RESUMEN
During sporulation in the budding yeast Saccharomyces cerevisiae, proper development of the prospore membrane is necessary for the formation of viable spores. The prospore membrane will eventually become the plasma membrane of the newly formed haploid spore and also serves as the template for the deposition of the spore wall. The prospore membrane is generated de novo during meiosis II and the growing edge of the prospore membrane is associated with the Leading Edge Protein (LEP) complex. We find that the Smk1 MAP kinase, along with its activator Ssp2, transiently localizes with the LEP during late meiosis II. SSP2 is required for the leading edge localization of Smk1; this localization is independent of the activation state of Smk1. Like other LEP components, the localization of Smk1 at the leading edge also depends on Ady3. Although prospore membrane development begins normally in smk1 and ssp2 mutants, late prospore membrane formation is disrupted, with the formation of ectopic membrane compartments. Thus, MAP kinase signaling plays an important role in the formation of the prospore membrane.
RESUMEN
T cells form an immune synapse (IS) with antigen-presenting cells (APCs) to detect antigens that match their TCR. Mitochondria, pannexin-1 (panx1) channels, and P2X4 receptors congregate at the IS where mitochondria produce the ATP that panx1 channels release in order to stimulate P2X4 receptors. P2X4 receptor stimulation causes cellular Ca2+ influx that up-regulates mitochondrial metabolism and localized ATP production at the IS. Here we show that P2Y11 receptors are essential players that sustain these T cell activation mechanisms. We found that P2Y11 receptors retract from the IS toward the back of cells where their stimulation by extracellular ATP induces cAMP/PKA signaling that redirects mitochondrial trafficking to the IS. P2Y11 receptors thus reinforce IS signaling by promoting the aggregation of mitochondria with panx1 ATP release channels and P2X4 receptors at the IS. This dual purinergic signaling mechanism involving P2X4 and P2Y11 receptors focuses mitochondrial metabolism to the IS where localized ATP production sustains synaptic activity in order to allow successful completion of T cell activation responses. Our findings have practical implications because rodents lack P2Y11 receptors, raising concerns as to the validity of rodent models to study treatment of infections and inflammatory conditions.
Asunto(s)
Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos/inmunología , Mitocondrias/metabolismo , Receptores Purinérgicos P2/metabolismo , Linfocitos T/inmunología , Comunicación Autocrina , Linfocitos T CD4-Positivos/inmunología , Señalización del Calcio , AMP Cíclico/metabolismo , Humanos , Células Jurkat , Microtúbulos/metabolismo , Receptores Purinérgicos P2X4 , Transducción de Señal , Células U937RESUMEN
T cells must migrate to encounter antigen-presenting cells and perform their roles in host defense. Here, we found that autocrine stimulation of the purinergic receptor P2Y11 regulates the migration of human CD4 T cells. P2Y11 receptors redistributed from the front to the back of polarized cells where they triggered intracellular cAMP/PKA signals that attenuated mitochondrial metabolism at the back. The absence of P2Y11 receptors at the front of cells resulted in hotspots of mitochondrial metabolism and localized ATP production that stimulated P2X4 receptors, Ca2+ influx, and pseudopod protrusion at the front. This regulatory function of P2Y11 receptors depended on their subcellular redistribution and autocrine stimulation by cellular ATP release and was perturbed by indiscriminate global stimulation. We conclude that excessive extracellular ATP-such as in response to inflammation, sepsis, and cancer-disrupts this autocrine feedback mechanism, which results in defective T cell migration, impaired T cell function, and loss of host immune defense.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Mitocondrias/metabolismo , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Células Jurkat , Microscopía Fluorescente/métodos , Agonistas Purinérgicos/farmacología , Antagonistas Purinérgicos/farmacología , Receptores Purinérgicos P2/metabolismoRESUMEN
Meiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes an STE20 family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citocinesis , Proteínas de Unión al GTP/metabolismo , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Ischemia and reperfusion injury following severe trauma or cardiac arrest are major causes of organ damage in intensive care patients. The brain is particularly vulnerable because hypoxia rapidly damages neurons due to their heavy reliance on oxidative phosphorylation. Therapeutic hypothermia can reduce ischemia-induced brain damage, but cooling procedures are slow and technically difficult to perform in critical care settings. It has been previously reported that injection of naturally occurring adenosine 5'-monophosphate (AMP) can rapidly induce hypothermia in mice. We studied the underlying mechanisms and found that AMP transiently reduces the heart rate, respiratory rate, body temperature, and the consciousness of adult male and female C57BL/6J mice. Adding AMP to mouse or human neuronal cell cultures dose-dependently reduced the membrane potential (ΔΨm) and Ca signaling of mitochondria in these cells. AMP treatment increased intracellular AMP levels and activated AMP-activated protein kinase, which resulted in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and of mitochondrial and cytosolic Ca signaling in resting and stimulated neurons. Pretreatment with an intraperitoneal injection of AMP almost doubled the survival time of mice under hypoxic (6% O2) or anoxic (<1% O2) conditions when compared to untreated mice. These findings suggest that AMP induces a hypometabolic state that slows mitochondrial respiration, reduces oxygen demand, and delays the processes that damage mitochondria in the brain and other organs following hypoxia and reperfusion. Further examination of these mechanisms may lead to new treatments that preserve organ function in critical care patients.
Asunto(s)
Adenosina Monofosfato/farmacología , Adenosina Monofosfato/uso terapéutico , Hipoxia/metabolismo , Hipoxia/prevención & control , Mitocondrias/metabolismo , Oxígeno/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Bacterial infections and sepsis are leading causes of morbidity and mortality in critically ill patients. Currently, there are no effective treatments available to improve clinical outcome in sepsis. Here, we elucidated a mechanism by which Escherichia coli (E. coli) bacteria impair neutrophil (PMN) chemotaxis and we studied whether this mechanism can be therapeutically targeted to improve chemotaxis and antimicrobial host defense. PMNs detect bacteria with formyl peptide receptors (FPR). FPR stimulation triggers mitochondrial ATP production and release. Autocrine stimulation of purinergic receptors exerts excitatory and inhibitory downstream signals that induce cell polarization and cell shape changes needed for chemotaxis. Here we show that the bacterial cell wall product LPS dose-dependently impairs PMN chemotaxis. Exposure of human PMNs to LPS triggered excessive mitochondrial ATP production and disorganized intracellular trafficking of mitochondria, resulting in global ATP release that disrupted purinergic signaling, cell polarization, and chemotaxis. In mice infected i.p. with E. coli, LPS treatment increased the spread of bacteria at the infection site and throughout the systemic circulation. Removal of excessive systemic ATP with apyrase improved chemotaxis of LPS-treated human PMNs in vitro and enhanced the clearance of E. coli in infected and LPS-treated mice. We conclude that systemic ATP accumulation in response to LPS is a potential therapeutic target to restore PMN chemotaxis and to boost the antimicrobial host immune defense in sepsis.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Escherichia coli/inmunología , Interacciones Huésped-Patógeno/inmunología , Lipopolisacáridos/inmunología , Neutrófilos/inmunología , Adenosina/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apirasa/metabolismo , Biomarcadores , Modelos Animales de Enfermedad , Humanos , Espacio Intracelular/metabolismo , Ratones , Mitocondrias/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/metabolismo , Peritonitis/inmunología , Peritonitis/microbiologíaRESUMEN
