Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 39
Filtrar
1.
PLoS Biol ; 21(1): e3001949, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36693044

RESUMO

The state of open science needs to be monitored to track changes over time and identify areas to create interventions to drive improvements. In order to monitor open science practices, they first need to be well defined and operationalized. To reach consensus on what open science practices to monitor at biomedical research institutions, we conducted a modified 3-round Delphi study. Participants were research administrators, researchers, specialists in dedicated open science roles, and librarians. In rounds 1 and 2, participants completed an online survey evaluating a set of potential open science practices, and for round 3, we hosted two half-day virtual meetings to discuss and vote on items that had not reached consensus. Ultimately, participants reached consensus on 19 open science practices. This core set of open science practices will form the foundation for institutional dashboards and may also be of value for the development of policy, education, and interventions.


Assuntos
Pesquisa Biomédica , Humanos , Consenso , Técnica Delphi , Inquéritos e Questionários , Projetos de Pesquisa
2.
Environ Sci Pollut Res Int ; 28(4): 4857-4878, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32949360

RESUMO

The Pietra del Pertusillo freshwater reservoir is a major artificial lake of environmental, biological, and ecological importance located in the Basilicata region, southern Italy. The reservoir arch-gravity dam was completed in 1963 for producing hydroelectric energy and providing water for human use, and nearby there are potential sources of anthropogenic pollution such as urban and industrial activities. For the first time, the minero-chemistry of the lake and fluvio-lacustrine sediments of the reservoir have been evaluated to assess the environmental quality. Moreover, the composition of fluvial sediments derived from the peri-lacual zone of the reservoir and of local outcropping bedrock were also studied to understand the factors affecting the behavior of elements in the freshwater reservoir, with particular attention paid to heavy metals. In Italy, specific regulatory values concerning the element threshold concentration for lake and river sediments do not exist, and for this reason, soil threshold values are considered the standard for sediments of internal waters. The evaluation of the environmental quality of reservoir sediments has been performed using enrichment factors obtained with respect to the average composition of a reconstructed local upper continental crust. We suggest this method as an innovative standard in similar conditions worldwide. In the studied reservoir sediments, the trace elements that may be of some environmental concern are Cr, Cu, Zn, As, and Pb although, at this stage, the distribution of these elements appears to be mostly driven by geogenic processes. However, within the frame of the assessment and the preservation of the quality of aquatic environments, particular attention has to be paid to As (which shows median value of 10 ppm, reaching a maximum value of 26 ppm in Quaternary sediments), constantly enriched in the lacustrine samples and especially in the fine-grained fraction (median = 8.5 ppm).


Assuntos
Metais Pesados , Poluentes Químicos da Água , China , Monitoramento Ambiental , Sedimentos Geológicos , Humanos , Itália , Metais Pesados/análise , Medição de Risco , Rios , Poluentes Químicos da Água/análise
4.
Oncotarget ; 6(5): 3375-93, 2015 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-25682866

RESUMO

We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth.


Assuntos
Matriz Extracelular/metabolismo , Complexo de Golgi/metabolismo , Neoplasias/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cortactina/metabolismo , Ativação Enzimática , Humanos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Podossomos/metabolismo , Interferência de RNA , Receptores de Peptídeos/genética , Transfecção , Proteínas de Transporte Vesicular/genética , Quinases da Família src/metabolismo
6.
J Cell Sci ; 127(Pt 5): 977-93, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24413173

RESUMO

Previous studies have demonstrated that membrane tubule-mediated transport events in biosynthetic and endocytic routes require phospholipase A2 (PLA2) activity. Here, we show that cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) is targeted to the membrane compartments of the clathrin-independent endocytic route through a C-terminal stretch of positively charged amino acids, which allows the enzyme to interact with phosphoinositide lipids [especially PI(4,5)P2] that are enriched in clathrin-independent endosomes. Ablation of cPLA2ε suppressed the formation of tubular elements that carry internalized clathrin-independent cargoes, such as MHC-I, CD147 and CD55, back to the cell surface and, therefore, caused their intracellular retention. The ability of cPLA2ε to support recycling through tubule formation relies on the catalytic activity of the enzyme, because the inactive cPLA2ε(S420A) mutant was not able to recover either tubule growth or transport from clathrin-independent endosomes. Taken together, our findings indicate that cPLA2ε is a new important regulator of trafficking processes within the clathrin-independent endocytic and recycling route. The affinity of cPLA2ε for this pathway supports a new hypothesis that different PLA2 enzymes use selective targeting mechanisms to regulate tubule formation locally during specific trafficking steps in the secretory and/or endocytic systems.


