RESUMO
Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.
Assuntos
Cerebelo , Proteínas de Transferência de Fosfolipídeos , Sinapses , Transmissão Sináptica , Vesículas Sinápticas , Animais , Vesículas Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Feminino , Masculino , Cerebelo/citologia , Sinapses/metabolismo , Sinapses/fisiologia , Células Cultivadas , Camundongos Knockout , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neurônios/fisiologia , Endocitose/fisiologiaRESUMO
Sexual reproduction involves the fusion of two gametes of opposite sex. Although the sperm-expressed fusogen HAPLESS 2 (HAP2) or GENERATIVE CELL SPECIFIC 1 (GCS1) plays a vital role in this process in many eukaryotic organisms and an understanding of its regulation is emerging in unicellular systems [J. Zhang et al., Nat. Commun. 12, 4380 (2021); J. F. Pinello et al. Dev. Cell 56, 3380-3392.e9 (2021)], neither HAP2/GCS1 interactors nor mechanisms for delivery and activation at the fusion site are known in multicellular plants. Here, we show that Arabidopsis thaliana HAP2/GCS1 interacts with two sperm DUF679 membrane proteins (DMP8 and DMP9), which are required for the EGG CELL 1 (EC1)-induced translocation of HAP2/GCS1 from internal storage vesicle to the sperm plasma membrane to ensure successful fertilization. Our studies in Arabidopsis and tobacco provide evidence for a conserved function of DMP8/9-like proteins as HAP2/GCS1 partner in seed plants. Our data suggest that seed plants evolved a DMP8/9-dependent fusogen translocation process to achieve timely acquisition of sperm fusion competence in response to egg cell-derived signals, revealing a previously unknown critical step for successful fertilization.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Sementes/metabolismo , Arabidopsis/metabolismo , Espermatozoides/metabolismo , Fertilização/fisiologiaRESUMO
Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.
Assuntos
Proteínas de Transporte , Proteínas de Drosophila/genética , Retículo Endoplasmático , Proteínas de Transporte Vesicular/genética , Animais , Membrana Celular/metabolismo , Drosophila melanogaster , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositóis , Saccharomyces cerevisiaeRESUMO
In contrast to synaptic vesicle exocytosis, secretory granule exocytosis follows a much longer time course, and thus allows for different prefusion states prior to stimulation. Indeed, total internal reflection fluorescence microscopy in living pancreatic ß cells reveals that, prior to stimulation, either visible or invisible granules fuse in parallel during both early (first) and late (second) phases after glucose stimulation. Therefore, fusion occurs not only from granules predocked to the plasma membrane but also from those translocated from the cell interior during ongoing stimulation. Recent findings suggest that such heterogeneous exocytosis is conducted by a specific set of multiple Rab27 effectors that appear to operate on the same granule; namely, exophilin-8, granuphilin, and melanophilin play differential roles in distinct secretory pathways to final fusion. Furthermore, the exocyst, which is known to tether secretory vesicles to the plasma membrane in constitutive exocytosis, cooperatively functions with these Rab27 effectors in regulated exocytosis. In this review, the basic nature of insulin granule exocytosis will be described as a representative example of secretory granule exocytosis, followed by a discussion of the means by which different Rab27 effectors and the exocyst coordinate to regulate the entire exocytic processes in ß cells.
Assuntos
Insulina , Proteínas rab de Ligação ao GTP , Insulina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Proteínas de Transporte Vesicular/metabolismo , ExocitoseRESUMO
Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here, we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size â¼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.
Assuntos
Exocitose , Vesículas Secretórias , Animais , Grânulos Citoplasmáticos/metabolismo , Drosophila , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Secretagogin (SCGN) is a hexa-EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.
Assuntos
Exocitose , Secretagoginas/química , Secretagoginas/metabolismo , Animais , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mutação , Ligação Proteica , Conformação Proteica , Secretagoginas/genética , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Peixe-ZebraRESUMO
The monomeric GTPase Rab27 regulates exocytosis of a broad range of vesicles in multicellular organisms. Several effectors bind GTP-bound Rab27a and/or Rab27b on secretory vesicles to execute a series of exocytic steps, such as vesicle maturation, movement along microtubules, anchoring within the peripheral F-actin network, and tethering to the plasma membrane, via interactions with specific proteins and membrane lipids in a local milieu. Although Rab27 effectors generally promote exocytosis, they can also temporarily restrict it when they are involved in the rate-limiting step. Genetic alterations in Rab27-related molecules cause discrete diseases manifesting pigment dilution and immunodeficiency, and can also affect common diseases such as diabetes and cancer in complex ways. Although the function and mechanism of action of these effectors have been explored, it is unclear how multiple effectors act in coordination within a cell to regulate the secretory process as a whole. It seems that Rab27 and various effectors constitutively reside on individual vesicles to perform consecutive exocytic steps. The present review describes the unique properties and in vivo roles of the Rab27 system, and the functional relationship among different effectors coexpressed in single cells, with pancreatic beta cells used as an example.Key words: membrane trafficking, regulated exocytosis, insulin granules, pancreatic beta cells.
