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1.
Proc Natl Acad Sci U S A ; 117(33): 20088-20099, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32732436

RESUMO

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.


Assuntos
Sinalização do Cálcio/fisiologia , Medula Espinal/metabolismo , Células Th17/metabolismo , Animais , Autoantígenos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Fluorescência Verde , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina , Linfócitos T Reguladores
2.
Nat Commun ; 11(1): 4276, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848151

RESUMO

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 µm apart) enhances LTP through a NMDA receptor-mediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 µm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.


Assuntos
Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Potenciais de Ação/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Modelos Neurológicos , Células Piramidais/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia
3.
Proc Natl Acad Sci U S A ; 117(31): 18849-18857, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690691

RESUMO

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.


Assuntos
Proteínas de Arabidopsis , Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio , Cálcio/metabolismo , Imunidade Vegetal/fisiologia , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Vacúolos/metabolismo
4.
J Pharmacol Sci ; 144(2): 83-88, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32709559

RESUMO

Astrocytes generate robust intracellular Ca2+ signals that are assumed to be key regulators of astrocytic function. Among various Ca2+ mobilization mechanisms, Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor (IP3R) has attracted attention as a major component of astrocytic Ca2+ signaling. Manipulation of astrocytic IP3-Ca2+ signaling, such as genetic deletion of the type 2 IP3R, has revealed multifaceted roles of astrocytic ER Ca2+ release in health and disease. Recent developments in Ca2+ imaging techniques including ER intraluminal Ca2+ imaging have been indispensable in determining the physiological and pathophysiological significance of astrocytic ER Ca2+ release via IP3Rs. Beneficial and detrimental roles of IP3R-dependent Ca2+ release in astrocytes have been revealed in wide variety of disorders in the brain, strongly suggesting astrocytic IP3-Ca2+ signaling as a novel and promising therapeutic target.


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Animais , Encefalopatias/etiologia , Encefalopatias/genética , Encefalopatias/terapia , Deleção de Genes , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Imagem Molecular , Terapia de Alvo Molecular
5.
Am J Physiol Renal Physiol ; 319(2): F245-F255, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567348

RESUMO

Ca2+ is an important second messenger that translates extracellular stimuli into intracellular responses. Although there has been significant progress in understanding Ca2+ dynamics in organs such as the brain, the nature of Ca2+ signals in the kidney is still poorly understood. Here, we show that by using a genetically expressed highly sensitive reporter (GCaMP6s), it is possible to perform imaging of Ca2+ signals at high resolution in the mouse kidney in vivo. Moreover, by applying machine learning-based automated analysis using a Ca2+-independent signal, quantitative data can be extracted in an unbiased manner. By projecting the resulting data onto the structure of the kidney, we show that different tubular segments display highly distinct spatiotemporal patterns of Ca2+ signals. Furthermore, we provide evidence that Ca2+ activity in the proximal tubule decreases with increasing distance from the glomerulus. Finally, we demonstrate that substantial changes in intracellular Ca2+ can be detected in proximal tubules in a cisplatin model of acute kidney injury, which can be linked to alterations in cell structure and transport function. In summary, we describe a powerful new tool to investigate how single cell behavior is integrated with whole organ structure and function and how it is altered in disease states relevant to humans.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Lesão Renal Aguda/metabolismo , Animais , Humanos , Rim/anatomia & histologia , Rim/metabolismo , Túbulos Renais Proximais/anatomia & histologia , Camundongos
6.
Mol Cell ; 78(6): 1055-1069, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32559424

RESUMO

Ca2+ ions are key second messengers in both excitable and non-excitable cells. Owing to the rather pleiotropic nature of Ca2+ transporters and other Ca2+-binding proteins, however, Ca2+ signaling has attracted limited attention as a potential target of anticancer therapy. Here, we discuss cancer-associated alterations of Ca2+ fluxes at specific organelles as we identify novel candidates for the development of drugs that selectively target Ca2+ signaling in malignant cells.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Neoplasias/metabolismo , Animais , Canais de Cálcio/metabolismo , Humanos , Mitocôndrias/metabolismo , Neoplasias/genética , Transdução de Sinais/fisiologia , Canais de Receptores Transientes de Potencial/metabolismo
7.
Life Sci ; 255: 117834, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454158