Assuntos
Clatrina/metabolismo , Endocitose , Fosfolipases A2 do Grupo IV/fisiologia , Sequência de Aminoácidos , Sinalização do Cálcio , Endossomos/metabolismo , Fosfolipases A2 do Grupo IV/química , Células HeLa , Humanos , Hidrólise , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico
7.
Biol Direct ; 8: 8, 2013 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-23557484

RESUMO

UNLABELLED: Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants - two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA. REVIEWER: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia.


Assuntos
Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Actinas/metabolismo , Animais , Humanos , Tubo Polínico/citologia , Tubo Polínico/metabolismo
8.
Eur J Cell Biol ; 92(3): 113-21, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23306026

RESUMO

Transformed cancer cells have an altered metabolism, characterized by a shift towards aerobic glycolysis, referred to as 'the Warburg phenotype'. A change in flux through mitochondrial OXPHOS and cytosolic pathways for ATP production and a gain of capacity for biomass production in order to sustain the needs for altered growth and morphodynamics are typically involved in this global rewiring of cancer cell metabolism. Characteristically, these changes in metabolism are accompanied by enhanced uptake of nutrients like glucose and glutamine. Here we focus on the relationship between cell metabolism and cell dynamics, in particular the formation and function of invadopodia, specialized structures for focal degradation of the extracellular matrix. Since we recently found presence of enzymes that are active in glycolysis and associated pathways in invadopodia, we hypothesize that metabolic adaptation and invadopodia formation are linked processes. We give an overview on the background for this idea and show for the first time that extracellular matrix degradation by invadopodia can be differentially manipulated, without effects on cell proliferation, by use of metabolic inhibitors or changes in nutrient composition of cell culture media. We conclude that cell metabolism and carbohydrate availability, especially pyruvate, are involved in fuelling of invadopodia formation and activity.


Assuntos
Matriz Extracelular/metabolismo , Neoplasias/metabolismo , Pseudópodes/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Fosforilação Oxidativa , Transdução de Sinais , Células Tumorais Cultivadas
9.
Cell Mol Life Sci ; 70(12): 2175-90, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23307072

RESUMO

Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.


Assuntos
Movimento Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioma/fisiopatologia , NAD/metabolismo , Invasividade Neoplásica/fisiopatologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Northern Blotting , Western Blotting , Glioma/metabolismo , Humanos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/farmacologia , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Imagem com Lapso de Tempo , Células Tumorais Cultivadas
10.
Environ Toxicol ; 28(4): 201-6, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21544921

RESUMO

Trifluralin, a herbicide used to protect many arable and horticultural crops, was evaluated for its potential toxicity on the mammalian ovary. To this end, adult female mice were fed or not (control) with a trifluralin-enriched diet (150 mg/kg body weight/day) during gestation and lactation. After weaning, 3-week-old female mice from either trifluralin-treated or control groups were used to evaluate whether the exposure to this herbicide in utero and during lactation could induce stress responses in the ovary. It was found that trifluralin exposure caused a significantly higher level of p53, but not of pRb, in the whole ovary, and in particular in granulosa cells. TUNEL staining showed that herbicide treatment did not increase the apoptotic index of the somatic compartment. Also oocyte fertilizability was unaffected, as metaphase II oocytes retrieved from treated mice were capable of forming male and female pronuclei after in vitro fertilization as control mice. However, trifluralin determined a slightly higher number of oocytes with cytoplasmic degeneration compared with control animals. In conclusion, our results suggest that exposure to a low trifluralin dose during pregnancy and lactation does not impair oocyte quality, but can induce a stress response in ovarian somatic cells.


Assuntos
Herbicidas/toxicidade , Ovário/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Trifluralina/toxicidade , Adulto , Animais , Apoptose/efeitos dos fármacos , Dieta , Feminino , Fertilização/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Técnicas In Vitro , Lactação , Masculino , Exposição Materna/efeitos adversos , Metáfase , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Ovário/citologia , Ovário/metabolismo , Gravidez , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo
11.
J Cell Sci ; 125(Pt 14): 3265-70, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22854039

RESUMO

Disabling mutations in the FGD1 gene cause faciogenital dysplasia (also known as Aarskog-Scott syndrome), a human X-linked developmental disorder that results in disproportionately short stature, facial, skeletal and urogenital anomalies, and in a number of cases, mild mental retardation. FGD1 encodes the guanine nucleotide exchange factor FGD1, which is specific for the Rho GTPase cell division cycle 42 (CDC42). CDC42 controls cytoskeleton-dependent membrane rearrangements, transcriptional activation, secretory membrane trafficking, G1 transition during the cell cycle and tumorigenic transformation. The cellular mechanisms by which FGD1 mutations lead to the hallmark skeletal deformations of faciogenital dysplasia remain unclear, but the pathology of the disease, as well as some recent discoveries, clearly show that the protein is involved in the regulation of bone development. Two recent studies unveiled new potential functions of FGD1, in particular, its involvement in the regulation of the formation and function of invadopodia and podosomes, which are cellular structures devoted to degradation of the extracellular matrix in tumour and endothelial cells. Here, we discuss the hypothesis that FGD1 might be an important regulator of events controlling extracellular matrix remodelling and possibly cell invasion in physiological and pathological settings. Additionally, we focus on how studying the cell biology of FGD1 might help us to connect the dots that link CDC42 signalling with remodelling of the extracellular matrix (ECM) in physiology and complex diseases, while, at the same time, furthering our understanding of the pathogenesis of faciogenital dysplasia.


Assuntos
Nanismo/genética , Matriz Extracelular/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Deformidades Congênitas da Mão/genética , Cardiopatias Congênitas/genética , Nanismo/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Face/anormalidades , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Genitália Masculina/anormalidades , Genitália Masculina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Deformidades Congênitas da Mão/metabolismo , Cardiopatias Congênitas/metabolismo , Humanos , Proteína cdc42 de Ligação ao GTP/metabolismo
12.
PLoS One ; 7(6): e37352, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22701565

RESUMO

BACKGROUND: Protein mono-ADP-ribosylation is a reversible post-translational modification that modulates the function of target proteins. The enzymes that catalyze this reaction in mammalian cells are either bacterial pathogenic toxins or endogenous cellular ADP-ribosyltransferases. The latter include members of three different families of proteins: the well characterized arginine-specific ecto-enzymes ARTCs, two sirtuins and, more recently, novel members of the poly(ADP-ribose) polymerase (PARP/ARTD) family that have been suggested to act as cellular mono-ADP-ribosyltransferases. Here, we report on the characterisation of human ARTD15, the only known ARTD family member with a putative C-terminal transmembrane domain. METHODOLOGY/PRINCIPAL FINDINGS: Immunofluorescence and electron microscopy were performed to characterise the sub-cellular localisation of ARTD15, which was found to be associated with membranes of the nuclear envelope and endoplasmic reticulum. The orientation of ARTD15 was determined using protease protection assay, and is shown to be a tail-anchored protein with a cytosolic catalytic domain. Importantly, by combining immunoprecipitation with mass spectrometry and using cell lysates from cells over-expressing FLAG-ARTD15, we have identified karyopherin-ß1, a component of the nuclear trafficking machinery, as a molecular partner of ARTD15. Finally, we demonstrate that ARTD15 is a mono-ADP-ribosyltransferase able to induce the ADP-ribosylation of karyopherin-ß1, thus defining the first substrate for this enzyme. CONCLUSIONS/SIGNIFICANCE: Our data reveal that ARTD15 is a novel ADP-ribosyltransferase enzyme with a new intracellular location. Finally, the identification of karyopherin-ß1 as a target of ARTD15-mediated ADP-ribosylation, hints at a novel regulatory mechanism of karyopherin-ß1 functions.