Assuntos
Exocitose , Proteínas rab de Ligação ao GTP , Membrana Celular/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
All animals must coordinate growth rate and timing of maturation to reach the appropriate final size. Here, we describe hobbit, a novel and conserved gene identified in a forward genetic screen for Drosophila animals with small body size. hobbit is highly conserved throughout eukaryotes, but its function remains unknown. We demonstrate that hobbit mutant animals have systemic growth defects because they fail to secrete insulin. Other regulated secretion events also fail in hobbit mutant animals, including mucin-like 'glue' protein secretion from the larval salivary glands. hobbit mutant salivary glands produce glue-containing secretory granules that are reduced in size. Importantly, secretory granules in hobbit mutant cells lack essential membrane fusion machinery required for exocytosis, including Synaptotagmin 1 and the SNARE SNAP-24. These membrane fusion proteins instead accumulate inside enlarged late endosomes. Surprisingly, however, the Hobbit protein localizes to the endoplasmic reticulum. Our results suggest that Hobbit regulates a novel step in intracellular trafficking of membrane fusion proteins. Our studies also suggest that genetic control of body size, as a measure of insulin secretion, is a sensitive functional readout of the secretory machinery.
Assuntos
Tamanho Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Insulina/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Glândulas Salivares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/genética , Proteínas do Grude Salivar de Drosophila/metabolismo , Secreção de Insulina , Tamanho do Órgão/genética , Transporte Proteico/genética , Vesículas Secretórias/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Sinaptotagmina I/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca2+). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca2+ sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca2+-dependent release has been unclear. Here, we report that the Ca2+-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca2+-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca2+-stimulated release. We propose a parsimonious model whereby Ca2+-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca2+ Our data further suggest Ca2+-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.
Assuntos
Cálcio/metabolismo , Exocitose , Fusão de Membrana/fisiologia , Multimerização Proteica/fisiologia , Sinaptotagmina I/metabolismo , Animais , Cátions Bivalentes/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Imunofluorescência , Proteínas de Fluorescência Verde/química , Microscopia Eletrônica , Mutação , Células PC12 , Ligação Proteica/fisiologia , Ratos , Proteínas Recombinantes/metabolismo , Sinaptotagmina I/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The remodeling of biological membranes is crucial for a vast number of cellular activities and is an inherently multiscale process in both time and space. Seminal work has provided important insights into nanometer-scale membrane deformations, and highlighted the remarkable variation and complexity in the underlying molecular machineries and mechanisms. However, how membranes are remodeled at the micron-scale, particularly in vivo, remains poorly understood. Here, we discuss how using regulated exocytosis of large (1.5-2.0 µm) membrane-bound secretory granules in the salivary gland of live mice as a model system, has provided evidence for the importance of the actomyosin cytoskeleton in micron-scale membrane remodeling in physiological conditions. We highlight some of these advances, and present mechanistic hypotheses for how the various biochemical and biophysical properties of distinct actomyosin networks may drive this process.
Assuntos
Actomiosina/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Animais , Exocitose/fisiologia , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismoRESUMO
BACKGROUND: The regulation of exocytosis is physiologically vital in cells and requires a variety of distinct proteins and lipids that facilitate efficient, fast, and timely release of secretory vesicle cargo. Growing evidence suggests that regulatory lipids act as important lipid signals and regulate various biological processes including exocytosis. Though functional roles of many of these regulatory lipids has been linked to exocytosis, the dynamic behavior of these lipids during membrane fusion at sites of exocytosis in cell culture remains unknown. METHODS: Total internal reflection fluorescence microscopy (TIRF) was used to observe the spatial organization and temporal dynamics (i.e. spatial positioning and timing patterns) of several lipids, and accessory proteins, like lipid kinases and protein kinases, in the form of protein kinase C (PRKC) associated with sites of exocytosis of matrix metalloproteinase-9 (MMP-9) in living MCF-7 cancer cells. RESULTS: Following stimulation with phorbol myristate acetate (PMA) to promote exocytosis, a transient accumulation of several distinct regulatory lipids, lipid kinases, and protein kinases at exocytic sites was observed. This transient accumulation centered at the time of membrane fusion is followed by a rapid diffusion away from the fusion sites. Additionally, the synthesis of these regulatory lipids, degradation of these lipids, and the downstream effectors activated by these lipids, are also achieved by the recruitment and accumulation of key enzymes at exocytic sites (during the moment of cargo release). This includes key enzymes like lipid kinases, protein kinases, and phospholipases that facilitate membrane fusion and exocytosis of MMP-9. CONCLUSIONS: This work suggests that these regulatory lipids and associated effector proteins are locally synthesized and/or recruited to sites of exocytosis, during membrane fusion and cargo release. More importantly, their enrichment at fusion sites serves as an important spatial and temporal organizing "element" defining individual exocytic sites.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Animais , Western Blotting , Exocitose/genética , Exocitose/fisiologia , Humanos , Células MCF-7 , Microscopia de Fluorescência , Proteína Quinase C/metabolismo , Vesículas Secretórias/metabolismoRESUMO
Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter-containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single-cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn2+ through modulation of neurotransmitter release.
Assuntos
Neurotransmissores/genética , Células PC12/metabolismo , Transmissão Sináptica/genética , Zinco/química , Animais , Transporte Biológico , RatosRESUMO
The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease (PD). In this review, we discuss recent results concerning its primary function, which appears to be on cell membranes. The pre-synaptic location of synuclein has suggested a role in neurotransmitter release and it apparently associates with synaptic vesicles because of their high curvature. Indeed, synuclein over-expression inhibits synaptic vesicle exocytosis. However, loss of synuclein has not yet been shown to have a major effect on synaptic transmission. Consistent with work showing that synuclein can promote as well as sense membrane curvature, recent analysis of synuclein triple knockout mice now shows that synuclein accelerates dilation of the exocytic fusion pore. This form of regulation affects primarily the release of slowly discharged lumenal cargo such as neural peptides, but presumably also contributes to maintenance of the release site. This article is part of the Special Issue "Synuclein".
Assuntos
Doença de Parkinson/metabolismo , alfa-Sinucleína/fisiologia , Animais , Axônios/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Exocitose/fisiologia , Humanos , Fusão de Membrana/fisiologia , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/patologia , Mutação de Sentido Incorreto , Terminações Pré-Sinápticas/química , Domínios Proteicos , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , alfa-Sinucleína/química , alfa-Sinucleína/deficiência , alfa-Sinucleína/genéticaRESUMO
Exocytosis of secretory granules entails budding from the trans-Golgi network, sorting and maturation of cargo proteins, and trafficking and fusion to the plasma membrane. Rab27a regulates the late steps in this process, such as granule recruitment to the fusion site, whereas Rab2a functions in the early steps, such as granule biogenesis and maturation. Here, we demonstrate that these two small GTPases simultaneously bind to Noc2 (also known as RPH3AL) in a GTP-dependent manner, although Rab2a binds only after Rab27a has bound. In pancreatic ß-cells, the ternary Rab2a-Noc2-Rab27a complex specifically localizes on perinuclear immature granules, whereas the binary Noc2-Rab27a complex localizes on peripheral mature granules. In contrast to the wild type, Noc2 mutants defective in binding to Rab2a or Rab27a fail to promote glucose-stimulated insulin secretion. Although knockdown of any component of the ternary complex markedly inhibits insulin secretion, only knockdown of Rab2a or Noc2, and not that of Rab27a, impairs cargo processing from proinsulin to insulin. These results suggest that the dual effector, Noc2, regulates the transition from Rab2a-mediated granule biogenesis to Rab27a-mediated granule exocytosis.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose , Proteínas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Mutantes/metabolismo , Ligação Proteica , Proteínas/química , Ratos , Proteínas rab27 de Ligação ao GTPRESUMO
Real-time imaging of regulated exocytosis in secreting organs can provide unprecedented temporal and spatial detail. Here, we highlight recent advances in 3D time-lapse imaging in Drosophila salivary glands at single-granule resolution. Using fluorescently labeled proteins expressed in the fly, it is now possible to image the dynamics of vesicle biogenesis and the cytoskeletal factors involved in secretion. 3D imaging over time allows one to visualize and define the temporal sequence of events, including clearance of cortical actin, fusion pore formation, mixing of the vesicular and plasma membranes and recruitment of components of the cytoskeleton. We will also discuss the genetic tools available in the fly that allow one to interrogate the essential factors involved in secretory vesicle formation, cargo secretion and the ultimate integration of the vesicular and plasma membranes. We argue that the combination of high-resolution real-time imaging and powerful genetics provides a platform to investigate the role of any factor in regulated secretion.
