RESUMEN
The coordination of cells or structures within the plane of a tissue is known as planar polarization. It is often governed by the asymmetric distribution of planar polarity proteins within cells. A number of quantitative methods have been developed to provide a readout of planar polarized protein distributions. However, previous planar polarity quantification methods can be affected by variation in cell geometry. Hence, we developed a novel planar polarity quantification method based on Principal Component Analysis (PCA) that is shape insensitive. Here, we compare this method with other state-of-the-art methods on simulated models and biological datasets. We found that the PCA method performs robustly in quantifying planar polarity independently of variation in cell geometry and other image conditions. We designed a user-friendly graphical user interface called QuantifyPolarity, equipped with three polarity methods for automated quantification of polarity. QuantifyPolarity also provides tools to quantify cell morphology and packing geometry, allowing the relationship of these characteristics to planar polarization to be investigated. This tool enables experimentalists with no prior computational expertise to perform high-throughput cell polarity and shape analysis automatically and efficiently.
Asunto(s)
Polaridad Celular/fisiología , Análisis de Componente Principal/métodos , Animales , Dípteros/fisiología , Femenino , Ensayos Analíticos de Alto Rendimiento/métodos , MasculinoRESUMEN
Planar polarity is a widespread phenomenon found in many tissues, allowing cells to coordinate morphogenetic movements and function. A common feature of animal planar polarity systems is the formation of molecular bridges between cells, which become polarised along a tissue axis. We propose that these bridges provide a general mechanism by which cells interpret different forms of tissue gradients to coordinate directional information. We illustrate this using a generalised and consistent modelling framework, providing a conceptual basis for understanding how different mechanisms of gradient function can generate planar polarity. We make testable predictions of how different gradient mechanisms can influence polarity direction.
Asunto(s)
Polaridad Celular/fisiología , Modelos Biológicos , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Cytokine receptors often act via the Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway to form a signalling cascade that is essential for processes such as haematopoiesis, immune responses and tissue homeostasis. In order to transduce ligand activation, cytokine receptors must dimerise. However, mechanisms regulating their dimerisation are poorly understood. In order to better understand the processes regulating cytokine receptor levels, and their activity and dimerisation, we analysed the highly conserved JAK/STAT pathway in Drosophila, which acts via a single receptor, known as Domeless. We performed a genome-wide RNAi screen in Drosophila cells, identifying MASK as a positive regulator of Domeless dimerisation and protein levels. We show that MASK is able to regulate receptor levels and JAK/STAT signalling both in vitro and in vivo We also show that its human homologue, ANKHD1, is also able to regulate JAK/STAT signalling and the levels of a subset of pathway receptors in human cells. Taken together, our results identify MASK as a novel regulator of cytokine receptor levels, and suggest functional conservation, which may have implications for human health.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Genoma de los Insectos , Interferencia de ARN , Receptores de Citocinas/genética , Receptores de Interleucina/química , Secuencias de Aminoácidos , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Humanos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Unión Proteica , Estabilidad Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de SeñalRESUMEN
Zebrafish (Danio rerio) are increasingly used to investigate spine development, growth, and for studying the etiology of spinal deformity, such as scoliosis. Here, we present a micro-computed tomography-based pipeline for visualizing the zebrafish skeleton. We describe steps for sample preparation, imaging, data management, and processing. We then detail analysis of vertebral and spine morphology using open-source software. This protocol will be useful for scientists using zebrafish to understand spine development and disease. For complete details on the use and execution of this protocol, please refer to Bearce et al. (2022).1.
Asunto(s)
Escoliosis , Animales , Escoliosis/diagnóstico por imagen , Pez Cebra , Microtomografía por Rayos X , Columna Vertebral/diagnóstico por imagenRESUMEN
BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genoma de los Insectos , Quinasas Janus/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Factores de Transcripción STAT/genética , Factores de Transcripción/genética , Animales , Células Cultivadas , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Biblioteca de Genes , Genes Reporteros , Estudio de Asociación del Genoma Completo , Quinasas Janus/metabolismo , Luciferasas , Plásmidos , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Although many signal transduction pathways have been implicated in the development of human disease, the identification of pathway targets and the biological processes that mediate disease progression remains challenging. One such disease-related pathway is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cascade whose constitutive misactivation by the JAK2 V617F mutation underlies most human myeloproliferative disorders. Here, we use transcript profiling of Drosophila haemocyte-like cells to identify JAK/STAT target genes, combined with an in vivo model for JAK-induced blood cell overproliferation, to identify the main effectors required for haematopoietic tumour development. The identified human homologues of the Drosophila effectors were tested for potential V617F-mediated transcriptional regulation in human HeLa cells and compared with small interfering RNA-derived data, quantify their role in regulating the proliferation of cancer-derived cell lines. Such an inter-species approach is an effective way to identify factors with conserved functions that might be central to human disease.
Asunto(s)
Perfilación de la Expresión Génica , Neoplasias Hematológicas/metabolismo , Quinasas Janus/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Transcripción Genética , Animales , Proliferación Celular , Análisis por Conglomerados , Drosophila melanogaster , Regulación de la Expresión Génica , Células HeLa , Neoplasias Hematológicas/patología , Hemocitos/citología , Humanos , Ratones , ARN Interferente Pequeño/metabolismoRESUMEN
The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.
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Ciclo Celular/fisiología , Microtúbulos/metabolismo , Mitosis/fisiología , Animales , Centrosoma/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Interferencia de ARN , Proteínas Ligasas SKP Cullina F-box/metabolismoRESUMEN
Classical activation of macrophages induces a wide range of signaling and vesicle trafficking events to produce a more aggressive cellular phenotype. The microtubule (MT) cytoskeleton is crucial for the regulation of immune responses. In the current study, we used a large scale proteomics approach to analyze the change in protein composition of the MT-associated protein (MAP) network by macrophage stimulation with the inflammatory cytokine interferon-gamma and the endotoxin lipopolysaccharide. Overall the analysis identified 409 proteins that bound directly or indirectly to MTs. Of these, 52 were up-regulated 2-fold or greater and 42 were down-regulated 2-fold or greater after interferon-gamma/lipopolysaccharide stimulation. Bioinformatics analysis based on publicly available binary protein interaction data produced a putative interaction network of MAPs in activated macrophages. We confirmed the up-regulation of several MAPs by immunoblotting and immunofluorescence analysis. More detailed analysis of one up-regulated protein revealed a role for HSP90beta in stabilization of the MT cytoskeleton during macrophage activation.
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Regulación de la Expresión Génica , Macrófagos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteómica/métodos , Animales , Biología Computacional/métodos , Citoesqueleto/metabolismo , Bases de Datos de Proteínas , Reacciones Falso Positivas , Humanos , Lipopolisacáridos/metabolismo , Activación de Macrófagos , Ratones , Modelos BiológicosRESUMEN
In developing tissues, sheets of cells become planar polarized, enabling coordination of cell behaviors. It has been suggested that "signaling" of polarity information between cells may occur either bidirectionally or monodirectionally between the molecules Frizzled (Fz) and Van Gogh (Vang). Using computational modeling we find that both bidirectional and monodirectional signaling models reproduce known non-autonomous phenotypes derived from patches of mutant tissue of key molecules but predict different phenotypes from double mutant tissue, which have previously given conflicting experimental results. Furthermore, we re-examine experimental phenotypes in the Drosophila wing, concluding that signaling is most likely bidirectional. Our modeling suggests that bidirectional signaling can be mediated either indirectly via bidirectional feedbacks between asymmetric intercellular protein complexes or directly via different affinities for protein binding in intercellular complexes, suggesting future avenues for investigation. Our findings offer insight into mechanisms of juxtacrine cell signaling and how tissue-scale properties emerge from individual cell behaviors.
RESUMEN
The core planar polarity pathway coordinates epithelial cell polarity during animal development, and loss of its activity gives rise to a range of defects, from aberrant morphogenetic cell movements to failure to correctly orient structures, such as hairs and cilia. The core pathway functions via a mechanism involving segregation of its protein components to opposite cells ends, where they form asymmetric intracellular complexes that couple cell-cell polarity. This segregation is a self-organizing process driven by feedback interactions between the core proteins themselves. Despite intense efforts, the molecular pathways underlying feedback have proven difficult to elucidate using conventional genetic approaches. Here we investigate core protein function during planar polarization of the Drosophila wing by combining quantitative measurements of protein dynamics with loss-of-function genetics, mosaic analysis, and temporal control of gene expression. Focusing on the key core protein Frizzled, we show that its stable junctional localization is promoted by the core proteins Strabismus, Dishevelled, Prickle, and Diego. In particular, we show that the stabilizing function of Prickle on Frizzled requires Prickle activity in neighboring cells. Conversely, Prickle in the same cell has a destabilizing effect on Frizzled. This destabilizing activity is dependent on the presence of Dishevelled and blocked in the absence of Dynamin and Rab5 activity, suggesting an endocytic mechanism. Overall, our approach reveals for the first time essential in vivo stabilizing and destabilizing interactions of the core proteins required for self-organization of planar polarity.
Asunto(s)
Tipificación del Cuerpo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Receptores Frizzled/genética , Proteínas con Dominio LIM/genética , Alas de Animales/crecimiento & desarrollo , Animales , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Receptores Frizzled/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas con Dominio LIM/metabolismo , Mutación con Pérdida de Función , Alas de Animales/metabolismoRESUMEN
Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms.
Asunto(s)
Proteínas de Drosophila/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endocitosis , Quinasas Janus/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Receptores de Interleucina/metabolismo , Factores de Transcripción STAT/metabolismo , Transcripción GenéticaRESUMEN
To understand how long-range patterning gradients are interpreted at the cellular level, we investigate how a gradient of expression of the Four-jointed kinase specifies planar polarised distributions of the cadherins Fat and Dachsous in the Drosophila wing. We use computational modelling to test different scenarios for how Four-jointed might act and test the model predictions by employing fluorescence recovery after photobleaching as an in vivo assay to measure the influence of Four-jointed on Fat-Dachsous binding. We demonstrate that in vivo, Four-jointed acts both on Fat to promote its binding to Dachsous and on Dachsous to inhibit its binding to Fat, with a bias towards a stronger effect on Fat. Overall, we show that opposing gradients of Fat and Dachsous phosphorylation are sufficient to explain the observed pattern of Fat-Dachsous binding and planar polarisation across the wing, and thus demonstrate the mechanism by which a long-range gradient is interpreted.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/anatomía & histología , Glicoproteínas de Membrana/fisiología , Animales , Dimerización , Humanos , FosforilaciónRESUMEN
BACKGROUND: The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. METHODS & FINDINGS: We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. CONCLUSIONS: Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential.
Asunto(s)
Drosophila melanogaster/metabolismo , Inhibidores Enzimáticos/farmacología , Janus Quinasa 2/metabolismo , Metotrexato/farmacología , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Drosophila melanogaster/genética , Janus Quinasa 2/genéticaRESUMEN
The JAK/STAT signaling pathway has essential roles in multiple developmental processes, including stem cell maintenance, immune responses, and cellular proliferation. As a result, it has been extensively studied in both vertebrate systems and lower complexity models, such as Drosophila. Given its connection with such a wide range of biological functions, it is no surprise that pathway misregulation is frequently associated with multiple human diseases including cancer. While the core components of the pathway, and a number of negative regulators, are well known and conserved in many organisms, more subtle levels of regulation and inter-pathway crosstalk are less well understood. With the emergence of RNA interference (RNAi) as a tool to knock down gene expression and so evaluate protein function, high-throughput screens have been developed to identify pathway regulators on a genome-wide scale. Here we discuss the approaches and methods employed thus far for identification of pathway regulators using RNAi in Drosophila. Furthermore, we discuss possible approaches for future screens and the significant potential for applying RNAi technology in vertebrate models.
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Técnicas Genéticas , Quinasas Janus/metabolismo , Interferencia de ARN , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , Animales , Línea Celular Tumoral , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Humanos , Transcripción Genética/genéticaRESUMEN
The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex.
Asunto(s)
Proteínas Portadoras/metabolismo , Centrosoma/metabolismo , Segregación Cromosómica , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Meiosis , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Femenino , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Cinetocoros/metabolismo , Larva/genética , Larva/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Mitosis , Datos de Secuencia Molecular , Complejos Multiproteicos , Interferencia de ARN , Eliminación de SecuenciaRESUMEN
Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs.