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BACKGROUND: TRPM4 is a broadly expressed, calcium-activated, monovalent cation channel that regulates immune cell function in mice and cell lines. Clinically, however, partial loss- or gain-of-function mutations in TRPM4 lead to arrhythmia and heart disease, with no documentation of immunologic disorders. OBJECTIVE: To characterize functional cellular mechanisms underlying the immune dysregulation phenotype in a proband with a mutated TRPM4 gene. METHODS: We employed a combination of biochemical, cell biological, imaging, omics analyses, flow cytometry, and gene editing approaches. RESULTS: We report the first human cases to our knowledge with complete loss of the TRPM4 channel, leading to immune dysregulation with frequent bacterial and fungal infections. Single-cell and bulk RNA sequencing point to altered expression of genes affecting cell migration, specifically in monocytes. Inhibition of TRPM4 in T cells and the THP-1 monocyte cell line reduces migration. More importantly, primary T cells and monocytes from TRPM4 patients migrate poorly. Finally, CRISPR knockout of TRPM4 in THP-1 cells greatly reduces their migration potential. CONCLUSION: Our results demonstrate that TRPM4 plays a critical role in regulating immune cell migration, leading to increased susceptibility to infections.
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Store operated Ca2+ entry (SOCE) is a ubiquitous signalling module with established roles in the immune system, secretion and muscle development. Recent evidence supports a complex role for SOCE in the nervous system. In this review we present an update of the current knowledge on SOCE function in the brain with a focus on its role as a regulator of brain activity and excitability.
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[This corrects the article DOI: 10.1371/journal.pbio.3000901.].
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Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.
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Canales de Calcio , Calcio , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteína ORAI1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The steroid hormone progesterone (P4) mediates many physiological processes through either nuclear receptors that modulate gene expression or membrane P4 receptors (mPRs) that mediate nongenomic signaling. mPR signaling remains poorly understood. Here we show that the topology of mPRß is similar to adiponectin receptors and opposite to that of G-protein-coupled receptors (GPCRs). Using Xenopus oocyte meiosis as a well-established physiological readout of nongenomic P4 signaling, we demonstrate that mPRß signaling requires the adaptor protein APPL1 and the kinase Akt2. We further show that P4 induces clathrin-dependent endocytosis of mPRß into signaling endosome, where mPR interacts transiently with APPL1 and Akt2 to induce meiosis. Our findings outline the early steps involved in mPR signaling and expand the spectrum of mPR signaling through the multitude of pathways involving APPL1.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Endocitosis , Endosomas/metabolismo , Femenino , Meiosis/fisiología , Oocitos/metabolismo , Progesterona/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas de Xenopus/fisiología , Xenopus laevisRESUMEN
Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.
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Canales de Calcio Tipo L/metabolismo , Hipertensión/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Moléculas de Interacción Estromal/metabolismo , Remodelación Vascular/fisiología , Animales , Antihipertensivos/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Insuficiencia Cardíaca , Humanos , Proteínas de la Membrana/genética , Miocitos del Músculo Liso , Proteínas de Neoplasias , Proteína ORAI1/genética , Ratas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1) hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.
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Hipertensión , Hipohidrosis , Animales , Niño , Humanos , Ratones , Calcio/metabolismo , Señalización del Calcio , Citocinas/metabolismo , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Sistema Cardiovascular/metabolismoRESUMEN
Store-operated Ca2+ entry (SOCE), mediated by the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) and the plasma membrane (PM) channel Orai1, is inhibited during mitosis. STIM1 phosphorylation has been suggested to mediate this inhibition, but it is unclear whether additional pathways are involved. Here, we demonstrate using various approaches, including a nonphosphorylatable STIM1 knock-in mouse, that STIM1 phosphorylation is not required for SOCE inhibition in mitosis. Rather, multiple pathways converge to inhibit Ca2+ influx in mitosis. STIM1 interacts with the cochaperone BAG3 and localizes to autophagosomes in mitosis, and STIM1 protein levels are reduced. The density of ER-PM contact sites (CSs) is also dramatically reduced in mitosis, thus physically preventing STIM1 and Orai1 from interacting to activate SOCE. Our findings provide insights into ER-PM CS remodeling during mitosis and a mechanistic explanation of the inhibition of Ca2+ influx that is required for cell cycle progression.
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Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Mitosis/fisiología , Proteínas de Neoplasias/metabolismo , Fosforilación/fisiología , Molécula de Interacción Estromal 1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Ciclo Celular/fisiología , Línea Celular , Línea Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Ratones , Proteína ORAI1/metabolismoRESUMEN
Store-operated Ca2+ entry (SOCE) represents a predominant Ca2+ influx pathway in non-excitable cells. SOCE is required for immune cell activation and is mediated by the plasma membrane (PM) channel ORAI1 and the endoplasmic reticulum (ER) Ca2+ sensor STIM1. Mutations in the Orai1 or STIM1 genes abolish SOCE leading to combined immunodeficiency (CID), muscular hypotonia, and anhidrotic ectodermal dysplasia. Here, we identify a novel autosomal recessive mutation in ORAI1 in a child with CID. The patient is homozygous for p.C126R mutation in the second transmembrane domain (TM2) of ORAI1, a region with no previous loss-of-function mutations. SOCE is suppressed in the patient's lymphocytes, which is associated with impaired T cell proliferation and cytokine production. Functional analyses demonstrate that the p.C126R mutation does not alter protein expression but disrupts ORAI1 trafficking. Orai1-C126R does not insert properly into the bilayer resulting in ER retention. Insertion of an Arg on the opposite face of TM2 (L135R) also results in defective folding and trafficking. We conclude that positive side chains within ORAI1 TM2 are not tolerated and result in misfolding, defective bilayer insertion, and channel trafficking thus abolishing SOCE and resulting in CID.
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Canalopatías/diagnóstico , Proteína ORAI1/genética , Enfermedades de Inmunodeficiencia Primaria/diagnóstico , Calcio/metabolismo , Proliferación Celular , Células Cultivadas , Canalopatías/genética , Canalopatías/inmunología , Citocinas/inmunología , Femenino , Humanos , Lactante , Mutación , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Transporte de Proteínas , Linfocitos T/inmunologíaRESUMEN
Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.
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Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Compuestos de Boro/química , Compuestos de Boro/farmacología , Calcio/química , Retículo Endoplásmico/química , Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/química , Molécula de Interacción Estromal 2/genéticaRESUMEN
Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRß (PAQR8) partner that is required for mPRß plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.
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Membrana Celular/metabolismo , Progesterona/metabolismo , Receptores de LDL/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animales , Membrana Celular/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Unión Proteica , Transporte de Proteínas , Receptores de LDL/genética , Receptores de Progesterona/genética , Transducción de Señal , Xenopus/genética , Proteínas de Xenopus/genéticaRESUMEN
Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway.
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Puntos de Control del Ciclo Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Profase Meiótica I , Oocitos/citología , Oocitos/metabolismo , Xenopus laevis/metabolismo , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Profase Meiótica I/efectos de los fármacos , Modelos Biológicos , Progesterona/farmacología , Fracciones Subcelulares/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
TPEN is an amino chelator of transition metals that is effective at the cellular and whole organism levels. Although TPEN of often used as a selective zinc chelators, it has affinity for copper and iron and has been shown to chelate both biologically. We have previously shown that TPEN selectively kills colon cancer cells based on its ability to chelate copper, which is highly enriched in colon cancer cells. The TPEN-copper complex is redox active thus allowing for increased ROS production in cancer cells and as such cellular toxicity. Here we generate TPEN derivatives with the goal of increasing its selectivity for copper while minimizing zinc chelation to reduce potential side effects. We show that one of these derivatives, TPEEN despite the fact that it exhibits reduced affinity for transition metals, is effective at inducing cell death in breast cancer cells, and exhibits less toxicity to normal breast cells. The toxicity effect of the both chelators coupled to the metal content of the different cell types reveals that they exhibit their toxicity through chelating redox active metals (iron and copper). As such TPEEN is an important novel chelators that can be exploited in anti-cancer therapies.
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Antineoplásicos/farmacología , Cobre/farmacología , Etilenodiaminas/farmacología , Compuestos Organometálicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cobre/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Etilenodiaminas/química , Humanos , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
The G2-M transition of the cell cycle requires the activation of members of the Cdc25 dual specificity phosphatase family. Using Xenopus oocyte maturation as a model system, we have previously shown that chelation of transition metals blocks meiosis progression by inhibiting Cdc25C activation. Here, using approaches that allow for the isolation of very pure and active recombinant Cdc25C, we show that Cdc25C does not bind zinc as previously reported. Additionally, we show that mutants in the disordered C-terminal end of Cdc25C are poor initiators of meiosis, likely due to their inability to localize to the proper sub-cellular location. We further demonstrate that the transition metal chelator, TPEN, acts on or upstream of polo-like kinases in the oocyte to block meiosis progression. Together our results provide novel insights into Cdc25C structure-function relationship and the role of transition metals in regulating meiosis.
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Meiosis/efectos de los fármacos , Oocitos/citología , Oocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Elementos de Transición/farmacología , Secuencia de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Codón/genética , Etilenodiaminas/farmacología , Proteínas Mutantes/metabolismo , Oocitos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/aislamiento & purificación , Proteínas de Xenopus/metabolismo , Fosfatasas cdc25/química , Fosfatasas cdc25/genética , Fosfatasas cdc25/aislamiento & purificación , Fosfatasas cdc25/metabolismoRESUMEN
Ca(2+)-activated Cl(-) channels (CaCCs) play important physiological functions in epithelia and other tissues. In frog oocytes the CaCC Ano1 regulates resting membrane potential and the block to polyspermy. Here, we show that Ano1 expression increases the oocyte surface, revealing a novel function for Ano1 in regulating cell morphology. Confocal imaging shows that Ano1 increases microvilli length, which requires ERM-protein-dependent linkage to the cytoskeleton. A dominant-negative form of the ERM protein moesin precludes the Ano1-dependent increase in membrane area. Furthermore, both full-length and the truncated dominant-negative forms of moesin co-localize with Ano1 to the microvilli, and the two proteins co-immunoprecipitate. The Ano1-moesin interaction limits Ano1 lateral membrane mobility and contributes to microvilli scaffolding, therefore stabilizing larger membrane structures. Collectively, these results reveal a newly identified role for Ano1 in shaping the plasma membrane during oogenesis, with broad implications for the regulation of microvilli in epithelia.
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Canales de Cloruro/metabolismo , Proteínas de Microfilamentos/genética , Oocitos/metabolismo , Oogénesis/genética , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Canales de Cloruro/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microfilamentos/metabolismo , Microvellosidades/genética , Oocitos/crecimiento & desarrollo , Mapas de Interacción de Proteínas/genética , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Ca2+ signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium-binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non-excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+ , there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store-operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store-operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept.
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Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Células Acinares/metabolismo , Animales , Humanos , Oocitos/metabolismoRESUMEN
Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3 R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3 Rs at distant sites, a process we refer to as "Ca2+ teleporting." Therefore, sustained SOCE activation in the presence of an IP3 -dependent "leak" pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095-1103, 2017. © 2016 Wiley Periodicals, Inc.
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Señalización del Calcio , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Oocitos/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
The key proteins mediating store-operated Ca(2+) entry (SOCE) are the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the plasma membrane Ca(2+)-selective channel Orai1. Here, we quantitatively dissect Orai1 trafficking dynamics and show that Orai1 recycles rapidly at the plasma membrane (Kex≃0.1â min(-1)), with â¼40% of the total Orai1 pool localizing to the plasma membrane at steady state. A subset of intracellular Orai1 localizes to a sub-plasmalemal compartment. Store depletion is coupled to Orai1 plasma membrane enrichment in a STIM1-dependent fashion. This is due to trapping of Orai1 into cortical ER STIM1 clusters, leading to its removal from the recycling pool and enrichment at the plasma membrane. Interestingly, upon high STIM1 expression, Orai1 is trapped into STIM1 clusters intracellularly, thus preventing its plasma membrane enrichment following store depletion. Consistent with this, STIM1 knockdown prevents trapping of excess Orai1 into limiting STIM1 clusters in the cortical ER. SOCE-dependent Ca(2+) influx shows a similar biphasic dependence on the Orai1:STIM1 ratio. Therefore, a STIM1-dependent Orai1 'trafficking trap' mechanism controls Orai1 plasma membrane enrichment and SOCE levels, thus modulating the SOCE 'bandwidth' for downstream signaling.
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Canales de Calcio/genética , Señalización del Calcio/genética , Calcio/metabolismo , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Animales , Células CHO , Canales de Calcio/biosíntesis , Membrana Celular/metabolismo , Cricetulus , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de Neoplasias/biosíntesis , Proteína ORAI1 , Transporte de Proteínas/genética , ARN Interferente Pequeño , Transducción de Señal , Molécula de Interacción Estromal 1RESUMEN
The Cdc25 family encodes dual specificity protein phosphatases that play critical roles in cell cycle progression. Activation of the Cdc25C represents a primary driver for meiosis progression in Xenopus oocytes. Given its central role in meiosis the Xenopus Cdc25C has been studied extensively, however purification of the recombinant protein is difficult thus preventing better characterization of its function. Here we describe methods to overcome these difficulties resulting in the production of high purity and yield recombinant Xenopus Cdc25C. We use a synthetic Xenopus Cdc25C gene that was codon optimized for expression in E. coli. We further combine an N-terminal His-tag with a C-terminal Strep-tag II, to isolate extremely pure full-length Cdc25C protein. The recombinant Xenopus Cdc25C is active both in vitro using a phosphatase assay and in vivo when injected into Xenopus oocytes. This new approach should be applicable to the purification of other members of the Cdc25 gene family.
Asunto(s)
Escherichia coli/genética , Proteínas de Xenopus/genética , Xenopus/metabolismo , Fosfatasas cdc25/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Oligopéptidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas de Xenopus/aislamiento & purificación , Proteínas de Xenopus/metabolismo , Fosfatasas cdc25/aislamiento & purificación , Fosfatasas cdc25/metabolismoRESUMEN
Vertebrate oocytes are naturally arrested at prophase of meiosis I for sustained periods of time before resuming meiosis in a process called oocyte maturation that prepares the egg for fertilization. Members of the constitutively active GPR3/6/12 family of G-protein coupled receptors represent important mediators of meiotic arrest. In the frog oocyte the GPR3/12 homolog GPRx (renamed GPR185) has been shown to sustain meiotic arrest by increasing intracellular cAMP levels through GαSßγ. Here we show that GPRx is enriched at the cell membrane (~80%), recycles through an endosomal compartment at steady state, and loses its ability to signal once trapped intracellularly. Progesterone-mediated oocyte maturation is associated with significant internalization of both endogenous and overexpressed GPRx. Furthermore, a GPRx mutant that does not internalize in response to progesterone is significantly more efficient than wild-type GPRx at blocking oocyte maturation. Collectively our results argue that internalization of the constitutively active GPRx is important to release oocyte meiotic arrest.