important effector organelles containing a toxic array of microbicidal proteins, whose controlled release is required to kill microorganisms while minimizing inflammation and damage to host tissue. We investigated the role of these granular responses in contributing to immune disarray in ALD. Neutrophil granular content and mobilization were measured by flow cytometric quantitation of cell-surface/intracellular markers, [secretory vesicles (CD11b), secondary granules (CD66b), and primary granules (CD63; myeloperoxidase)] before and after bacterial stimulation in 29 patients with ALD cirrhosis (15 abstinent; 14 actively drinking) compared with healthy controls (HC). ImageStream Flow Cytometry characterized localization of granule subsets within the intracellular and cell-surface compartments. The plasma cytokine environment was analyzed using ELISA/cytokine bead array. Circulating neutrophils were primed in the resting state with upregulated surface expression of CD11b (P = 0.0001) in a cytokine milieu rich in IL-8 (P < 0.001) and lactoferrin (P = 0.035). Neutrophils showed exaggerated mobilization to the cell surface of primary granules at baseline (P = 0.001) and in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (P = 0.009) and Escherichia coli (P = 0.0003) in ALD. There was no deficit in granule content or mobilization to the cell membrane in any granule subset observed. Paradoxically, active alcohol consumption abrogated the hyperresponsive neutrophil granular responses compared with their abstinent counterparts. Neutrophils are preprimed at baseline with augmented effector organelle mobilization in response to bacterial stimulation; neutrophil degranulation is not a mechanism leading to innate immunoparesis in ALD.NEW & NOTEWORTHY Neutrophil granule release is dysregulated in patients with alcohol-related cirrhosis (ALD) with augmented effector organelle mobilization and microbiocidal protein release. Neutrophil granules are upregulated in ALD at baseline and demonstrate augmented responses to bacterial challenge. The granular responses in ALD did not contribute to the observed functional deficit in innate immunity but rather were dysregulated and hyperresponsive, which may induce bystander damage to host tissue. Paradoxically, active alcohol consumption abrogated the excessive neutrophil granular responses to bacterial stimulus compared with their abstinent counterparts.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Cirrose Hepática Alcoólica/patologia , Neutrófilos/fisiologia , Adulto , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Cirrose Hepática Alcoólica/metabolismo , Masculino , Pessoa de Meia-Idade , Vesículas Secretórias/fisiologia
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