Previous studies have shown that T cell receptor (TCR) and CD28 coreceptor stimulation involves rapid ATP release, autocrine purinergic feedback via P2X receptors, and mitochondrial ATP synthesis that promote T cell activation. Here, we show that ADP formation and autocrine stimulation of P2Y1 receptors are also involved in these purinergic signaling mechanisms. Primary human CD4 T cells and the human Jurkat CD4 T cell line express P2Y1 receptors. The expression of this receptor increases following T cell stimulation. Inhibition of P2Y1 receptors impairs the activation of mitochondria, as assessed by mitochondrial Ca2+ uptake, and reduces cytosolic Ca2+ signaling in response to TCR/CD28 stimulation. We found that the addition of exogenous ADP or overexpression of P2Y1 receptors significantly increased IL-2 mRNA transcription in response to TCR/CD28 stimulation. Conversely, antagonists or silencing of P2Y1 receptors reduced IL-2 mRNA transcription and attenuated T cell functions. We conclude that P2Y1 and P2X receptors have non-redundant, synergistic functions in the regulation of T cell activation. P2Y1 receptors may represent potential therapeutic targets to modulate T cell function in inflammation and host defense.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Activación de Linfocitos/fisiología , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Transducción de Señal/inmunología , Comunicación Autocrina , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , HumanosRESUMEN
OBJECTIVES: Monocytes and macrophages produce interleukin-1ß by inflammasome activation which involves adenosine triphosphate release, pannexin-1 channels, and P2X7 receptors. However, interleukin-1ß can also be produced in an inflammasome-independent fashion. Here we studied if this mechanism also involves adenosine triphosphate signaling and how it contributes to inflammasome activation. DESIGN: In vitro studies with human cells and randomized animal experiments. SETTING: Preclinical academic research laboratory. SUBJECTS: Wild-type C57BL/6 and pannexin-1 knockout mice, healthy human subjects for cell isolation. INTERVENTIONS: Human monocytes and U937 macrophages were treated with different inhibitors to study how purinergic signaling contributes to toll-like receptor-induced cell activation and interleukin-1ß production. Wild-type and pannexin-1 knockout mice were subjected to cecal ligation and puncture to study the role of purinergic signaling in interleukin-1ß production and host immune defense. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor agonists triggered mitochondrial adenosine triphosphate production and adenosine triphosphate release within seconds. Inhibition of mitochondria, adenosine triphosphate release, or P2 receptors blocked p38 mitogen-activated protein kinase and caspase-1 activation and interleukin-1ß secretion. Mice lacking pannexin-1 failed to activate monocytes, to produce interleukin-1ß, and to effectively clear bacteria following cecal ligation and puncture. CONCLUSIONS: Purinergic signaling has two separate roles in monocyte/macrophage activation, namely to facilitate the initial detection of danger signals via toll-like receptors and subsequently to regulate nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 inflammasome activation. Further dissection of these mechanisms may reveal novel therapeutic targets for immunomodulation in critical care patients.
Asunto(s)
Adenosina Trifosfato/inmunología , Infecciones/inmunología , Inflamasomas/inmunología , Activación de Macrófagos/inmunología , Monocitos/inmunología , Animales , Técnicas de Cultivo de Célula , Conexinas/farmacología , Modelos Animales de Enfermedad , Compuestos Heterocíclicos con 3 Anillos , Humanos , Immunoblotting , Interleucina-1beta/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/farmacología , Transducción de Señal , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inhibidoresRESUMEN
T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine stromal-derived factor-1α (SDF-1α) triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.
Asunto(s)
Adenosina Trifosfato/inmunología , Comunicación Autocrina/inmunología , Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Mitocondrias/inmunología , Receptores Purinérgicos P2X4/inmunología , Adenosina Trifosfato/genética , Animales , Comunicación Autocrina/genética , Linfocitos T CD4-Positivos/citología , Humanos , Inflamación/genética , Inflamación/inmunología , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Mitocondrias/genética , Receptores Purinérgicos P2X4/genéticaRESUMEN
PURPOSE: Sepsis remains an unresolved clinical problem with high in-hospital mortality. Despite intensive research over decades, no treatments for sepsis have become available. Here we explore the role of ATP in the pathophysiology of sepsis. ATP is not only a universal energy carrier but it also acts as an extracellular signaling molecule that regulates immune function. ATP stimulates a large family of purinergic receptors found on the cell surface of virtually all mammalian cells. In severe sepsis and septic shock, ATP is released in large amounts into the extracellular space where it acts as a "danger" signal. In this review, we focus on the roles of ATP as a key regulator of immune cell function and as a disruptive signal that contributes to immune dysfunction in sepsis. METHODS: We summarized the current understanding of the pathophysiology of sepsis, with special emphasis on the emerging role of systemic ATP as a disruptive force that promotes morbidity and mortality in sepsis. FINDINGS: Over the past two decades, the discovery that regulated ATP release and purinergic signaling represent a novel regulatory mechanism in immune cell physiology has opened up new possibilities in the treatment of sepsis. Immune cells respond to stimulation with the release of cellular ATP, which regulates cell functions in autocrine and paracrine fashions. In sepsis, large amounts of systemic ATP produced by tissue damage and inflammation disrupt these regulatory purinergic signaling mechanisms, leading to immune dysfunction that promotes the pathophysiologic processes involved in sepsis. IMPLICATIONS: The knowledge of these ATP-dependent signaling processes is likely to reveal exciting new avenues in the treatment of the unresolved clinical problem of sepsis.
Asunto(s)
Receptores Purinérgicos/inmunología , Sepsis/inmunología , Transducción de Señal/inmunología , Animales , Humanos , RatonesRESUMEN
During sporulation in Saccharomyces cerevisiae, a double lipid bilayer called the prospore membrane is formed de novo, growing around each meiotic nucleus and ultimately closing to create four new cells within the mother cell. Here we show that SPS1, which encodes a kinase belonging to the germinal center kinase III family, is involved in prospore membrane development and is required for prospore membrane closure. We find that SPS1 genetically interacts with SPO77 and see that loss of either gene disrupts prospore membrane closure in a similar fashion. Specifically, cells lacking SPS1 and SPO77 produce hyperelongated prospore membranes from which the leading edge protein complex is not removed from the prospore membrane in a timely fashion. The SPS1/SPO77 pathway is required for the proper phosphorylation and stability of Ssp1, a member of the leading edge protein complex that is removed and degraded when the prospore membrane closes. Genetic dissection of prospore membrane closure finds SPS1 and SPO77 act in parallel to a previously described pathway of prospore membrane closure that involves AMA1, an activator of the meiotic anaphase promoting complex.
Asunto(s)
Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Esporas Fúngicas/genética , Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Meiosis/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Transporte de Proteínas/genética , Proteolisis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/metabolismoRESUMEN
Green fluorescent protein (GFP) has become an invaluable tool in biological research. Many GFP variants have been created that differ in brightness, photostability, and folding robustness. We have created two hybrid GFP variants, Envy and Ivy, which we placed in a vector for the C-terminal tagging of yeast proteins by PCR-mediated recombination. The Envy GFP variant combines mutations found in the robustly folding SuperfolderGFP and GFPγ, while the Ivy GFP variant is a hybrid of GFPγ and the yellow-green GFP variant, Clover. We compared Envy and Ivy to EGFP, SuperfolderGFP and GFPγ and found that Envy is brighter than the other GFP variants at both 30°C and 37°C, while Ivy is the most photostable. Envy and Ivy are recognized by a commonly used anti-GFP antibody, and both variants can be immunoprecipitated using the GFP TRAP Camelidae antibody nanotrap technology. Because Envy is brighter than the other GFP variants and is as photostable as GFPγ, we suggest that Envy should be the preferred GFP variant, while Ivy may be used in cases where photostability is of the utmost importance.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Plásmidos/genética , Saccharomyces cerevisiae/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Mediciones Luminiscentes , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMEN
Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411-415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae.