Signalisation cellulaire | EPFL Graph Search

La signalisation cellulaire est un système complexe de communication qui régit les processus fondamentaux des cellules et coordonne leur activité. La capacité des cellules à percevoir leur micro-environnement et à y répondre correctement est à la base de leur développement et de celui des organismes multicellulaires, de la cicatrisation et du système immunitaire, ainsi que de l'homéostasie tissulaire normale. Des dysfonctionnements dans le traitement de l'information cellulaire peuvent être responsables de maladies telles que le cancer, les maladies auto-immunes et le diabète. L'allostérie à grande distance est souvent un aspect important de ces processus. L'approche traditionnelle de la biologie a mis l'accent sur l'étude des aspects spécifiques aux différentes voies de signalisation cellulaire, tandis que les recherches en biologie des systèmes visent à mieux comprendre la structure sous-jacente aux réseaux de signalisation cellulaire et l'impact des modifications de ces réseaux sur

Notch Signaling in Pigment Cells and Generation of Mouse Models for Ocular Diseases

Bhushan Sarode

Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival and differentiation. In melanocytes, Notch signaling is essential for the maintenance of melanocytes and the melanocyte stem cell population. Whereas, in pigmented eye structures, specifically in ciliary body and iris, the role of Notch signaling in their structure and their development is largely unknown. In the first part, I have tried to identify possible Notch target genes in melanocytes which might indicate why the melanocyte stem cell population disappears in the absence of Notch. For that purpose, I carried out microarray analysis on Notch deficient melanoblasts and identified putative Notch targets. I have furthermore established mouse melanocyte cell lines from mice which could be used as a valuable tool to study melanocyte biology. In the second part, I have elucidated the functional significance of the Notch cascade in pigmented eye structures. The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intraocular pressure and regulate exposure of the internal eye structures to light respectively. In this part I have analyzed the role of the Notch signaling by performing both loss- and gain-of-function experiments for Notch in the murine ocular pigmented epithelium using Mart-1::Cre mice. Loss of canonical Notch2 signaling results in normal iris development but ciliary body aplasia. In contrast, Notch gain-of-function induces the reciprocal phenotype and causes aniridia and ciliary body hyperplasia. The loss and gain-of-function models I have generated develop ocular hypotony and ocular hypertension respectively, mimicking the human diseases phthisis bulbi and closed angle glaucoma. Importantly, the ocular hypertension that occurs following Notch overexpression is responsive to hypotensive drugs, and thus can serve as a platform to evaluate novel hypotensive agents that may be used to treat closed angle glaucoma. In summary, the present study has identified novel putative Notch target genes which will provide us information to study the pigmentation and related disorder. The data obtained in the second chapter provide novel insight into the role of Notch signaling during development of the ciliary body and iris and thus represent a conceptual advance in our understanding of how pigmented structures of the eye are formed. In addition, the genetic models presented in this study represent robust tools with which to study the etiology, pathology and treatment of diseases related to aberrant intraocular pressure, such as phthisis bulbi and closed angle glaucoma.

EPFL2013

Identification of genetic interactors of ypa2 in Schizosaccharomyces pombe

Manuela Moraru

In this study, the fission yeast Schizosaccharomyces pombe was used as a model system to study the cellular signaling that underlies cell cycle progression. Phosphorylation plays an important part in the regulation of this process. Kinases catalyze the transfer of a phosphate group to a substrate, which may regulate its activity. Protein phosphatases oppose the activity of protein kinases; the balance of their activities regulates signaling flux in many signaling pathways. The Septation Initiation Network (SIN) is a signaling cascade that regulates cytokinesis in fission yeast. SIN signaling is triggered by a GTPase and is transduced by three protein kinases; phosphorylation plays an important role in controlling SIN signalling. Numerous genetic screens and biochemical analyses have identified phosphatases and kinases that regulate the SIN. The protein phosphatases Calcineurin/Protein Phosphatase 2B (PP2B) and Flp1p promote SIN signaling, while PP2A is a SIN inhibitor. PP2A is conserved and is activated by the chaperone Phosphatase Two A Phosphatase Activator (PTPA). S. pombe has two PTPA-related genes, ypa1 and ypa2; the deletion mutant of the latter rescues SIN mutants which cannot undergo cytokinesis, consistent with PP2A opposing SIN signaling. The phenotype of the deletion mutant of ypa2, indicates that it is involved in the control of cell polarity, cell growth, mitotic commitment and cytokinesis. In this study, we characterized the hypomorphic allele ypa2-S2 which rescues SIN mutants, but is otherwise largely normal. ypa2-s2 was used as bait in a synthetic genetic array screen, to identify genes whose products compensate for reduced function of Ypa2p. A large number of hits were identified, of which approximately ninety percent are novel genetic interactors of ypa2. Validation studies indicated that eighty percent of the interactions seen in the high-throughput screen can be reconstructed. The identified genes regulate several biological processes, including signal transduction and protein phosphorylation. This prompted us to further characterize the roles of Ckb1p and Flp1p, in regulating cytokinesis signaling. Ckb1p is a regulatory subunit for Casein Kinase II (CK II), which was previously implicated in polar growth. The phenotype of the mutant ypa2-S2 ckb1-â indicates that Ckb1p, similar to Ypa2p, contributes to the regulation of mitotic commitment and cytokinesis signaling. Furthermore, ckb1-â showed negative genetic interaction with PP2A and SIN mutants. Flp1p is a protein phosphatase that was reported to regulate mitotic commitment and to promote SIN signaling. The double mutant between ypa2-S2 and flp1-â showed cell separation defects and phenotypic analysis of double mutants between flp1-â and PP2A mutants indicates that these phosphatases cooperate in cell separation. Overall, this work has uncovered novel facets of the regulation of cytokinesis in S. pombe, implicating CK II in controlling SIN signaling, and uncovering cooperative interactions between different phosphatases in controlling cytokinesis.

EPFL2016

Activin Signalling in Human Melanoma Cells

Susanna Eveliina Kallioinen

Melanoma, the most fatal of all skin cancers, by unknown mechanisms overcomes the cytostatic effects of TGFβ, while maintaining the TGFβ signaling needed for its tumor promoting functions. Besides TGFβ, ectopic induction of the TGFβ family member Nodal has been reported in human melanoma to correlate with progression to metastatic growth. The same authors reported that a Smad2 morpholino antisense oligonucleotide delays Nodal-induced tumor growth of the aggressive melanoma cell line C8161 in xenograft assays. To investigate the underlying molecular mechanism, we compared C8161 melanoma cells with isogenic poorly metastatic C81-61 cells reportedly lacking Nodal expression. Unexpectedly, reverse transcription PCR, Western blot analysis using specific antibodies, and activity assays revealed that metastatic C8161 cells and many other human melanoma cell lines and tumors do not express Nodal, but frequently upregulate the related ligand Activin A. However, siRNA knockdown of Activin A did not reduce invasiveness of C8161 cells in matrigel, nor did the overexpression of Activin A in C81-61 cells increase it. The activity of Smad2,3 signaling was evaluated in the melanoma cell lines by a seriesWestern blot and reporter assays. The Smad signaling pathway was found to be attenuated in both C8161 and C81-61 cell lines, as only a small fraction of total Smad2,3 was detected in the nuclear fraction of the melanoma cells. In addition, little Smad3 signaling activity was observed by a Smad3-dependent luciferase reporter. To evaluate paracrine effects, human melanoma cells that do or do not express exogenous Activin A or Nodal were xenografted into nude mice. Neither Activin A nor Nodal enhanced metastatic growth in this assay. Inhibition of Activin A did also not alter metastasis formation. However, in the presence of the human fragment crytallisable region (Fc), Activin A seems to enhanced metastatic growth. Further studies are required to confirm these results, and to find the mechanism by which Activin A enhances metastatic potential.

EPFL2012