Assuntos
Drosophila/fisiologia , Exocitose , Glândulas Salivares/ultraestrutura , Vesículas Secretórias/ultraestrutura , Imagem com Lapso de Tempo/métodos , Animais , Citoesqueleto/metabolismo , Humanos , Imageamento Tridimensional , Fusão de Membrana , Microscopia de Fluorescência , Biologia Molecular/métodos , Glândulas Salivares/metabolismoRESUMO
A micromolar concentration of zinc has been shown to significantly change the dynamics of exocytosis as well as the vesicle contents in a model cell line, providing direct evidence that zinc regulates neurotransmitter release. To provide insight into how zinc modulates these exocytotic processes, neurotransmitter release and vesicle content were compared with single cell amperometry and intracellular impact vesicle cytometry with a range of zinc concentrations. Additionally, time-of-flight secondary ion mass spectrometry (ToF-SIMS) images of lipid distributions in the cell membrane after zinc treatment correlate to changes in exocytosis. By combining electrochemical techniques and mass spectrometry imaging, we proposed a mechanism by which zinc changes the fusion pore and the rate of neurotransmitter release by changing lipid distributions and results in the modulation of synaptic strength and plasticity.
Assuntos
Lipídeos/química , Zinco/química , Animais , Catecolaminas/análise , Membrana Celular/metabolismo , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Etilenodiaminas/química , Exocitose/efeitos dos fármacos , Células PC12 , Análise de Componente Principal , Ratos , Espectrometria de Massas por Ionização por Electrospray , Zinco/farmacologiaRESUMO
SummaryProlyl endopeptidase (PREP) is a post-proline cleaving enzyme. It is involved in the regulation of multiple inositol polyphosphate phosphatase activity implicated in the pathway of inositol 1,4,5-trisphosphate, resulting in the modulation of cytosolic Ca2+ levels. Besides its peptidase activity, PREP was identified as a binding partner of tubulin, suggesting that it may participate in microtubule-associate processes. In this paper, we evaluated the expression of PREP mRNA and protein by polymerase chain reaction and western blot analyses and its co-localization with tubulin by immunofluorescence in adult mouse seminal vesicles. We showed that both proteins are cytoplasmic: tubulin is localized at the apical half part of the cell, while PREP has a more diffuse localization, showing a prominent distribution at the apical cytoplasm. These findings support our hypothesis of a specific role for PREP in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular its association with tubulin filaments. Moreover, it may regulate Ca2+ levels, and promote the final step of vesicular exocytosis, namely the fusion of the vesicles with the plasma membrane. These results strongly suggest that there is a pivotal role for PREP in vesicle exocytosis, as well as in the physiology of mouse seminal vesicles.
Assuntos
Exocitose , Glândulas Seminais/enzimologia , Serina Endopeptidases/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Prolil Oligopeptidases , Ligação Proteica , Serina Endopeptidases/genéticaRESUMO
Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) complex mediating fast Ca(2+)-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b-deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b-deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b-deficient mouse may represent a diabesity model.
Assuntos
Doenças Metabólicas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Adiposidade , Animais , Glicemia/metabolismo , Peso Corporal , Dislipidemias/patologia , Ingestão de Energia , Metabolismo Energético , Comportamento Alimentar , Feminino , Homeostase , Hipertrofia , Hipotálamo/metabolismo , Insulina/metabolismo , Secreção de Insulina , Leptina/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Doenças Metabólicas/sangue , Camundongos Obesos , Fenótipo , Receptores para Leptina/metabolismo , Proteína 25 Associada a Sinaptossoma/deficiênciaRESUMO
The neocortex represents one of the largest estates of the human brain. This structure comprises ~30-40 billions of neurones and even more of non-neuronal cells. Astrocytes, highly heterogeneous homoeostatic glial cells, are fundamental for housekeeping of the brain and contribute to information processing in neuronal networks. Gray matter astrocytes tightly enwrap synapses, contact blood vessels and, naturally, are also in contact with the extracellular space, where convection of fluid takes place. Thus astrocytes receive signals from several distinct extracellular domains and can get excited by numerous mechanisms, which regulate cytosolic concentration of second messengers, such as Ca2+ and cAMP. Excited astrocytes often secrete diverse substances (generally referred to as gliosignalling molecules) that include classical neurotransmitters such as glutamate and ATP or neuromodulators such as D-serine or neuropeptides. Astrocytic secretion occurs through several mechanisms: by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release of gliosignalling molecules appears fundamentally similar to that operating in neurones, since it depends on the SNARE proteins-dependent merger of the vesicle membrane with the plasmalemma. However, the coupling between the stimulus and astroglial vesicular secretion is at least one order of magnitude slower than that in neurones. Here we review mechanisms of astrocytic excitability and the molecular, anatomical and physiological properties of vesicular apparatus mediating the release of gliosignalling molecules in health and in the neurodegenerative pathology.
Assuntos
Astrócitos/metabolismo , Doenças Neurodegenerativas/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Astrócitos/patologia , Exocitose , Humanos , Neocórtex/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Proteínas SNARE/metabolismoRESUMO
Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.