RESUMEN
Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1-containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM-FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.
Asunto(s)
Anexinas , Transferencia Resonante de Energía de Fluorescencia , Anexinas/genética , Anexinas/metabolismo , Proteínas S100/química , Proteínas S100/metabolismoRESUMEN
Knowledge about precise numbers of specific molecules is necessary for understanding and verification of biological pathways. The RAD51 protein is central in the repair of DNA double-strand breaks (DSBs) by homologous recombination repair and understanding its role in cellular pathways is crucial to design mechanistic DNA repair models. Here, we determined the number of RAD51 molecules in several human cell lines including primary fibroblasts. We showed that between 20000 to 100000 of RAD51 molecules are available per human cell that theoretically can be used for simultaneously loading at least 7 DSBs. Interestingly, the amount of RAD51 molecules does not significantly change after the induction of DNA damage using bleomycin or γ-irradiation in cells but an accumulation of RAD51 on the chromatin occurs. Furthermore, we generated an EGFP-RAD51 fusion under the control of HSV thymidine kinase promoter sequences yielding moderate protein expression levels comparable to endogenously expressed RAD51. Initial characterizations suggest that these low levels of ectopically expressed RAD51 are compatible with cell cycle progression of human cells. Hence, we provide parameters for the quantitative understanding and modeling of RAD51-involving processes.
Asunto(s)
Roturas del ADN de Doble Cadena , Daño del ADN/genética , Recombinasa Rad51/genética , Reparación del ADN por Recombinación/genética , Ciclo Celular/genética , Línea Celular , Proliferación Celular/genética , Cromatina/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Humanos , Cultivo Primario de Células , Timidina Quinasa/genéticaRESUMEN
Numerous studies exploring oncogenic Ras or manipulating physiological Ras signalling have established an irrefutable role for Ras as driver of cell cycle progression. Despite this wealth of information the precise signalling timeline and effectors engaged by Ras, particularly during G1, remain obscure as approaches for Ras inhibition are slow-acting and ill-suited for charting discrete Ras signalling episodes along the cell cycle. We have developed an approach based on the inducible recruitment of a Ras-GAP that enforces endogenous Ras inhibition within minutes. Applying this strategy to inhibit Ras stepwise in synchronous cell populations revealed that Ras signaling was required well into G1 for Cyclin D induction, pocket protein phosphorylation and S-phase entry, irrespective of whether cells emerged from quiescence or G2/M. Unexpectedly, Erk, and not PI3K/Akt or Ral was activated by Ras at mid-G1, albeit PI3K/Akt signalling was a necessary companion of Ras/Erk for sustaining cyclin-D levels and G1/S transition. Our findings chart mitogenic signaling by endogenous Ras during G1 and identify limited effector engagement restricted to Raf/MEK/Erk as a cogent distinction from oncogenic Ras signalling.
Asunto(s)
Ciclina D/metabolismo , Fase G1/fisiología , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fase S/fisiología , Proteínas ras/metabolismo , Proliferación Celular/fisiología , Células HEK293 , Células HeLa , Humanos , Multimerización de Proteína , Transducción de Señal/fisiología , Transducción Genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/farmacología , Proteínas ras/antagonistas & inhibidoresRESUMEN
X-linked lymphoproliferative disease (XLP-1) is an often-fatal primary immunodeficiency associated with the exuberant expansion of activated CD8(+) T cells after Epstein-Barr virus (EBV) infection. XLP-1 is caused by defects in signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), an adaptor protein that modulates T cell receptor (TCR)-induced signaling. SAP-deficient T cells exhibit impaired TCR restimulation-induced cell death (RICD) and diminished TCR-induced inhibition of diacylglycerol kinase α (DGKα), leading to increased diacylglycerol metabolism and decreased signaling through Ras and PKCθ (protein kinase Cθ). We show that down-regulation of DGKα activity in SAP-deficient T cells restores diacylglycerol signaling at the immune synapse and rescues RICD via induction of the proapoptotic proteins NUR77 and NOR1. Pharmacological inhibition of DGKα prevents the excessive CD8(+) T cell expansion and interferon-γ production that occur in SAP-deficient mice after lymphocytic choriomeningitis virus infection without impairing lytic activity. Collectively, these data highlight DGKα as a viable therapeutic target to reverse the life-threatening EBV-associated immunopathology that occurs in XLP-1 patients.
Asunto(s)
Diacilglicerol Quinasa/antagonistas & inhibidores , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/patología , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Muerte Celular/efectos de los fármacos , Citocinas/biosíntesis , Diacilglicerol Quinasa/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos , Recuento de Linfocitos , Trastornos Linfoproliferativos/tratamiento farmacológico , Proteínas de Transporte de Membrana/metabolismo , Ratones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/deficiencia , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria/metabolismo , Tiazoles/farmacología , Proteínas ras/metabolismoRESUMEN
A series of four donor aryl alkynyl substituted thiazole derivatives 3a-d and three similar aryl donor-acceptor systems 6a-c have been synthesized. All compounds bear different electron-donating groups in the 5-position of the thiazole core. The influence of both electron donor strength and the additional phenylethynyl unit on photophysical properties, i.e. UV/Vis absorption, fluorescence emission and fluorescence lifetime, has been evaluated. Additionally, theoretical calculations have been performed at the CAM-B3LYP/6-31+G(d,p) level and good agreement with the experimental data has been achieved. The new derivatives synthesized via palladium catalyzed cross coupling are characterised by moderately strong emission between 474 and 538 nm (ΦF = 0.35-0.39) and Stokes' shifts ranging from 0.54 to 0.79 eV (4392-6351 cm(-1)). The smaller chromophores of type 6 exhibit modest to high fluorescence emission (ΦF = 0.45-0.76) between 470 and 529 nm and their Stokes' shifts range from 0.59 to 0.65 eV (4765-5251 cm(-1)).
Asunto(s)
Alquinos/química , Tiazoles/química , Electrones , Estructura Molecular , Teoría CuánticaRESUMEN
Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.
Asunto(s)
Autofagia , Lisosomas/fisiología , Proteínas/genética , Paraplejía Espástica Hereditaria/patología , Animales , Células Cultivadas , Cerebelo/patología , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Corteza Motora/patología , Células de Purkinje/patología , Paraplejía Espástica Hereditaria/genéticaRESUMEN
The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.
Asunto(s)
Membrana Celular/química , Compuestos Férricos/química , Hidroxiapatitas/química , Fenómenos Magnéticos , Nanopartículas del Metal/química , Nanotecnología/métodos , Membrana Celular/ultraestructura , Dextranos/química , Humanos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Espectrometría RamanRESUMEN
Members of the Ras family of small guanosine triphosphate phosphohydrolases are GDP/GTP-binding proteins that function as pivotal molecular switches in multiple cell biological processes. The prototypical Ras family members K-Ras, N-Ras, and H-Ras, in particular, have been the focus of intense research for the last 30 years owing to their critical function as signalling nodes in the control of cell growth and proliferation and as drivers of oncogenic transformation. One aspect that has attracted much attention in recent times is the spatial control of Ras activity, which is dictated largely by a series of posttranslational modifications that do effectively govern the subcellular distribution and trafficking of Ras. Accordingly, strong emphasis has been placed on developing methodological microscopy-based approaches for the visualization of active Ras-GTP complexes at subcellular resolution. Here we describe the use of a collection of fluorescent affinity probes for the real-time visualization of Ras-GTP in live cells. These probes are multivalent and thus feature high avidity/affinity to Ras-GTP, which obviates the over-expression of Ras and enables one to image endogenous Ras-GTP formation. In addition, this chapter details the use of automated segmentation strategies for the unbiased quantification of probe-derived fluorescence at individual subcellular sites like the plasma membrane and endomembranes.
Asunto(s)
Microscopía Confocal/métodos , Proteínas ras/metabolismo , Animales , Adhesión Celular , Línea Celular , Supervivencia Celular , Colorantes Fluorescentes/metabolismo , Genes Reporteros/genética , Aparato de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Plásmidos/genética , Factores de Tiempo , Transfección , Proteínas ras/genéticaRESUMEN
Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.
Asunto(s)
Proteínas Portadoras/genética , Endosomas/metabolismo , Lisosomas/metabolismo , Degeneración Retiniana/genética , Paraplejía Espástica Hereditaria/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Modelos Animales de Enfermedad , Endosomas/patología , Humanos , Lisosomas/genética , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Mutación , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Paraplejía Espástica Hereditaria/metabolismo , Paraplejía Espástica Hereditaria/patologíaRESUMEN
Olfactory cyclic nucleotide-gated (CNG) ion channels are essential contributors to signal transduction of olfactory sensory neurons. The activity of the channels is controlled by the cyclic nucleotides guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP). The olfactory CNG channels are composed of two CNGA2 subunits, one CNGA4 and one CNGB1b subunit, each containing a cyclic nucleotide-binding domain. Using patch-clamp fluorometry, we measured ligand binding and channel activation simultaneously and showed that cGMP activated olfactory CNG channels not only by binding to the two CNGA2 subunits but also by binding to the CNGA4 subunit. In a channel in which the CNGA2 subunits were compromised for ligand binding, cGMP binding to CNGA4 was sufficient to partly activate the channel. In contrast, in heterotetrameric channels, the CNGB1b subunit did not bind cGMP, but channels with this subunit showed activation by cAMP. Thus, the modulatory subunits participate actively in translating ligand binding to activation of heterotetrameric olfactory CNG channels and enable the channels to differentiate between cyclic nucleotides.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Nervio Olfatorio/metabolismo , Subunidades de Proteína/metabolismo , Transducción de Señal/fisiología , Animales , Fluorometría , Microscopía Fluorescente , Técnicas de Placa-Clamp , Unión Proteica , RatasRESUMEN
Chain length, size, composition, surface charge, and other properties of polymeric materials affect their recognition and uptake by cells and must be optimized to deliver polymers selectively to their target. However, it is often not possible to precisely modify selected properties without changing other parameters. To overcome these difficulties, well-defined poly(pentafluorostyrene)-based polymers are prepared that can be grafted via thiol/para-fluorine "click" reaction with 1-thio-ß-D-glucose and 1-thio-ß-D-galactose. Fluorescence microscopy and flow cytometry show that nanoparticles are taken up by HepG2 cells to a higher degree than the respective water-soluble polymers, and that internalization of both galactosylated homo- and nanoprecipitated block copolymers is enhanced.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Nanopartículas , Polímeros/metabolismo , Poliestirenos/química , Carcinoma Hepatocelular/patología , Citometría de Flujo , Glicosilación , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Microscopía Electrónica de Rastreo , Espectrometría de FluorescenciaRESUMEN
PURPOSE: Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. METHODS: Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted. RESULTS: RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. CONCLUSIONS: The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.
Asunto(s)
Lipofuscina/metabolismo , Retina/metabolismo , Drusas Retinianas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Espectrometría de Fluorescencia/métodos , Anciano de 80 o más Años , Femenino , Fluorescencia , Humanos , Microscopía Confocal , Persona de Mediana Edad , Drusas Retinianas/patología , Donantes de TejidosRESUMEN
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetrameric membrane proteins that generate electrical rhythmicity in specialized neurons and cardiomyocytes. The channels are primarily activated by voltage but are receptors as well, binding the intracellular ligand cyclic AMP. The molecular mechanism of channel activation is still unknown. Here we analyze the complex activation mechanism of homotetrameric HCN2 channels by confocal patch-clamp fluorometry and kinetically quantify all ligand binding steps and closed-open isomerizations of the intermediate states. For the binding affinity of the second, third and fourth ligand, our results suggest pronounced cooperativity in the sequence positive, negative and positive, respectively. This complex interaction of the subunits leads to a preferential stabilization of states with zero, two or four ligands and suggests a dimeric organization of the activation process: within the dimers the cooperativity is positive, whereas it is negative between the dimers.
Asunto(s)
Canales Iónicos/metabolismo , Animales , AMP Cíclico/farmacología , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/química , Ligandos , Miocitos Cardíacos , Neuronas , Técnicas de Placa-Clamp , Canales de Potasio , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Subunidades de ProteínaRESUMEN
The investigation of the plasma membrane with intercorrelated multiparameter techniques is a prerequisite for understanding its function. Presented here, is a simultaneous electrochemical and topographic study of the cell membrane using a miniaturized amperometric enzymatic biosensor. The fabrication of this biosensor is also reported. The biosensor combines a scanning force microscopy (AFM) gold-coated cantilever and an enzymatic transducer layer of peroxidases (PODs). When these enzymes are brought in contact with the substrate, the specific redox reaction produces an electric current. The intensity of this current is detected simultaneously with the surface imaging. For sensor characterization, hydroquinone-2-carboxylic acid (HQ) is selected as an intrinsic source of H(2)O(2). HQ has been electrochemically regenerated by the reduction of antraquinone-2-carboxylic acid (AQ). The biosensor reaches the steady state value of the current intensity in 1 ± 0.2s.
Asunto(s)
Técnicas Biosensibles/instrumentación , Membrana Celular/química , Membrana Celular/ultraestructura , Microscopía de Fuerza Atómica/instrumentación , Animales , Técnicas Biosensibles/métodos , Células CHO , Cricetinae , Cricetulus , Técnicas Electroquímicas , Enzimas Inmovilizadas , Diseño de Equipo , Oro , Hidroquinonas/química , Microscopía de Fuerza Atómica/métodos , Oxidación-Reducción , PeroxidasasRESUMEN
Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Animales , Bovinos , GMP Cíclico/metabolismo , Espacio Intracelular/metabolismo , Activación del Canal Iónico , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , TermodinámicaRESUMEN
Ras transmits manifold signals from the TCR at various crossroads in the life of a T cell. For example, selection programs in the thymus or the acquisition of a state of hypo-responsiveness known as anergy are just some of the T cell features known to be controlled by TCR-sparked signals that are intracellularly propagated by Ras. These findings raise the question of how Ras can transmit such a variety of signals leading to the shaping of equally many T cell traits. Because Ras proteins transit through endomembrane compartments on their way to the plasma membrane (PM), compartmentalized Ras activation at distinct subcellular sites represents a potential mechanism for signal diversification in TCR signaling. This hypothesis has been nurtured by studies in T cells engineered to overexpress Ras that reported distinct activation of Ras at the PM and Golgi. Contrary to this scenario, we report in this study that activation of endogenous Ras, imaged in live Jurkat T cells using novel affinity probes for Ras-GTP, proceeds only at the PM even upon enforced signal flux through the diacylglycerol/RasGRP1 pathway. Physiological engagement of the TCR at the immunological synapse in primary T cells caused focalized Ras-GTP accumulation also only at the PM. Analysis of palmitoylation-deficient Ras mutants, which are confined to endomembranes, confirmed that the TCR does not activate Ras in that compartment and revealed a critical function for palmitoylation in N-Ras/H-Ras activation. These findings identify the PM as the only site of TCR-driven Ras activation and document that endomembranes are not a signaling platform for Ras in T cells.
Asunto(s)
Membrana Celular/metabolismo , Lipoilación/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Colorantes Fluorescentes/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Células Jurkat , Lipoilación/genética , Proteína Oncogénica p21(ras)/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Ratas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/biosíntesis , Factores de Intercambio de Guanina Nucleótido ras/genética , Proteínas ras/biosíntesisRESUMEN
HCN pacemaker channels are tetramers mediating rhythmicity in neuronal and cardiac cells. The activity of these channels is controlled by both membrane voltage and the ligand cAMP, binding to each of the four channel subunits. The molecular mechanism underlying channel activation and the relationship between the two activation stimuli are still unknown. Using patch-clamp fluorometry and a fluorescent cAMP analog, we show that full ligand-induced activation appears already with only two ligands bound to the tetrameric channel. Kinetic analysis of channel activation and ligand binding suggests direct interaction between the voltage sensor and the cyclic nucleotide-binding domain, bypassing the pore. By exploiting the duality of activation in HCN2 channels by voltage and ligand binding, we quantify the increase of the binding affinity and overall free energy for binding upon channel activation, proving thus the principle of reciprocity between ligand binding and conformational change in a receptor protein.
Asunto(s)
Fenómenos Biofísicos/genética , Canales Iónicos/metabolismo , Ligandos , Animales , Fenómenos Biofísicos/efectos de los fármacos , AMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Fluorometría/métodos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Canales Iónicos/genética , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Microinyecciones/métodos , Oocitos , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Conformación Proteica/efectos de los fármacos , Factores de Tiempo , Xenopus laevisRESUMEN
Various fluorophore-based microscopic methods, comprising Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods, however, remains difficult. In this study, we report the successful application of combined BiFC-FRET-fluorescence lifetime imaging microscopy and BiFC-FRET-acceptor photobleaching measurements to visualize the formation of ternary soluble N-ethylmaleimide-sensitive factor attachment receptor complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Hojas de la Planta/metabolismo , Proteínas SNARE/metabolismo , Proteínas Luminiscentes/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
New multifunctional copoly(2-oxazoline) nanoparticles were prepared for cell studies. The polymer contains double-bond side chains as potential reaction sites for "thio"-click reactions as well as a fluorescein label covalently bound to the polymer backbone. Using the nanoprecipitation technique, spherical nanoparticles of 200-800 nm were obtained. Confocal laser scanning microscopy measurements revealed the cellular uptake of the nanoparticles.
RESUMEN
The pharmaceutically important polymer P(MAA-r-MMA)(1:2) (EUDRAGIT(®) S100) was investigated concerning its behavior to form nanoparticles via nanoprecipitation. The particles obtained were characterized regarding their size, shape, and characteristics using DLS, SEM, and AUC. Furthermore, the P(MAA-r-MMA)(1:2) copolymer was modified with different markers in order to achieve polymer-based nanocarrier systems, which are detectable and may be useful for controlled drug delivery devices to monitor the drug pathways. The particles were labeled by physical entrapment as well as by covalent attachment of various markers, e.g., radicals, fluorescent-, and near-infrared dyes, to the polymer. Physical entrapment of radicals into the polymeric units was performed by co-nanoprecipitation of P(MAA-r-MMA)(1:2) and a radical marker. By means of covalent binding of the markers to the polymer, a stable and more defined labeling of the particles was also performed, leading only to a low degree of modification of the pharmaceutical polymer. After nanoprecipitation, the resulting labeled particles were characterized by SEM and DLS, whereas their biocompatibility was proven by in vitro studies. In order to ensure the possibility of detection of the particles inside the body for drug delivery-, sensor-, and imaging applications, the polymeric carriers were also investigated by electron spin resonance, fluorescence, as well as near-infrared spectroscopy.