Assuntos
ADP Ribose Transferases/metabolismo , Retículo Endoplasmático/enzimologia , Membrana Nuclear/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , beta Carioferinas/metabolismo , Retículo Endoplasmático/ultraestrutura , Imunofluorescência , Humanos , Espectrometria de Massas , Microscopia Eletrônica , Membrana Nuclear/ultraestrutura , Poli(ADP-Ribose) Polimerases/genética , Estrutura Terciária de Proteína
13.
Eur J Cell Biol ; 91(11-12): 969-77, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22699001

RESUMO

The aim of this study was to analyze the potential role of NG2 chondroitin sulfate proteoglycan in amoeboid morphology and invasiveness of cancer cells. In the highly metastatic amoeboid cell lines A3 and A375M2, siRNA-mediated down-regulation of NG2 induced an amoeboid-mesenchymal transition associated with decreased invasiveness in 3D collagen and inactivation of the GTPase Rho. Conversely, the expression of NG2 in mesenchymal sarcoma K2 cells as well as in A375M2 cells resulted in an enhanced amoeboid phenotype associated with increased invasiveness and elevated Rho-GTP levels. Remarkably, the amoeboid-mesenchymal transition in A375M2 cells triggered by NG2 down-regulation was associated with increased extracellular matrix-degrading ability, although this was not sufficient to compensate for the decreased invasive capability caused by down-regulated Rho/ROCK signaling. Conversely, in K2 cells with overexpression of NG2, the ability to degrade the extracellular matrix was greatly reduced. Taken together, we suggest that NG2-mediated activation of Rho leading to effective amoeboid invasiveness is a possible mechanism through which NG2 could contribute to tumor cell invasion and metastasis.


Assuntos
Movimento Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/química , Regulação para Baixo , Matriz Extracelular/metabolismo , Humanos , Proteínas de Membrana/genética , Conformação Molecular , Invasividade Neoplásica , Neoplasias/patologia , RNA Interferente Pequeno , Ratos , Regulação para Cima , Quinases Associadas a rho/metabolismo
14.
J Cell Sci ; 125(Pt 16): 3858-69, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22595522

RESUMO

Filamins are an important family of actin-binding proteins that, in addition to bundling actin filaments, link cell surface adhesion proteins, signaling receptors and channels to the actin cytoskeleton, and serve as scaffolds for an array of intracellular signaling proteins. Filamins are known to regulate the actin cytoskeleton, act as mechanosensors that modulate tissue responses to matrix density, control cell motility and inhibit activation of integrin adhesion receptors. In this study, we extend the repertoire of filamin activities to include control of extracellular matrix (ECM) degradation. We show that knockdown of filamin increases matrix metalloproteinase (MMP) activity and induces MMP2 activation, enhancing the ability of cells to remodel the ECM and increasing their invasive potential, without significantly altering two-dimensional random cell migration. We further show that within filamin A, the actin-binding domain is necessary, but not sufficient, to suppress the ECM degradation seen in filamin-A-knockdown cells and that dimerization and integrin binding are not required. Filamin mutations are associated with neuronal migration disorders and a range of congenital malformations characterized by skeletal dysplasia and various combinations of cardiac, craniofacial and intestinal anomalies. Furthermore, in breast cancers loss of filamin A has been correlated with increased metastatic potential. Our data suggest that effects on ECM remodeling and cell invasion should be considered when attempting to provide cellular explanations for the physiological and pathological effects of altered filamin expression or filamin mutations.


Assuntos
Proteínas Contráteis/metabolismo , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Metaloproteinase 2 da Matriz/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proteínas Contráteis/deficiência , Proteínas Contráteis/genética , Ativação Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrossarcoma/enzimologia , Fibrossarcoma/genética , Filaminas , Técnicas de Silenciamento de Genes , Humanos , Integrinas/metabolismo , Metaloproteinase 14 da Matriz , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica , Fenótipo , Estrutura Terciária de Proteína
15.
Eur J Cell Biol ; 91(11-12): 961-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22564726

RESUMO

Invadopodia are proteolytically active protrusions formed by invasive tumoral cells when grown on an extracellular matrix (ECM) substratum. A current challenge is to understand how proteolytic activity is so precisely localised at discrete sites of the plasma membrane to produce focalised ECM degradation at invadopodia. Indeed, a number of components including metalloproteases need to be directed to invadopodia to ensure proper segregation of proteolytic activities. We recently found invadopodia to feature the properties of cholesterol-rich membrane domains (a.k.a. lipid drafts) and that ECM degradation depends on the tight control of cholesterol homeostasis. Since apically directed polarised sorting and transport in epithelial cells relies on segregation of proteins into lipid rafts at the Golgi complex, we hypothesised that invadopodia-dependent ECM degradation might also rely on lipid raft-dependent polarised transport routes. To investigate this issue we undertook a three-pronged approach. First, we found that microtubule depolymerisation, which is known to disrupt polarised transport in polarised cells, strongly inhibited invadopodia formation, while not affecting overall protein transport. In the second approach we found that glycosylphosphatidylinositol-anchored green fluorescent protein (an apical model protein), but not vesicular stomatitis virus G-protein or influenza virus hemagglutinin (both model basolateral model cargoes), was transported to sites of ECM degradation. Finally, RNAi-mediated knock-down of proteins known to specifically regulate polarised apical or basolateral transport in epithelial cells, such as caveolin 1 and annexin XIIIB or clathrin, respectively, demonstrated that the selective inhibition of the apical, but not the basolateral, transport route impairs invadopodia formation and ECM degradation. Taken together, our findings suggest that invadopodia are apical-like membrane domains, where signal transduction and local membrane remodelling events might be temporally and spatially confined via selective raft-dependent apical transport routes.


Assuntos
Estruturas da Membrana Celular/ultraestrutura , Matriz Extracelular/metabolismo , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Anexinas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Estruturas da Membrana Celular/metabolismo , Clatrina/genética , Clatrina/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Microtúbulos/metabolismo , Transporte Proteico , RNA Interferente Pequeno , Transdução de Sinais , Proteínas do Envelope Viral/metabolismo
16.
Toxicol Appl Pharmacol ; 260(2): 155-61, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22369882

RESUMO

The ethylene-bis-dithiocarbamate mancozeb is a widely used fungicide with low reported toxicity in mammals. In mice, mancozeb induces embryo apoptosis, affects oocyte meiotic spindle morphology and impairs fertilization rate even when used at very low concentrations. We evaluated the toxic effects of mancozeb on the mouse and human ovarian somatic granulosa cells. We examined parameters such as cell morphology, induction of apoptosis, and p53 expression levels. Mouse granulosa cells exposed to mancozeb underwent a time- and dose-dependent modification of their morphology, and acquired the ability to migrate but not to proliferate. The expression level of p53, in terms of mRNA and protein content, decreased significantly in comparison with unexposed cells, but no change in apoptosis was recorded. Toxic effects could be attributed, at least in part, to the presence of ethylenthiourea (ETU), the main mancozeb catabolite, which was found in culture medium. Human granulosa cells also showed dose-dependent morphological changes and reduced p53 expression levels after exposure to mancozeb. Altogether, these results indicate that mancozeb affects the somatic cells of the mammalian ovarian follicles by inducing a premalignant-like status, and that such damage occurs to the same extent in both mouse and human GC. These results further substantiate the concept that mancozeb should be regarded as a reproductive toxicant.


Assuntos
Apoptose/fisiologia , Fungicidas Industriais/toxicidade , Células da Granulosa/efeitos dos fármacos , Maneb/toxicidade , Folículo Ovariano/efeitos dos fármacos , Zineb/toxicidade , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Células da Granulosa/ultraestrutura , Humanos , Camundongos , Microscopia Confocal , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteína Supressora de Tumor p53/metabolismo
17.
Mol Cell Biol ; 31(22): 4430-41, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21911474

RESUMO

Podosomes are dynamic actin-rich adhesion plasma membrane microdomains endowed with extracellular matrix-degrading activities. In aortic endothelial cells, podosomes are induced by transforming growth factor ß (TGF-ß), but how this occurs is largely unknown. It is thought that, in endothelial cells, podosomes play a role in vessel remodeling and/or in breaching anatomical barriers. We demonstrate here that, in bovine aortic endothelial cells, that the Cdc42-specific guanine exchange factor (GEF) Fgd1 is expressed and regulated by TGF-ß to induce Cdc42-dependent podosome assembly. Within 15 min of TGF-ß stimulation, Fgd1, but none of the other tested Cdc42 GEFs, undergoes tyrosine phosphorylation, associates with Cdc42, and translocates to the subcortical cytoskeleton via a cortactin-dependent mechanism. Small interfering RNA-mediated Fgd1 knockdown inhibits TGF-ß-induced Cdc42 activation. Fgd1 depletion also reduces podosome formation and associated matrix degradation and these defects are rescued by reexpression of Fgd1. Although overexpression of Fgd1 does not promote podosome formation per se, it enhances TGF-ß-induced matrix degradation. Our results identify Fgd1 as a TGF-ß-regulated GEF and, as such, the first GEF to be involved in the process of cytokine-induced podosome formation. Our findings reveal the involvement of Fgd1 in endothelial cell biology and open up new avenues to study its role in vascular pathophysiology.


Assuntos
Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Vasos Sanguíneos/metabolismo , Bovinos , Cortactina/metabolismo , Nanismo , Matriz Extracelular/ultraestrutura , Face/anormalidades , Doenças Genéticas Ligadas ao Cromossomo X , Genitália Masculina/anormalidades , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Deformidades Congênitas da Mão , Cardiopatias Congênitas , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/biossíntese , Proteína cdc42 de Ligação ao GTP/genética
18.
Eur J Cell Biol ; 90(2-3): 115-27, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-20609496

RESUMO

When highly invasive cancer cells are cultured on an extracellular matrix substrate, they extend proteolytically active membrane protrusions, termed invadopodia, from their ventral surface into the underlying matrix. Our understanding of the molecular composition of invadopodia has rapidly advanced in the last few years, but is far from complete. To accelerate component discovery, we resorted to a proteomics approach by applying DIfference Gel Electrophoresis (DIGE) to compare invadopodia-enriched sub-cellular fractions with cytosol and cell body membrane fractions and the whole cell lysate. The fractionation procedure was validated through step-by-step monitoring of the enrichment in typical actin-related invadopodia-associated proteins. After statistical analysis, 129 protein spots were selected for peptide mass fingerprinting analysis; of these 76 were successfully identified and found to correspond to 58 proteins belonging to different functional classes including aerobic glycolysis and other metabolic pathways, protein synthesis, degradation and folding, cytoskeletal components and membrane-associated proteins. Finally, validation of a number of identified proteins was carried out by a combination of immuno-blotting on cell fractions and immunofluorescence localization at invadopodia. These results reveal newly identified components of invadopodia and open further avenues to the molecular study of invasive growth behavior of cancer cells.


Assuntos
Extensões da Superfície Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Fracionamento Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Imunofluorescência/métodos , Humanos , Immunoblotting/métodos , Proteômica/métodos , Reprodutibilidade dos Testes
19.
Trends Cell Biol ; 20(2): 64-70, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19931459

RESUMO

Recent years have witnessed growing interest in the biology of invadopodia, proteolytically active protrusions formed by invasive tumor cells when cultured on an extracellular matrix (ECM). Although substantial progress has been made towards defining their basic elements and features, the need remains to understand how these components are recruited and, ultimately, how ECM degradation is so precisely localized. According to recent evidence, invadopodia are raft-like membrane domains where cholesterol levels are tightly regulated, and active transport of protease-delivering carriers is required for their function. On this basis we hypothesize that the correct delivery of cargo to invadopodia is ensured by a polarized, cholesterol-dependent trafficking mechanism, similar to that of the apical domain of epithelial cells.


Assuntos
Polaridade Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Matriz Extracelular/metabolismo , Invasividade Neoplásica/fisiopatologia , Animais , Movimento Celular/fisiologia , Extensões da Superfície Celular/ultraestrutura , Colesterol/metabolismo , Endocitose/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Células Tumorais Cultivadas
20.
Mol Biol Cell ; 20(19): 4140-52, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19641024

RESUMO

Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.


Assuntos
Junções Aderentes/efeitos dos fármacos , Caderinas/metabolismo , Cavéolas/metabolismo , Caveolina 1/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Junções Aderentes/metabolismo , Animais , Western Blotting , Caderinas/genética , Cavéolas/ultraestrutura , Caveolina 1/genética , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica , Mutação , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA