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1.
J Microsc ; 287(3): 114-137, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35810393

RESUMO

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes has limited the volume size and number of samples that can be imaged. Recent improvements in methodology are currently driving a revolution in volume EM, making possible the structural imaging of whole organs and small organisms. In turn, these recent developments in image acquisition have created or stressed bottlenecks in other parts of the pipeline, like sample preparation, image analysis and data management. While the progress in image analysis is stunning due to the advent of automatic segmentation and server-based annotation tools, several challenges remain. Here we discuss recent trends in volume EM, emerging methods for increasing throughput and implications for sample preparation, image analysis and data management.


A key concept in biology is that the structure of tissues, cells and their components (cell organelles) often relates to their function. With electron microscopy (EM), it is possible to reveal this structure with nanometre resolution and therefore infer about its function. Electron microscopy of tissues knows a long history of method development, starting in the 1940s. Method development has largely determined the possibilities and scope of electron microscopy. In the 2000s, innovative techniques were developed that allowed routine imaging of tissues in 3D with a higher degree of automation. Nevertheless, conventional electron microscopy techniques remain unsuited for imaging of tissue with nanometre resolution on a millimetre scale because of their low inherent throughput. Here we analyse trends in volume electron microscopy (EM of tissues in 3D) by reviewing the application, acquisition parameters and data information from over 100 publications in the field. We see an expansion of interest from the conventional applications in neuroscience to other fields, such as cell biology. Additionally, the size of data sets is growing rapidly. From here, we review in detail how certain developments in methodology from the past 10 years have tried to overcome the low acquisition throughput of electron microscopes, by making these techniques more robust during long acquisitions, but also much faster by parallelisation. We find that these new developments have big implications for sample preparation, processing and analysis of the images and data management. We therefore also describe the new developments in these separate domains. We illustrate how novel sample preparation protocols have been developed specifically for larger volumes, how the introduction of machine learning has accelerated automated segmentation of volume EM data and that there is an ongoing transition from local to remote data storage and management. We also touch upon the tools that researchers use to analyse and annotate EM data. We conclude that the potential of volume EM remains high and the new developments open up possibilities for novel biological studies. We promote the sharing of resources and tools between researchers and institutions to maximise the potential from the new developments in volume electron microscopy.


Assuntos
Processamento de Imagem Assistida por Computador , Manejo de Espécimes , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica , Proteínas , Manejo de Espécimes/métodos
2.
FEBS Lett ; 596(19): 2497-2512, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35644832

RESUMO

Microscopic analysis of molecules and physiology in living cells and systems is a powerful tool in life sciences. While in vivo subcellular microscopic analysis of healthy and diseased human organs remains impossible, zebrafish larvae allow studying pathophysiology of many organs using in vivo microscopy. Here, we review the potential of the larval zebrafish pancreas in the context of islets of Langerhans and Type 1 diabetes. We highlight the match of zebrafish larvae with the expanding toolbox of fluorescent probes that monitor cell identity, fate and/or physiology in real time. Moreover, fast and efficient modulation and localization of fluorescence at a subcellular level, through fluorescence microscopy, including confocal and light sheet (single plane illumination) microscopes tailored to in vivo larval research, is addressed. These developments make the zebrafish larvae an extremely powerful research tool for translational research. We foresee that living larval zebrafish models will replace many cell line-based studies in understanding the contribution of molecules, organelles and cells to organ pathophysiology in whole organisms.


Assuntos
Ilhotas Pancreáticas , Peixe-Zebra , Animais , Corantes Fluorescentes , Humanos , Larva , Microscopia de Fluorescência
3.
Nat Commun ; 12(1): 3379, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099719

RESUMO

GATA3 is as a lineage-specific transcription factor that drives the differentiation of CD4+ T helper 2 (Th2) cells, but is also involved in a variety of processes such as immune regulation, proliferation and maintenance in other T cell and non-T cell lineages. Here we show a mechanism utilised by CD4+ T cells to increase mitochondrial mass in response to DNA damage through the actions of GATA3 and AMPK. Activated AMPK increases expression of PPARG coactivator 1 alpha (PPARGC1A or PGC1α protein) at the level of transcription and GATA3 at the level of translation, while DNA damage enhances expression of nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2). PGC1α, GATA3 and NRF2 complex together with the ATR to promote mitochondrial biogenesis. These findings extend the pleotropic interactions of GATA3 and highlight the potential for GATA3-targeted cell manipulation for intervention in CD4+ T cell viability and function after DNA damage.


Assuntos
Linfócitos T CD4-Positivos/citologia , Dano ao DNA , Fator de Transcrição GATA3/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Cultura Primária de Células
4.
Front Aging ; 2: 681428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35821991

RESUMO

Mitochondrial health and cellular metabolism can heavily influence the onset of senescence in T cells. CD8+ EMRA T cells exhibit mitochondrial dysfunction and alterations to oxidative phosphorylation, however, the metabolic properties of senescent CD8+ T cells from people living with type 2 diabetes (T2D) are not known. We show here that mitochondria from T2D CD8+ T cells had a higher oxidative capacity together with increased levels of mitochondrial reactive oxgen species (mtROS), compared to age-matched control cells. While fatty acid uptake was increased, fatty acid oxidation was impaired in T2D CD8+ EMRA T cells, which also showed an accumulation of lipid droplets and decreased AMPK activity. Increasing glucose and fatty acids in healthy CD8+ T cells resulted in increased p-p53 expression and a fragmented mitochondrial morphology, similar to that observed in T2D CD8+ EMRA T cells. The resulting mitochondrial changes are likely to have a profound effect on T cell function. Consequently, a better understanding of these metabolic abnormalities is crucial as metabolic manipulation of these cells may restore correct T cell function and help reduce the impact of T cell dysfunction in T2D.

6.
Elife ; 92020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32985972

RESUMO

Experience influences behavior, but little is known about how experience is encoded in the brain, and how changes in neural activity are implemented at a network level to improve performance. Here we investigate how differences in experience impact brain circuitry and behavior in larval zebrafish prey capture. We find that experience of live prey compared to inert food increases capture success by boosting capture initiation. In response to live prey, animals with and without prior experience of live prey show activity in visual areas (pretectum and optic tectum) and motor areas (cerebellum and hindbrain), with similar visual area retinotopic maps of prey position. However, prey-experienced animals more readily initiate capture in response to visual area activity and have greater visually-evoked activity in two forebrain areas: the telencephalon and habenula. Consequently, disruption of habenular neurons reduces capture performance in prey-experienced fish. Together, our results suggest that experience of prey strengthens prey-associated visual drive to the forebrain, and that this lowers the threshold for prey-associated visual activity to trigger activity in motor areas, thereby improving capture performance.


Assuntos
Aprendizagem/fisiologia , Comportamento Predatório/fisiologia , Prosencéfalo/fisiologia , Vias Visuais/fisiologia , Peixe-Zebra/fisiologia , Animais
7.
J Invest Dermatol ; 140(4): 806-815.e5, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31518559

RESUMO

The skin is our interface with the outside world, and consequently it is exposed to a wide range of microbes and allergens. Recent studies have indicated that allergen-specific skin-resident memory T (TRM) cells play a role in allergic contact dermatitis (ACD). However, the composition and dynamics of the epidermal T-cell subsets during ACD are not known. Here we show that exposure of the skin to the experimental contact allergen DNFB results in a displacement of the normally occurring dendritic epidermal T cells (DETC) concomitant with an accumulation of epidermal CD8+CD69+CD103+ TRM cells in mice. By studying knockout mice, we provide evidence that CD8+ T cells are required for the displacement of the DETC and that DETC are not required for recruitment of CD8+ TRM cells to the epidermis following allergen exposure. We demonstrate that the magnitude of the allergic reaction correlates with the number of CD8+ epidermal TRM cells, which again correlates with allergen dose and number of allergen exposures. Finally, in an attempt to elucidate why CD8+ epidermal TRM cells persist in the epidermis, we show that CD8+ epidermal TRM cells have a higher proliferative capability and are bioenergetically more stable, displaying a higher spare respiratory capacity than DETC.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Memória Imunológica , Animais , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/patologia , Dermatite Alérgica de Contato/patologia , Modelos Animais de Doenças , Epiderme/patologia , Camundongos , Camundongos Knockout
8.
Aging Cell ; 19(2): e13067, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31788930

RESUMO

The susceptibility of human CD4+ and CD8+ T cells to senesce differs, with CD8+ T cells acquiring an immunosenescent phenotype faster than the CD4+ T cell compartment. We show here that it is the inherent difference in mitochondrial content that drives this phenotype, with senescent human CD4+ T cells displaying a higher mitochondrial mass. The loss of mitochondria in the senescent human CD8+ T cells has knock-on consequences for nutrient usage, metabolism and function. Senescent CD4+ T cells uptake more lipid and glucose than their CD8+ counterparts, leading to a greater metabolic versatility engaging either an oxidative or a glycolytic metabolism. The enhanced metabolic advantage of senescent CD4+ T cells allows for more proliferation and migration than observed in the senescent CD8+ subset. Mitochondrial dysfunction has been linked to both cellular senescence and aging; however, it is still unclear whether mitochondria play a causal role in senescence. Our data show that reducing mitochondrial function in human CD4+ T cells, through the addition of low-dose rotenone, causes the generation of a CD4+ T cell with a CD8+ -like phenotype. Therefore, we wish to propose that it is the inherent metabolic stability that governs the susceptibility to an immunosenescent phenotype.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Senescência Celular/imunologia , Imunossenescência/fisiologia , Mitocôndrias/metabolismo , Trifosfato de Adenosina/metabolismo , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Movimento Celular/imunologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Glucose/metabolismo , Glicólise/imunologia , Humanos , Antígenos Comuns de Leucócito/sangue , Antígenos Comuns de Leucócito/metabolismo , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Rotenona/farmacologia
9.
Vaccine ; 38(3): 635-643, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31727505

RESUMO

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Vacinas Bacterianas/administração & dosagem , Chlamydia trachomatis/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Interferon Tipo I , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/imunologia , Composição de Medicamentos/métodos , Feminino , Imunidade Celular/imunologia , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucosa/efeitos dos fármacos , Mucosa/imunologia
10.
Nat Protoc ; 14(3): 864-900, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30804570

RESUMO

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca2+ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.


Assuntos
Holografia/métodos , Optogenética/métodos , Fótons , Sequência de Aminoácidos , Animais , Compostos Azo/química , Feminino , Células HEK293 , Humanos , Ligantes , Domínios Proteicos , Ratos Sprague-Dawley , Receptores de Glutamato/química , Estereoisomerismo
11.
Nat Chem Biol ; 14(7): 655-663, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29867144

RESUMO

The unusually high demand for metals in the brain, along with insufficient understanding of how their dysregulation contributes to neurological diseases, motivates the study of how inorganic chemistry influences neural circuitry. We now report that the transition metal copper is essential for regulating rest-activity cycles and arousal. Copper imaging and gene expression analysis in zebrafish identifies the locus coeruleus-norepinephrine (LC-NE) system, a vertebrate-specific neuromodulatory circuit critical for regulating sleep, arousal, attention, memory and emotion, as a copper-enriched unit with high levels of copper transporters CTR1 and ATP7A and the copper enzyme dopamine ß-hydroxylase (DBH) that produces NE. Copper deficiency induced by genetic disruption of ATP7A, which loads copper into DBH, lowers NE levels and hinders LC function as manifested by disruption in rest-activity modulation. Moreover, LC dysfunction caused by copper deficiency from ATP7A disruption can be rescued by restoring synaptic levels of NE, establishing a molecular CTR1-ATP7A-DBH-NE axis for copper-dependent LC function.


Assuntos
Cobre/metabolismo , Locus Cerúleo/metabolismo , Norepinefrina/metabolismo , Animais , Cobre/química , Locus Cerúleo/química , Norepinefrina/química , Peixe-Zebra
12.
Ageing Res Rev ; 47: 24-30, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29902528

RESUMO

The impact of cellular senescence during ageing is well established, however senescence is now recognised to play a role in a variety of age related and metabolic diseases, such as cancer, autoimmune and cardiovascular diseases. It is therefore crucial to gain a better understanding of the mechanisms that control cellular senescence. In recent years our understanding of the intimate relationship between cell metabolism, cell signalling and cellular senescence has greatly improved. In this review we discuss the differing roles of glucose and protein metabolism in both senescent fibroblast and CD8+ T-cells, and explore the impact cellular metabolism has on the senescence-associated secretory phenotype (SASP) of these cell types.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Senescência Celular/fisiologia , Fibroblastos/metabolismo , Animais , Linfócitos T CD8-Positivos/patologia , Fibroblastos/patologia , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Linfócitos T/patologia
13.
Biochemistry ; 57(11): 1733-1747, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29465990

RESUMO

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.


Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila/química , Luz , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Halorhodospira halophila/genética , Ligação de Hidrogênio , Fotorreceptores Microbianos/genética , Relação Estrutura-Atividade
14.
Aging Cell ; 17(1)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29024417

RESUMO

Cellular senescence is accompanied by a senescence-associated secretory phenotype (SASP). We show here that primary human senescent CD8+ T cells also display a SASP comprising chemokines, cytokines and extracellular matrix remodelling proteases that are unique to this subset and contribute to age-associated inflammation. We found the CD8+ CD45RA+ CD27- EMRA subset to be the most heterogeneous, with a population aligning with the naïve T cells and another with a closer association to the effector memory subset. However, despite the differing processes that give rise to these senescent CD8+ T cells once generated, they both adopt a unique secretory profile with no commonality to any other subset, aligning more closely with senescence than quiescence. Furthermore, we also show that the SASP observed in senescent CD8+ T cells is governed by p38 MAPK signalling.


Assuntos
Linfócitos T CD8-Positivos/citologia , Senescência Celular/genética , Citocinas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Adulto , Citocinas/metabolismo , Dano ao DNA/genética , Voluntários Saudáveis , Humanos , Sistema de Sinalização das MAP Quinases/genética , Pessoa de Meia-Idade , Fenótipo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
15.
J Phys Chem Lett ; 8(18): 4498-4503, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28872878

RESUMO

Iron-sulfur proteins play essential roles in various biological processes. Their electronic structure and vibrational dynamics are key to their rich chemistry but nontrivial to unravel. Here, the first ultrafast transient absorption and impulsive coherent vibrational spectroscopic (ICVS) studies on 2Fe-2S clusters in Rhodobacter capsulatus ferreodoxin VI are characterized. Photoexcitation initiated populations on multiple excited electronic states that evolve into each other in a long-lived charge-transfer state. This suggests a potential light-induced electron-transfer pathway as well as the possibility of using iron-sulfur proteins as photosensitizers for light-dependent enzymes. A tyrosine chain near the active site suggests potential hole-transfer pathways and affirms this electron-transfer pathway. The ICVS data revealed vibrational bands at 417 and 484 cm-1, with the latter attributed to an excited-state mode. The temperature dependence of the ICVS modes suggests that the temperature effect on protein structure or conformational heterogeneities needs to be considered during cryogenic temperature studies.


Assuntos
Proteínas Ferro-Enxofre/química , Conformação Proteica , Rhodobacter capsulatus/fisiologia , Temperatura , Espectroscopia de Ressonância de Spin Eletrônica , Ferredoxinas , Guanina/análogos & derivados , Proteínas Ferro-Enxofre/fisiologia , Oxirredução , Fotoquímica , Análise Espectral , Enxofre/química , Vibração
16.
Neuron ; 91(3): 587-601, 2016 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-27397519

RESUMO

Inter-individual behavioral variation is thought to increase fitness and aid adaptation to environmental change, but the underlying mechanisms are poorly understood. We find that variation between individuals in neuromodulatory input contributes to individuality in short-term habituation of the zebrafish (Danio Rerio) acoustic startle response (ASR). ASR habituation varies greatly between individuals, but differences are stable over days and are heritable. Acoustic stimuli that activate ASR-command Mauthner cells also activate dorsal raphe nucleus (DRN) serotonergic neurons, which project to the vicinity of the Mauthner cells and their inputs. DRN neuron activity decreases during habituation in proportion to habituation and a genetic manipulation that reduces serotonin content in DRN neurons increases habituation, whereas serotonergic agonism or DRN activation with ChR2 reduces habituation. Finally, level of rundown of DRN activity co-segregates with extent of behavioral habituation across generations. Thus, variation between individuals in neuromodulatory input contributes to individuality in a core adaptive behavior. VIDEO ABSTRACT.


Assuntos
Núcleo Dorsal da Rafe/citologia , Núcleo Dorsal da Rafe/fisiologia , Habituação Psicofisiológica/fisiologia , Individualidade , Reflexo de Sobressalto/fisiologia , Neurônios Serotoninérgicos/fisiologia , Peixe-Zebra/fisiologia , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Apomorfina/farmacologia , Núcleo Dorsal da Rafe/efeitos dos fármacos , Núcleo Dorsal da Rafe/metabolismo , Habituação Psicofisiológica/efeitos dos fármacos , Quipazina/farmacologia , Reflexo de Sobressalto/efeitos dos fármacos , Rodopsina/biossíntese , Rodopsina/genética , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo
17.
Immunity ; 44(3): 597-608, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26944200

RESUMO

The cationic polysaccharide chitosan is an attractive candidate adjuvant capable of driving potent cell-mediated immunity, but the mechanism by which it acts is not clear. We show that chitosan promotes dendritic cell maturation by inducing type I interferons (IFNs) and enhances antigen-specific T helper 1 (Th1) responses in a type I IFN receptor-dependent manner. The induction of type I IFNs, IFN-stimulated genes and dendritic cell maturation by chitosan required the cytoplasmic DNA sensor cGAS and STING, implicating this pathway in dendritic cell activation. Additionally, this process was dependent on mitochondrial reactive oxygen species and the presence of cytoplasmic DNA. Chitosan-mediated enhancement of antigen specific Th1 and immunoglobulin G2c responses following vaccination was dependent on both cGAS and STING. These findings demonstrate that a cationic polymer can engage the STING-cGAS pathway to trigger innate and adaptive immune responses.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Quitosana/administração & dosagem , Células Dendríticas/fisiologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Nucleotidiltransferases/metabolismo , Células Th1/imunologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular , Células Cultivadas , DNA/metabolismo , Células Dendríticas/efeitos dos fármacos , Feminino , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Imunoglobulina G/metabolismo , Interferon Tipo I/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Nucleotidiltransferases/genética , Espécies Reativas de Oxigênio/metabolismo , Vacinas/administração & dosagem
18.
Elife ; 52016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26929991

RESUMO

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.


Assuntos
Luz , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos da radiação , Potenciais de Ação , Animais , Hipocampo/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Peixe-Zebra/embriologia
19.
Immunity ; 44(2): 368-79, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26885859

RESUMO

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.


Assuntos
Interferon gama/metabolismo , Macrófagos/fisiologia , Glicoproteínas de Membrana/metabolismo , Mycobacterium tuberculosis/imunologia , Receptores de Interleucina-1/metabolismo , Tuberculose Pulmonar/imunologia , Animais , Autofagia/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Células HEK293 , Humanos , Imunidade Inata/genética , Sistema de Sinalização das MAP Quinases/genética , Macrófagos/microbiologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Polimorfismo Genético , Ligação Proteica/genética , RNA Interferente Pequeno/genética , Receptores de Interferon/metabolismo , Receptores de Interleucina-1/genética , Tuberculose Pulmonar/genética , Receptor de Interferon gama
20.
Nat Methods ; 12(9): 852-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26167640

RESUMO

Circuit mapping requires knowledge of both structural and functional connectivity between cells. Although optical tools have been made to assess either the morphology and projections of neurons or their activity and functional connections, few probes integrate this information. We have generated a family of photoactivatable genetically encoded Ca(2+) indicators that combines attributes of high-contrast photolabeling with high-sensitivity Ca(2+) detection in a single-color protein sensor. We demonstrated in cultured neurons and in fruit fly and zebrafish larvae how single cells could be selected out of dense populations for visualization of morphology and high signal-to-noise measurements of activity, synaptic transmission and connectivity. Our design strategy is transferrable to other sensors based on circularly permutated GFP (cpGFP).


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas Luminescentes/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Optogenética/métodos , Animais , Rastreamento de Células/métodos , Células Cultivadas , Drosophila , Luz , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Engenharia de Proteínas/métodos , Ratos , Peixe-Zebra
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