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The Cx43 CT mimetic peptide, αCT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a TRITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, αCT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, αCT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1) , and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of αCT1, αCT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of αCT1 in the treatment of vascular edema. / Doctor of Philosophy / Endothelial cells make up blood vessels within the heart and regulate the exchange of fluids between the circulation and heart tissue. In many forms of heart disease, the cardiac endothelium is disrupted, resulting in a damaging leakage and buildup of fluids within the heart. This work explores how a small peptide, derived from a naturally occurring molecule, may help to prevent fluid-associated damage to the heart by stabilizing the blood endothelium.

According to the American Cancer Society, Cancer is the second most common cause of death in the United States, only exceeded by heart disease. Over the past decade, deciphering the complex structure of individual cells and understanding the symptoms of cancer disease has been a highly emphasized research area. The exact cause of Cancer and the genetic heterogeneity that determines the severity of the disease and its response to treatment has been a great challenge. Researchers from the engineering discipline have increasingly made use of recent technological innovations, namely the Atomic Force Microscope (AFM), to better understand cell physics and provide a means for cell biomechanical profiling. The presented work's research objective is to establish a fundamental framework for the development of novel biosensors for cell separation and disease diagnosis. By using AFM nanoindentation, several studies were conducted to identify key distinctions in the trends of cell viscoelasticity between healthy, nontumorigenic cells and their malignant, highly tumorigenic counterparts. The possibility of identifying useful 'biomarkers' was also investigated. Due to the lack of an available human ovarian cell line, experiments were done on a recently developed mouse ovarian surface epithelial (MOSE) cell line, which resembles to human cell characteristics and represents early, intermediate, and late stages of the ovarian cancer. Material properties were extracted via Hertz model contact theory. The experimental results illustrate that the elasticity of late stage MOSE cells were 50% less than that of the early stage. Cell viscosity also decreased by 65% from early to late stage, indicating that the increase in cell deformability directly correlates with increasing levels of malignancy. Various cancer treatment and component-specific drugs were used to identify the causes for the changes in cell biomechanical behavior, depicting that the decrease in the concentration levels of cell structural components, predominantly the actin filament framework, is directly associated with the changes in cell biomechanical property. The investigation of MOSE cells being subject to multiple mechanical loads illustrated that healthy cells react to shear forces by stiffening up to 25% of their original state. On the other hand, cancerous cells are void of such response and at times show signs of decreasing rigidity. Finally, deformation studies on MOSE cancer stem cells have shown that these cells carry a unique elasticity profile among other cell stage phenotypes that could allow for their detection. The results herein carry great potential into contributing to cell separation methods and analysis, furthering the understanding of cell mechanism dynamics. While prior literature emphasizes on the elastic modulus of cells, the study of cell viscosity and other key material properties holds a critical place in the realistic modeling of these complex microstructures. A comprehensive study of individual cells holds a great amount of promise in the development of effective clinical research in the fight against cancer. / Master of Science

Yes / The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner. / This work was supported by SFB 670 and DFG NO 113/22. K.B. was supported by a fellowship from the NRW International Graduate School “From Embryo to Old Age: the Cell Biology and Genetics of Health and Disease” (IGSDHD), Cologne.

Yes / The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics. / Cancer Research UK (to L.M.M. [A17196], R.H.I. [A19257], and S.W.G.T.) and NIH grants P01-GM103723 and P41-EB002025 (to K.M.H.). N.R.P. is supported by a Pancreatic Cancer Research Fund grant (to L.M.M.). Funding to Prof. Rottner by the Deutsche Forschungsgemeinschaft (grant RO2414/3-2).

Yes / Efficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif. / Cancer Research UK grants A15672, A24450, and multidisciplinary grant A20017.

Intensive Forschung hat in den vergangenen Jahrzehnten zu einer sehr detaillierten Charakterisierung des Geschmackssystems der Säugetiere geführt. Dennoch sind mit den bislang eingesetzten Methoden wichtige Fragestellungen unbeantwortet geblieben. Eine dieser Fragen gilt der Unterscheidung von Bitterstoffen. Die Zahl der Substanzen, die für den Menschen bitter schmecken und in Tieren angeborenes Aversionsverhalten auslösen, geht in die Tausende. Diese Substanzen sind sowohl von der chemischen Struktur als auch von ihrer Wirkung auf den Organismus sehr verschieden. Während viele Bitterstoffe potente Gifte darstellen, sind andere in den Mengen, die mit der Nahrung aufgenommen werden, harmlos oder haben sogar positive Effekte auf den Körper. Zwischen diesen Gruppen unterscheiden zu können, wäre für ein Tier von Vorteil. Ein solcher Mechanismus ist jedoch bei Säugetieren nicht bekannt. Das Ziel dieser Arbeit war die Untersuchung der Verarbeitung von Geschmacksinformation in der ersten Station der Geschmacksbahn im Mausgehirn, dem Nucleus tractus solitarii (NTS), mit besonderem Augenmerk auf der Frage nach der Diskriminierung verschiedener Bitterstoffe. Zu diesem Zweck wurde eine neue Untersuchungsmethode für das Geschmackssystem etabliert, die die Nachteile bereits verfügbarer Methoden umgeht und ihre Vorteile kombiniert. Die Arc-catFISH-Methode (cellular compartment analysis of temporal activity by fluorescent in situ hybridization), die die Charakterisierung der Antwort großer Neuronengruppen auf zwei Stimuli erlaubt, wurde zur Untersuchung geschmacksverarbeitender Zellen im NTS angewandt. Im Zuge dieses Projekts wurde erstmals eine stimulusinduzierte Arc-Expression im NTS gezeigt. Die ersten Ergebnisse offenbarten, dass die Arc-Expression im NTS spezifisch nach Stimulation mit Bitterstoffen auftritt und sich die Arc exprimierenden Neurone vornehmlich im gustatorischen Teil des NTS befinden. Dies weist darauf hin, dass Arc-Expression ein Marker für bitterverarbeitende gustatorische Neurone im NTS ist. Nach zweimaliger Stimulation mit Bittersubstanzen konnten überlappende, aber verschiedene Populationen von Neuronen beobachtet werden, die unterschiedlich auf die drei verwendeten Bittersubstanzen Cycloheximid, Chininhydrochlorid und Cucurbitacin I reagierten. Diese Neurone sind vermutlich an der Steuerung von Abwehrreflexen beteiligt und könnten so die Grundlage für divergentes Verhalten gegenüber verschiedenen Bitterstoffen bilden. / Intense research in the past decades has led to a detailed understanding of the mammalian taste system. Some important issues, however, have remained unanswered with the established methods that have been applied so far. One of these questions is whether different bitter substances can be distinguished. There are thousands of compounds which taste bitter to humans and elicit innate aversive behavior in animals. Moreover, these bitter substances are very heterogeneous regarding their structure as well as their effect on the organism. While many bitter tastants are potent poisons, others are harmless or even have beneficial effects in the amounts that are typically ingested. The ability to discriminate between those groups of bitter tastants could be an evolutionary advantage. Such a mechanism, however, is not known for mammals. The aim of this thesis was to study the processing of taste information in the first station of gustatory processing in the mouse brain, the nucleus of the solitary tract (NTS). Of particular interest was the question concerning discrimination of bitter tastants. To this end a new method was established for the taste system combining the advantages of methods used before while circumventing their disadvantages. The Arc catFISH method (cellular compartment analysis of temporal activity by fluorescent in situ hybridization), which allows the characterization of responses of large neuron populations to two stimuli, was used to analyze taste-processing cells in the NTS. In the course of this project a stimulus-induced Arc expression in the NTS was shown for the first time. The results demonstrated that Arc expression in the NTS appears specifically after stimulation with bitter tastants and that the Arc expressing neurons are located primarily in the gustatory part of the NTS. This indicates that Arc expression is a marker for bitter-processing gustatory neurons in the NTS. Upon stimulating twice with bitter compounds, distinct, yet overlapping neuron populations were identified, that reacted differently to the three bitter substances cycloheximide, quinine hydrochloride, and cucurbitacin I. Presumably these neurons are involved in the regulation of aversive reflexes and could form a basis for divergent behavior towards different bitter substances.

Le complexe linker of nucleoskeleton and cytoskeleton (LINC) est un complexe protéique conservé au cours de l’évolution, reliant les compartiments cytoplasmiques et nucléaires au travers la membrane nucléaire. Bien que les données récentes montrent une de ce complexe dans la régulation de la morphologie nucléaire et de la méiose, son implication dans l’organisation de la chromatine a été moins étudié chez les plantes. Le premier objectif de ce travail était de développer un plugin NucleusJ ImageJ dédié à la caractérisation de la morphologie nucléaire et de l’organisation de la chromatine en 3D. NucleusJ calcul 15 paramètres, y compris la forme et la taille des noyaux ainsi que des objets intra-nuclaires et leur position dans le noyau. Une documentation pour ce programme est disponible pour son utilisation, ainsi que des qu’un jeu données de noyaux pour tester ce programme. Plusieurs améliorations sont en cours pour développer une nouvelle version de ce plugin. Dans une deuxième partie de ce travail, des méthodes d’imagerie 3D ont été utilisées pour étudier la morphologie nucléaire et l’organisation de la chromatine dans les noyaux interphasiques chez Arabidopsis thaliana dans lequel les domaines d’htrochromatique sont groupés en régions detectable appelés chromocentres. Ces chromocentres forment un environnement répressif contribuant la rpression transcriptionnelle de séquences répétées permettant la stabilité du génome. Des mesures quantitatives de la position 3D de chromocentres dans le noyau montrent que la plupart chromocentres sont situés proximité de la périphérie du noyau, mais que cette distance peut être modifiée par le volume nucléaire ou dans certains mutants affectant le complexe LINC. Ce complexe LINC est proposé pour contribuer l’organisation de la chromatine et à son positionnement, de plus la mutation de ce complexe est associée une dérégulation l’inactivation de la transcription, ainsi qu’a une décompaction des séquences hétérochromatiques. La dernière partie de ce travail tire profit de séquences gnomiques disponibles et les données de RNA-seq pour explorer l’évolution des protines de la NE chez les plantes. Au Final, le travail réalisé durant cette thèse associe la génétique, la biologie moléculaire, la bioinformatique et de l’imagerie afin de mieux comprendre la contribution de l’enveloppe nucléaire dans l’organisation de la morphologie du noyaux et de la chromatine et suggère l’implication fonctionnelle du complexe LINC dans ces processus. / The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The first aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphology and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with different nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromocentres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC complex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes advantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional nuclear envelope. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes.

Le cœur humain est un organe composé de différents types cellulaires tels que les cardiomyocytes, les fibroblastes, les muscles lisses et les cellules endothéliales. Ces cellules se diversifient grâce à des mécanismes moléculaires spécifiques en acquérant leurs propriétés fonctionnelles spécifiques. L’embryon de Drosophile est un modèle simple et adapté pour étudier la diversification des cellules cardiaques et leurs propriétés spécifiques. Le but du projet est d’améliorer notre connaissance sur les acteurs moléculaires qui contrôlent la diversification des cellules cardiaques. Pour atteindre cet objectif nous avons appliqué la méthode TRAP-rc ("rare cell Translation Ribosome Affinity Purification") suivie du séquençage ARN pour identifier les ARN messagers en cours de traduction spécifiques des cellules cardiaques Tin et Lb (Tin CBs et Lb CBs) à deux stades de développement corrélés avec la morphogenèse du cœur embryonnaire. Dans une première analyse focalisée sur l'analyse des données issues des TRAP-Seq sur cellules Tin nous avons mis en évidence que CAP et MSP-300 sont impliqués dans la migration des cardioblasts pendant la fermeture du cœur. En parallèle, nous avons également identifié deux autres gènes impliqués dans la morphogenèse, kon-tiki et dGrip qui semblent contrôler la cohésion des CBs au cours de la migration. En outre, nous avons trouvé qu'au stade 16, environ 60% des gènes enrichis sont communs entre les populations Tin et Lb. Parmi ces gènes, Src42, sqa et flr participent à la régulation du cytosquelette d'actine et nos analyses ont permis de démontrer qu'ils avaient également des fonctions dans la morphogenèse cardiaque. Nous avons également identifié des groupes de gènes plus spécifiques à chacune des populations ciblées. Une catégorie fonctionnelle fortement associée à la population Lb, comprend les gènes qui régulent l'épissage des ARN messagers et certains de ces gènes semblent être requis au cours de la morphogenèse cardiaque. Enfin, nous avons comparé nos données de TRAP-seq cardiaque avec des données de TRAP-Seq issues du muscle somatique (de l'équipe), et ainsi identifié près de 90 gènes qui présentent des isoformes protéiques spécifiques à chaque tissu notamment impliquées dans la formation de l'unité contractile sarcomérique. Ceci suggère que des mécanismes d'épissage spécifiques sont mis en place dans différents types cellulaires pour moduler les fonctions de certaines protéines musculaires. A travers ce projet, nous avons identifié de nouveaux acteurs généraux de la migration collective des cardioblastes au cours de la fermeture du cœur mais également de nouveaux gènes potentiellement impliqués dans l’acquisition des propriétés spécifiques dessous populations cardiaques Tin et Lb et de tissus musculaires distincts. Nous espérons que les données générées permettront dans le futur de mieux comprendre les mécanismes de la cardiogenèse des vertébrés ainsi que l’étiologie de maladies cardiaques. / Cardiac cells diversification is required for the formation of a functional heart. Human heart is a multi-lineage organ that develops through progressive diversification of progenitors derived from different heart fields. This process is underlined by numerous changes in the expression of a repertory of genes that allow cells to acquire their own identity and functions. The Drosophila embryo is a relatively simple model to study the diversification of cardiac cells and their properties. The goal of this project is to identify the repertories of genes that control the formation of different types of cardiac cells. To reach this objective we applied Translation Ribosome Affinity Purification (TRAP) method followed by RNA sequencing in order to identify mRNA engaged in translation specific to two cardiac cell types (Tinman (Tin) and Labybird (Lb) expressing cells), at two different time windows. We obtained a list of enriched genes for the different types of cardiac cells and time points. In a first part, we focused our attention on the Tindatasets and found that two genes, CAP and Msp300, are involved in cardioblasts migration during the heart closure. Then we identified two other candidate genes kontiki and dGrip that seem to contribute to maintain cohesion between CBs during heartmorphogenesis. Moreover by comparing our spatial datasets, we found that for the same time point, around 60% of Tin CBs enriched genes are common with Lb CBs enriched population and within this group we identified evolutionary conserved genes such as Src42, flr and sqa known to be involved in the cytoskeleton organization and in the actinpolymerization and depolymerisation. Our premiminary analyses show that they seem to be required for correct cardiac morphogenesis. We also identified sets of genes more specific for each cardiac cell population. Indeed, Lb CBs datasets show that in early stage there is the enrichment of genes mostly involved in transcriptional regulation and RNA splicing and some of these genes (prp8 and prp38) are involved in cardiac development. In parallel, we compared our TRAP-Seq dataset in the cardiac system with the TRAP-seqon muscle cells, and identified close to 90 genes that present cardiac or muscular specific isoforms. It is known that the alternative splicing, by increasing proteins diversity, contributes to the acquisition of specific cell properties. Furthermore, some cardiomyopathies are associated to defects in the alternative splicing of genes encoding sarcomeric proteins that we found in our dataset such as Tropomyosin and Zasp52. With this project, we have identified new actors of collective cardioblast migration and a set of genes with potential role in the acquisition of individual properties of Tin and Lbcardiac cells or of specific type of muscle tissue. We hope that our data could provide new insights into the genetic control of vertebrate cardiogenesis and into etiology of cardiac diseases.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.