RESUMEN
The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.
Asunto(s)
Moléculas de Adhesión Celular , Proteínas de Drosophila , Drosophila melanogaster , Vía de Señalización Hippo , Proteínas de la Membrana , Animales , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismoRESUMEN
Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. Loss of RASSF8 elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, the depletion of which phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Epitelio/metabolismo , Proteínas Represoras/metabolismo , Alas de Animales/metabolismo , Uniones Adherentes/metabolismo , Animales , Proteínas Portadoras , Citoplasma/metabolismo , Morfogénesis/fisiología , Pupa/metabolismoRESUMEN
Hippo signalling integrates diverse stimuli related to epithelial architecture to regulate tissue growth and cell fate decisions. The Hippo kinase cascade represses the growth-promoting transcription co-activator Yorkie. The FERM protein Expanded is one of the main upstream Hippo signalling regulators in Drosophila as it promotes Hippo kinase signalling and directly inhibits Yorkie. To fulfil its function, Expanded is recruited to the plasma membrane by the polarity protein Crumbs. However, Crumbs-mediated recruitment also promotes Expanded turnover via a phosphodegron-mediated interaction with a Slimb/ß-TrCP SCF E3 ligase complex. Here, we show that the Casein Kinase 1 (CKI) family is required for Expanded phosphorylation. CKI expression promotes Expanded phosphorylation and interaction with Slimb/ß-TrCP. Conversely, CKI depletion in S2 cells impairs Expanded degradation downstream of Crumbs. In wing imaginal discs, CKI loss leads to elevated Expanded and Crumbs levels. Thus, phospho-dependent Expanded turnover ensures a tight coupling of Hippo pathway activity to epithelial architecture.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Drosophila , Regulación de la Expresión Génica , Fosforilación , Unión Proteica , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas con Repetición de beta-Transducina/metabolismoRESUMEN
Polarity is a shared feature of most cells. In epithelia, apical-basal polarity often coexists, and sometimes intersects with planar cell polarity (PCP), which orients cells in the epithelial plane. From a limited set of core building blocks (e.g. the Par complexes for apical-basal polarity and the Frizzled/Dishevelled complex for PCP), a diverse array of polarized cells and tissues are generated. This suggests the existence of little-studied tissue-specific factors that rewire the core polarity modules to the appropriate conformation. In Drosophila sensory organ precursors (SOPs), the core PCP components initiate the planar polarization of apical-basal determinants, ensuring asymmetric division into daughter cells of different fates. We show that Meru, a RASSF9/RASSF10 homologue, is expressed specifically in SOPs, recruited to the posterior cortex by Frizzled/Dishevelled, and in turn polarizes the apical-basal polarity factor Bazooka (Par3). Thus, Meru belongs to a class of proteins that act cell/tissue-specifically to remodel the core polarity machinery.
Asunto(s)
División Celular Asimétrica , Polaridad Celular , Proteínas de Drosophila/fisiología , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Transporte Vesicular/fisiología , Animales , Perfilación de la Expresión GénicaRESUMEN
BACKGROUND: Coordinated multicellular growth during development is achieved by the sensing of spatial and nutritional boundaries. The conserved Hippo (Hpo) signaling pathway has been proposed to restrict tissue growth by perceiving mechanical constraints through actin cytoskeleton networks. The actin-associated LIM proteins Zyxin (Zyx) and Ajuba (Jub) have been linked to the control of tissue growth via regulation of Hpo signaling, but the study of Zyx has been hampered by a lack of genetic tools. RESULTS: We generated a zyx mutant in Drosophila using TALEN endonucleases and used this to show that Zyx antagonizes the FERM-domain protein Expanded (Ex) to control tissue growth, eye differentiation, and F-actin accumulation. Zyx membrane targeting promotes the interaction between the transcriptional co-activator Yorkie (Yki) and the transcription factor Scalloped (Sd), leading to activation of Yki target gene expression and promoting tissue growth. Finally, we show that Zyx's growth-promoting function is dependent on its interaction with the actin-associated protein Enabled (Ena) via a conserved LPPPP motif and is antagonized by Capping Protein (CP). CONCLUSIONS: Our results show that Zyx is a functional antagonist of Ex in growth control and establish a link between actin filament polymerization and Yki activity.
Asunto(s)
Actinas/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Proteínas de la Membrana/fisiología , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Zixina/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Femenino , Genes de Insecto , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Proteínas Nucleares/genética , Tamaño de los Órganos/genética , Tamaño de los Órganos/fisiología , Organogénesis/genética , Organogénesis/fisiología , Transactivadores/genética , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Proteínas Señalizadoras YAP , Zixina/genéticaRESUMEN
A gene expression oscillator called the segmentation clock controls somite segmentation in the vertebrate embryo. In zebrafish, the oscillatory transcriptional repressor genes her1 and her7 are crucial for genesis of the oscillations, which are thought to arise from negative autoregulation of these genes. The period of oscillation is predicted to depend on delays in the negative-feedback loop, including, most importantly, the transcriptional delay - the time taken to make each molecule of her1 or her7 mRNA. her1 and her7 operate in parallel. Loss of both gene functions, or mutation of her1 combined with knockdown of Hes6, which we show to be a binding partner of Her7, disrupts segmentation drastically. However, mutants in which only her1 or her7 is functional show only mild segmentation defects and their oscillations have almost identical periods. This is unexpected because the her1 and her7 genes differ greatly in length. We use transgenic zebrafish to measure the RNA polymerase II elongation rate, for the first time, in the intact embryo. This rate is unexpectedly rapid, at 4.8 kb/minute at 28.5°C, implying that, for both genes, the time taken for transcript elongation is insignificant compared with other sources of delay, explaining why the mutants have similar clock periods. Our computational model shows how loss of her1 or her7 can allow oscillations to continue with unchanged period but with reduced amplitude and impaired synchrony, as manifested in the in situ hybridisation patterns of the single mutants.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , ARN Polimerasa II/metabolismo , Somitos/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Fluoresceínas/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación/métodos , Modelos Biológicos , Modelos Teóricos , Mutación , Oscilometría/métodos , ARN Polimerasa II/genética , Temperatura , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
The specification of tissue size during development involves the coordinated action of many signalling pathways responding to organ-intrinsic signals, such as morphogen gradients, and systemic cues, such as nutrient status. The conserved Hippo (Hpo) pathway, which promotes both cell-cycle exit and apoptosis, is a major determinant of size control. The pathway core is a kinase cassette, comprising the kinases Hpo and Warts (Wts) and the scaffold proteins Salvador (Sav) and Mats, which inactivates the pro-growth transcriptional co-activator Yorkie (Yki). We performed a split-TEV-based genome-wide RNAi screen for modulators of Hpo signalling. We characterize the Drosophila salt-inducible kinases (Sik2 and Sik3) as negative regulators of Hpo signalling. Activated Sik kinases increase Yki target expression and promote tissue overgrowth through phosphorylation of Sav at Ser 413. As Sik kinases have been implicated in nutrient sensing, this suggests a link between the Hpo pathway and systemic growth control.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas 14-3-3/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Nucleares/metabolismo , Tamaño de los Órganos , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Transactivadores/metabolismo , Alas de Animales/enzimología , Alas de Animales/crecimiento & desarrollo , Proteínas Señalizadoras YAPRESUMEN
Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1-q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor beta (OSMRbeta), in three families. OSMRbeta is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.
Asunto(s)
Amiloidosis Familiar/genética , Subunidad beta del Receptor de Oncostatina M/genética , Secuencia de Aminoácidos , Amiloidosis Familiar/patología , Brasil , Técnicas de Cultivo de Célula , Cromosomas Humanos Par 5 , Análisis Mutacional de ADN , Femenino , Genes Dominantes , Humanos , Queratinocitos , Masculino , Datos de Secuencia Molecular , Mutación Missense , Subunidad beta del Receptor de Oncostatina M/química , Linaje , Homología de Secuencia , Sudáfrica , Reino UnidoRESUMEN
Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.
Asunto(s)
Terapia Genética/efectos adversos , Lentivirus/genética , Neoplasias Hepáticas/etiología , Animales , Animales Recién Nacidos , Feto , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , VIH-1/genética , Hígado/patología , Neoplasias Hepáticas/patología , Ratones , Ratones TransgénicosRESUMEN
Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.