<p>Polarization is important for the function and morphology of many different cell types. The keys regulators of polarity in eukaryotes are the Rho-family GTPases. In the budding yeast Saccharomyces cerevisiae, which must polarize in order to bud and to mate, the master regulator is the highly conserved Rho GTPase, Cdc42. During polarity establishment, active Cdc42 accumulates at a site on the plasma membrane characterizing the “front” of the cell where the bud will emerge. The orientation of polarization is guided by upstream cues that dictate the site of Cdc42 clustering. However, in the absence of upstream cues, yeast can still polarize in a random direction during symmetry breaking. Symmetry breaking suggests cells possess an autocatalytic polarization mechanism that can amplify stochastic fluctuations of polarity proteins through a positive feedback mechanism.</p><p> Two different positive feedback mechanisms have been proposed to polarize Cdc42 in budding yeast. One model posits that Cdc42 activation must be localized to a site at the plasma membrane. Another model posits that Cdc42 delivery must be localized to a particular site at the plasma membrane. Although both mechanisms could work in parallel to polarize Cdc42, it is unclear which mechanism is critical to polarity establishment. We directly tested the predictions of the two positive feedback models using genetics and live microscopy. We found that localized Cdc42 activation is necessary for polarity establishment.</p><p> While this explains how active Cdc42 localizes to a particular site at the plasma membrane, it does not address how Cdc42 concentrates at that site. Several different mechanisms have been proposed to concentrate Cdc42. The GDI can extract Cdc42 from membranes and selective mobilize GDP-Cdc42 in the cytoplasm. It was proposed that selectively mobilizing GDP-Cdc42 in combination with local activation could locally concentrate total Cdc42 at the polarity site. Although the GDI is important for rapid Cdc42 accumulation at the polarity site, it is not essential to Cdc42 concentration. It was proposed that delivery of Cdc42 by actin-mediated vesicle can act as a backup pathway to concentrate Cdc42. However, we found no evidence for an actin-dependent concentrating pathway. Live microscopy experiments reveal that prenylated proteins are not restricted to membranes, and can enter the cytoplasm. We found that the GDI-independent concentrating pathway still requires Cdc42 to exchange between the plasma membrane and the cytoplasm, which is supported by computational modeling. In the absence of the GDI, we found that Cdc42 GAP became essential for polarization. We propose that the GAP limits GTP-Cdc42 leak into the cytoplasm, which would be prohibitive to Cdc42 polarization.</p> / Dissertation

Les cellules positionnent leur plan de division de manière précise et prévisible. En particulier au tout début de l’embryogenèse, la cellule-œuf suit un patron de clivage extrêmement reproductible, mais néanmoins sensible aux perturbations (manipulation de la forme de la cellule,…), ce qui suggère une plasticité intrinsèque du système. Au cours de ma thèse, je me suis intéressée aux signaux qui déterminent la position des plans de division embryonnaires, et à leur compétition. Dans un premier temps, j’ai développé un modèle pour prédire le positionnement du plan de division à partir de la forme de la cellule, et de la présence éventuelle de polarité maternelle à la membrane ou d’une distribution inhomogène de yolk/organelles dans le cytoplasme. Ce modèle est basé sur les forces de traction exercées par les microtubules des astres interphasiques sur le fuseau mitotique/noyau. Sous l’hypothèse que ces forces dépendent de la longueur des microtubules (dynéine dans le cytoplasme) et sont modulées par la polarité membranaire, il est alors possible de trouver la position d’équilibre du fuseau, qui détermine le futur plan de division. J’ai également reproduit les formes et réarrangements des cellules (blastomères) dans l’embryon après la division, à l’aide d’un programme (The Surface Evolver) qui minimise l’énergie de surface sous différentes contraintes : ici les volumes, tensions de surface et éventuels confinements. En bouclant la génération des formes des blastomères avec la prédiction de leurs divisions (les formes permettent de prédire la division, qui permet de générer les formes des cellules filles, etc…), j’ai pu reproduire de manière quantitative quatre patrons de clivage représentatifs (poisson-zèbre, xenope, oursin, ascidie), jusqu’au stade 8 à 16 cellules, in silico. J’ai également testé le modèle sur des expériences classiques de perturbation dans ces quatre systèmes (Hertwig, Hörstadius, ablation de la polarité,…), et reproduit les observations de la littérature. Cette première partie suggère que ces systèmes sont auto-organisés et que la détermination du plan de division dépend principalement d’un nombre restreint de signaux. Dans un second temps, j’ai cherché à caractériser la compétition entre les signaux de forme et de polarité maternelle chez l’embryon d’oursin, de manière quantitative. Ce projet comprend une part importante d’imagerie 3D (position des centrosomes et division, polarité, forme des blastomères), ainsi que des expériences visant à tester le rôle de la forme/taille des blastomères et de la polarité (séparation des blastomères, microchambres de différentes formes, inhibition de la polarité,…). Les résultats obtenus sont comparés aux prédictions du modèle, cette fois basées sur la forme imagée des blastomères. Ces résultats expérimentaux confirment les hypothèses de l’étude in silico, et permettent d’évaluer la robustesse du système biologique pour affiner le modèle. / Cells position their cleavage plane in a precise and predictable way. In particular, during the early embryogenesis, the cleavage pattern of the egg cell is extremely reproducible, yet sensitive to perturbation (shape manipulation,…), which suggests an intrinsic plasticity of the system. My PhD project is about the signals that determine the positions of the cleavage planes in the embryo, and their competition. First, I developed a model to predict division plane positioning from cell shape and possible additional cortical maternal polarity or inhomogeneous yolk/organelles distribution within the cytoplasm. This model is based on pulling forces exerted by interphase astral microtubules on the mitotic spindle/nucleus. Under the hypothesis that these forces depend on microtubule lengths (dynein in the cytoplasm), and are modulated by cortical polarity, it is then possible to find the equilibrium position of the spindle, that sets the future division plane. In addition, I reproduced the shapes and rearrangement of cells (blastomeres) within the embryo, with a program (The Surface Evolver) that minimizes surface energy under various constraints : here cell volumes, surface tensions and possible confinements. The modeling framework I used consisted in a loop between cell shape generation and division plane prediction (cell shape allows to predict cell division, that gives the daughter cells volumes and positions to generate the next cell shapes, and so on…). I could quantitatively reproduce four representative cleavage patterns (zebrafish, xenopus, sea urchin, ascidian), up to the 8 to 16-cell stage, in silico. I also tested the model on classic perturbation experiments in these four systems (Hertwig, Hörstadius, polarity ablation,…), and reproduced the observations of the literature. This first part suggests an auto-organization of these systems, and that the determination of the cleavage plane mainly depends on a limited number of signals. Second, I aimed at characterizing the competition between shape and maternal polarity cues, in a quantitative manner. This project comprises 3D imaging (positions of the centrosomes and division planes, polarity, blastomere shape), as well as experiments assessing the roles of blastomere shape/size and of polarity (blastomere separation, microchambers of different shapes, polarity inhibition,…). The results are compared to the predictions of the model, that now inputs the imaged blastomere shapes. These experimental results confirm the hypotheses of the in silico study, and allow assessing the robustness of the biological system to refine the model.

京都大学 / 新制・論文博士 / 博士(生命科学) / 乙第13431号 / 論生博第26号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 石川 冬木, 教授 原田 浩 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

As an organism develops, multiple cellular processes need to occur in order to specify and organize tissue. One essential process is the establishment of cell polarity, which drives cell fate specification and stem cell differentiation. Another key process is programmed cell death, which is important for tissue remodeling and clearing damaged or diseased cells from the body. A loss in cell polarity can lead to defects in tissue organization and carcinogenesis. Defects in programmed cell death can lead to autoimmune diseases and cancer. However, hyperactive programmed cell death can lead to neurodegeneration. The Drosophila ovary, which is composed of germline and somatic cells, is an excellent model to study both cell polarity and cell death. In the germ cells, oocyte fate is specified and maintained through the asymmetric localization of cell cycle and cell polarity RNAs, proteins, and organelles, such as mitochondria, to and within the oocyte. Additionally the somatic follicle cells, which surround the germ cells, require a specific apical-basal polarity to function. During oogenesis, programmed cell death can be induced within the ovary to prevent oogenesis from maturing under low nutrient, high stress or crowded conditions. When this occurs, the germline is cleared from the ovary by a process known as engulfment. Somatic follicle cells surrounding the germline synchronously enlarge and engulf the corpses of the dying germline cells. It is unknown what triggers the enlargement of the follicle cells. Previous research has shown that the apical side of a follicle cell is heavily marked by cell polarity proteins, to specify the apical side away from the lateral and basal sides. Since many important genes regulating both cell polarity and engulfment are conserved between Drosophila and other eukaryotes, we can study the establishment and maintenance of cell polarity and its role during engulfment to obtain a better understanding of these processes in mammals and their relevance to diseases. This dissertation investigates the role of cell polarity in both the specification of oocyte cell fate, and the organization and enlargement of the follicle cells during engulfment in the ovary. / 2016-11-03T00:00:00Z

Notch est le récepteur d’une voie de communication intercellulaire, conservée au cours de l’évolution et impliquée dans de nombreux processus développementaux. Chez Drosophila melanogaster, la spécification et la division des précurseurs des organes sensoriels (SOPs) sont gouvernées par l’activation différentielle de la voie de signalisation Notch. Cette activation est dépendante de l’interaction entre le récepteur Notch et les ligands Delta/Serrate et LAG-2. Cette interaction favorise le clivage protéolytique du récepteur Notch puis la libération et la translocation du domaine intracellulaire dans le noyau de la cellule receveuse du signal. L’activation de Notch est étroitement régulée dans le temps et dans l’espace et est sous le contrôle du trafic intracellulaire. Toutefois, la localisation exacte de l’interaction entre le ligand et le récepteur demeure encore débattue.Précédemment, la protéine Stratum, prédite pour avoir un rôle de facteur d’échange nucléotidique (GEF), fut identifiée comme régulateur de la voie de signalisation Notch. Ici, nous montrons que la perte de Stratum induit des phénotypes Notch associés à une délocalisation du co-facteur de Notch, Sanpodo, au pôle apical des cellules et dans le réseau trans- golgien, avec Notch et Delta. De plus, nous montrons que Rab8 est délocalisée en absence de Stratum et la perte de Rab8 récapitule les phénotypes Notch observés dans le mutant strat. Ensemble, nous résultats indiquent que Stratum et Rab8 régulent la voie de signalisation Notch en contrôlant à la fois le tri et le transport polarisé de Notch, Sanpodo et Delta à la sortie de l’appareil de Golgi. / Notch is the receptor of an evolutionarily conserved intercellular communication pathway, involved in numerous developmental processes. In Drosophila melanogaster, the specification and the division of sensory organ precursors (SOPs) are governed by the differential activation of the Notch signalling pathway. This activation depends on the interaction between the Notch receptor and its ligands Delta/Serrate and LAG-2. This interaction induces the proteolytic cleavage of the Notch receptor, the release and the translocation of the intracellular domain in the nucleus of the signal-receiving cell. The Notch activation is tightly regulated in time and in space and is controlled by intracellular trafficking. However, the exact localisation of the interaction remains debated.Previously, Stratum, predicted to be a guanine exchange factor (GEF), was identified as a regulator of the Notch signalling pathway. Here we show that the loss of Stratum induces Notch phenotypes associated with a mislocalization of the Notch co- factor, Sanpodo, at the apical pole of cells and in the trans-golgi network, with Notch and Delta. Moreover we show that Rab8 is mislocalized in the absence of Stratum and the loss of Rab8 recapitules Notch phenotypes observed in the strat mutant. Together our results indicate that Stratum and Rab8 regulate the Notch signalling pathway by controlling both the sorting and the transport of Notch, Sanpodo and Delta at the exit of the Golgi apparatus.

In several human cancers, ErbB2 over-expression facilitates the formation of constitutively active homodimers resistant to internalization which results in progressive signal amplification from the receptor, conducive to cell survival, proliferation, or metastasis. Here we report on studies of the influence of ErbB2 over-expression on localization and signaling in polarized Caco-2 and MDCK cells, two established models to study molecular trafficking. In these cells, ErbB2 is not over-expressed and shares basolateral localization with ErbB3. Over-expression of ErbB2 by transient transfection resulted in partial separation of the receptors by relocalization of ErbB2, but not ErbB3, to the apical surface, as shown by biotinylation of the apical or basolateral surfaces. These results were confirmed by immunofluorescence and confocal microscopy. Polarity controls indicated that the relocalization of ErbB2 is not the result of depolarization of the cells. Biotinylation and confocal microscopy also showed that apical, but not basolateral ErbB2 is activated at tyrosine 1139. This phosphotyrosine binds adaptor protein Grb2, as confirmed by immunoprecipitation. However, we found that it does not initiate the canonical Grb2-Ras-Raf-Erk pathway. Instead, our data supports the activation of a survival pathway via Bcl-2. The effects of ErbB2 over-expression were abrogated by the humanized anti-ErbB2 monoclonal antibody Herceptin added only from the apical side. The ability of apical ErbB2 to initiate an altered downstream cascade suggests that subcellular localization of the receptor plays an important role in regulating ErbB2 signaling in polarized epithelia.

Indiana University-Purdue University Indianapolis (IUPUI) / Glioblastoma is a lethal cancer that arises from support cells in the nervous system and kills around 20,000 people in the United States each year. While much is known about the highly malignant primary glioblastoma, the natural history of lower grade glioma (LGG) is less understood. While the majority of LGGs are initiated by a mutation in isocitrate dehydrogenase, the events leading to their malignant progression into a grade IV tumor are not known. Analysis of primary tumor sample data has revealed that low transcript levels of Angiomotin-like-1 (AmotL1) strongly associate with poor outcomes of patients with these cancers. Follow-up RNA-sequencing of human embryonic astrocytes with AmotL1 silencing revealed the downregulation of many transcripts that encode proteins mediating gap junctions (GJ) between astrocytes, especially connexin-43 (Cx43). Cx43 protein oligomerizes to form functional channels comprising the astrocytic GJ. AmotL1 knockdown through RNA interference decreases Cx43 transcript and protein levels while increasing its distribution to GJs. This suggests increased GJ formation and intercellular communication, as similar localization patterns are observed in differentiated astrocytes. Astrocytes with AmotL1 knockdown also display a pronounced pancake-like morphology, suggesting that the actin cytoskeleton is affected. Imaging reveals that cells with reduced AmotL1 have characteristic losses in both stress fibers and focal actin under the cell body but notable increases in cortical F-actin. Consistent with previous studies, AmotL1 may promote increases in the number and thickness of F-actin fibers. Because actin binding to related angiomotins is inhibited by phosphorylation from the LATs kinases, I define the effects of expressing wildtype AmotL1 versus mutants that mimic or prevent phosphorylation by LATs1/2. Interestingly, expression of AmotL1 S262D in combination with NEDD4-1, a ubiquitin ligase, results in a profound loss of actin stress fibers. Dependence on NEDD4-1 suggests that this phenotype is due to the induced degradation of proteins that promote F-actin, e.g. RhoA. These results directly support a model in which phosphorylated AmotL1 specifically inhibits F-actin formation as opposed to unphosphorylated AmotL1 which is known to promote stress fiber formation. Thus, in addition to regulating polarity and YAP/TAZ transcriptional co-activators, AmotL1 plays major functions in dictating cellular F-actin dynamics. / 2021-01-01

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20213号 / 理博第4298号 / 新制||理||1617(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)講師 嶋田 知生, 教授 長谷 あきら, 教授 鹿内 利治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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