RESUMEN
Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.
Asunto(s)
Regeneración Nerviosa/fisiología , Tejido Nervioso/lesiones , Tejido Nervioso/fisiopatología , Octopodiformes/fisiología , Recuperación de la Función/fisiología , Animales , Axones/fisiología , Conducta Animal , Fenómenos Electrofisiológicos , Femenino , Masculino , Octopodiformes/anatomía & histología , Respiración , Piel/inervaciónRESUMEN
The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling.
Asunto(s)
ARN Helicasas DEAD-box/genética , Daño del ADN/genética , Reparación del ADN/inmunología , Histonas/metabolismo , Ribonucleasa III/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Reparación del ADN/genética , Humanos , Proteínas Nucleares/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Ribonucleasa Pancreática/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.
Asunto(s)
Endocitosis , Epitelio/metabolismo , Fenómenos Biomecánicos , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/metabolismo , Aparato de Golgi/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Biomarcadores de Tumor , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Endosomas/metabolismo , Exocitosis , Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/metabolismo , Humanos , Invasividad Neoplásica , Pronóstico , Transporte de Proteínas , Proteolisis , Recurrencia , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
RAB5 is a small GTPase that belongs to the wide family of Rab proteins and localizes on early endosomes. In its active GTP-bound form, RAB5 recruits downstream effectors that, in turn, are responsible for distinct aspects of early endosome function, including their movement along microtubules. We previously reported that, at the onset of mitosis, RAB5positive vesicles cluster around the spindle poles and, during metaphase, move along spindle microtubules. RNAi-mediated depletion of the three RAB5 isoforms delays nuclear envelope breakdown at prophase and severely affects chromosome alignment and segregation. Here we show that depletion of the Kinesin-2 motor complex impairs long-range movement of RAB5 endosomes in interphase cells and prevents localization of these vesicles at the spindle during metaphase. Similarly to the effect caused by RAB5 depletion, functional ablation of Kinesin-2 delays nuclear envelope breakdown resulting in prolonged prophase. Altogether these findings suggest that endosomal transport at the onset of mitosis is required to control timing of nuclear envelope breakdown.
Asunto(s)
Endosomas/fisiología , Cinesinas/genética , Cinesinas/metabolismo , Huso Acromático/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Humanos , Interfase , Metafase , Microtúbulos/metabolismo , Interferencia de ARNRESUMEN
Protein acetylation is mediated by histone acetyltransferases (HATs) and deacetylases (HDACs), which influence chromatin dynamics, protein turnover and the DNA damage response. ATM and ATR mediate DNA damage checkpoints by sensing double-strand breaks and single-strand-DNA-RFA nucleofilaments, respectively. However, it is unclear how acetylation modulates the DNA damage response. Here we show that HDAC inhibition/ablation specifically counteracts yeast Mec1 (orthologue of human ATR) activation, double-strand-break processing and single-strand-DNA-RFA nucleofilament formation. Moreover, the recombination protein Sae2 (human CtIP) is acetylated and degraded after HDAC inhibition. Two HDACs, Hda1 and Rpd3, and one HAT, Gcn5, have key roles in these processes. We also find that HDAC inhibition triggers Sae2 degradation by promoting autophagy that affects the DNA damage sensitivity of hda1 and rpd3 mutants. Rapamycin, which stimulates autophagy by inhibiting Tor, also causes Sae2 degradation. We propose that Rpd3, Hda1 and Gcn5 control chromosome stability by coordinating the ATR checkpoint and double-strand-break processing with autophagy.
Asunto(s)
Autofagia , Roturas del ADN de Doble Cadena , Histona Desacetilasas/metabolismo , Saccharomyces cerevisiae , Acetilación/efectos de los fármacos , Aminopeptidasas/metabolismo , Autofagia/efectos de los fármacos , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Inestabilidad Cromosómica , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Quinasas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Ácido Valproico/farmacologíaRESUMEN
BACKGROUND: Polycystin-1 (PC-1) is a large plasma membrane receptor, encoded by the PKD1 gene, which is mutated in most cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). The disease is characterized by renal cysts. The precise function of PC-1 remains elusive, although several studies suggest that it can regulate the cellular shape in response to external stimuli. We and others reported that PC-1 regulates the actin cytoskeleton and cell migration. RESULTS: Here we show that cells over-expressing PC-1 display enhanced adhesion rates to the substrate, while cells lacking PC-1 have a reduced capability to adhere. In search for the mechanism responsible for this new property of PC-1 we found that this receptor is able to regulate the stability of the microtubules, in addition to its capability to regulate the actin cytoskeleton. The two cytoskeletal components are acting in a coordinated fashion. Notably, we uncovered that PC-1 regulation of the microtubule cytoskeleton impacts on the turnover rates of focal adhesions in migrating cells and we link all these properties to the capability of PC-1 to regulate the activation state of Focal Adhesion Kinase (FAK). CONCLUSIONS: In this study we show several new features of the PC-1 receptor in modulating microtubules and adhesion dynamics, which are essential for its capability to regulate migration.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Adhesiones Focales/metabolismo , Microtúbulos/metabolismo , Canales Catiónicos TRPP/metabolismo , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Perros , Recuperación de Fluorescencia tras Fotoblanqueo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células de Riñón Canino Madin Darby , Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Canales Catiónicos TRPP/antagonistas & inhibidores , Canales Catiónicos TRPP/genéticaRESUMEN
Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Microtúbulos/metabolismo , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab5/antagonistas & inhibidores , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma.
Asunto(s)
Células Madre Mesenquimatosas , Adipogénesis , Diferenciación Celular , Células Cultivadas , Colorantes/metabolismo , Lípidos , Minerales , Osteogénesis , Células Madre , Tetraciclinas/metabolismoRESUMEN
Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.
Asunto(s)
Ataxia Telangiectasia , Proteínas Serina-Treonina Quinasas , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cromatina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteómica , Proteínas de Unión al ADN/metabolismo , Fosforilación , Daño del ADN , Citoesqueleto/metabolismoRESUMEN
Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
Asunto(s)
Neoplasias Colorrectales , Neoplasias Peritoneales , Humanos , Matriz Extracelular Descelularizada , Peritoneo , Neoplasias Peritoneales/metabolismo , Neoplasias Peritoneales/secundario , Neoplasias Peritoneales/terapia , Organoides , Neoplasias Colorrectales/metabolismoRESUMEN
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endosomas/metabolismo , Helicobacter pylori/química , Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Glicosilfosfatidilinositoles/metabolismo , Células HeLa , Humanos , Transporte de ProteínasRESUMEN
Infection with Helicobacter pylori is responsible for gastritis and gastroduodenal ulcers but is also a high risk factor for the development of gastric adenocarcinoma and lymphoma. The most pathogenic H. pylori strains (i.e., the so-called type I strains) associate the CagA virulence protein with an active VacA cytotoxin but the rationale for this association is unknown. CagA, directly injected by the bacterium into colonized epithelium via a type IV secretion system, leads to cellular morphological, anti-apoptotic and proinflammatory effects responsible in the long-term (years or decades) for ulcer and cancer. VacA, via pinocytosis and intracellular trafficking, induces epithelial cell apoptosis and vacuolation. Using human gastric epithelial cells in culture transfected with cDNA encoding for either the wild-type 38 kDa C-terminal signaling domain of CagA or its non-tyrosine-phosphorylatable mutant form, we found that, depending on tyrosine-phosphorylation by host kinases, CagA inhibited VacA-induced apoptosis by two complementary mechanisms. Tyrosine-phosphorylated CagA prevented pinocytosed VacA to reach its target intracellular compartments. Unphosphorylated CagA triggered an anti-apoptotic activity blocking VacA-induced apoptosis at the mitochondrial level without affecting the intracellular trafficking of the toxin. Assaying the level of apoptosis of gastric epithelial cells infected with wild-type CagA(+)/VacA(+)H. pylori or isogenic mutants lacking of either CagA or VacA, we confirmed the results obtained in cells transfected with the CagA C-ter constructions showing that CagA antagonizes VacA-induced apoptosis. VacA toxin plays a role during H. pylori stomach colonization. However, once bacteria have colonized the gastric niche, the apoptotic action of VacA might be detrimental for the survival of H. pylori adherent to the mucosa. CagA association with VacA is thus a novel, highly ingenious microbial strategy to locally protect its ecological niche against a bacterial virulence factor, with however detrimental consequences for the human host.
Asunto(s)
Antígenos Bacterianos/metabolismo , Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/patogenicidad , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Western Blotting , Línea Celular , Helicobacter pylori/metabolismo , Humanos , Inmunoprecipitación , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes de Fusión , Transfección , Tirosina/metabolismoRESUMEN
Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/genética , Oogénesis/genética , Ribonucleoproteínas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Gránulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Homeostasis/genética , Longevidad/genética , Neuronas/citología , Neuronas/metabolismo , Oocitos/citología , Oocitos/metabolismo , Transición de Fase , Profilinas/genética , Profilinas/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Actinas/metabolismo , Polaridad Celular/genética , Polaridad Celular/fisiología , Células Epiteliales/metabolismo , Femenino , Humanos , Morfogénesis/genética , Morfogénesis/fisiología , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas de Unión al GTP rab/genéticaRESUMEN
The mechanism whereby extracellular Ca(2+) exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca(2+)-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca(2+)-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd(3+), La(3+) and neomycin, elicited a heterogeneous intracellular Ca(2+) signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP(3)) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na(+)/Ca(2+) exchanger upon substitution of extracellular Na(+) unmasked the Ca(2+) signal triggered by an increase in extracellular Ca(2+) levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca(2+) response to the CaSR agonist La(3+). These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca(2+) from intracellular InsP(3)-sensitive stores.
Asunto(s)
Señalización del Calcio/fisiología , Células Endoteliales/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Sensibles al Calcio/genética , Animales , Señalización del Calcio/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Estrenos/farmacología , Gadolinio/farmacología , Indoles/farmacología , Lantano/farmacología , Meglumina/farmacología , Neomicina/farmacología , Fenilalanina/farmacología , Pirrolidinonas/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Sensibles al Calcio/agonistas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/fisiología , Espermina/farmacología , Triptófano/farmacología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/genética , Reparación del ADN , ADN/genética , Subunidad 1 del Complejo Mediador/metabolismo , Transcripción Genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Subunidad 1 del Complejo Mediador/genética , Osteoblastos/citología , Osteoblastos/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
EPS15 and its homologous EPS15L1 are endocytic accessory proteins. Studies in mammalian cell lines suggested that EPS15 and EPS15L1 regulate endocytosis in a redundant manner. However, at the organismal level, it is not known to which extent the functions of the two proteins overlap. Here, by exploiting various constitutive and conditional null mice, we report redundant and nonredundant functions of the two proteins. EPS15L1 displays a unique nonredundant role in the nervous system, whereas both proteins are fundamental during embryo development as shown by the embryonic lethality of -Eps15/Eps15L1-double KO mice. At the cellular level, the major process redundantly regulated by EPS15 and EPS15L1 is the endocytosis of the transferrin receptor, a pathway that sustains the development of red blood cells and controls iron homeostasis. Consequently, hematopoietic-specific conditional Eps15/Eps15L1-double KO mice display traits of microcytic hypochromic anemia, due to a cell-autonomous defect in iron internalization.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Endocitosis/fisiología , Anemia Hipocrómica/genética , Animales , Escala de Evaluación de la Conducta , Desarrollo Embrionario/fisiología , Eritrocitos/metabolismo , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Genes Letales/fisiología , Hipocampo/citología , Hierro/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Receptores de Transferrina/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Sinapsis/metabolismoRESUMEN
The inner wall of excised rat aorta was scraped by a microelectrode and Ca2+ signals were investigated by fluorescence microscopy in endothelial cells (ECs) directly coupled with injured cells. The injury caused an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i), followed by a long-lasting decay phase due to Ca2+ influx from extracellular space. The immediate response was mainly due to activation of purinergic receptors, as shown by the effect of P2X and P2Y receptors agonists and antagonists, such as suramin, alpha,beta-MeATP, MRS-2179 and 2-MeSAMP. Inhibition of store-operated Ca2+ influx did not affect either the peak response or the decay phase. Furthermore, the latter was: (i) insensitive to phospholipase C inhibition, (ii) sensitive to the gap junction blockers, palmitoleic acid, heptanol, octanol and oleamide, and (iii) sensitive to La3+ and Ni2+, but not to Gd3+. Finally, ethidium bromide or Lucifer Yellow did not enter ECs facing the scraped area. These results suggest that endothelium scraping: (i) causes a short-lasting stimulation of healthy ECs by extracellular nucleotides released from damaged cells and (ii) uncouples the hemichannels of the ECs facing the injury site; these hemichannels do not fully close and allow a long-lasting Ca2+ entry.
Asunto(s)
Señalización del Calcio/fisiología , Endotelio Vascular/lesiones , Endotelio Vascular/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Aorta Abdominal/citología , Aorta Torácica/citología , Endotelio Vascular/efectos de los fármacos , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Ratas , Receptores Purinérgicos P2/metabolismo , Suramina/farmacologíaRESUMEN
The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.