RESUMO

AIMS: Hydrogen sulfide (H2S) is shown in ocular tissues and suggested to involve in the regulation of retinal circulation. However, the mechanism of H2S-induced relaxation on retinal artery is not clarified yet. Herein, we aimed to evaluate the role of several calcium (Ca2+) signaling and Ca2+ sensitization mechanisms in the relaxing effect of H2S donor, NaHS, on retinal arteries. MATERIALS AND METHODS: Relaxing effects of NaHS (10-5-3 × 10-3M) were determined on precontracted retinal arteries in Ca2+ free medium as well as in the presence of the inhibitors of Ca2+ signaling and Ca2+ sensitization mechanisms. Additively, Ca2+ sensitivity of the contractile apparatus were evaluated by CaCl2-induced contractions in the presence of NaHS (3 × 10-3M). Functional experiments were furtherly assessed by protein and/or mRNA expressions, as appropriate. KEY FINDINGS: The relaxations to NaHS were preserved in Ca2+ free medium while NaHS pretreatment decreased the responsiveness to CaCl2. The inhibitors of plasmalemmal Ca2+-ATPase, sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, Na+-Ca2+ ion-exchanger and myosin light chain kinase (MLCK) unchanged the relaxations to NaHS. Likewise, Ca2+ sensitization mechanisms including, rho kinase, protein kinase C and tyrosine kinase were unlikely to mediate the relaxation to NaHS in retinal artery. Whereas, a marked reduction was determined in NaHS-induced relaxations in the presence of MLCP inhibitor, calyculin A. Supportively, NaHS pretreatment significantly reduced phosphorylation of MYPT1-subunit of MLCP. SIGNIFICANCE: The relaxing effect of NaHS in retinal artery is likely to be related to the activation of MLCP and partly, to decrement in Ca2+ sensitivity of contractile apparatus.


Assuntos
Cálcio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Artéria Retiniana/metabolismo , Animais , Cloreto de Cálcio/administração & dosagem , Sinalização do Cálcio/fisiologia , Bovinos , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Masculino , Fosforilação/fisiologia , Sulfetos/administração & dosagem , Sulfetos/farmacologia
8.
Gene ; 752: 144765, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32413480

RESUMO

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.


Assuntos
Acoplamento Excitação-Contração/genética , Penaeidae/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Evolução Biológica , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Evolução Molecular , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Especificidade da Espécie , Transcriptoma/genética
9.
Life Sci ; 255: 117758, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32407845

RESUMO

AIMS: NLR family pyrin domain containing 3 (NLRP3) inflammasome activation contributes to the development of diabetic cardiovascular complications. CD38 regulates vascular inflammation through cyclic ADP-ribose (cADPR)-mediated Ca2+ signaling in vascular smooth muscle cells (VSMCs). Ca2+ mobilization may modulate inflammasome activation by impacting mitochondrial function. However, it remains unclear whether CD38 regulates NLRP3 inflammasome activation in VSMCs through cADPR-dependent Ca2+ release under diabetic condition. Main methods and key findings: In VSMCs, we observed that high glucose (HG, 30 mM) enhanced CD38 protein expression and ADP ribosyl cyclase activity. Moreover, along with less abundance of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and their colocalization, the expression of active caspase-1(p20) and IL-1ß were significantly inhibited by CD38 gene deficiency with siRNA transfection in VSMCs. Further, CD38 regulated the release of intracellular cADPR-mediated Ca2+ and mitochondrial DNA (mtDNA) to the cytosol, which was associated with NLRP3 inflammasome activation and VSMCs proliferation and collagen I synthesis. Finally, we found that CD38 inhibitors, nicotinamide and telmisartan significantly improved the endothelium-independent contraction and vascular remodeling, which was also associated with the inhibition of NLRP3 inflammasome in the aorta media in the diabetic mice. SIGNIFICANCE: Our data suggested that CD38/cADPR-mediated Ca2+ signaling contributed to the mitochondrial damage, consequently released mtDNA to the cytosol, which was related with NLRP3 inflammasome activation and VSMCs remodeling in diabetic mice.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Inflamassomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Sinalização do Cálcio/fisiologia , ADP-Ribose Cíclica/metabolismo , DNA Mitocondrial/metabolismo , Diabetes Mellitus Experimental/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/patologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia
10.
Nat Commun ; 11(1): 2679, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471994

RESUMO

The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4R269C) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4R269C triggers increased intracellular Ca2+ through a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca2+ and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca2+-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca2+ responses, the importance of tightly regulated Ca2+ dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.


Assuntos
Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Drosophila melanogaster/metabolismo , Doenças Neurodegenerativas/genética , Canais de Cátion TRPV/genética , Animais , Animais Geneticamente Modificados , Axônios/patologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/patologia , Asas de Animais/crescimento & desenvolvimento
11.
Sci China Life Sci ; 63(6): 875-885, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32266609

RESUMO

Brain-to-brain interfaces (BtBIs) hold exciting potentials for direct communication between individual brains. However, technical challenges often limit their performance in rapid information transfer. Here, we demonstrate an optical brain-to-brain interface that transmits information regarding locomotor speed from one mouse to another and allows precise, real-time control of locomotion across animals with high information transfer rate. We found that the activity of the genetically identified neuromedin B (NMB) neurons within the nucleus incertus (NI) precisely predicts and critically controls locomotor speed. By optically recording Ca2+ signals from the NI of a "Master" mouse and converting them to patterned optogenetic stimulations of the NI of an "Avatar" mouse, the BtBI directed the Avatar mice to closely mimic the locomotion of their Masters with information transfer rate about two orders of magnitude higher than previous BtBIs. These results thus provide proof-of-concept that optical BtBIs can rapidly transmit neural information and control dynamic behaviors across individuals.


Assuntos
Interfaces Cérebro-Computador , Encéfalo/fisiologia , Locomoção/fisiologia , Imagem Óptica/métodos , Animais , Controle Comportamental , Comportamento Animal/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Simulação por Computador , Dependovirus/metabolismo , Células HEK293 , Humanos , Cinética , Camundongos , Modelos Biológicos , Neurocinina B/análogos & derivados , Neurocinina B/fisiologia , Neurônios/fisiologia , Núcleos da Rafe/fisiologia , Máquina de Vetores de Suporte , Transfecção
12.
Invest Ophthalmol Vis Sci ; 61(4): 2, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32271891

RESUMO

Purpose: Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs. Methods: Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea. Results: The transient receptor potential vanilloid (TRPV) transcriptome in the mouse corneal epithelium is dominated by Trpv4, followed by Trpv2, Trpv3, and low levels of Trpv1 mRNAs. TRPV4 protein was localized to basal and intermediate epithelial strata, keratocytes, and the endothelium in contrast to the cognate TRPV1, which was confined to intraepithelial afferents and a sparse subset of CECs. The TRPV4 agonist GSK1016790A induced cation influx and calcium elevations, which were abolished by the selective blocker HC067047. Hypotonic solutions, membrane strain, and moderate heat elevated [Ca2+]CEC with swelling- and temperature-, but not strain-evoked signals, sensitive to HC067047. GSK1016790A and swelling evoked calcium-dependent ATP release, which was suppressed by HC067027 and the hemichannel blocker probenecid. Conclusions: These results demonstrate that cation influx via TRPV4 transduces osmotic and thermal but not strain inputs to CECs and promotes hemichannel-dependent ATP release. The TRPV4-hemichannel-ATP signaling axis might modulate corneal pain induced by excessive mechanical, osmotic, and chemical stimulation.


Assuntos
Epitélio Anterior/metabolismo , Mecanotransdução Celular/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Cultivadas , Eletrofisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pressão Osmótica , Técnicas de Patch-Clamp , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Canais de Cátion TRPV/genética
13.
PLoS Biol ; 18(4): e3000700, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32330125

RESUMO

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A-mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A-dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release-activated Ca2+ channel 1 (Orai1) within ER-plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.


Assuntos
Canais Iônicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação Estromal/genética
14.
Nat Commun ; 11(1): 1906, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312988

RESUMO

Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.


Assuntos
Astrócitos/citologia , Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Sinapses/metabolismo , Animais , Encéfalo , Cálcio/metabolismo , Hipocampo , Imageamento Tridimensional , Masculino , Camundongos , Microscopia , Neurônios/metabolismo
15.
Nat Commun ; 11(1): 1143, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123168

RESUMO

By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions.


Assuntos
Sinalização do Cálcio/fisiologia , Optogenética/métodos , Linfócitos T/metabolismo , Animais , Proteínas de Arabidopsis/genética , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular , Movimento Celular , Quimiocinas/metabolismo , Criptocromos/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fótons , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Célula Única/métodos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Linfócitos T/citologia
16.
PLoS Comput Biol ; 16(3): e1007605, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32119665

RESUMO

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.


Assuntos
Canais de Cálcio/metabolismo , Ouriços-do-Mar/metabolismo , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Quimiotaxia/fisiologia , Biologia Computacional/métodos , Íons/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Modelos Teóricos , Transdução de Sinais , Motilidade Espermática/fisiologia
17.
PLoS Comput Biol ; 16(3): e1007650, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163407

RESUMO

Calcium imaging has been widely used for measuring spiking activities of neurons. When using calcium imaging, we need to extract summarized information from the raw movie beforehand. Recent studies have used matrix deconvolution for this preprocessing. However, such an approach can neither directly estimate the generative mechanism of spike trains nor use stimulus information that has a strong influence on neural activities. Here, we propose a new deconvolution method for calcium imaging using marked point processes. We consider that the observed movie is generated from a probabilistic model with marked point processes as hidden variables, and we calculate the posterior of these variables using a variational inference approach. Our method can simultaneously estimate various kinds of information, such as cell shape, spike occurrence time, and tuning curve. We apply our method to simulated and experimental data to verify its performance.


Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Algoritmos , Animais , Cálcio/metabolismo , Simulação por Computador , Camundongos , Modelos Neurológicos , Modelos Teóricos , Neurônios/fisiologia , Processamento de Sinais Assistido por Computador
18.
J Neurosci ; 40(15): 3052-3062, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32132265

RESUMO

Maintenance of cardiorespiratory homeostasis depends on autonomic reflexes controlled by neuronal circuits of the brainstem. The neurophysiology and neuroanatomy of these reflex pathways are well understood, however, the mechanisms and functional significance of autonomic circuit modulation by glial cells remain largely unknown. In the experiments conducted in male laboratory rats we show that astrocytes of the nucleus of the solitary tract (NTS), the brain area that receives and integrates sensory information from the heart and blood vessels, respond to incoming afferent inputs with [Ca2+]i elevations. Astroglial [Ca2+]i responses are triggered by transmitters released by vagal afferents, glutamate acting at AMPA receptors and 5-HT acting at 5-HT2A receptors. In conscious freely behaving animals blockade of Ca2+-dependent vesicular release mechanisms in NTS astrocytes by virally driven expression of a dominant-negative SNARE protein (dnSNARE) increased baroreflex sensitivity by 70% (p < 0.001). This effect of compromised astroglial function was specific to the NTS as expression of dnSNARE in astrocytes of the ventrolateral brainstem had no effect. ATP is considered the principle gliotransmitter and is released by vesicular mechanisms blocked by dnSNARE expression. Consistent with this hypothesis, in anesthetized rats, pharmacological activation of P2Y1 purinoceptors in the NTS decreased baroreflex gain by 40% (p = 0.031), whereas blockade of P2Y1 receptors increased baroreflex gain by 57% (p = 0.018). These results suggest that glutamate and 5-HT, released by NTS afferent terminals, trigger Ca2+-dependent astroglial release of ATP to modulate baroreflex sensitivity via P2Y1 receptors. These data add to the growing body of evidence supporting an active role of astrocytes in brain information processing.SIGNIFICANCE STATEMENT Cardiorespiratory reflexes maintain autonomic balance and ensure cardiovascular health. Impaired baroreflex may contribute to the development of cardiovascular disease and serves as a robust predictor of cardiovascular and all-cause mortality. The data obtained in this study suggest that astrocytes are integral components of the brainstem mechanisms that process afferent information and modulate baroreflex sensitivity via the release of ATP. Any condition associated with higher levels of "ambient" ATP in the NTS would be expected to decrease baroreflex gain by the mechanism described here. As ATP is the primary signaling molecule of glial cells (astrocytes, microglia), responding to metabolic stress and inflammatory stimuli, our study suggests a plausible mechanism of how the central component of the baroreflex is affected in pathological conditions.


Assuntos
Astrócitos/fisiologia , Barorreflexo/fisiologia , Núcleo Solitário/fisiologia , Trifosfato de Adenosina/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Masculino , Neurônios Aferentes/metabolismo , Neurotransmissores/metabolismo , Neurotransmissores/fisiologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Proteínas SNARE/fisiologia , Serotonina/farmacologia , Estimulação do Nervo Vago
19.
J Neurosci ; 40(16): 3152-3164, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32156830

RESUMO

Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.


Assuntos
Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Animais Geneticamente Modificados , Antiporters/genética , Sinalização do Cálcio/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Trocador de Sódio e Cálcio/genética
20.
Nat Rev Nephrol ; 16(6): 337-351, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32127698

RESUMO

The kidney is a remarkable organ that accomplishes the challenge of removing waste from the body and simultaneously regulating electrolyte and water balance. Pro-urine flows through the nephron in a highly dynamic manner and adjustment of the reabsorption rates of water and ions to the variable tubular flow is required for electrolyte homeostasis. Renal epithelial cells sense the tubular flow by mechanosensation. Interest in this phenomenon has increased in the past decade since the acknowledgement of primary cilia as antennae that sense renal tubular flow. However, the significance of tubular flow sensing for electrolyte handling is largely unknown. Signal transduction pathways regulating flow-sensitive physiological responses involve calcium, purinergic and nitric oxide signalling, and are considered to have an important role in renal electrolyte handling. Given that mechanosensation of tubular flow is an integral role of the nephron, defective tubular flow sensing is probably involved in renal disease. Studies investigating tubular flow and electrolyte transport differ in their methodology, subsequently hampering translational validity. This Review provides the basis for understanding electrolyte disorders originating from altered tubular flow sensing as a result of pathological conditions.


Assuntos
Sinalização do Cálcio/fisiologia , Túbulos Renais/metabolismo , Óxido Nítrico/metabolismo , Receptores Purinérgicos/metabolismo , Reabsorção Renal/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Desequilíbrio Hidroeletrolítico/metabolismo , Água Corporal/metabolismo , Cílios , Eletrólitos/metabolismo , Células Epiteliais , Taxa de Filtração Glomerular , Humanos , Pelve Renal , Mecanotransdução Celular , Microfluídica , Transdução de Sinais
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