RESUMO
Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.
Assuntos
Cromatina/metabolismo , Lâmina Nuclear/metabolismo , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Cromatina/química , Cromossomos/química , Cromossomos/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Hibridização in Situ Fluorescente , InterfaseRESUMO
GDE2 (also known as GDPD5) is a multispanning membrane phosphodiesterase with phospholipase D-like activity that cleaves select glycosylphosphatidylinositol (GPI)-anchored proteins and thereby promotes neuronal differentiation both in vitro and in vivo GDE2 is a prognostic marker in neuroblastoma, while loss of GDE2 leads to progressive neurodegeneration in mice; however, its regulation remains unclear. Here, we report that, in immature neuronal cells, GDE2 undergoes constitutive endocytosis and travels back along both fast and slow recycling routes. GDE2 trafficking is directed by C-terminal tail sequences that determine the ability of GDE2 to cleave GPI-anchored glypican-6 (GPC6) and induce a neuronal differentiation program. Specifically, we define a GDE2 truncation mutant that shows aberrant recycling and is dysfunctional, whereas a consecutive deletion results in cell-surface retention and gain of GDE2 function, thus uncovering distinctive regulatory sequences. Moreover, we identify a C-terminal leucine residue in a unique motif that is essential for GDE2 internalization. These findings establish a mechanistic link between GDE2 neuronal function and sequence-dependent trafficking, a crucial process gone awry in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.
Assuntos
Neuroblastoma , Fosfolipases , Animais , Diferenciação Celular/genética , Glicosilfosfatidilinositóis/genética , Camundongos , Diester Fosfórico Hidrolases/genéticaRESUMO
Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas Mad2/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Reparo de DNA por Recombinação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/metabolismo , Humanos , Switching de Imunoglobulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mad2/deficiência , Proteínas Mad2/genética , Camundongos , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with ß1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.
Assuntos
Proteínas de Transporte/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Profilinas/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Canais de Cloreto/química , Sequência Conservada , Cristalografia por Raios X , Ativação Enzimática , Forminas , Células HeLa , Humanos , Integrinas/metabolismo , Modelos Moleculares , Profilinas/química , Ligação Proteica , Conformação Proteica , Transporte ProteicoRESUMO
Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS), characterized by inflammation-mediated demyelination, axonal injury and neurodegeneration. The mechanisms underlying impaired neuronal function are not fully understood, but evidence is accumulating that the presence of the gliotic scar produced by reactive astrocytes play a critical role in these detrimental processes. Here, we identified astrocytic Transient Receptor Potential cation channel, subfamily M, member 7 (TRPM7), a Ca2+ -permeable nonselective cation channel, as a novel player in the formation of a gliotic scar. TRPM7 was found to be highly expressed in reactive astrocytes within well-characterized MS lesions and upregulated in primary astrocytes under chronic inflammatory conditions. TRPM7 overexpressing astrocytes impaired neuronal outgrowth in vitro by increasing the production of chondroitin sulfate proteoglycans, a key component of the gliotic scar. These findings indicate that astrocytic TRPM7 is a critical regulator of the formation of a gliotic scar and provide a novel mechanism by which reactive astrocytes affect neuronal outgrowth.
Assuntos
Astrócitos/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Esclerose Múltipla/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Canais de Cátion TRPM/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/patologia , Neurônios/patologia , Proteínas Serina-Treonina Quinases/genética , Ratos , Canais de Cátion TRPM/genéticaRESUMO
We developed single-image fluorescence lifetime imaging microscopy (siFLIM), a method for acquiring quantitative lifetime images from a single exposure. siFLIM takes advantage of a new generation of dedicated cameras that simultaneously record two 180°-phase-shifted images, and it allows for video-rate lifetime imaging with minimal phototoxicity and bleaching. siFLIM is also inherently immune to artifacts stemming from rapid cellular movements and signal transients.
Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Imagem com Lapso de Tempo/métodos , Cálcio/metabolismo , Calibragem , Corantes Fluorescentes/química , Células HeLa , Histamina/farmacologia , Humanos , FótonsRESUMO
PURPOSE: To determine the correlation of preoperative fascia thickness (FT) and intraoperative fascia preservation (FP) with erectile function (EF) after nerve-sparing robot-assisted radical prostatectomy (RARP). METHODS: Our analysis included 106 patients, with localized prostate cancer and no erectile dysfunction (ED) before RARP, assessed with preoperative 3 Tesla (3 T) multiparametric magnetic resonance imaging (MRI). FP score was defined as the extent of FP from the base to the apex of the prostate, quantitatively assessed by the surgeon. Median fascia thickness (MFT) per patient was defined as the sum of the median FT of 12 MRI regions. Preserved MFT (pMFT) was the sum of the saved MFT. The percentage of pFMT (ppMFT) was also calculated. Fascia surface (FS) was measured on MRI and it was combined with FP score resulting in preserved FS (pFS) and percentage of pFS (ppFS). RESULTS: FP score, pMFT, ppMFT, pFS and ppFS were significantly lower (p < 0.0001) in patients with ED. In the multivariate regression analysis, lower FP score [odds ratio (OR) 0.721, p = 0.03] and lower ppMFT (OR 0.001, p = 0.027) were independent predictors of ED. ROC analysis showed the highest area under the curve for ppMFT (0.787) and FP score (0.767) followed by pMFT (0.755) and ppFS (0.743). CONCLUSIONS: MRI-determined periprostatic FT combined with intraoperative FP score are correlated to postprostatectomy EF. Based on the hypothesis that a thicker fascia forms a protective layer for the nerves, we recommend assessing FT preoperatively to counsel men for the odds of preserving EF after RARP.
Assuntos
Disfunção Erétil/epidemiologia , Fáscia/diagnóstico por imagem , Nervos Periféricos , Complicações Pós-Operatórias/epidemiologia , Prostatectomia/métodos , Neoplasias da Próstata/cirurgia , Procedimentos Cirúrgicos Robóticos/métodos , Área Sob a Curva , Fáscia/anatomia & histologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Razão de Chances , Tamanho do Órgão , Tratamentos com Preservação do Órgão , Prognóstico , Neoplasias da Próstata/patologia , Curva ROCRESUMO
Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo. Here, we show that TRPM7 contributes to maintaining a mesenchymal phenotype in breast cancer cells by tensional regulation of the EMT transcription factor SOX4. The functional consequences of SOX4 knockdown closely mirror those produced by TRPM7 knockdown. By traction force measurements, we demonstrate that TRPM7 reduces cytoskeletal tension through inhibition of myosin II activity. Moreover, we show that SOX4 expression and downstream mesenchymal markers are inversely regulated by cytoskeletal tension and matrix rigidity. Overall, our results identify SOX4 as a transcription factor that is uniquely sensitive to cellular tension and indicate that TRPM7 may contribute to breast cancer progression by tensional regulation of SOX4.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição SOXC/metabolismo , Canais de Cátion TRPM/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição SOXC/genética , Canais de Cátion TRPM/genética , Resistência à TraçãoRESUMO
Cyclic AMP is a ubiquitous second messenger that orchestrates a variety of cellular functions over different timescales. The mechanisms underlying specificity within this signaling pathway are still not well understood. Several lines of evidence suggest the existence of spatial cAMP gradients within cells, and that compartmentalization underlies specificity within the cAMP signaling pathway. However, to date, no studies have visualized cAMP gradients in three spatial dimensions (3D: x, y, z).This is in part due to the limitations of FRET-based cAMP sensors, specifically the low signal-to-noise ratio intrinsic to all intracellular FRET probes. Here, we overcome this limitation, at least in part, by implementing spectral imaging approaches to estimate FRET efficiency when multiple fluorescent labels are used and when signals are measured from weakly expressed fluorescent proteins in the presence of background autofluorescence and stray light. Analysis of spectral image stacks in two spatial dimensions (2D) from single confocal slices indicates little or no cAMP gradients formed within pulmonary microvascular endothelial cells (PMVECs) under baseline conditions or following 10 min treatment with the adenylyl cyclase activator forskolin. However, analysis of spectral image stacks in 3D demonstrates marked cAMP gradients from the apical to basolateral face of PMVECs. Results demonstrate that spectral imaging approaches can be used to assess cAMP gradients-and in general gradients in fluorescence and FRET-within intact cells. Results also demonstrate that 2D imaging studies of localized fluorescence signals and, in particular, cAMP signals, whether using epifluorescence or confocal microscopy, may lead to erroneous conclusions about the existence and/or magnitude of gradients in either FRET or the underlying cAMP signals. Thus, with the exception of cellular structures that can be considered in one spatial dimension, such as neuronal processes, 3D measurements are required to assess mechanisms underlying compartmentalization and specificity within intracellular signaling pathways.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Masculino , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Razão Sinal-RuídoRESUMO
Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.
Assuntos
Endotelinas/metabolismo , Lisofosfolipídeos/metabolismo , Melanoma/metabolismo , Podossomos/metabolismo , Receptores de Ácidos Lisofosfatídicos/agonistas , Proteína cdc42 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Melanoma/enzimologia , Melanoma/patologia , Microscopia Confocal , Microscopia de Fluorescência , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Podossomos/enzimologia , Podossomos/patologia , Interferência de RNA , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem com Lapso de Tempo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/agonistas , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Transient receptor potential (TRP) channels comprise a family of cation channels implicated in a variety of cellular processes, including proliferation, cell migration and cell survival. As a consequence, members of this ion family play prominent roles during embryonic development, tissue maintenance and cancer progression. Although most TRP channels are non-selective, many cellular responses, mediated by TRP channels, appear to be calcium-dependent. In addition, there is mounting evidence for channel-independent roles for TRP channels. In this review, we will discuss how both these channel-dependent and -independent mechanisms affect cellular programs essential during embryonic development, and how perturbations in these pathways contribute to a variety of pathologies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.
Assuntos
Cálcio/metabolismo , Desenvolvimento Embrionário/fisiologia , Homeostase/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Citoesqueleto/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Modelos Biológicos , Família Multigênica , Especificidade de Órgãos , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles. We find that mDia1 is an epidermal growth factor (EGF)-regulated actin nucleator involved in membrane ruffling using a combination of knockdown and rescue experiments. At the molecular level, mDia1 polymerizes linear actin filaments, activating the Arp2/3 complex, and localizes within nascent and mature membrane ruffles. We employ functional complementation experiments and optogenetics to show that mDia1 cooperates with the Arp2/3 complex in initiating lamellipodia and ruffles. Finally, we show that genetic and pharmacological interference with this cooperation hampers ruffling and cell migration. Thus, we propose that the lamellipodium- and ruffle-initiating machinery consists of two actin nucleators that act sequentially to regulate membrane protrusion and cell migration.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Estruturas da Membrana Celular/metabolismo , Pseudópodes/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Estruturas da Membrana Celular/genética , Chlorocebus aethiops , Forminas , Células HeLa , Humanos , Pseudópodes/genéticaRESUMO
Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.
Assuntos
Autoantígenos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Colágenos não Fibrilares/metabolismo , Pele/metabolismo , Autoantígenos/genética , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/genética , Distonina , Hemidesmossomos/genética , Hemidesmossomos/ultraestrutura , Humanos , Integrina beta4/genética , Queratinócitos/ultraestrutura , Queratinas/genética , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Colágenos não Fibrilares/genética , Pele/ultraestrutura , Colágeno Tipo XVIIRESUMO
Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to ß1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of ß1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of ß1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking.
Assuntos
Canais de Cloreto/metabolismo , Integrina beta1/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Receptores ErbB/metabolismo , Adesões Focais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Lisofosfolipídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Soro , Transdução de Sinais/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Imagem Molecular/métodos , Carbocianinas/química , Linhagem Celular Tumoral , Técnicas Citológicas/métodos , Corantes Fluorescentes/metabolismo , Humanos , Irídio/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismoRESUMO
We demonstrated that increasing intracellular cAMP concentrations result in the inhibition of migration of PANC-1 and other pancreatic ductal adenocarcinoma (PDAC) cell types. The rise of cAMP was accompanied by rapid and reversible cessation of ruffling, by inhibition of focal adhesion turnover and by prominent loss of paxillin from focal adhesions. All these phenomena develop rapidly suggesting that cAMP effectors have a direct influence on the cellular migratory apparatus. The role of two primary cAMP effectors, exchange protein activated by cAMP (EPAC) and protein kinase A (PKA), in cAMP-mediated inhibition of PDAC cell migration and migration-associated processes was investigated. Experiments with selective activators of EPAC and PKA demonstrated that the inhibitory effect of cAMP on migration, ruffling, focal adhesion dynamics and paxillin localisation is mediated by PKA, whilst EPAC potentiates migration.
Assuntos
Carcinoma Ductal Pancreático/patologia , Movimento Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Paxilina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/enzimologia , Linhagem Celular Tumoral , Extensões da Superfície Celular/efeitos dos fármacos , Colforsina/farmacologia , Adesões Focais/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologia , Transporte Proteico/efeitos dos fármacosRESUMO
The outcome of cell signaling depends not only on signal strength but also on temporal progression. We use Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to investigate intracellular signaling dynamics. We examined the ß1 receptor-Gαs-cAMP signaling axis using both widefield frequency domain FLIM (fdFLIM) and fast confocal time-correlated single photon counting (TCSPC) setups. Unexpectedly, we observed that fdFLIM revealed transient cAMP responses in HeLa and Cos7 cells, contrasting with sustained responses as detected with TCSPC. Investigation revealed no light-induced effects on cAMP generation or breakdown. Rather, folic acid present in the imaging medium appeared to be the culprit, as its excitation with blue light sensitized degradation of ß1 agonists. Our findings highlight the impact of subtle phototoxicity on experimental outcomes, advocating confocal TCSPC for reliable analysis of response kinetics and stressing the need for full disclosure of chemical formulations by scientific vendors.
RESUMO
Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/classificação , Microscopia de Fluorescência/métodos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Fluorescência Verde/análise , Dados de Sequência MolecularRESUMO
DNA interstrand crosslinks (ICLs) pose a major obstacle for DNA replication and transcription if left unrepaired. The cellular response to ICLs requires the coordination of various DNA repair mechanisms. Homologous recombination (HR) intermediates generated in response to ICLs, require efficient and timely conversion by structure-selective endonucleases. Our knowledge on the precise coordination of this process remains incomplete. Here, we designed complementary genetic screens to map the machinery involved in the response to ICLs and identified FIRRM/C1orf112 as an indispensable factor in maintaining genome stability. FIRRM deficiency leads to hypersensitivity to ICL-inducing compounds, accumulation of DNA damage during S-G2 phase of the cell cycle, and chromosomal aberrations, and elicits a unique mutational signature previously observed in HR-deficient tumors. In addition, FIRRM is recruited to ICLs, controls MUS81 chromatin loading, and thereby affects resolution of HR intermediates. FIRRM deficiency in mice causes early embryonic lethality and accelerates tumor formation. Thus, FIRRM plays a critical role in the response to ICLs encountered during DNA replication.
Assuntos
Dano ao DNA , Reparo do DNA , Animais , Camundongos , Replicação do DNA , Recombinação Homóloga , DNARESUMO
Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. Knockdown of phospholipase Cbeta3 (PLCbeta3) inhibits PtdIns(4,5)P(2) hydrolysis and keeps Cx43 channels open after receptor activation. Using a translocatable 5-phosphatase, we show that PtdIns(4,5)P(2) depletion is sufficient to close Cx43 channels. When PtdIns(4,5)P(2) is overproduced by PtdIns(4)P 5-kinase, Cx43 channel closure is impaired. We find that the Cx43 binding partner zona occludens 1 (ZO-1) interacts with PLCbeta3 via its third PDZ domain. ZO-1 is essential for PtdIns(4,5)P(2)-hydrolyzing receptors to inhibit cell-cell communication, but not for receptor-PLC coupling. Our results show that PtdIns(4,5)P(2) is a key regulator of Cx43 channel function, with no role for other second messengers, and suggest that ZO-1 assembles PLCbeta3 and Cx43 into a signaling complex to allow regulation of cell-cell communication by localized changes in PtdIns(4,5)P(2).