RESUMEN
Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is â¼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to â¼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero , Humanos , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Mensajero/ultraestructura , Microscopía por Crioelectrón , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Polimerasa II/ultraestructura , Transcripción Genética , Metiltransferasas/química , Metiltransferasas/metabolismo , Metiltransferasas/ultraestructura , Modelos QuímicosRESUMEN
RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.
Asunto(s)
ARN Polimerasa II , Factores de Transcripción , Animales , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , ARN Polimerasa III/genética , Transcripción Genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , ADNRESUMEN
The RNA-binding protein tristetraprolin (TTP) is a potent activator of mRNA decay, specifically for transcripts bearing AU-rich elements (AREs) in their 3'-untranslated regions. TTP functions as a mediator for mRNA decay by interacting with the decay machinery and recruiting it to the target ARE-mRNA. In this study, we report a weak, but direct interaction between TTP and the human decapping enzyme DCP2, which impacts the stability of ARE transcripts. The TTP-DCP2 interaction is unusual as it involves intrinsically disordered regions (IDRs) of both binding partners. We show that the IDR of DCP2 has a propensity for oligomerization and liquid-liquid phase separation in vitro. Binding of TTP to DCP2 leads to its partitioning into phase-separated droplets formed by DCP2, suggesting that molecular crowding might facilitate the weak interaction between the two proteins and enable assembly of a decapping-competent mRNA-protein complex on TTP-bound transcripts in cells. Our studies underline the role of weak interactions in the cellular interaction network and their contribution towards cellular functionality.
Asunto(s)
Endorribonucleasas/química , Estabilidad del ARN , Tristetraprolina/química , Regiones no Traducidas 3' , Endorribonucleasas/metabolismo , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMEN
Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Proteínas HSP90 de Choque Térmico , Enfermedad de Alzheimer/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas tau/metabolismoRESUMEN
The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3'-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD). This pathway also involves the RNA helicase UPF1, which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway. Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway. We demonstrate the involvement of UPF2, a core NMD factor and a known activator of UPF1, in SMD. UPF2 acts as an adaptor between Stau1 and UPF1, stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP. Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Transactivadores/metabolismo , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.
Asunto(s)
Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Intravital/métodos , Imagen de Lapso de Tiempo/métodos , Proteínas de Unión al GTP rho/genética , Animales , Antineoplásicos/farmacología , Huesos/citología , Huesos/metabolismo , Movimiento Celular/efectos de los fármacos , Dasatinib/farmacología , Clorhidrato de Erlotinib/farmacología , Femenino , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Regulación de la Expresión Génica , Intestino Delgado/metabolismo , Intestino Delgado/ultraestructura , Microscopía Intravital/instrumentación , Glándulas Mamarias Animales/irrigación sanguínea , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/ultraestructura , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/ultraestructura , Mecanotransducción Celular , Ratones , Ratones Transgénicos , Neutrófilos/metabolismo , Neutrófilos/ultraestructura , Osteocitos/metabolismo , Osteocitos/ultraestructura , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestructura , Imagen de Lapso de Tiempo/instrumentación , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Sleep is a behavior that is found in all animals that have a nervous system and that have been studied carefully. In Caenorhabditis elegans larvae, sleep is associated with a turning behavior, called flipping, in which animals rotate 180° about their longitudinal axis. However, the molecular and neural substrates of this enigmatic behavior are not known. Here, we identified the conserved NK-2 homeobox gene ceh-24 to be crucially required for flipping. ceh-24 is required for the formation of processes and for cholinergic function of sublateral motor neurons, which separately innervate the four body muscle quadrants. Knockdown of cholinergic function in a subset of these sublateral neurons, the SIAs, abolishes flipping. The SIAs depolarize during flipping and their optogenetic activation induces flipping in a fraction of events. Thus, we identified the sublateral SIA neurons to control the three-dimensional movements of flipping. These neurons may also control other types of motion.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Homeodominio/metabolismo , Actividad Motora , Neuronas Motoras/fisiología , Sueño , Animales , Larva/fisiologíaRESUMEN
Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many non-histone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1A methylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B-mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosomal profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.
Asunto(s)
Lisina/metabolismo , Metiltransferasas/genética , Factor 1 de Elongación Peptídica/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Aminoacil-ARN de Transferencia/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Regulación de la Expresión Génica , Guanosina Trifosfato/metabolismo , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Especificidad de Órganos , Factor 1 de Elongación Peptídica/química , Factor 1 de Elongación Peptídica/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ratas , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosome-profiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5' end and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3' end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Ingeniería Genética/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Ribosomas/genética , Humanos , Oligonucleótidos/genética , Biosíntesis de Proteínas/genética , Ribosomas/química , Transcriptoma/genéticaRESUMEN
E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.
Asunto(s)
Cadherinas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Imagen Óptica/métodos , Microambiente Tumoral , Animales , Cadherinas/genética , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Especificidad de Órganos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias Humanas/metabolismo , Miocitos Cardíacos/metabolismo , Vía de Señalización Wnt , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Línea Celular , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/citología , Humanos , Factor de Transcripción MSX1/genética , Factor de Transcripción MSX1/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Miocitos Cardíacos/citología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismoRESUMEN
PURPOSE: We evaluate the risk of a second urinary diversion in patients after radical cystectomy and urinary diversion. MATERIALS AND METHODS: We retrospectively analyzed the records of 1,614 patients who underwent urinary diversion from January 1986 to March 2009. The primary diversion was neobladder in 71.9% of male patients and 42.3% of female patients, conduit in 17.6% and 38.6%, and ureterocutaneostomy in 9.5% and 12.5%, respectively. The outcome of interest was the need for a second urinary diversion. RESULTS: A total of 51 second/third diversions in 48 patients formed the study population. Mean time from primary to second diversion was 57 months (range 0 to 286). The indication for cystectomy was oncologic in 28 patients and nononcologic in 23. Conversions were continent to continent (14), incontinent to continent (14), continent to incontinent (13) and incontinent to incontinent (10). Twelve patients had tumor recurrence impacting the initial diversion. In 8 patients the indication was abscess necrosis of the diversion or radiogenic damage. Six patients with renal failure required conversion. All patients with conversion from incontinent to continent had a strong desire to avoid a stoma. Four patients died perioperatively and short bowel syndrome developed in 1 patient. CONCLUSIONS: A second urinary diversion was required in 1.8% of patients with bladder cancer with a heterogenous etiology vs 25% when the underlying disease was nononcologic. Only men with apex sparing cystectomy and women whose bladder had not been removed achieved excellent functional outcomes for later orthotopic reconstruction.
Asunto(s)
Cistectomía/métodos , Derivación Urinaria/métodos , Enfermedades Urológicas/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reoperación , Estudios Retrospectivos , Factores de Riesgo , Resultado del TratamientoRESUMEN
The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.
Asunto(s)
Neutrófilos/enzimología , Proteínas de Unión al GTP rac/metabolismo , Animales , Activación Enzimática , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones , Neutrófilos/citología , Transducción de Señal , Análisis Espacio-Temporal , Proteínas de Unión al GTP rac/químicaRESUMEN
Sleep-like states are characterized by massively reduced behavioral activity. Little is known about genetic control of sleep-like behavior. It is also not clear how general activity levels during wake-like behavior influence activity levels during sleep-like behavior. Mutations that increase wake-like activity are generally believed to also increase activity during sleep-like behavior and mutations that decrease wake-like activity are believed to have decreased activity during sleep-like behavior. We studied sleep-like behavior during lethargus in larvae of Caenorhabditis elegans. We looked through a small set of known mutants with altered activity levels. As expected, mutants with increased activity levels typically showed less sleep-like behavior. Among these hyperactive mutants was a gain-of-function mutant of the conserved heterotrimeric G protein subunit Galphaq gene egl-30. We found, however, that an unusual semidominant hypoactive mutant of egl-30 also had reduced sleep-like behavior. While movement was severely reduced and impaired in the semidominant egl-30 mutant, sleep-like behavior was severely reduced: the semidominant egl-30 mutant lacked prolonged periods of complete immobility, reduced spontaneous neural activity less, and reduced responsiveness to stimulation less. egl-30 is a well-known regulator of behavior. Our results suggest that egl-30 controls not only general activity levels, but also differences between wake-like and sleep-like behavior.
Asunto(s)
Animales Modificados Genéticamente/fisiología , Conducta Animal/fisiología , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación de la Expresión Génica , Larva/metabolismo , Letargia/fisiopatología , Mutación/genética , Sueño/fisiología , Animales , Proteínas de Caenorhabditis elegans/genética , Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Larva/citología , Mecanotransducción Celular , Neuronas/fisiología , Transducción de Señal , VigiliaRESUMEN
In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.
Asunto(s)
Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Movimiento Celular , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Interferencia de ARN , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/química , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de SeñalRESUMEN
Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.
Asunto(s)
Antineoplásicos/farmacología , Transferencia Resonante de Energía de Fluorescencia/métodos , Imagenología Tridimensional/métodos , Neoplasias Pancreáticas/metabolismo , Pirimidinas/farmacología , Tiazoles/farmacología , Familia-src Quinasas/metabolismo , Animales , Técnicas Biosensibles/métodos , Células Cultivadas , Dasatinib , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Microscopía FluorescenteRESUMEN
Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation-promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP's arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.
Asunto(s)
Actinas/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/fisiología , Metaloproteinasa 14 de la Matriz/metabolismo , Seudópodos/patología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Western Blotting , Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Femenino , Transferencia Resonante de Energía de Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Invasividad Neoplásica , Multimerización de Proteína , Transporte de Proteínas , Seudópodos/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Células Tumorales Cultivadas , Proteína Neuronal del Síndrome de Wiskott-Aldrich/antagonistas & inhibidores , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Although Rac and its activator Tiam1 are known to stimulate cell-cell adhesion, the mechanisms regulating their activity in cell-cell junction formation are poorly understood. Here, we identify ß2-syntrophin as a Tiam1 interactor required for optimal cell-cell adhesion. We show that during tight-junction (TJ) assembly ß2-syntrophin promotes Tiam1-Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1-Rac activity is necessary for TJ assembly. We further demonstrate that ß2-syntrophin localizes more basally than Par-3 at cell-cell junctions, thus generating an apicobasal Rac activity gradient at developing cell-cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, ß2-syntrophin depletion perturbs Tiam1 and Rac localization at cell-cell junctions and causes defects in apical lumen formation. We conclude that ß2-syntrophin and Par-3 fine-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a la Distrofina/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Polaridad Celular/efectos de los fármacos , Perros , Doxiciclina/farmacología , Proteínas Asociadas a la Distrofina/genética , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Inmunohistoquímica , Espectrometría de Masas , Proteínas de la Membrana/genética , Microscopía Fluorescente , Uniones Estrechas/efectos de los fármacos , Proteínas de Unión al GTP rac/genéticaRESUMEN
Sleep is characterized by reduced muscle activity resulting in reduced movement and a typical posture compatible with relaxed muscles. Prior to each molt, C. elegans larvae go through a phase of behavioral quiescence called Lethargus. Lethargus has sleep-like properties, but a specific posture has not yet been described. Do C. elegans larvae relax their muscles during sleep and do they assume a typical posture? We measured worm posture and body wall muscle activity using calcium imaging across the sleep-wake-like cycle. We found that worms were less curved and had less muscle activity during the sleep-like state. We conclude that during Lethargus, muscle activity is reduced, resulting in a relaxed body posture typical for a sleep-like state.