Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
1.
EMBO J ; 40(16): e107901, 2021 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-34169542

RESUMEN

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.


Asunto(s)
Calcio/metabolismo , Diferenciación Celular , Células T Asesinas Naturales/metabolismo , Timo/citología , Animales , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitocondrias/metabolismo
2.
Nature ; 499(7457): 238-42, 2013 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-23792561

RESUMEN

The STIM1-ORAI1 pathway of store-operated Ca(2+) entry is an essential component of cellular Ca(2+) signalling. STIM1 senses depletion of intracellular Ca(2+) stores in response to physiological stimuli, and relocalizes within the endoplasmic reticulum to plasma-membrane-apposed junctions, where it recruits and gates open plasma membrane ORAI1 Ca(2+) channels. Here we use a genome-wide RNA interference screen in HeLa cells to identify filamentous septin proteins as crucial regulators of store-operated Ca(2+) entry. Septin filaments and phosphatidylinositol-4,5-bisphosphate (also known as PtdIns(4,5)P2) rearrange locally at endoplasmic reticulum-plasma membrane junctions before and during formation of STIM1-ORAI1 clusters, facilitating STIM1 targeting to these junctions and promoting the stable recruitment of ORAI1. Septin rearrangement at junctions is required for PtdIns(4,5)P2 reorganization and efficient STIM1-ORAI1 communication. Septins are known to demarcate specialized membrane regions such as dendritic spines, the yeast bud and the primary cilium, and to serve as membrane diffusion barriers and/or signalling hubs in cellular processes such as vesicle trafficking, cell polarity and cytokinesis. Our data show that septins also organize the highly localized plasma membrane domains that are important in STIM1-ORAI1 signalling, and indicate that septins may organize membrane microdomains relevant to other signalling processes.


Asunto(s)
Calcio/metabolismo , Factores de Transcripción NFATC/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Septinas/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Genoma Humano , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Transporte de Proteínas , Septinas/deficiencia , Septinas/genética , Transducción de Señal , Molécula de Interacción Estromal 1
3.
Proc Natl Acad Sci U S A ; 112(51): E7083-92, 2015 Dec 22.
Artículo en Inglés | MEDLINE | ID: mdl-26644574

RESUMEN

The stromal interaction molecule (STIM)-ORAI calcium release-activated calcium modulator (ORAI) pathway controls store-dependent calcium entry, a major mechanism of physiological calcium signaling in mammalian cells. The core elements of the pathway are the regulatory protein STIM1, located in the endoplasmic reticulum (ER) membrane, the calcium channel ORAI1 in the plasma membrane, and sites of close contact between the ER and the plasma membrane that permit the two proteins to interact. Research on calcium signaling has centered on STIM1, ORAI1, and a few proteins that directly modulate STIM-ORAI function. However, little is known about proteins that organize ER-plasma membrane junctions for STIM-ORAI-dependent calcium signaling. Here, we report that an ER-resident membrane protein identified in a previous genome-wide RNAi screen, transmembrane protein 110 (TMEM110), regulates the long-term maintenance of ER-plasma membrane junctions and the short-term physiological remodeling of the junctions during store-dependent calcium signaling.


Asunto(s)
Canales de Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Señalización del Calcio , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Células HeLa , Humanos , Células Jurkat , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteína ORAI1 , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2
4.
EMBO J ; 30(19): 3895-912, 2011 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-21847095

RESUMEN

Cell polarization enables restriction of signalling into microdomains. Polarization of lymphocytes following formation of a mature immunological synapse (IS) is essential for calcium-dependent T-cell activation. Here, we analyse calcium microdomains at the IS with total internal reflection fluorescence microscopy. We find that the subplasmalemmal calcium signal following IS formation is sufficiently low to prevent calcium-dependent inactivation of ORAI channels. This is achieved by localizing mitochondria close to ORAI channels. Furthermore, we find that plasma membrane calcium ATPases (PMCAs) are re-distributed into areas beneath mitochondria, which prevented PMCA up-modulation and decreased calcium export locally. This nano-scale distribution-only induced following IS formation-maximizes the efficiency of calcium influx through ORAI channels while it decreases calcium clearance by PMCA, resulting in a more sustained NFAT activity and subsequent activation of T cells.


Asunto(s)
Señalización del Calcio , Calcio/química , Linfocitos T/citología , Canales de Calcio/metabolismo , Membrana Celular/enzimología , Citoesqueleto/metabolismo , Electrofisiología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Sinapsis Inmunológicas , Células Jurkat , Activación de Linfocitos , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , Proteína ORAI1 , Estructura Terciaria de Proteína
5.
Cancer Discov ; 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39189614

RESUMEN

TET2 mutations (mTET2) are common genetic events in myeloid malignancies and clonal hematopoiesis (CH). These mutations arise in the founding clone and are implicated in many clinical sequelae associated with oncogenic feedforward inflammatory circuits. However, the direct downstream effector of mTET2 responsible for the potentiation of this inflammatory circuit is unknown. To address this, we performed scRNA and scATAC-seq in COVID-19 patients with and without TET2-mutated CH reasoning that the inflammation from COVID-19 may highlight critical downstream transcriptional targets of mTET2. Using this approach, we identified MALAT1, a therapeutically tractable lncRNA, as a central downstream effector of mTET2 that is both necessary and sufficient to induce the oncogenic pro-inflammatory features of mTET2 in vivo. We also elucidate the mechanism by which mTET2 upregulate MALAT1 and describe an interaction between MALAT1 and P65 which leads to RNA "shielding" from PP2A dephosphorylation thus preventing resolution of inflammatory signaling.

6.
Immunol Rev ; 231(1): 132-47, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19754894

RESUMEN

Cell polarization is a key feature of T-cell function. The immunological synapse (IS) between T cells and antigen-presenting cells is a beautiful example of how polarization of cells is used to guide cell function. Receptors, signal transducers, the cytoskeleton, and organelles are enriched at or depleted from the IS after its formation, and in many cases these re-localizations have already been linked with certain T-cell functions. One key step for T-cell activation is a rise in the cytoplasmic calcium concentration. Whereas it is undisputed that the IS initiates and controls calcium signals in T cells, very little is known about the role of T-cell polarization for calcium signals and calcium-dependent signal transduction. We briefly summarize the basic commonly agreed principles of IS-dependent calcium signal generation but then focus on the less well understood influence of polarization on calcium signals. The discussion of the role of polarization for calcium signals leads to a model how the IS controls local and global calcium signals and calcium-dependent T-cell functions. We develop a theoretical formalism based on existing spatiotemporal calcium dynamic simulations to better understand the model in the future and allow further predictions which can be tested by fast, high resolution live-cell microscopy.


Asunto(s)
Señalización del Calcio , Sinapsis Inmunológicas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Polaridad Celular , Forma de la Célula , Humanos , Activación de Linfocitos , Linfocitos T/citología
7.
Clin Cancer Res ; 29(15): 2919-2932, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-37223910

RESUMEN

PURPOSE: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. EXPERIMENTAL DESIGN: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. RESULTS: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. CONCLUSIONS: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.


Asunto(s)
Leucemia Mielomonocítica Crónica , MicroARNs , Humanos , Línea Celular Tumoral , Proteínas , MicroARNs/genética , MicroARNs/metabolismo
8.
J Biol Chem ; 286(18): 16186-96, 2011 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-21402693

RESUMEN

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca(2+) homeostasis and Ca(2+)-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca(2+) entry (SOCE) and voltage-operated Ca(2+) entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca(2+) released-activated Ca(2+) (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca(2+)-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca(2+) uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca(2+) uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca(2+) uptake, suggesting that they favor the Ca(2+)-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca(2+) buffering conditions but not in strong Ca(2+) buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca(2+)-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca(2+).


Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Canales de Calcio/metabolismo , Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Línea Celular Tumoral , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Musculares/patología , Miocitos del Músculo Liso/patología , Ratas , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patología
9.
J Immunol ; 184(1): 184-90, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19949095

RESUMEN

Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.


Asunto(s)
Calcio/metabolismo , Sinapsis Inmunológicas/fisiología , Activación de Linfocitos/inmunología , Mitocondrias/ultraestructura , Linfocitos T/metabolismo , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Humanos , Células Jurkat , Microscopía Fluorescente , Proteína ORAI1 , Técnicas de Placa-Clamp , Linfocitos T/citología , Linfocitos T/inmunología
10.
Sci Rep ; 9(1): 10839, 2019 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-31346209

RESUMEN

ORAI1 Ca2+ channels in the plasma membrane (PM) are gated by STIM1 at endoplasmic reticulum (ER)-PM junctions to effect store-dependent Ca2+ entry into cells, but little is known about how local STIM-ORAI signalling at junctions is coordinated with overall cellular architecture. Filamentous septins can specify cytoskeletal rearrangements and have been found recently to modulate STIM-ORAI signalling. Here we show by super-resolution imaging of ORAI1, STIM1, and septin 4 in living cells that septins facilitate Ca2+ signalling indirectly. Septin 4 does not colocalize preferentially with ORAI1 in resting or stimulated cells, assemble stably at ER-PM junctions, or specify a boundary that directs or confines ORAI1 to junctions. Rather, ORAI1 is recruited to junctions solely through interaction with STIM proteins, while septins regulate the number of ER-PM junctions and enhance STIM1-ORAI1 interactions within junctions. Thus septins communicate with STIM1 and ORAI1 through protein or lipid intermediaries, and are favorably positioned to coordinate Ca2+ signalling with rearrangements in cellular architecture.


Asunto(s)
Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteína ORAI1/metabolismo , Septinas/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Calcio/metabolismo , Células HeLa , Humanos
11.
FEBS Lett ; 581(18): 3557-62, 2007 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-17624329

RESUMEN

Lymphocyte signaling and activation leads to the influx of extracellular Ca(2+) via the activation of Ca(2+) release activated Ca(2+) (CRAC) channels in the plasma membrane. Activation of CRAC channels occurs following emptying of the endoplasmic reticulum intracellular Ca(2+) stores. One model to explain the coupling of store-emptying to CRAC activation is the secretion-like conformational coupling model. This model proposes that store depletion increases junctions between the endoplasmic reticulum and the plasma membrane in a manner that could be regulated by the cortical actin cytoskeleton. Here, we show that stabilization or depolymerization of the actin cytoskeleton failed to affect CRAC activation. We therefore conclude that rearrangement of the actin cytoskeleton is dispensable for store-operated Ca(2+) entry in T-cells.


Asunto(s)
Actinas/metabolismo , Canales de Calcio/metabolismo , Citoesqueleto/metabolismo , Linfocitos T/metabolismo , Actinas/antagonistas & inhibidores , Calcio/metabolismo , Electrofisiología , Humanos , Células Jurkat , Técnicas de Placa-Clamp
12.
Cell Calcium ; 60(5): 309-321, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27451384

RESUMEN

Ca2+ microdomains and spatially resolved Ca2+ signals are highly relevant for cell function. In T cells, local Ca2+ signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca2+ is sufficient to significantly increase the cytosolic Ca2+ concentration. To test this hypothesis we developed a whole cell model for Ca2+ homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca2+ concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca2+ concentration and decreases the local Ca2+ concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca2+ changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca2+ dynamics in T-cells during polarization.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Citoesqueleto/metabolismo , Sinapsis Inmunológicas/metabolismo , Mitocondrias/metabolismo , Rotación , Señalización del Calcio/inmunología , Células Cultivadas , Humanos , Células Jurkat
13.
Nat Cell Biol ; 17(10): 1339-47, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26322679

RESUMEN

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca(2+) signalling. Store-operated Ca(2+) entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca(2+) influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca(2+)-NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.


Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteoma/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Células COS , Chlorocebus aethiops , Perfilación de la Expresión Génica , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Microscopía Confocal , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteómica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Molécula de Interacción Estromal 1
14.
Cell Calcium ; 36(2): 99-109, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15193858

RESUMEN

Fura-2 is the most common dye to measure cytosolic Ca2+ concentrations ([Ca2+]i). To facilitate simultaneous imaging of many cells while preserving their cytosolic environment, fura-2 is often loaded into the cytosol in its membrane-permeant ester form. It has been reported that small amounts of fura-2 accumulate in intracellular compartments, an effect that is usually neglected. We show that either focal or non-focal stimulation methods induce large [Ca2+]i gradients in T-lymphocytes during both, Ca2+ release and Ca2+ influx across the plasma membrane. Interfering with mitochondrial Ca2+ homeostasis and by labeling mitochondria with MitoTracker, we demonstrate that [Ca2+]i gradients co-localize with mitochondria and are attributable to mitochondrial fura-2 sequestration. Gradients could not be avoided by different loading protocols, compromising measurements of "real" [Ca2+]i gradients following T-cell stimulation. They were observed in human blood and lamina propria lymphocytes, Jurkat T-cells, mast cells, but not to the same extent in HEK-293 cells. Finally, we show that T-lymphocytes can be efficiently loaded with the membrane-impermeant fura-2 salt by electroporation and by osmotic lysis of pinocytic vesicles, which result in the loss of [Ca2+]i gradients. These methods are therefore suitable to study localized Ca2+ signals in large populations of T-cells while preserving their cytosolic integrity.


Asunto(s)
Señalización del Calcio/fisiología , Citosol/metabolismo , Fura-2/metabolismo , Mitocondrias/metabolismo , Linfocitos T/metabolismo , Presentación de Antígeno/fisiología , Humanos , Células Jurkat , Transducción de Señal/fisiología , Factores de Tiempo
15.
Nat Commun ; 5: 5164, 2014 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-25296861

RESUMEN

The ER-resident regulatory protein STIM1 triggers store-operated Ca(2+) entry by direct interaction with the plasma membrane Ca(2+) channel ORAI1. The mechanism of channel gating remains undefined. Here we establish that STIM1 gates the purified recombinant ORAI1 channel in vitro, and use Tb(3+) luminescence and, separately, disulfide crosslinking to probe movements of the pore-lining helices. We show that interaction of STIM1 with the cytoplasmic face of the human ORAI1 channel elicits a conformational change near the external entrance to the pore, detectable at the pore Ca(2+)-binding residue E106 and the adjacent pore-lining residue V102. We demonstrate that a short nonpolar segment of the pore including V102 forms a barrier to ion flux in the closed channel, implicating the STIM1-dependent movement in channel gating. Our data explain the close coupling between ORAI1 channel gating and ion selectivity, and open a new avenue to dissect the gating, modulation and inactivation of ORAI-family channels.


Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Membrana Celular/metabolismo , Humanos , Proteína ORAI1 , Estructura Secundaria de Proteína , Molécula de Interacción Estromal 1
16.
Cell Calcium ; 52(1): 57-63, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22425631

RESUMEN

Energy supply is the most prominent function of mitochondria, but in addition, mitochondria are indispensable for a multitude of other important cellular functions including calcium (Ca(2+)) signaling and buffering, the supply of metabolites and the sequestration of apoptotic factors. The efficiency of those functions highly depends on the proper positioning of mitochondria within the cytosol. In lymphocytes, mitochondria preferentially localize into the vicinity (∼200nm) of the immune synapse (IS). This localization is regulated by motor-based cytoskeleton-mediated transport, the fusion/fission dynamics of mitochondria, and probably also through tethering with the ER. IS formation also induces the accumulation of CRAC/ORAI1 Ca(2+) channels, the CRAC/ORAI channel activator STIM1, K(+) channels and plasma membrane Ca(2+) ATPase (PMCA) within the IS. Such a large agglomeration of Ca(2+) binding organelles and proteins highlights the IS as a critical cellular compartment for Ca(2+) dependent lymphocyte activation. At the IS, Ca(2+) microdomains generated beneath open CRAC/ORAI channels provide a rapid, robust and reliable mechanism for driving cellular responses in mast cells and T cells. Here, we discuss the relevance of motor-based mitochondrial transport, fusion, fission and tethering for mitochondrial localization in T cells and the importance of subplasmalemmal mitochondria to control local CRAC/ORAI1-dependent Ca(2+) microdomains at the IS for efficient T lymphocyte activation.


Asunto(s)
Mitocondrias/metabolismo , Linfocitos T/metabolismo , Calcio/química , Canales de Calcio/metabolismo , Señalización del Calcio , Metabolismo Energético , Humanos , Sistema Inmunológico/metabolismo , Activación de Linfocitos , Linfocitos T/inmunología
17.
Cell Calcium ; 45(2): 109-22, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18789821

RESUMEN

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.


Asunto(s)
Señalización del Calcio/inmunología , Forma de la Célula , Sinapsis Inmunológicas/inmunología , Espacio Intracelular/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Actinas/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Células Jurkat , Mitocondrias/metabolismo
18.
J Med Virol ; 80(5): 798-802, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18360913

RESUMEN

Viral hepatitis ranks as the fifth cause of morbidity for infectious diseases in Cuba. Epidemics are observed frequently in the population, the hepatitis A virus being the main agent responsible for such epidemics. Previous reports also confirmed the circulation of the hepatitis E virus. From 1998 to 2003, 258 serum samples were collected by the Reference Laboratory on Viral Hepatitis during 33 outbreaks of acute viral hepatitis as well as from 39 sporadic clinical cases. Sera were tested for anti-HAV and anti-HEV IgM by EIA. Overall of the 33 outbreaks studied sera from 12 (36.4%) were positive for anti-HAV IgM only, from 7 (21.2%) were positive for anti-HEV IgM only, and from 14 (42.4%) were positive for antibodies to both viruses. Individually of the 258 sera collected, 137 (53.1%) were positives for anti-HAV IgM, 20 (7.8%) were positives for anti-HEV IgM, 33 (12.8%) were positives for both markers and 68 (26.4%) were negative to both. Of the clinical cases, 4 (10.3%) were positives for anti-HAV IgM, 13 (33.3%) were positives for anti-HEV IgM and 5 (12.8%) were positives for both markers. Seventeen (43.6%) sera were negatives for all viral hepatitis markers available (A-E). A high positivity for HEV was found in outbreaks tested with the kit produced by CIGB. In particular HEV seems to infect individuals of all ages. The results demonstrate the co-circulation of and co-infection with two enterically transmitted viruses; however a higher positivity was observed for anti-HAV than to anti-HEV (53.1% vs. 7.8%) in outbreaks.


Asunto(s)
Hepatitis A/complicaciones , Hepatitis A/epidemiología , Hepatitis E/complicaciones , Hepatitis E/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Comorbilidad , Cuba/epidemiología , Brotes de Enfermedades , Anticuerpos Antihepatitis/sangre , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina M/sangre , Lactante , Recién Nacido , Persona de Mediana Edad
19.
Proc Natl Acad Sci U S A ; 104(36): 14418-23, 2007 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-17726106

RESUMEN

T helper (Th) cell activation is required for the adaptive immune response. Formation of the immunological synapse (IS) between Th cells and antigen-presenting cells is essential for Th cell activation. IS formation induces the polarization and redistribution of many signaling molecules; however, very little is known about organelle redistribution during IS formation in Th cells. We show that formation of the IS induced cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. Using total internal reflection microscopy, we found that upon stimulation, the distance between the IS and mitochondria was decreased to values<200 nm. Consequently, mitochondria close to the IS took up more Ca2+ than the ones farther away from the IS. The redistribution of mitochondria to the IS was necessary to maintain Ca2+ influx across the plasma membrane and Ca2+-dependent Th cell activation. Our results suggest that mitochondria are part of the signaling complex at the IS and that their localization close to the IS is required for Th cell activation.


Asunto(s)
Activación de Linfocitos/inmunología , Mitocondrias/metabolismo , Sinapsis/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Calcio/metabolismo , Línea Celular , Proliferación Celular , Humanos , Microscopía Electrónica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología
20.
Eur J Immunol ; 37(10): 2723-33, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17899547

RESUMEN

Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca(2+) dependence of proliferation and apoptosis in primary human CD4(+) T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca(2+) entry through Ca(2+) channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca(2+)](i)) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4(+) T cells. We also show that proliferation was very Ca(2+) sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca(2+)](i) levels of 90-120 nM. We conclude that very small changes in [Ca(2+)](i) can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.


Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Calcio/fisiología , Proliferación Celular , Activación de Linfocitos/inmunología , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Humanos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA