RESUMO
Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial-mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sinalização do Cálcio , Cálcio/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Hipóxia Celular , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Vimentina/biossínteseRESUMO
The use of normal immersion oil, developed for 23 degrees C, at 37 degrees C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live-cell imaging at 37 degrees C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23 degrees C counterparts.
Assuntos
Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Óleos , Temperatura , Animais , Células Cultivadas , Rim/citologia , CamundongosRESUMO
The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging Property (SIP) charts (Brakenhoff et al., 2005). It is shown that the integrated intensity of a z-stack from a biological sample, imaged under identical conditions as the layer, can be calibrated in terms of fluorescence layer units of the calibration layer. The imaging after such calibration becomes, as a first approximation, independent of the microscope system and imaging conditions. This is demonstrated on axially integrated images of standard fluorescent beads and standard BPAE Fluorocells. Corrections on the microscope imaging conditions include shading effects, imaging with different magnifications and objectives, and using different microscope systems. It is also shown that with the present approach the actual underlying three-dimensional (3D) fluorescence data set itself can be corrected for variations in point spread function (PSF) imaging efficiency over the imaging data cube. Realizing such calibration between imaging conditions or systems requires basically only the 2D fluorescer molecule density of the reference layers and the section distances with which the layer data are collected.
Assuntos
Calibragem , Aumento da Imagem/métodos , Microscopia Confocal , Microscopia de Fluorescência , Animais , Calibragem/normas , Bovinos , Células Cultivadas , Células Endoteliais , Microscopia Confocal/métodos , Microscopia Confocal/normas , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência/normas , Artéria Pulmonar/citologia , Padrões de Referência , Fatores de TempoRESUMO
The proportion of enterococcal infections caused by ampicillin-resistant Enterococcus faecium (AREfm) in a European hospital increased from 2% in 1994 to 32% in 2005, with prevalence rates of AREfm endemicity of up to 35% in at least six hospital wards. Diabetes mellitus, three or more admissions in the preceding year, and use of beta-lactams and fluoroquinolones, were all associated with AREfm colonisation. Of 217 AREfm isolates that were genotyped, 97% belonged to clonal complex 17 (CC17). This ecological change mimics events preceding the emergence of vancomycin-resistant E. faecium (VREF) in the USA and may presage the emergence of CC17 VREF in European hospitals.
Assuntos
Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/epidemiologia , Resistência a Ampicilina , Farmacorresistência Bacteriana Múltipla , Ecologia , Enterococcus faecalis/genética , Enterococcus faecium/genética , Humanos , Repetições Minissatélites , Resistência a VancomicinaRESUMO
BACKGROUND: Control of vancomycin-resistant Enterococcus faecium (VRE) in European hospitals is hampered because of widespread asymptomatic carriage of VRE by healthy Europeans. In 2000, our hospital (The University Medical Center Utrecht, Utrecht, The Netherlands) was confronted with a large outbreak of VRE. INTERVENTION: On the basis of genotyping (by pulsed-field gel electrophoresis), epidemic and nonepidemic VRE strains were distinguished, and infection-control measures were exclusively targeted toward epidemic VRE. The outbreak was retrospectively divided into 3 periods of different infection-control measures. Compliance with use of alcohol-based hand rubs was enforced during all periods. Period I involved active surveillance, isolation of carriers, and cohorting (duration, 4 months); preemptive isolation of high-risk patients for VRE colonization was added in period II (7 months); and cohorting and preemptive isolation were abandoned in period III (18 months). METHODS: When the outbreak was identified, 27 patients in 6 wards were colonized; 93% were colonized with an epidemic VRE strain. Detection rates of nonepidemic VRE were 3.5%, 3.0%, and 2.9% among 683, 810, and 977 screened patients in periods I, II, and III, respectively, comparable to a prevalence of 2% (95% confidence interval [CI], 1%-3.5%) among 600 nonhospitalized persons. The relative risks of detecting epidemic VRE in periods II and III, compared with period I, were 0.67 (95% CI, 0.41-1.10) for period II and 0.02 (95% CI, 0.002-0.6) for period III. Infection-control measures were withheld for patients colonized with nonepidemic VRE (76 [54%] of 140 patients with a test result positive for VRE). Use of alcohol-based hand rubs increased by 31%-275% in outbreak wards. CONCLUSION: Genotyping-targeted infection control, isolation of VRE carriers, enhancement of hand-hygiene compliance, and preemptive isolation successfully controlled nosocomial spread of epidemic VRE infection.
Assuntos
Surtos de Doenças/prevenção & controle , Enterococcus faecium/classificação , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções por Bactérias Gram-Positivas/prevenção & controle , Isolamento de Pacientes , Resistência a Vancomicina , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Genótipo , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Higiene , Testes de Sensibilidade MicrobianaRESUMO
Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.
RESUMO
In May 2000, the first outbreak of vancomycin-resistant Enterococcus faecium (VREF) was detected in the University Medical Center Utrecht in the nephrology ward. The question arose why some VREF strains spread among hospitalized patients, whereas other strains do not. Thirty patients who were found to be colonized with VREF between May and November 2000 were included in the study. Molecular typing confirmed that 19 of them carried an identical epidemic strain which harbored the esp gene while 11 were colonized by nonepidemic strains that were all esp negative. Acquisition of the outbreak strain was significantly associated with diabetes mellitus, renal transplantation, and extensive use of antibiotics, especially cephalosporins, in the 2-month period before the first isolation of VREF. To establish the duration of colonization, prospective surveillance of VREF carriage for a 6-month period starting from the first isolation of VREF was realized for 20 patients. After 6 months, VREF was still recovered from 60% of carriers of the outbreak strain versus 20% of carriers of nonepidemic strains (P < 0.01). However, antibiotic use during the follow-up period was significantly higher by carriers of the outbreak strain than by carriers of nonepidemic strains. The fact that the outbreak strain was recovered for a longer period of time than nonepidemic strains may facilitate dissemination of the strain. The results support a careful restrictive antibiotic policy for wards at risk for spread of VREF and implementation of isolation precautions for patients who are colonized with esp-positive outbreak strains.
Assuntos
Enterococcus/classificação , Enterococcus/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/transmissão , Resistência a Vancomicina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Eletroforese em Gel de Campo Pulsado , Feminino , Hospitais com mais de 500 Leitos , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Filogenia , Sorotipagem/métodos , Vancomicina/farmacologiaRESUMO
Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.
Assuntos
Canais de Cloreto/metabolismo , Proteínas de Ligação a DNA/fisiologia , Neurônios/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Potenciais de Ação , Animais , Linhagem Celular , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP , Neurônios/fisiologiaRESUMO
Agonist-induced intracellular Ca(2+) signals following phospholipase C (PLC) activation display a variety of patterns, including transient, sustained, and oscillatory behavior. These Ca(2+) changes have been well characterized, but detailed kinetic analyses of PLC activation in single living cells is lacking, due to the absence of suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLCdelta1 tagged with cyan and yellow fluorescent proteins as a sensitive readout of phosphatidylinositol bisphosphate metabolism for use both in cell populations and in single cells. Fluorescence resonance energy transfer requires significantly less excitation intensity, enabling prolonged and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat cells, and can be scaled to record from cell populations as well as single neurites. Characterization of responses to various agonists by this method reveals that stimuli that elicit very similar Ca(2+) mobilization responses can exhibit widely different kinetics of PLC activation, and that the latter appears to follow receptor activation more faithfully than the cytosolic Ca(2+) transient.
Assuntos
Fosfatidilinositóis/metabolismo , Fosfolipases Tipo C/metabolismo , Sequência de Bases , Cálcio/metabolismo , Membrana Celular/metabolismo , Tamanho Celular , Primers do DNA , Ativação Enzimática , Cinética , Proteínas Luminescentes/metabolismo , Transporte Proteico , Espectrometria de Fluorescência , Células Tumorais Cultivadas , Fosfolipases Tipo C/efeitos dos fármacosRESUMO
A density gradient electrophoresis (DGE) apparatus (2.2 x, 14 cm) was constructed for the rapid separation of milligram quantities of proteins. By using binary buffers according to Bier (Electrophoresis 1993, 14, 1011-1018) proteins were rate-zonally separated in less than 60 min. Acidic proteins were separated in a pH 8.6, 56 microS/cm buffer, and basic proteins in a pH 5.4, 76 microS/cm buffer. Thus the A (pI 5.15) and B (pI 5.30) forms of beta-lactoglobulin as well as the sialylated glycoforms of apotransferrin were well separated at pH 8.6. The isoforms of myoglobin (pI 6.9 and 7.35, respectively), RNAse A (pI 9.45) and cytochrome c (pI 10.0) and lysozyme (pI 11) were separated at pH 5.4 within 80 min. On a 7 cm DGE column, subcellular organelles derived from HeLa cells were separated in standard electrophoresis buffer (655 microS/cm) for 90 min at 10 mA. Using a new low conductivity buffer (193 microS/cm) 20 min was sufficient to separate late endosomes, lysosomes, endoplasmic reticulum, early endosomes, plasma membrane, clathrin-coated pits, proteasomes, and clathrin-coated vesicles within a single run directly from a postnuclear supernatant.
Assuntos
Eletroforese/métodos , Organelas/química , Proteínas/isolamento & purificação , Fracionamento Celular , Membrana Celular/química , Clatrina/metabolismo , Vesículas Revestidas/química , Eletroforese/instrumentação , Endossomos/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/química , Substâncias Macromoleculares , Frações Subcelulares/química , Células Tumorais CultivadasRESUMO
Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."
Assuntos
Drosophila/genética , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transmissão Sináptica/fisiologia , Animais , Mapeamento Cromossômico , Clonagem Molecular , Drosophila/enzimologia , Eletrofisiologia , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Lítio/farmacologia , Masculino , Dados de Sequência Molecular , Mutação/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurotransmissores/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Vesículas Sinápticas/metabolismoRESUMO
BACKGROUND: Phospholipase D (PLD) hydrolyzes phospholipids to generate phosphatidic acid (PA) and a free headgroup. PLDs occur as both intracellular and secreted forms; the latter can act as potent virulence factors. Exogenous PLD has growth-factor-like properties, in that it induces proto-oncogene transcription, mitogenesis and cytoskeletal changes in target cells. The underlying mechanism is unknown, although it is generally assumed that PLD action is mediated by PA serving as a putative second messenger. RESULTS: In quiescent fibroblasts, exogenous PLD (from Streptomyces chromofuscus) stimulated accumulation of the GTP-bound form of Ras, activation of mitogen-activated protein (MAP) kinase and DNA synthesis, through the pertussis-toxin-sensitive inhibitory G protein Gi. Furthermore, PLD mimicked bioactive lysophospholipids (but not PA) in inducing Ca2+ mobilization, membrane depolarization and Rho-mediated neurite retraction. PLD action was mediated by Iysophosphatidic acid (LPA) derived from Iysophosphatidylcholine acting on cognate G-protein-coupled LPA receptor(s). There was no evidence for the involvement of PA in mediating the effects of exogenous PLD. CONCLUSIONS: Our results provide a molecular explanation for the multiple cellular responses to exogenous PLDs. These PLDs generate bioactive LPA from pre-existing Iysophosphatidylcholine in the outer membrane leaflet, resulting in activation of G-protein-coupled LPA receptors and consequent activation of Ras, Rho and Ca2+ signaling pathways. Unscheduled activation of LPA receptors may underlie, at least in part, the known pathogenic effects of exogenous PLDs.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/biossíntese , Proteínas de Membrana/metabolismo , Fosfolipase D/farmacologia , Receptores Acoplados a Proteínas G , Proteínas ras/metabolismo , Animais , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Fosfolipase D/metabolismo , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores de Ácidos Lisofosfatídicos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoB de Ligação ao GTPRESUMO
Gap junctions mediate cell-cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell-cell communication is rapidly disrupted by G protein-coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1-2 h of receptor stimulation. In contrast, a desensitization-defective G protein-coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein-coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell-cell communication.
Assuntos
Comunicação Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular , Conexina 43/metabolismo , Eletrodos , Células HeLa , Humanos , Camundongos , Técnicas de Patch-Clamp , Proteínas Tirosina Quinases/antagonistas & inibidores , Ratos , Quinases da Família srcRESUMO
Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors are phosphorylated by kinases that mediate agonist-dependent receptor deactivation. Although many receptor kinases have been isolated, the corresponding phosphatases, necessary for restoring the ground state of the receptor, have not been identified. Drosophila RDGC (retinal degeneration C) is a phosphatase required for rhodopsin dephosphorylation in vivo. Loss of RDGC caused severe defects in the termination of the light response as well as extensive light-dependent retinal degeneration. These phenotypes resulted from the hyperphosphorylation of rhodopsin because expression of a truncated rhodopsin lacking the phosphorylation sites restored normal photoreceptor function. These results suggest the existence of a family of receptor phosphatases involved in the regulation of G protein-coupled signaling cascades.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Proteínas de Ligação ao GTP/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismo , Animais , Animais Geneticamente Modificados , Arrestina/metabolismo , Escuridão , Drosophila , Eletrorretinografia , Luz , Mutação , Fosfoproteínas Fosfatases/genética , Fosforilação , Retina/metabolismo , Degeneração Retiniana , Transdução de SinaisRESUMO
Phospholipase C (PLC) is the focal point for two major signal transduction pathways: one initiated by G protein-coupled receptors and the other by tyrosine kinase receptors. Active PLC hydrolyzes phosphatidylinositol bisphosphate (PIP2) into the two second messengers inositol 1,4,5-trisphosphate (InsP3) and diacyl glycerol (DAG). DAG activates protein kinase C, and InsP3 mobilizes calcium from intracellular stores via the InsP3 receptor. Changes in [Ca2+]i regulate the function of a wide range of target proteins, including ion channels, kinases, phosphatases, proteases, and transcription factors (Berridge, 1993). In the mouse, there are three InsP3R genes, and type 1 InsP3R mutants display ataxia and epileptic seizures (Matsumoto et al., 1996). In Drosophila, only one InsP3 receptor (InsP3R) gene is known, and it is expressed ubiquitously throughout development (Hasan and Rosbash, 1992; Yoshikawa et al., 1992; Raghu and Hasan, 1995). Here, we characterize Drosophila InsP3R mutants and demonstrate that the InsP3R is essential for embryonic and larval development. Interestingly, maternal InsP3R mRNA is sufficient for progression through the embryonic stages, but larval organs show asynchronous and defective cell divisions, and imaginal discs arrest early and fail to differentiate. We also generated adult mosaic animals and demonstrate that phototransduction, a model PLC pathway thought to require InsP3R, does not require InsP3R for signaling.
Assuntos
Canais de Cálcio/fisiologia , Drosophila melanogaster/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Visão Ocular/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Drosophila melanogaster/crescimento & desenvolvimento , Genes de Insetos , Receptores de Inositol 1,4,5-Trifosfato , Larva/citologia , Mutagênese , Retina/citologia , Deleção de Sequência , Fosfolipases Tipo C/fisiologiaRESUMO
SUMMARY: Drosophila phototransduction is a G protein-coupled, calcium-regulated signaling cascade that serves as a model system for the dissection of phospholipase C (PLC) signaling in vivo. The Drosophila light-activated conductance is constituted in part by the transient receptor potential (trp) ion channel, yet trp mutants still display a robust response demonstrating the presence of additional channels. The transient receptor potential-like (trpl) gene encodes a protein displaying 40% amino acid identity with TRP. Mammalian homologs of TRP and TRPL recently have been isolated and postulated to encode components of the elusive I(crac) conductance. We now show that TRP and TRPL localize to the membrane of the transducing organelle, together with rhodopsin and PLC, consistent with a role in PLC signaling during phototransduction. To determine the function of TRPL in vivo, we isolated trpl mutants and characterized them physiologically and genetically. We demonstrate that the light-activated conductance is composed of TRP and TRPL ion channels and that each can be activated on its own. We also use genetic and electrophysiological tools to study the contribution of each channel type to the light response and show that TRP and TRPL can serve partially overlapping functions.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Hormônios de Inseto/metabolismo , Proteínas de Insetos , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio/genética , Canais de Cálcio/efeitos da radiação , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/efeitos da radiação , Drosophila/genética , Drosophila/efeitos da radiação , Feminino , Hormônios de Inseto/genética , Hormônios de Inseto/efeitos da radiação , Luz , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/efeitos da radiação , Dados de Sequência Molecular , Mutação , Fotobiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Transdução de Sinais , Canais de Potencial de Receptor TransitórioRESUMO
Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Neuritos/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Linhagem Celular , Tamanho Celular , Ceramidas/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Humanos , Lisofosfolipídeos/farmacologia , Neuritos/metabolismo , Neuritos/ultraestrutura , Receptores de Superfície Celular/fisiologia , Receptores de Lisofosfolipídeos , Transdução de Sinais/fisiologia , Esfingosina/farmacologia , Suramina/farmacologia , Proteínas rho de Ligação ao GTPRESUMO
Serum stimulation of quiescent fibroblasts leads to a dramatic depolarization of the plasma membrane; however, the identity of the active serum factor(s) and the underlying mechanism are unknown. We find that this serum activity is attributable to albumin-bound lysophosphatidic acid (LPA) acting on its own G protein-coupled receptor, and that membrane depolarization is due to activation of an anion conductance mediating Cl- efflux. This depolarizing Cl- current can also be activated by thrombin and neuropeptide receptors; it is distinct from volume-regulated Cl- currents. Activation of the Cl- current consistently follows stimulation of phospholipase C and coincides with remodelling of the actin cytoskeleton, which is regulated by the Ras-related GTPase Rho. However, the response is not due to Ca2+/protein kinase C signalling and requires neither Rho nor Ras activation. The results indicate that in quiescent fibroblasts, LPA and other G protein-coupled receptor agonists evoke membrane depolarization by activating a new type of Cl- channel through a signalling pathway that is closely associated with phosphoinositide hydrolysis, yet independent of known second messengers.
Assuntos
Canais de Cloreto/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Albumina Sérica/fisiologia , Animais , Tamanho Celular , Células Cultivadas , Cloretos/fisiologia , Fibroblastos , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Ativação do Canal Iônico , Potenciais da Membrana , Ratos , Receptores de Ácidos Lisofosfatídicos , Fosfolipases Tipo C/fisiologia , Proteínas rho de Ligação ao GTPRESUMO
Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells, which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered, the rank order of potency is oleoyl-LPA > arachidonoyl-LPA > linolenoyl-LPA > linoleoyl-LPA > stearoyl-LPA = palmitoyl-LPA > myristoyl-LPA. The shorter-chain species, lauroyl- and decanoyl-LPA, show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses > 15 microM. Similarly, the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate, a putative messenger with some structural similarities to LPA, elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however, cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that, although many features of the LPA structure are important for optimal activity, the phosphate group is most critical, suggesting that this moiety is directly involved in receptor activation.