RESUMO
Damaged mitochondria need to be cleared to maintain the quality of the mitochondrial pool. Here, we report mitocytosis, a migrasome-mediated mitochondrial quality-control process. We found that, upon exposure to mild mitochondrial stresses, damaged mitochondria are transported into migrasomes and subsequently disposed of from migrating cells. Mechanistically, mitocytosis requires positioning of damaged mitochondria at the cell periphery, which occurs because damaged mitochondria avoid binding to inward motor proteins. Functionally, mitocytosis plays an important role in maintaining mitochondrial quality. Enhanced mitocytosis protects cells from mitochondrial stressor-induced loss of mitochondrial membrane potential (MMP) and mitochondrial respiration; conversely, blocking mitocytosis causes loss of MMP and mitochondrial respiration under normal conditions. Physiologically, we demonstrate that mitocytosis is required for maintaining MMP and viability in neutrophils in vivo. We propose that mitocytosis is an important mitochondrial quality-control process in migrating cells, which couples mitochondrial homeostasis with cell migration.
Assuntos
Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Movimento Celular/fisiologia , Citoplasma/metabolismo , Exocitose/fisiologia , Feminino , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Organelas/metabolismoRESUMO
Root system architecture (RSA), the distribution of roots in soil, plays a major role in plant survival. RSA is shaped by multiple developmental processes that are largely governed by the phytohormone auxin, suggesting that auxin regulates responses of roots that are important for local adaptation. However, auxin has a central role in numerous processes, and it is unclear which molecular mechanisms contribute to the variation in RSA for environmental adaptation. Using natural variation in Arabidopsis, we identify EXOCYST70A3 as a modulator of the auxin system that causes variation in RSA by acting on PIN4 protein distribution. Allelic variation and genetic perturbation of EXOCYST70A3 lead to alteration of root gravitropic responses, resulting in a different RSA depth profile and drought resistance. Overall our findings suggest that the local modulation of the pleiotropic auxin pathway can gives rise to distinct RSAs that can be adaptive in specific environments.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Alelos , Apomorfina/análogos & derivados , Apomorfina/farmacologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secas , Exocitose , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismoRESUMO
Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4+ and CD8+ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8+ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4+ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Células Matadoras Naturais/imunologia , Leishmania donovani/fisiologia , Leishmaniose Visceral/imunologia , Malária/imunologia , Proteínas de Membrana/metabolismo , Plasmodium/fisiologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Exocitose , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Vesículas Secretórias/metabolismoRESUMO
The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.
Assuntos
Exocitose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Complexo de Golgi/metabolismo , Modelos Moleculares , Vesículas Secretórias/metabolismoRESUMO
A variety of signals finely tune insulin secretion by pancreatic ß cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in ß cells is critical for insulin secretion. Mice lacking COP1 in ß cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human ß-cell pathophysiology. In normal ß cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/metabolismo , Exocitose , Deleção de Genes , Glucose/metabolismo , Humanos , Hiperglicemia/metabolismo , Secreção de Insulina , Camundongos , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.
Assuntos
Infecções por Escherichia coli/imunologia , Lisossomos/microbiologia , Canais de Cátion TRPC/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/fisiologia , Animais , Autofagia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Exocitose , Lisossomos/enzimologia , Lisossomos/metabolismo , Camundongos , Bexiga Urinária/imunologia , Bexiga Urinária/microbiologia , Bexiga Urinária/patologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologiaRESUMO
Steroid hormones are a large family of cholesterol derivatives regulating development and physiology in both the animal and plant kingdoms, but little is known concerning mechanisms of their secretion from steroidogenic tissues. Here, we present evidence that in Drosophila, endocrine release of the steroid hormone ecdysone is mediated through a regulated vesicular trafficking mechanism. Inhibition of calcium signaling in the steroidogenic prothoracic gland results in the accumulation of unreleased ecdysone, and the knockdown of calcium-mediated vesicle exocytosis components in the gland caused developmental defects due to deficiency of ecdysone. Accumulation of synaptotagmin-labeled vesicles in the gland is observed when calcium signaling is disrupted, and these vesicles contain an ABC transporter that functions as an ecdysone pump to fill vesicles. We propose that trafficking of steroid hormones out of endocrine cells is not always through a simple diffusion mechanism as presently thought, but instead can involve a regulated vesicle-mediated release process.
Assuntos
Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Vesículas Secretórias/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Difusão , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Glândulas Endócrinas/metabolismo , Exocitose , Técnicas de Silenciamento de Genes , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Larva/citologia , Larva/metabolismo , Sinaptotagminas/metabolismoRESUMO
The lysosome has long been viewed as the recycling center of the cell. However, recent discoveries have challenged this simple view and have established a central role of the lysosome in nutrient-dependent signal transduction. The degradative role of the lysosome and its newly discovered signaling functions are not in conflict but rather cooperate extensively to mediate fundamental cellular activities such as nutrient sensing, metabolic adaptation, and quality control of proteins and organelles. Moreover, lysosome-based signaling and degradation are subject to reciprocal regulation. Transcriptional programs of increasing complexity control the biogenesis, composition, and abundance of lysosomes and fine-tune their activity to match the evolving needs of the cell. Alterations in these essential activities are, not surprisingly, central to the pathophysiology of an ever-expanding spectrum of conditions, including storage disorders, neurodegenerative diseases, and cancer. Thus, unraveling the functions of this fascinating organelle will contribute to our understanding of the fundamental logic of metabolic organization and will point to novel therapeutic avenues in several human diseases.
Assuntos
Lisossomos/metabolismo , Animais , Doença , Exocitose , Humanos , Transdução de SinaisRESUMO
Most functions of eukaryotic cells are controlled by cellular membranes, which are not static entities but undergo frequent budding, fission, fusion, and sculpting reactions collectively referred to as membrane dynamics. Consequently, regulation of membrane dynamics is crucial for cellular functions. A key mechanism in such regulation is the reversible recruitment of cytosolic proteins or protein complexes to specific membranes at specific time points. To a large extent this recruitment is orchestrated by phosphorylated derivatives of the membrane lipid phosphatidylinositol, known as phosphoinositides. The seven phosphoinositides found in nature localize to distinct membrane domains and recruit distinct effectors, thereby contributing strongly to the maintenance of membrane identity. Many of the phosphoinositide effectors are proteins that control membrane dynamics, and in this review we discuss the functions of phosphoinositides in membrane dynamics during exocytosis, endocytosis, autophagy, cell division, cell migration, and epithelial cell polarity, with emphasis on protein effectors that are recruited by specific phosphoinositides during these processes.
Assuntos
Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Autofagia , Endocitose , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Exocitose , HumanosRESUMO
The spermatozoon is a highly differentiated and polarized cell, with two main structures: the head, containing a haploid nucleus and the acrosomal exocytotic granule, and the flagellum, which generates energy and propels the cell; both structures are connected by the neck. The sperm's main aim is to participate in fertilization, thus activating development. Despite this common bauplan and function, there is an enormous diversity in structure and performance of sperm cells. For example, mammalian spermatozoa may exhibit several head patterns and overall sperm lengths ranging from â¼30 to 350 µm. Mechanisms of transport in the female tract, preparation for fertilization, and recognition of and interaction with the oocyte also show considerable variation. There has been much interest in understanding the origin of this diversity, both in evolutionary terms and in relation to mechanisms underlying sperm differentiation in the testis. Here, relationships between sperm bauplan and function are examined at two levels: first, by analyzing the selective forces that drive changes in sperm structure and physiology to understand the adaptive values of this variation and impact on male reproductive success and second, by examining cellular and molecular mechanisms of sperm formation in the testis that may explain how differentiation can give rise to such a wide array of sperm forms and functions.
Assuntos
Exocitose/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Testículo/citologia , Animais , Evolução Biológica , Humanos , Masculino , Mamíferos/fisiologia , Espermatozoides/citologiaRESUMO
Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.
Assuntos
Astrócitos , Sistema Nervoso Central , Ácido Glutâmico , Transdução de Sinais , Adulto , Humanos , Astrócitos/classificação , Astrócitos/citologia , Astrócitos/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/metabolismo , Transmissão Sináptica , Cálcio/metabolismo , Exocitose , Análise da Expressão Gênica de Célula Única , Proteína Vesicular 1 de Transporte de Glutamato/deficiência , Proteína Vesicular 1 de Transporte de Glutamato/genética , Deleção de Genes , Córtex Cerebral/citologia , Córtex Cerebral/metabolismoRESUMO
Transmitter release is a fast Ca(2+)-dependent process triggered in response to membrane depolarization. It involves two major calcium-binding proteins, the voltage-gated calcium channel (VGCC) and the vesicular protein synaptotagmin (syt1). Ca(2+) binding triggers transmitter release with a time response of conformational changes that are too fast to be accounted for by Ca(2+) binding to syt1. In contrast, conformation-triggered release, which engages Ca(2+) binding to VGCC, better accounts for the fast rate of the release process. Here, we summarize findings obtained from heterologous expression systems, neuroendocrine cells, and reconstituted systems, which reveal the molecular mechanism by which Ca(2+) binding to VGCC triggers exocytosis prior to Ca(2+) entry into the cell. This review highlights the molecular aspects of an intramembrane signaling mechanism in which a signal is propagated from the channel transmembrane (TM) domain to the TM domain of syntaxin 1A to trigger transmitter release. It discusses fundamental problems of triggering transmitter release by syt1 and suggests a classification of docked vesicles that might explain synchronous transmitter release, spontaneous release, and facilitation of transmitter release.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Exocitose/fisiologia , Células Neuroendócrinas/metabolismo , Transmissão Sináptica/fisiologia , Sinaptotagminas/metabolismo , Animais , Canais de Cálcio/fisiologia , Humanos , Modelos Biológicos , Células Neuroendócrinas/fisiologiaRESUMO
Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.
Assuntos
Quimiocina CCL21 , Células Dendríticas , Exocitose , Receptores CCR7 , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Quimiocina CCL21/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Células Dendríticas/metabolismo , Receptores CCR7/metabolismo , Receptores CCR7/genética , Junções Intercelulares/metabolismo , Migração Transendotelial e Transepitelial , Endotélio Linfático/metabolismo , Endotélio Linfático/citologia , Células Endoteliais/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Técnicas de Cocultura , Células Cultivadas , Movimento CelularRESUMO
The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.
Assuntos
Processamento Alternativo , Relógios Circadianos/genética , Exocitose , Glucose/metabolismo , Secreção de Insulina/genética , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/fisiologia , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/fisiologia , Células Cultivadas , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Nucleares/fisiologia , Obesidade/genética , Obesidade/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
In recent years, sensory neuroscientists have made major efforts to dissect the structure and function of ribbon synapses which process sensory information in the eye and ear. This review aims to summarize our current understanding of two key aspects of ribbon synapses: 1) their mechanisms of exocytosis and endocytosis and 2) their molecular anatomy and physiology. Our comparison of ribbon synapses in the cochlea and the retina reveals convergent signaling mechanisms, as well as divergent strategies in different sensory systems.
Assuntos
Cóclea/fisiologia , Retina/fisiologia , Sinapses/fisiologia , Animais , Endocitose , Exocitose , Humanos , Transmissão SinápticaRESUMO
In a recent issue of Nature,Wan et al. (2018) show that glucose-stimulated ß cells secrete insulin B chain peptides relevant to autoimmunity in type 1 diabetes. Peptides such as insulin B:12-20 are released into circulation, where they can be directly and broadly presented by antigen-presenting cells throughout the lymphatic system.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Ilhotas Pancreáticas , Autoimunidade , Exocitose , Humanos , Insulina/imunologia , Tecido Linfoide , PeptídeosRESUMO
Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.
Assuntos
Astrócitos/metabolismo , Proteínas do Olho/metabolismo , Ácido Glutâmico/metabolismo , Canais Iônicos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Bestrofinas , Células Cultivadas , Exocitose , Proteínas do Olho/genética , Células HEK293 , Humanos , Canais Iônicos/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Canais de Potássio de Domínios Poros em Tandem/genética , Alinhamento de Sequência , Transdução de SinaisRESUMO
Synaptic transmission involves cell-to-cell communication at the synaptic junction between two neurons, and chemical and electrical forms of this process have been extensively studied. In the brain, excitatory glutamatergic synapses are often made on dendritic spines that enlarge during learning1-5. As dendritic spines and the presynaptic terminals are tightly connected with the synaptic cleft6, the enlargement may have mechanical effects on presynaptic functions7. Here we show that fine and transient pushing of the presynaptic boutons with a glass pipette markedly promotes both the evoked release of glutamate and the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins8-12-as measured by Förster resonance transfer (FRET) and fluorescence lifetime imaging-in rat slice culture preparations13. Both of these effects persisted for more than 20 minutes. The increased presynaptic FRET was independent of cytosolic calcium (Ca2+), but dependent on the assembly of SNARE proteins and actin polymerization in the boutons. Notably, a low hypertonic solution of sucrose (20 mM) had facilitatory effects on both the FRET and the evoked release without inducing spontaneous release, in striking contrast with a high hypertonic sucrose solution (300 mM), which induced exocytosis by itself14. Finally, spine enlargement induced by two-photon glutamate uncaging enhanced the evoked release and the FRET only when the spines pushed the boutons by their elongation. Thus, we have identified a mechanosensory and transduction mechanism15 in the presynaptic boutons, in which the evoked release of glutamate is enhanced for more than 20 min.
Assuntos
Exocitose , Ácido Glutâmico , Animais , Ácido Glutâmico/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ratos , Proteínas SNARE/metabolismo , Sacarose/metabolismo , Sacarose/farmacologia , Sinapses/metabolismoRESUMO
In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.