The role of two members of the Ly6 superfamily in the organization of septate junctions during Drosophila melanogaster development

La superfamille Ly6 est une famille de gènes présente dans le génome de la plupart des métazoaires, y compris l'Humain. Ces gènes codent principalement pour des glycoprotéines attachées à la membrane par une ancre GPI (Glycosylphosphatidylinositol), mais aussi pour des ligands solubles. Les membres de cette famille se caractérisent par la présence d'un domaine extracellulaire, appelé domaine Ly6, dont la structuration est assurée par 8 à 10 cystéines présents dans des positions conservées. La grande variabilité du reste de la séquence des protéines Ly6 leur permet d'exercer des fonctions divergentes, hautement spécialisées. Même si certaines fonctions des protéines Ly6 ont été élucidées chez divers organismes, on connait très peu sur leurs rôles potentiels pendant le développement animal. Durant ma thèse, j'ai utilisé la drosophile comme système modèle afin d'étendre nos connaissances sur les fonctions de ces protéines dans un contexte développemental. Notre travail a permis d'identifier l'ensemble des 36 membres de la superfamille Ly6 chez Drosophila melanogaster. J'ai étudié plus particulièrement le rôle de deux membres de cette famille au cours du développement. La caractérisation fonctionnelle des mutants pour ces deux gènes, appelés boudin et coiled, a montré qu'ils sont tous les deux requis pour la morphogenèse trachéale et l'organisation des jonctions septées dans les tissus épithéliaux. Les jonctions septées sont des structures d'adhérence cellulaire, analogues aux jonctions serrées des vertébrés. Elles permettent aux épithéliums d'exercer leur fonction de barrière paracellulaire qui régule le passage des solutés et des ions. Les jonctions septées de la drosophile sont aussi similaires aux jonctions paranodales des vertébrés, présentes au contact entre axones et cellules de Schwann, et nos résultats montrent que boudin et coiled sont également requis pour l'organisation des jonctions septées dans le système nerveux. D'autre part, nous avons montré que la protéine Boudin est capable de diffuser d'une cellule à l'autre pour réguler la formation des jonctions septées. Ce mode d'action "cellulaire non-autonome " n'avait jamais été décrit pour des protéines qui participent à l'organisation des jonctions septées. L'étude du mode de diffusion et du trafficking de Boudin permettra de mieux comprendre comment cette protéine exerce sa fonction. Enfin, un autre challenge sera d'identifier les partenaires fonctionnels de Boudin et Coiled pour élucider les mécanismes moléculaires par lesquels ces protéines contrôlent le maintien et l'organisation des jonctions septées.The Ly6 superfamily is a large family of genes present in most metazoan genomes, including 45 members in Humans. These genes mainly encode for extracellular glycoproteins attached to the cell membrane by a GPI anchor (Glycosylphosphatidylinositol), but also for soluble ligands. They are characterized by the presence of an extracellular domain, called Ly6 domain, whose structure is provided by 8 to 10 cysteines present in conserved positions. The great variability exhibited by the Ly6 primary sequences allows these proteins to exert highly divergent roles. Although their function has been elucidated in various organisms, we still know very little about their potential roles during animal development. During my PhD, I used the Drosophila model system to extend our knowledge about the functions of these proteins in a developmental context. Our work has permitted the identification of 36 members of the Ly6 superfamily in Drosophila melanogaster, and I have characterized at a functional level two of these genes during development. Phenotypic analysis of mutants for these two genes, called boudin and coiled, has shown that both of them are required for tracheal morphogenesis and organization of septate junctions in epithelial tissues. Septate junctions are cell adhesion structures analogous to vertebrate tight junctions. They allow epithelia to perform their barrier function and regulate the passage of solutes and ions through the paracellular space. Septate junctions in Drosophila are similar to the vertebrate paranodal junctions, present at the contact between axons and Schwann cells, and our results show that boudin and coiled are also required for the organization of septate junctions in the fly nervous system. On the other hand, we have shown that the protein Boudin is able to diffuse from one cell to another to regulate septate junction formation. This non cell autonomous mode of action had never been described for proteins involved in septate junction organization. Studying the diffusion mechanisms and the trafficking of Boudin is important to better understand how this protein performs its function. Finally, another challenge will be to identify functional partners of Boudin and Coiled to elucidate the molecular mechanisms by which these proteins control the maintenance and the organization of septate junction structures

Multispecies Analysis of Expression Pattern Diversification in the Recently Expanded Insect Ly6 Gene Family

The deposited article version is a "MBE Advance Access" published on March 4, 2015" provided by Oxford University Press, and it contains attached the supplementary materials within the pdf.The deposited article is a post-print version.Some supplementary materials are not present in the uploaded version of the article.Gene families often consist of members with diverse expression domains reflecting their functions in a wide variety of tissues. However, how the expression of individual members, and thus their tissue-specific functions, diversified during the course of gene family expansion is not well understood. In this study, we approached this question through the analysis of the duplication history and transcriptional evolution of a rapidly expanding subfamily of insect Ly6 genes. We analyzed different insect genomes and identified seven Ly6 genes that have originated from a single ancestor through sequential duplication within the higher Diptera. We then determined how the original embryonic expression pattern of the founding gene diversified by characterizing its tissue-specific expression in the beetle Tribolium castaneum, the butterfly Bicyclus anynana, and the mosquito Anopheles stephensi and those of its duplicates in three higher dipteran species, representing various stages of the duplication history (Megaselia abdita, Ceratitis capitata, and Drosophila melanogaster). Our results revealed that frequent neofunctionalization episodes contributed to the increased expression breadth of this subfamily and that these events occurred after duplication and speciation events at comparable frequencies. In addition, at each duplication node, we consistently found asymmetric expression divergence. One paralog inherited most of the tissue-specificities of the founder gene, whereas the other paralog evolved drastically reduced expression domains. Our approach attests to the power of combining a well-established duplication history with a comprehensive coverage of representative species in acquiring unequivocal information about the dynamics of gene expression evolution in gene families.IAEA Seibersdorf (Austria); USDA; Duke University; McGill University; DRGC (Kyoto); DSHB (Iowa); Toulouse RIO Imaging Platform; Fundação para a Ciência e a Tecnologia grants: (SFRH/BPD/75139/2010, ANR-13-ISV7-0001-01, ANR-13-ISV7-0001-02, FCT-ANR/BIA-ANM/0003/2013); Fundação Calouste Gulbenkian; Instituto Gulbenkian de Ciência; Agence Nationale de la Recherche (ANR).info:eu-repo/semantics/publishedVersio

The Ly6 Protein Coiled Is Required for Septate Junction and Blood Brain Barrier Organisation in Drosophila

Background: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. Methodology/Principal Findings: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. Conclusion/Significance: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose tha

Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes

Light on life: immunoscore immune-checkpoint, a predictor of immunotherapy response

ABSTRACTIn the last decade, a plethora of immunotherapeutic strategies have been designed to modulate the tumor immune microenvironment. In particular, immune checkpoint (IC) blockade therapies present the most promising advances made in cancer treatment in recent years. In non-small cell lung cancer (NSCLC), biomarkers predicting response to IC treatments are currently lacking. We have recently identified Immunoscore-IC, a powerful biomarker that predicts the efficiency of immune-checkpoint inhibitors (ICIs) in NSCLC patients. Immunoscore-IC is an in vitro diagnostic assay that quantifies densities of PD-L1+, CD8+ cells, and distances between CD8+ and PD-L1+ cells in the tumor microenvironment. Immunoscore-IC can classify responder vs non-responder NSCLC patients for ICIs therapy and is revealed as a promising predictive marker of response to anti-PD-1/PD-L1 immunotherapy in these patients. Immunoscore-IC has also shown a significant predictive value, superior to the currently used PD-L1 marker. In colorectal cancer (CRC), the addition of atezolizumab to first-line FOLFOXIRI plus bevacizumab improved progression-free survival (PFS) in patients with previously untreated metastatic CRC. In the AtezoTRIBE trial, Immunoscore-IC emerged as the first biomarker with robust predictive value in stratifying pMMR metastatic CRC patients who critically benefit from checkpoint inhibitors. Thus, Immunoscore-IC could be a universal biomarker to predict response to PD-1/PD-L1 checkpoint inhibitor immunotherapy across multiple cancer indications. Therefore, cancer patient stratification (by Immunoscore-IC), based on the presence of T lymphocytes and PD-L1 potentially provides support for clinicians to guide them through combination cancer treatment decisions

FLAG-Cold subcellular localisation in the wing epithelium and in S2 cells.

<p>(A–D″) Confocal images of third larval instar wing discs expressing the FLAG-Cold protein (shown in red in left panels, greyscale in the mid panels) in the <i>apGAL4</i> domain. The A–A″ and C–C″ show x-y planar views of the apical part of the epithelium, whereas D–D″ shows a more basal region and B–B″ a z-section. Accumulation of FLAG-Cold was seen in a cell apical region containing weak levels of Nrg-GFP (B–B″ and C–C″ white arrowheads) and no Pdi-GFP (A–A″, white arrowheads). Regions containing high levels of Nrg-GFP and corresponding to the SJ did not show FLAG-Cold accumulation (B–B″ and C–C″, red arrows). FLAG-Cold was also seen in internal vesicles (D–D″, white arrows). E–F″ Confocal images of S2 cells expressing FLAG-Cold (red in left panel, greyscale in the mid panel) and Moesin-GFP (GFP fluorescence shown in green, left panel). In permeabilised cells (E–E″), both FLAG-Cold and Moesin-GFP were detected by antibodies in the cell interior and at the membrane (anti-GFP shown in blue, left panel and in greyscale, right panel). In these conditions, we also observed internal vesicles accumulating FLAG-Cold (open arrowheads). In non permeabilised cells (F–F″), FLAG-Cold was detected at the membrane (arrowhead), whereas the cell interior was not accessible to the antibodies.</p

Kune is endocytosed and degraded during epidermal morphogenesis.

<p>(A-H) Confocal views of the ventral epidermis corresponding to live wild type stage 14 (A,C,E,G) or stage 16 (B,D,F,H) embryos expressing mCheKune (magenta and b/w in bottom panels) and EGFPKune (A,B), YFP-Rab5 (C,D), YFP-Rab7 (E,F) or Lamp1-YFP (G,H), all shown in green (top panels) and in b/w (middle panels). Vesicles containing both tagged Kune versions (labelled with circles) are visible in both stages (A,B). mCheKune is also detected in YFP-Rab5 early endosomes (C,D), YFP-Rab7 late endosomes (E,F) and Lamp-1-YFP lysosomes (G,H). Scale bar: 10 μm.</p

Boudin trafficking reveals the dynamic internalisation of specific septate junction components in <i>Drosophila</i>

<div><p>The maintenance of paracellular barriers in invertebrate epithelia depends on the integrity of specific cell adhesion structures known as septate junctions (SJ). Multiple studies in <i>Drosophila</i> have revealed that these junctions have a stereotyped architecture resulting from the association in the lateral membrane of a large number of components. However, little is known about the dynamic organisation adopted by these multi-protein complexes in living tissues. We have used live imaging techniques to show that the Ly6 protein Boudin is a component of these adhesion junctions and can diffuse systemically to associate with the SJ of distant cells. We also observe that this protein and the claudin Kune-kune are endocytosed in epidermal cells during embryogenesis. Our data reveal that the SJ contain a set of components exhibiting a high membrane turnover, a feature that could contribute in a tissue-specific manner to the morphogenetic plasticity of these adhesion structures.</p></div

Structure and expression pattern of the <i>cold</i> gene.

<p>(A) <i>coiled/CG2813</i> genomic region, showing the localisation of the <i>cold PBac</i> insertions used in this work. (B). Protein sequence alignment corresponding to the Ly6 domain of different insect Cold orthologues. Invariant residues are shown in red. The 12 invariant cysteines and C-terminal asparagines, are marked respectively by red circles and a red square. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017763#s4" target="_blank">Materials and Methods</a> for the species full names. (C,E,F) <i>In situ</i> hybridisation showing the <i>cold</i> mRNA distribution at different embryonic stages. Note at stage 5 an uniform signal corresponding to a maternal transcript and progressive accumulation of <i>cold</i> mRNA in the foregut and hindgut primordia at stage 11 (arrows), and in foregut, hindgut (arrows), trachea and salivary gland (arrowheads) of stage 13 embryos. (D) No signal was observed using a <i>cold</i> sense RNA probe. (G,H) Stage 13 <i>PBac^1001277</i> embryos expressing a YFP-Cold fusion protein and stained for FasIII. The YFP signal was detected in the trachea and epidermis (white arrows) and in salivary gland, fore and hindgut (white arrowheads). (I) Ventral cord of a stage 16 embryo revealing <i>cold</i> transcript accumulation in cells associated with the nerve tracks (arrows). (J,K) Stage 16 embryos stained for Repo (red) and the YFP-Cold fusion protein (green), which is expressed in a subset of glial cells placed in the ventral cord surface (arrowheads) and associated with the exiting nerves (arrows).</p

Exogenous mCheBou is captured and endocytosed in the embryonic epidermis.

<p>(A) Confocal optical sections showing the distribution of mCheBou in the ventral epidermis of a live stage 14 <i>bou</i> rescued embryo. After photobleaching (time 0) of an area delimited by a black square, the mCheBou signal gradually reappears in the cell cortex. Cells entirely bleached at time 0 are labelled with coloured dots, to facilitate their visualisation. (B) Confocal image showing the mCheBou distribution in the ventral epidermis of a live stage 14 wild type embryo, imaged using the same parameters as the rescued embryos shown in A. Only a faint signal is seen in the cell contours (red arrow). (C,D) mCheBou distribution (green in top panels, b/w in middle panels) in the ventral epidermis of live stage 14 <i>bou</i> rescued embryos expressing ubiquitously the early (YFP-Rab5, C) or late (YFP-Rab7, D) endosome markers (shown in magenta in top panels and on b/w, bottom panels). Examples of vesicles positive for mCheBou and each marker are labelled with circles. Scale bars: 10 μm.</p