Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination

AbstractThe retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development

Effect of Inclination of Twin Jets Impinging a Heated Wall

This study examines the interaction of twin oblique turbulent slot-jets of different directions (divergent, convergent or parallel) impinging a heated wall. A comparison of the results is done between the cases of perpendicular jets and three cases of twinned jets (parallel, convergent and divergent).The twin slot jets are located on a confining adiabatic wall at a distance of 8 slot jet width. Convective heat is investigated numerically examining the effect of Reynolds number (Re) and jet inclination angle (). This problem is relevant to a wide range of practical applications including nuclear engineering devices, manufacturing, material processing, electronic cooling, drying paper or textile, tempering of glass, etc. The numerical investigation is performed using two dimensional large eddy simulations (LES) approach with Smagorinsky sub-grid scale (SGS) models. The results show the presence of a complex flow resulting from the interaction of the two jets. When the impingement angle is reduced from 0° (perpendicular impingement) to 60°, the position of the stagnation points are modified and therefore the peaks of the Nusselt number locations on the impingement surface and their magnitude, vary. For largest Reynolds number Nusselt number is enhanced for all types of inclination. The averaged Nusselt number shows that the perpendicular impingement gives better heat transfer than that of the oblique jets. The poor heat transfer is obtained for the parallel oblique jets. For the same angle, divergent jets give smallest heat transfer than the convergent jets

Continental crust subduction-related structures: contrasting evolutions vs. contrasting memories

In this contribution, we will describe examples from the subducted continental crust of the Western Alps, Austroalpine domain. We will integrate results from structural geology, petrology/geochemistry, and modelling, at different scales from lithosphere to atomic scale, emphasising the roles of contrasting tectono-metamorphic evolutions versus the efficiency of rocks to memorize their promenade along the subduction plate interface or close to it. We will compare the evolution of continental crust slices found at different positions within the subduction architecture and recording specific structural features and metamorphic parageneses. We emphasise that the combined use of structural and metamorphic criteria is the only nowadays reliable method to fully investigate such an heterogeneous system

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

Molecular Structure and Dimeric Organization of the Notch Extracellular Domain as Revealed by Electron Microscopy

Background: The Notch receptor links cell fate decisions of one cell to that of the immediate cellular neighbor. In humans, malfunction of Notch signaling results in diseases and congenital disorders. Structural information is essential for gaining insight into the mechanism of the receptor as well as for potentially interfering with its function for therapeutic purposes. Methodology/Principal Findings: We used the Affinity Grid approach to prepare specimens of the Notch extracellular domain (NECD) of the Drosophila Notch and human Notch1 receptors suitable for analysis by electron microscopy and three-dimensional (3D) image reconstruction. The resulting 3D density maps reveal that the NECD structure is conserved across species. We show that the NECD forms a dimer and adopts different yet defined conformations, and we identify the membrane-proximal region of the receptor and its ligand-binding site. Conclusions/Significance: Our results provide direct and unambiguous evidence that the NECD forms a dimer. Our studies further show that the NECD adopts at least three distinct conformations that are likely related to different functional states of the receptor. These findings open the way to now correlate mutations in the NECD with its oligomeric state and conformation

Presenilin Controls CBP Levels in the Adult Drosophila Central Nervous System

Background: Dominant mutations in both human Presenilin (Psn) genes have been correlated with the formation of amyloid plaques and development of familial early-onset Alzheimer’s disease (AD). However, a definitive mechanism whereby plaque formation causes the pathology of familial and sporadic forms of AD has remained elusive. Recent discoveries of several substrates for Psn protease activity have sparked alternative hypotheses for the pathophysiology underlying AD. CBP (CREB-binding protein) is a haplo-insufficient transcriptional co-activator with histone acetly-transferase (HAT) activity that has been proposed to be a downstream target of Psn signaling. Individuals with altered CBP have cognitive deficits that have been linked to several neurological disorders. Methodology/Principal Findings: Using a transgenic RNA-interference strategy to selectively silence CBP, Psn, and Notch in adult Drosophila, we provide evidence for the first time that Psn is required for normal CBP levels and for maintaining specific global acetylations at lysine 8 of histone 4 (H4K8ac) in the central nervous system (CNS). In addition, flies conditionally compromised for the adult-expression of CBP display an altered geotaxis behavior that may reflect a neurological defect. Conclusions/Significance: Our data support a model in which Psn regulates CBP levels in the adult fly brain in a manner that is independent of Notch signaling. Although we do not understand the molecular mechanism underlying th

A Gain-of-Function Germline Mutation in Drosophila ras1 Affects Apoptosis and Cell Fate during Development

The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. Here we report the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, we used ras1R68Q to identify novel genes that interact with Ras and suppress cell death

Drosophila cbl Is Essential for Control of Cell Death and Cell Differentiation during Eye Development

Activation of cell surface receptors transduces extracellular signals into cellular responses such as proliferation, differentiation and survival. However, as important as the activation of these receptors is their appropriate spatial and temporal down-regulation for normal development and tissue homeostasis. The Cbl family of E3-ubiquitin ligases plays a major role for the ligand-dependent inactivation of receptor tyrosine kinases (RTKs), most notably the Epidermal Growth Factor Receptor (EGFR) through ubiquitin-mediated endocytosis and lysosomal degradation.Here, we report the mutant phenotypes of Drosophila cbl (D-cbl) during eye development. D-cbl mutants display overgrowth, inhibition of apoptosis, differentiation defects and increased ommatidial spacing. Using genetic interaction and molecular markers, we show that most of these phenotypes are caused by increased activity of the Drosophila EGFR. Our genetic data also indicate a critical role of ubiquitination for D-cbl function, consistent with biochemical models.These data may provide a mechanistic model for the understanding of the oncogenic activity of mammalian cbl genes

Uif, a Large Transmembrane Protein with EGF-Like Repeats, Can Antagonize Notch Signaling in Drosophila

<div><h3>Background</h3><p>Notch signaling is a highly conserved pathway in multi-cellular organisms ranging from flies to humans. It controls a variety of developmental processes by stimulating the expression of its target genes in a highly specific manner both spatially and temporally. The diversity, specificity and sensitivity of the Notch signaling output are regulated at distinct levels, particularly at the level of ligand-receptor interactions.</p> <h3>Methodology/Principal Findings</h3><p>Here, we report that the <em>Drosophila</em> gene <em>uninflatable</em> (<em>uif</em>), which encodes a large transmembrane protein with eighteen EGF-like repeats in its extracellular domain, can antagonize the canonical Notch signaling pathway. Overexpression of Uif or ectopic expression of a neomorphic form of Uif, Uif*, causes Notch signaling defects in both the wing and the sensory organ precursors. Further experiments suggest that ectopic expression of Uif* inhibits Notch signaling <em>in cis</em> and acts at a step that is dependent on the extracellular domain of Notch. Our results suggest that Uif can alter the accessibility of the Notch extracellular domain to its ligands during Notch activation.</p> <h3>Conclusions/Significance</h3><p>Our study shows that Uif can modulate Notch activity, illustrating the importance of a delicate regulation of this signaling pathway for normal patterning.</p> </div