RESUMEN

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.

Asunto(s)

Macrófagos/química , Microscopía de Fuerza Atómica/métodos , Microscopía Fluorescente/métodos , Actinas/química , Actinas/metabolismo , Animales , Fenómenos Biomecánicos , Fluorescencia , Células HeLa , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Fagocitosis , Células RAW 264.7

RESUMEN

Cellular components sequestered by autophagosomes during macroautophagy must be degraded and their components recycled in order to maintain homeostasis. To this end cells orchestrate the fusion of autophagosomes with lysosomes, degradative organelles that are rich in hydrolases. Most of the lysosomal enzymes function optimally at low pH, and products of macromolecular catabolism are cotransported with protons across the autolysosomal membrane. These functions are facilitated by the ability of lysosomes to pump protons inward, acidifying their lumen. Clearly, proper homeostasis of the luminal pH is crucial for autolysosomal function. We describe a method for the measurement of the absolute pH of individual autolysosomes in live cells. This technique involves measurement of the fluorescence of a pH-sensitive probe initially delivered to lysosomes and subsequently determined to have reached autolysosomes. By measuring the fluorescence at two separate wavelengths and calculating their ratio, potential artifacts introduced by photobleaching or by changes in autolysosome size, shape, or positioning are minimized. Combining such ratio determinations with an in situ calibration procedure enables absolute measurements of pH, which are superior to the qualitative estimates obtained with fluorescent weak bases such as LysoTracker.

Asunto(s)

Lisosomas/química , Imagen Óptica/métodos , Fagosomas/química , Autofagia , Ácidos Carboxílicos/química , Dextranos/química , Colorantes Fluorescentes/química , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/ultraestructura , Microscopía Fluorescente/métodos , Fagosomas/ultraestructura , Programas Informáticos

RESUMEN

We present the construction of the optical part of the ToF (time-of-flight) subdetector prototype for the AFP (ATLAS Forward Proton) detector. The ToF detector in conjunction with a 3D silicon pixel tracker will tag and measure protons originating in central exclusive interactions p + p → p + X + p, where the two outgoing protons are scattered in the very forward directions. The ToF is required to reduce so-called pileup backgrounds that arise from multiple proton interactions in the same bunch crossing at high luminosity. The background can fake the signal of interest, and the extra rejection from the ToF allows the proton tagger to operate at the high luminosity required for measurement of the processes. The prototype detector uses fused silica bars emitting Cherenkov radiation as a relativistic particle passes through it. The emitted Cherenkov photons are detected by a micro-channel plate multi-anode Photomultiplier Tube (MCP-PMT) and processed by fast electronics.

RESUMEN

Phagocytosis holds a central position in the development of a successful innate immune response and in the initiation of the corresponding adaptive response. The destruction of invading pathogens and the presentation of their antigens to lymphoid cells require acidification of the phagosomal lumen. The present review discusses the mechanism of phagosome acidification, with particular reference to the two components of the protonmotive force: the chemical (pH) gradient and the electrical potential across the phagosomal membrane. A method for the in situ measurement of the electrical potential across the phagosomal membrane is described. In addition, we discuss the finding that acidification is not only a consequence, but also a critical determinant of phagosome maturation. Luminal acidification appears to function as a timing device controlling the transition between early and late phagosomes.

Asunto(s)

Ácidos/metabolismo , Fagosomas/metabolismo , Electricidad , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Fenobarbital/metabolismo

RESUMEN

The regulation of volume is fundamental to life. There exist numerous conditions that can produce perturbations of cell volume. The cell has developed mechanisms to directly counteract these perturbations so as to maintain its physiological volume. Directed influx of the major extracellular cation, sodium, serves to counteract a decreased cell volume through the subsequent osmotically coupled movement of water to the intracellular space. This process, termed regulatory volume increase is often mediated by the ubiquitous sodium/hydrogen ion exchanger, NHE1. Similarly, the maintenance of intravascular volume is essential for the maintenance of blood pressure and consequently the proper perfusion of vital organs. Numerous mechanisms exist to counterbalance alterations in intravascular volume, not the least of which is the renal absorption of sodium filtered at the glomerulus. Two-thirds of filtered sodium and water are absorbed in the renal proximal tubule, a mechanism that intimately involves the apical sodium/hydrogen ion exchanger, NHE3. This isoform is fundamental to the maintenance and regulation of intravascular volume and blood pressure. In this article, the effects of cell volume on the activity of these different isoforms, NHE1 and NHE3, will be described and the consequences of their activity on intracellular and intravascular volume will be explored.

Asunto(s)

Túbulos Renales Proximales/metabolismo , Músculo Liso Vascular/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Presión Sanguínea/fisiología , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Tamaño de la Célula , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico , Intercambiador 1 de Sodio-Hidrógeno , Intercambiador 3 de Sodio-Hidrógeno , Equilibrio Hidroelectrolítico

RESUMEN

Influenza vaccination is a key intervention to reduce morbidity and mortality provoked by this disease. To date, the challenge of improving its efficacy remains unmet. The immunogenic synthetic peptide GK1 from Taenia crassiceps cysticerci was tested herein in its capacity as adjuvant, co-administered with the inactivated anti-influenza vaccine before and after challenge with influenza virus in both young and aged mice. Co-administration of GK1 with the influenza vaccine increased levels of anti-influenza antibodies in aged mice before and after infection, reduced the local inflammation that accompanied influenza vaccination itself and favored virus clearance after infection in both young and aged mice.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Vacunas contra la Influenza/inmunología , Péptidos/farmacología , Hidróxido de Aluminio/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Femenino , Inmunoglobulina G/sangre , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Saponinas/farmacología , Vacunación

RESUMEN

Cells of the innate immune system engulf invading microorganisms into plasma membrane-derived vacuoles called phagosomes. Newly formed phagosomes gradually acquire microbicidal properties by a maturation process which involves sequential and coordinated rounds of fusion with endomembranes and concomitant fission. Some pathogens interfere with this maturation sequence and thereby evade killing by the immune cells, managing to survive intracellularly as parasites. Phosphoinositides seem to be intimately involved in the processes of phagosome formation and maturation, and initial observations suggest that the ability of some microorganisms to survive intracellularly is associated with alterations in phosphoinositide metabolism. This chapter presents a brief overview of phosphoinositides in cells of the immune system, their metabolism in the context of phagocytosis and phagosome maturation and their possible derangements during infectious pathogenosis.

Asunto(s)

Fagocitosis/fisiología , Fagosomas/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Humanos , Modelos Biológicos , Transducción de Señal

RESUMEN

Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Asunto(s)

Proteínas de la Membrana/metabolismo , Fagocitosis/fisiología , Fagosomas/química , Fagosomas/fisiología , Fosfatidilinositoles/metabolismo , Transducción de Señal/fisiología , Proteínas de Transporte Vesicular , Bacterias/patogenicidad , Endocitosis , Fusión de Membrana , Fagosomas/microbiología , Proteínas SNARE , Relación Estructura-Actividad

RESUMEN

Phagocytosis by macrophages and neutrophils involves the spatial and temporal reorganisation of the actin-based cytoskeleton at sites of particle ingestion. Local polymerisation of actin filaments supports the protrusion of pseudopodia that eventually engulf the particle. Here we have investigated in detail the cytoskeletal events initiated upon engagement of Fc receptors in macrophages. Ena/vasodilator-stimulated phosphoprotein (VASP) proteins were recruited to phagosomes forming around opsonised particles in both primary and immortalised macrophages. Not only did the localisation of Ena/VASP proteins coincide, spatially and temporally, with the phagocytosis-induced reorganisation of actin filaments, but their recruitment to the phagocytic cup was required for the remodelling of the actin cytoskeleton, extension of pseudopodia and efficient particle internalisation. We also report that SLP-76, Vav and profilin were recruited to forming phagosomes. Upon induction of phagocytosis, a large molecular complex, consisting in part of Ena/VASP proteins, the Fyn-binding/SLP-76-associated protein (Fyb/SLAP), Src-homology-2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76), Nck, and the Wiskott-Aldrich syndrome protein (WASP), was formed. Our findings suggest that activation of Fcgamma receptors triggers two signalling events during phagocytosis: one through Fyb/SLAP that leads to recruitment of VASP and profilin; and another through Nck that promotes the recruitment of WASP. These converge to regulate actin polymerisation, controlling the assembly of actin structures that are essential for the process of phagocytosis.

Asunto(s)

Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Oncogénicas/metabolismo , Fagocitosis/fisiología , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/genética , Línea Celular , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/fisiología , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Monocitos/citología , Monocitos/metabolismo , Fagosomas/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Proteína del Síndrome de Wiskott-Aldrich , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

An 8-years-old boy was admitted with fever of unknown origin, cervical lymphadenopathy and hepatosplenomegaly and weight loss. His mother's HIV infection was diagnosed two weeks before his hospitalization, so he was diagnosed as perinatally acquired AIDS. Serology and serial cultures were negative for viral infections, toxoplasmosis, chagas, tuberculosis and atypical mycobacterium. The patient met clinical and laboratory criteria for hemophagocytic syndrome (HS) that was confirmed on bone marrow aspirate and biopsy. A cervical lymph node biopsy was performed which was diagnosed as Hodgkin's disease (HD) diffuse fibrosis lymphocyte depletion subtype. EBERs in situ hybridization and LMP-1 immunohistochemistry on the lymph node biopsy established the EBV association. On the basis of a sequence of appearance of the clinical, laboratory and histological signs, HIV, EBV or HD may have triggered HS as the last fatal event in this pediatric patient.

Asunto(s)

Infecciones por Virus de Epstein-Barr/complicaciones , Histiocitosis de Células no Langerhans/etiología , Enfermedad de Hodgkin/complicaciones , Linfoma Relacionado con SIDA/complicaciones , Niño , Resultado Fatal , VIH-1 , Histiocitosis de Células no Langerhans/virología , Enfermedad de Hodgkin/virología , Humanos , Masculino

RESUMEN

Engulfment of particles by phagocytes involves remodeling of the plasma membrane. We review recent work that suggests that focal exocytosis of endomembranes plays an important role in pseudopod extension during phagocytosis.

Asunto(s)

Membrana Celular/fisiología , Fagocitosis/fisiología , Fagosomas/fisiología , Actinas , Animales , Membrana Celular/microbiología , Exocitosis/fisiología , Humanos , Lisosomas/fisiología , Proteínas Opsoninas , Receptores de IgG/fisiología , Salmonella typhimurium/fisiología

RESUMEN

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Asunto(s)

Fagocitosis/fisiología , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Androstadienos/farmacología , Animales , Células Cultivadas , Inhibidores Enzimáticos/metabolismo , Fibroblastos/metabolismo , Genes Reporteros , Humanos , Inmunoglobulina G/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lisosomas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Microinyecciones , Fagosomas/ultraestructura , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Wortmanina

RESUMEN

Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and in the biosynthesis of bile acids, cholesterol, and ether phospholipids. Even though maintenance of a stable acid-base milieu is essential for proper peroxisomal function, the determination of the peroxisomal pH (pH(p)) remains inconclusive, and little is known about its regulation. To measure the pH of intact peroxisomes in situ, we used the peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver a pH-sensitive mutant of the green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper targeting was verified by colocalization with the peroxisomal marker catalase. Peroxisomes were visualized by imaging fluorescence microscopy, and ratiometric measurements were combined with calibration using ionophores or a null-point method to estimate pH(p). The pH(p) was between 6.9 and 7.1, resembling the cytosolic pH. Manipulation of the cytosolic pH in intact cells or after permeabilization of the plasmalemma with streptolysin O revealed that pH(p) changed in parallel, suggesting that the peroxisomal membrane is highly permeable to H(+) (equivalents). We conclude that peroxisomes do not regulate their pH independently, but instead their large H(+) permeability effectively connects them with the buffer reservoir of the cytoplasm and with the homeostatic mechanisms that control cytosolic pH.

Asunto(s)

Colorantes Fluorescentes/metabolismo , Proteínas Luminiscentes/metabolismo , Macrólidos , Peroxisomas/metabolismo , Transporte de Proteínas/fisiología , Animales , Antibacterianos/farmacología , Bicarbonatos/metabolismo , Células CHO , Dióxido de Carbono/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Células Cultivadas , Cricetinae , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Ionóforos/farmacología , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Nigericina/farmacología , Peroxisomas/química , Señales de Clasificación de Proteína , Transfección

RESUMEN

In the search for more potent and less toxic immunomodulators, adamantylamide dipeptide (AdDP) was synthesized by the covalent union of amantadine with the L-alanyl-D-isoglutamine residue of muramyldipeptide (MDP). The present experiments demonstrate the ability of AdDP, co-administered with a protein immunogen, to raise or enhance a humoral response in immunized animals. BALB/c mice were immunized either by the intraperitoneal (ip) or oral route with ovalbumin (Ova) alone or combined with either AdDP or CpG oligonucleotide (ODN-CpG), a proved adjuvant. A clear adjuvant dose-response relationship was observed on the increment of Ova-specific serum antibody titers when AdDP was used as adjuvant, irrespectively of the administration route. The IgG isotype analysis showed that AdDP promotes a consistent increment in IgG1 antibodies associated with a dominant Th2 response pattern. When administered by the oral route, AdDP was at least as efficient as ODN-CpG as adjuvant. Similar results were obtained in rabbits immunized by the oral route, suggesting that the adjuvanticity of AdDP is not restricted to the murine system. In conclusion, AdDP was shown to be a powerful and non-toxic adjuvant at both systemic and mucosal levels, which makes it a promising tool for vaccine development.

Asunto(s)

Adyuvantes Inmunológicos , Amantadina/análogos & derivados , Amantadina/inmunología , Dipéptidos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/toxicidad , Administración Oral , Amantadina/administración & dosificación , Amantadina/toxicidad , Animales , Islas de CpG/inmunología , Dipéptidos/administración & dosificación , Dipéptidos/toxicidad , Relación Dosis-Respuesta Inmunológica , Heces/química , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Inyecciones Intraperitoneales , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Mucosa Bucal/inmunología , Ovalbúmina/inmunología , Conejos , Especificidad de la Especie , Células Th2/inmunología , Células Th2/metabolismo

RESUMEN

Group A rotaviruses are the major cause of severe gastroenteritis in young children worldwide. Because rotavirus vaccination appeared imminent, a nationwide surveillance program was organized between October 1996 and October 1998 in the largest Argentine cities. Surveillance for disease burden, rotavirus detection, and rotavirus typing was undertaken at nine locations. Results showed rotavirus to be associated with 42% of diarrhea admissions. Although the prevalent G types changed from year to year, common G types were found in 96% of the cases and were usually associated with common P types. Uncommon G types, G9 and G5, were found at low prevalence and uncommon G/P combinations occurred at almost every study site. These data suggest that a rotavirus vaccine could substantially decrease the rotavirus disease burden in Argentina, but that introduction of a vaccine should be accompanied by a concurrent surveillance system.

Asunto(s)

Vigilancia de la Población , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/virología , Rotavirus/aislamiento & purificación , Distribución por Edad , Argentina/epidemiología , Preescolar , Diarrea/epidemiología , Diarrea/virología , Genotipo , Humanos , Lactante , Recién Nacido , Rotavirus/clasificación , Rotavirus/inmunología , Estaciones del Año , Serotipificación

RESUMEN

The Na(+)/H(+) exchanger NHE3 isoform mediates the entry of Na(+) into epithelial cells of the kidney and gastrointestinal tract. Hormones and pharmacological agents that activate cAMP-dependent protein kinase A (PKA) are potent inhibitors of native and ectopically expressed NHE3 in epithelial and Chinese hamster ovary AP-1 cells, respectively. Previous studies have shown that acute inhibition is coupled to direct phosphorylation of the exchanger, but this only partly accounts for the observed effects. In this report, we show that inhibition of NHE3 activity by forskolin, an activator of adenylate cyclase, occurs without changes in surface expression of the exchanger but is associated with altered cytoskeletal structure. This effect resembles that obtained with cytochalasin D or latrunculin B, actin disrupting agents that also inhibit NHE3. Such similarities prompted us to further investigate the relationship between PKA-induced inhibition of the exchanger and changes in the actin cytoskeleton. Inhibition of NHE3 by cytochalasin D does not require PKA, because the inhibitory effect is preserved in a mutant NHE3 that is not phosphorylated by PKA and in cells pretreated with the PKA inhibitor H89. In contrast, involvement of actin in the effect of cAMP on the exchanger is supported by the following observations: (i) jasplakinolide, an F-actin stabilizer, prevents the inhibition caused by forskolin, and (ii) constitutively active forms of RhoA and Rho kinase interfere with actin disruption by forskolin and also decrease inhibition of the transporter. These results suggest that reorganization of the cytoskeleton by PKA is involved in mediating inhibition of NHE3.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoesqueleto/fisiología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Actinas/metabolismo , Animales , Células CHO , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Fracciones Subcelulares/metabolismo

RESUMEN

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.

Asunto(s)

Cloruros/metabolismo , Proteínas de la Membrana/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células CHO , Tamaño de la Célula , Quimotripsina/metabolismo , Cricetinae , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Concentración Osmolar , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Ratas , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Factores de Tiempo

RESUMEN

The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.

Asunto(s)

MAP Quinasa Quinasa 4 , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células COS , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Fibroblastos , Humanos , Concentración de Iones de Hidrógeno , Soluciones Hipertónicas , Immunoblotting , MAP Quinasa Quinasa 1 , Ratones , Proteína Quinasa 8 Activada por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mutación/genética , Concentración Osmolar , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Transfección , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.

Asunto(s)

Estructuras de la Membrana Celular/metabolismo , Macrófagos/metabolismo , Fagocitosis/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de IgG/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Animales , Proteínas Sanguíneas/genética , Línea Celular , Macrófagos/citología , Ratones , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , Fosfoproteínas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido

RESUMEN

Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

Asunto(s)

Proteína Coat de Complejo I/fisiología , Macrófagos/fisiología , Receptores de IgG/fisiología , Animales , Línea Celular , Ratones , Fagocitosis/fisiología