Dissertations / Theses on the topic 'Cell migration Nuclear envelope'

Protein kinase ATR (Ataxia Telangiectasia and Rad3-related) is a key regulator of genomic integrity. In addition to its vital, well-understood role in maintaining replication fork stability, ATR is also involved in mediating mechanical stress response at the nuclear envelope preventing potential threats to the genome. Our data from sub-cellular distribution and interactome analysis of ATR suggests that ATR contributes to several cellular processes in multiple organelles such as mitochondria, actin cytoskeleton, Golgi and nuclear envelope. At the nuclear envelope ATR is present on both inner and outer nuclear membranes, on the nuclear pores and bound to perinuclear chromatin and to perinuclear actin fibers. In this study we show that ATR regulates nuclear membrane integrity by maintaining nuclear morphology and optimal membrane tension, by counteracting mechanical force imbalances at the NE and by coordinating nuclear events with nuclear and cell migration. We report a novel role of ATR in preventing and protecting nuclear envelope damage and DNA damage caused by mechanical constrains acting on the nucleus. Further we show that by maintaining nuclear envelope integrity ATR facilitates cell migration on 2D surfaces and by regulating nuclear membrane components and by limiting nuclear envelope damage it aids cell survival during confined 3D migrations. Loss of ATR dampens neuronal migration during development and cancer cells lacking ATR are inefficient in extravasation, do not survive circulation and fail to successfully metastasize into the host environment. Therefore, by promoting cell survival in altering mechanical microenvironment and during metastasis and invasion, ATR assists tumor development, suggesting a dual role for this kinase in tumorigenisis

The eukaryotic nuclear envelope (NE), separates the nucleoplasm from cytoplasm and is made up of two concentric lipid membranes, the outer and the inner nuclear membranes (ONM and INM), the nuclear pore complexes (NPCs) and an underlying filamentous nuclear lamina. The INM contains hundreds of unique transmembrane proteins of which only a handful have been characterized. In this thesis, I aimed to understand the functional organization of proteins in the nuclear envelope and I focused on investigating the functions of a recently identified INM transmembrane protein, Samp1. We have developed a novel and robust approach, MCLIP, to identify specific protein-protein interactions taking place in live cells. Using MCLIP, we have shown that Samp1 interacts with proteins of the LINC complex, the nuclear lamina and components of the mitotic spindle. Samp1's specific interactions with a variety of binding partners, suggest that Samp1 plays important roles both in interphase and in mitosis.  We have also shown that Samp1 can provide a binding site at the INM for the GTPase Ran, a master regulator of protein interactions in interphase and in mitosis. Furthermore, we have also investigated the role of Samp1 in cell differentiation using two independent model systems. In human iPSCs, ectopic expression of Samp1 promoted differentiation despite pluripotent culture conditions. In C2C12 myoblast, depletion of Samp1 completely blocked differentiation into myotubes. The two studies complement each other and suggest that Samp1 has a strong differentiation promoting activity. Taken together, the findings in this thesis, give insights on the unexpected and unforeseen roles played by a transmembrane protein in different fundamental cellular process.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.

The postulated model for the insulin - stimulated induction of mRNA efflux (Purrello et al., 1983) is based on the demonstrated binding of insulin to intracellular membrane structures (see chapter 2, section 2.2.1), and the in vitro effect of insulin on nuclear envelope phosphorylation, NTPase activity, and mRNA efflux (see chapter 5, section 5.1). These independent observations have led to the development of a model for the direct induction by insulin, at the level of the nucleus, of mRNA efflux (figure 1.1). However, the specific intracellular insulin binding has been inf erred from kinetic or morphological studies which have not identified a discrete membrane - bound polypeptide(s) as an insulin docking molecule in situ (Goldfine, 1981). Also, the stimulation of NTPase activity has only been established by monitoring the level of general ATP hydrolysis of nuclear envelope fractions in the presence and absence of insulin (Purrello et al., 1983). The scope of this thesis has been to further the understanding of this mechanism by attempting to a) unequivocally identify a specific nuclear envelope - associated insulin docking polypeptide in situ and b) to demonstrate that insulin directly affects the ATP - binding of nuclear envelope ATP - binding proteins. The latter would demonstrate a primary effect of insulin i.e. the modulation of the ATP - binding capacity of identified NTPases / protein kinases (or their release from some inactive storage form), and not a general phenomenon such as elevated ATP.

The nuclear envelope (NE) provides stiffness to the nucleus, protects the genome, and regulates the mechanotransduction process via its network of proteins. These roles govern gene transcription and cell survival and/or differentiation. Considering that these proteins transmit cytoplasmic signalling to the nucleus through interactions with transcription factors, the identification and control of these interactions could play an important role in the regulation of cell differentiation and survival. In mesenchymal stem cells (MSCs), it has been demonstrated that NE proteins are crucial to the differentiation process. Mutations in some of them are linked to envelopathies, in which mesenchymal tissues are differentially affected. In addition, during the ageing process, their level is decreased, which would explain in part some of the age-related changes in bone and muscle. Lamin A, emerin and MAN1 are the most studied NE proteins in terms of their involvement in the pathogenesis of envelopathies. In this thesis we hypothesized that these NE proteins are involved in the differentiation of MSCs into bone and fat, playing a role in the pathogenesis of age-related bone loss. Therefore, this research thesis reports new evidence on the role of these three proteins during osteoblastic and adipogenic differentiation of bone marrow-derived human MSCs.

The eukaryotic cell nucleus is characterized by a defined spatial organization of the chromatin, which relies on the physical tethering of many genomic loci to the inner surface of the nuclear envelope. This interaction is mainly mediated by lamins and lamin-associated proteins, which create a protein network at the nuclear periphery called nuclear lamina. Man1 is a member of a lamin-associated protein family known as LEM-domain proteins, which are characterized by the presence of a highly conserved domain, called LEM, that mediates the interaction with the chromatin. Data obtained with the yeast Man1 homolog Src1 underline the importance of this protein in different processes of the cell cycle, such as chromosome segregation, nuclear pores assembly, gene expression, chromatin organization and maintenance of genome stability, while in animal models, the function of Man1 has been associated to the regulation of developmental signalling pathways during embryogenesis. In this study, truncated recombinant mutants of Man1, containing the LEM domain, were shown to inhibit nuclear assembly and alter nuclear pore formation when added to Xenopus laevis cell-free extracts. Moreover, Xenopus nuclei assembled in the presence of Man1 truncated fragments were characterized by defects in chromatin organization, DNA replication and accumulation of DNA damage and, as a consequence, they failed to progress through mitosis. Furthermore, mouse embryonic stem cells (mESCs) depleted for Man1 showed evident signs of spontaneous differentiation, indicating inability in the maintenance of stem cell features. Intriguingly, preliminary analysis of Man1-knockout mESCs transcriptional profile showed an alteration of gene expression at the level of pericentromeric and telomeric regions, underlining a potential link between Man1 and genomic stability of these particular regions. In conclusion, this study illustrates the importance of Man1 in ensuring the proper chromatin organization necessary to support different cellular and DNA metabolic processes.

While a large body of work has focused on the transcriptional regulation of cellular identity, the role of the mechanical properties of cells and the importance of their physical interactions with the local environment remains less well understood. In this project, we explored the impact of cytoskeleton-generated forces exerted on the nucleus in the context of early embryonic stem (ES) cell fate decisions. We chose to perturb force generating components in the cytoskeleton – notably the molecular motor non-muscle myosin II - and key structural and chromatin binding proteins in the nuclear envelope, notably, the lamins (LMNA), Lamin B receptor (LBR) and components of the LINC complex (nesprins/KASH). The structural proteins in the nuclear envelope regulate both the mechanical response of the nucleus to force and the stabilization of peripheral heterochromatin (repressed genes). Our hypothesis is that reducing forces transmitted directly to chromatin or increasing tethering of peripheral heterochromatin to the nuclear envelope would restrict access to lineage specific genes sequestered at the nuclear lamina and thereby either impair, or delay, differentiation. We found phenotypes in the capacity of mouse ES cells to specify to the neural lineage following our perturbations: overexpression of LMNA, LBR and KASH proteins resulted in a significant fraction of cells that did not express the neuroectoderm marker Sox1 after four days of differentiation, while inhibiting non-muscle myosin II delayed Sox1 expression in the entire population. Overexpression of LMNA and LBR did not affect the ability of the cells to exit the naive pluripotent state, which raises the possibility that the perturbations are halting the cells in a formative phase prior to lineage specification. Future work will focus on looking at genome-wide transcriptional changes accompanying differentiation combined with an analysis of spatial information of differentially regulated genes.

Thesis (Ph.D.)--Cleveland State University, 2009.
Abstract. Title from PDF t.p. (viewed on Feb. 1, 2010). Includes bibliographical references (p. 104-114). Available online via the OhioLINK ETD Center and also available in print.

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
Microglia are active sensors of the brain and respond promptly to even minor disturbance in their microenvironment. A feature of this response is the accumulation of these cells at the site of lesion. Neonatal jaundice is a common condition of the newborn and may determine injury to neurons and glial cells, such as microglia, when levels of unconjugated bilirubin (UCB) are excessive. With the objective to evaluate whether microglia have a protective or deleterious role, we decided to assess, using the Boyden chamber, the chemotactic effect of free unbound UCB (fUCB), as well as the migration ability of UCB-treated microglia in the absence or in the presence of chemotatic compounds, such as ATP and S100B. Also, we intended to evaluate the effect of glycoursodeoxycholic acid (GUDCA) as a modulator. To characterize our usual model of microglia isolation, phenotypic evaluation of cultures with different days in vitro (DIV) was performed by estimating cell morphology, nuclear factor-kappaB (NF-κB) activation and phagocytic ability. We observed that fUCB did not act as a chemotactic compound for microglia and that cells treated with UCB showed decreased migration ability. Co-incubation with GUDCA prevented this effect and enhanced microglia migration. However, reduced effects were observed in the presence of ATP and abolished when using S100B. Isolated microglia with 2 DIV showed features of activation, but presentedramified morphology of the “resting” state, less NF-κB activation and increased phagocytosis at 13 DIV. Data indicate that microoglia exposure to UCB leads to a reduced migration ability and that co-incubation with GUDCA prevents this deleterious effect, resulting in an increased migration. Characterization of microglia phenotypes, along the time in culture, point to 13 DIV cells as the most suitable for studies intended to evaluate microglia reactivity to UCB, and probably to other stimuli.

Vesicular trafficking within cells is an important process for tissue development and homeostasis. A key step of vesicular trafficking is the fusion between two membranes, a process in which SNARE (Soluble NSF Attachment Protein Receptors) proteins play a fundamental role. SNAP29 (SyNaptosomal Associated Proteins 29) is a ubiquitous SNARE, regulating membrane fusion in different trafficking compartments and in different contexts in non dividing cells. We isolated a loss of function mutant in usnp, the gene encoding the Drosophila homolog of the human protein SNAP29 (Snap29 hereafter), that, when made homozygous in developing epithelial organs, disrupts epithelial architecture. In vivo, we find that Snap29 interacts with multiple SNARE proteins, localizes to a number of trafficking organelles, and is required for proper Golgi Apparatus morphology. In addition, we show that Snap29 is required for fusion of autophagosomes with lysosomes together with Syx17 and Vamp7, and that lack of Snap29 results in excess secretion, suggesting that Snap29 might act negatively in regulation of vesicle fusion at the plasma membrane. Interestingly, at the onset of mitosis, when trafficking compartments re-shape to allow the formation of the mitotic spindle, Snap29 is found at the outer KT in Drosophila S2 cells and localizes at spindle microtubules and centrosomes in mammalian cells. Depletion of Snap29 in Drosophila and mammalian cells leads to spindle assembly defects, associated to pro-metaphase delay in mammalian cells, and to the formation of daughter cells containing mininuclei. Mechanistically, lack of SNAP29 correlates with absence at KT of ZWINT-1 and ZWILCH, a component of RZZ complex, and with weak KTs-MTs attachments. In addition, we find that SPINDLY, the adaptor for recruitment to KTs of dynein/dynactin and MAD1, a component of the Spindle Assembly Checkpoint machinery, fail to be removed from KTs at the end of metaphase in SNAP29 depleted mammalian cells forced to reassemble the spindle after treatment with microtubules depolymerization drug. Finally, we show that cell division is impaired in Snap29 mutant tissues in vivo, that autophagy defects are not the cause of the altered epithelial tissues architecture in Snap29 mutants and that the trafficking and cell division function of Snap29 are molecularly distinct. All together our findings support a role of Snap29 at key steps of membrane trafficking and in cell division. Our study contribute to shed light on the pathogenesis of CEDNIK, a human congenital syndrome caused by SNAP29 inactivation. In addition to this, we propose that the function of SNAP29 in cell division might be evolutionarly related to that of complexes tethering MTs to vesicular organelles in interphase. We surmise that such function could be potentially relevant to development of aneuploidy in tumor-like masses.

Des malformations de l’enveloppe nucléaire sont observées dans des maladies de dystrophie musculaire ainsi que dans certains types de cancer tels que le cancer du sein ou du rein. Les mécanismes impliqués dans le maintien de la morphologie de l’enveloppe nucléaire sont basés sur des interactions protéo-lipidiques. Il a été démontré récemment que les propriétés physiques de lipides spécifiques jouent un rôle dans le processus de formation de l’enveloppe nucléaire chez le modèle de l’oursin. Afin de comprendre l’implication des lipides dans la régulation de l’architecture de l’enveloppe nucléaire nous avons utilisé les méthodes quantitatives de spectrométrie de masse et de résonance magnétique nucléaire (RMN) afin d’étudier la composition lipidique et les propriétés dynamiques de l’enveloppe nucléaire. L’extraction de noyaux a été effectuée à partir de cellules humaines de rein, les HEK 293T. Une extraction physique basée sur un traitement de pression et un gradient de sucrose a été optimisée afin d’obtenir une large quantité de noyaux intacts (lipides de l’enveloppe nucléaire) requis pour nos expériences. Ces noyaux purifiés comprennent un minimum de débris cellulaires, de réticulum endoplasmique et de l’appareil de golgi. Les lipides nucléaires sont ensuite extraits en utilisant une méthode Folch modifiée. Des expériences de RMN des liquides ont montré que l’enveloppe nucléaire était composée de nombreux types de phospholipides et dont la phosphatidylcholine était le lipide majoritaire. Par ailleurs, nous avons observé une grande quantité de phosphatidilinositol en comparaison avec d’autres types de membranes. Les expériences de spectrométrie de masse ont permis de mettre en évidence que la membrane nucléaire était composée majoritairement de lipides très insaturés et de chaines comportant en moyenne 34 atomes de carbone. Les analyses de RMN du solide effectuées sur des membranes reconstituées à partir des lipides nucléaires ont permis de mettre en évidence des propriétés physiques atypiques. La température de transition de phase fluide-gel est particulièrement faible et large à -10°C ±15°C. Ce phénomène est certainement dû à la présence de nombreuses espèces de lipides et de nombreuse insaturations. Par ailleurs, à 25°C, les membranes reconstituées de lipides nucléaires sont plus rigides que des membranes modèles classiques ce qui implique une plus large épaisseur de membrane. Enfin, les liposomes de membranes reconstituées ont montré une très importante déformation en ellipsoide de type prolate, dans les champs magnétiques, ce qui est particulièrement rare pour des membranes biologiques et ce qui suggère une importante élasticité de courbure de la membrane
Muscular dystrophy diseases, breast and kidney cancers are linked to malformation of the nuclear envelope (NE). Mechanisms involved in the maintenance of NE morphology are based on proteo-lipid interactions. Recently, the physical properties of specific lipids has highlighted their essential role in NE assembly process of a sea urchin model. To provide molecular insight in how the nuclear architecture is regulated, we used Mass spectrometry and Nuclear Magnetic Resonance (NMR) as quantitative methods to investigate the lipid composition and the dynamics of the NE. Nuclei extraction were performed from HEK 293T human kidney cells. A physical extraction based on a pressure treatment and a sucrose gradient was used and improved considerably the nuclei yield, and afforded obtaining a high quantity of intact nuclei (NE lipids) required for experiments, with a minimum of cell debris, or the ER and Golgi compartments. The nuclear lipids were then extracted from the pure nuclei using a modified Folch method. Liquid-state NMR experiments showed that the NE is composed of a complex mixture of phospholipids and with phosphatitylcholine present in a higher proportion compared to other membrane organelles. Furthermore, an unusual proportion of phosphatidylinositol has been found at the NE. Mass spectrometry experiments have shown that the composition of phospholipids is dominated by species with greatky unsaturated chains with an average length of 34 carbons. Reconstructed nuclear lipid extract membranes were analysed by solid-state NMR and exhibit atypical physical properties. The lamellar gel-fluid phase transition temperature was found very low and broad at -10  15°C, possibly due to the presence of numerous lipid species and unsaturated acyl chains. Furthermore, at 25°C, reconstructed nuclear lipid membrane was found to be more rigid than classical model membranes suggesting a larger bilayer thickness. Finally, reconstructed nuclear lipid liposomes have shown a very important prolate deformation in a magnetic field, which is unusual for biological membranes and suggests an important curvature elasticity for the membrane

Les cellules possèdent une capacitéfondamentale à leur survie : la migration. Del’embryogénèse aux métastases tumorales, lorsde la migration, les cellules doivent se faufiler àtravers des mailles sub-nucléaires pour atteindreleur localisation cible. Pour ce faire, ellespeuvent adapter leur mode locomotion ou leurspropriétés mécaniques à l’environnement quiles entoure. La cellule ainsi que son noyausubissent d’importantes déformations lors de lamigration en milieu confiné. Le noyau étantl’organelle le plus gros et le plus rigide, il peutlimiter la capacité migratoire de la cellule. Sespropriétés mécaniques sont donc décisives afinde migrer à travers un environnement complexe.Dans la littérature, les signaux moléculairespendant le processus migratoire ont étéabondamment décrits, mais la modélisationmécanique d’une cellule en migration peut-ellenous révéler de nouveaux éléments sur lesmécanismes sous-jacents ?La migration cellulaire est un procédé d’unecomplexité mécano-biologique telle, que tous sesaspects ne peuvent être modélisés à ce jour. Nousen choisissons donc trois que nousdévelopperons ici. Nous nous intéressonsd’abord à l’interaction mécanique entre le noyauet le cytoplasme lors d’une constriction de lacellule, puisque la plasticité du noyau sembleavoir un rôle primordial. Nous étudions ensuitele chimneying, un mode migratoire sansadhésion dont le mécanisme repose sur desforces de friction couplées à la poroélasticité ducytoplasme. Enfin, les substrats avec des micropilierssont depuis peu utilisés pour étudier lespropriétés mécaniques de la cellule et de sonnoyau, mais la mécanique de ce phénomène estpeu comprise. Nous modélisons le processus parlequel le noyau se déforme afin de déterminer s’ilest poussé ou tiré dans l’espace inter-piliers
One of the fundamental properties incells is their ability to migrate. Fromembryogenesis to tumor metastasis, migratingcells must overcome mechanical obstacles toreach their intended location, squeezing throughsub-cellular and sub-nuclear gaps. It can be doneby adapting the locomotion mode to thesurrounding environment or by tuning the cell’sown mechanical properties. Migrating in aconfined space leads to intensive deformation ofthe cell and thus its nucleus. Being the largestand stiffest organelle, the nucleus can hamperthe migratory process. Its mechanical propertieshence are key to a successful migration in acomplex environment. Molecular signals behindcell migration have been extensively studied inthe literature, but what can computationalmechanics modeling unveil about themechanisms behind cell migration?Cell migration is such a complex mechanobiologicalprocess, that all aspects cannot bemodeled at once for now. We choose threedistinct situations for in-depth study. We firstseek to understand the mechanical interplaybetween the nucleus and the cytoplasm, sincenuclear plasticity seems decisive for migrationthrough sub-nuclear gaps. Second, weinvestigate the mechanics of chimneying, aspecific confined migratory mode, in which noadhesion in needed for the cell to move forward.Poroelasticity, coupled with friction, appears asthe key to successful locomotion. Eventually,cell spreading on micro-pillared substrates hasrecently been developed to study nuclearmechanical properties. The mechanism behindthis process being however unclear, we designeda large deformation model to determine whetherthe nucleus is being pushed or pulled in theinter-pillars gaps

La forme du noyau peut varier significativement au cours du développement ou lors de processus pathologiques en raison des forces mécaniques émanant du microenvironnement ou générées par le cytosquelette. L’impact de la morphologie nucléaire sur la machinerie transcriptionnelle n’est cependant pas connu. En utilisant plusieurs approches afin de manipuler la morphologie nucléaire, nous avons observé que des changements de forme de l’enveloppe nucléaire régulent l’activité de AP1 et TEAD. Nous avons montré que l’aplatissement du noyau augmente la phosphorylation de c-Jun et la translocation de YAP, conduisant à une augmentation de la transcription des gènes cibles de AP1 et TEAD. Nous avons également observé que l’aplatissement du noyau se produit au cours du cycle cellulaire et favorise la prolifération via l’activation de TEAD et AP1 qui stimulent la progression de la phase G1 à la phase S
.The shape of the cell nucleus can vary considerably during developmental and pathological processes as a consequence of the mechanical forces emanating from the microenvironment or generated by the cytoskeleton. However the impact of nuclear morphology on the transcriptional machinery is not known. Using a combination of tools to manipulate the nuclear morphology, we observed that changes in nuclear shape regulate the activity of AP1 and TEAD. We showed that nuclear flattening increases c-Jun phosphorylation and YAP nuclear translocation, leading to transcriptional induction of AP1 and TEAD-target genes. Surprisingly, we found that nuclear compression is necessary and sufficient to mediate c-Jun and YAP activation in response to cell- generated contractility or cell spreading. We additionally observed that nuclear flattening occurs during the cell cycle and promotes proliferation via TEAD and AP1- dependent G1 to S progression

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that link the extracellular matrix to the actin cytoskeleton. Uniquely, these adhesion receptors mediate inside-out signal transduction, whereby extracellular adhesion is activated from within the cell by talin, a large cytoskeletal protein that binds to the cytoplasmic tail of the β integrin subunit via its PTB-like F3 domain. Features of the interface between talin1 and small β3 fragments only have been described previously. Through NMR studies of full-length integrin β tails, we have found that β tails differ widely in their interactions with different talin isoforms. The muscle-specific β1D/talin2 complex exhibited particularly high affinity, leading to the X-ray crystal structure of the β1D tail/talin2 F2-F3 complex. Further NMR and biological experiments demonstrated that integrin activation is induced by a concerted series of interactions between the talin F3 domain and the β tail and between the talin F2 domain and the cell membrane. Additional studies revealed the structural determinants of tight talin2/β1D binding and the basis of more general differences between β1 and β3 talin binding. NMR studies were also performed on tyrosine-phosphorylated integrin tails binding to the PTB domains of talin1 and Dok1, an inhibitor of integrin activation; these revealed that phosphorylation can inhibit integrin activation by increasing the affinity of the β tail for talin competitors. Key residues governing this switch were identified, and proteins were engineered with reversed affinities, offering potentially useful biological tools. Taken together, these results reveal the remarkable complexity of structural features that enable talin and its competitors to mediate this important form of transmembrane signalling.

The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC.

Osteoimmunology as an interdisciplinary research principle governing the cross-talk between the skeletal and immune systems. Better understanding of how these two systems operate is likely to lay the groundwork for biological therapies of bone related diseases. Mesenchymal stromal cells (MSCs) are self-renewing and pluripotent cells capable to differentiate into osteoblasts. Previous studies have shown that undifferentiated MSCs possess remarkable immunoregulatory properties through inhibiting the proliferation and activation of the major immune cell populations. However, it is unknown whether MSCs have the same immunoregulatory properties during osteogenic differentiation. We have reported for the first time that undifferentiated MSCs and osteogenically differentiated MSCs (OMSCs) have different effect on cell recruitment. Macrophages as the precursors of osteoclasts and immune cells were used as the effector cells for cell migration. We found that cell motility was enhanced by the OMSC conditioned medium (OMSC-CM) compared to the MSC conditioned medium (MSC-CM). It was noted that OMSCs also regulated the local immune responses by modulating cell morphology and elicited significant effects on macrophage activation, resulting in the up-regulation of pro-inflammatory cytokine expression. These results indicate that OMSCs are actively involved in bone formation by regulation of cell migration and activation. In the second part of our study, the potential factors secreted by OMSCs contributing to cell migration were evaluated through cytokine array analysis. Our results have demonstrated that OMSCs could regulate macrophage motility through the secretion of VEGF, which is involved in bone formation due to the fact that impaired bone formation was observed with the neutralization of VEGF in skull defects. In addition, VEGF secreted by OMSCs slightly activated the macrophages by up-regulation of pro-inflammatory cytokine expression. This study explores the key linkage between the skeletal and immune systems and expands the scope of osteoimmunology. The last part of our study has investigated the contribution of secreted factors from OMSCs to immune cell activation through the study of relationship between the vascular endothelial growth factor (VEGF) and the receptor activator of nuclear factor kappa-Β ligand (RANKL). Our data suggest that the inhibition of VEGF impaired osteogenesis and RANKL expression. In addition, RANKL was closely correlated to iNOS positive cells in vivo. In vitro study found that RANKL could trigger iNOS expression in macrophages which were involved in osteogenic differentiation of MSCs. RANKL induced macrophages showed different expression pattern in terms of cytokine expression compared with LPS and IFN-γ. This study emphasizes the involvement of VEGF and RANKL in macrophage polarization under physiological conditions in comparison to LPS and IFN-γ, which are derived from pathogens. Taken together, our study reveals a quite variable and multifaceted profile of osteogenically differentiated MSCs which enhance bone formation through induction of macrophage migration and activation. These findings enrich our understanding of osteoimmunology and bone remodelling, which will pave the theoretical principles for cell- based therapy in bone defects.

Lors de la division cellulaire, une cellule mère doit dupliquer (interphase) puis ségréger son matériel génétique de façon égale entre les deux cellules filles (mitose). Entre ces deux étapes, la cellule subit une réorganisation drastique gouvernée par l’acteur majeur Cdk1-Cycline B, conduisant à l’entrée en mitose. L’activation de cette kinase est régulée par une boucle d’auto-amplification où les premières molécules de Cdk1-Cycline B stimulent l’activation des suivantes. Il a été montré que la kinase Plk1 initie cette boucle d’auto-amplification en stimulant les activateurs et en réprimant les inhibiteurs de Cdk1-Cycline B en amont. Pour que cette kinase soit totalement active, elle doit être elle-même activée par Aurora A, en présence de son co-activateur Bora. Il est crucial de comprendre comment tous ces acteurs se coordonnent dans l’espace et dans le temps pour déclencher l’entrée en mitose car un dérèglement pourrait amener à une ségrégation de l’ADN anarchique, conduisant à la formation de tumeurs et l’apparition de cancers. Au cours de ma thèse, j’ai tout d’abord contribué à la mise en évidence d’un mécanisme conservé d’activation de Plk1 dans les cellules humaines et chez C. elegans (PLK-1), impliquant le co-activateur Bora ou SPAT-1 chez C. elegans. Nous avons montré que la phosphorylation de SPAT-1 par Cdk1-Cycline B induit son interaction avec PLK-1, ce qui promeut la phosphorylation de PLK-1 par Aurora A et donc son activation in vitro. Ce mécanisme phospho-dépendant de SPAT-1 est important in vivo pour contrôler dans le temps l’entrée en mitose. De plus, l’activation de Plk1 in vitro avec les protéines humaines suggèrent fortement une conservation du mécanisme. Nous avons ensuite montré que la phosphorylation de Bora et de SPAT-1 par Cdk1 sur les résidus S41, S112, S137 et S119, S190, T229 respectivement, est nécessaire à leur interaction avec Plk1/PLK-1, déclenchant ensuite l’activation de Plk1/PLK-1 et l’entrée en mitose. Ces résultats démontrent que Bora/SPAT-1 phosphorylée fait partie de la boucle d’auto-amplification de Cdk1-Cycline B via l’activation de Plk1, permettant à terme d’activer de façon irréversible les acteurs de l’entrée en mitose. Par la suite, je me suis focalisée sur le rôle de PLK-1 dans la rupture de l’enveloppe nucléaire en utilisant l’embryon de C. elegans comme système modèle. Après avoir démontré que PLK-1 est cruciale pour la rupture de l’enveloppe nucléaire dans les embryons, j’ai observé une localisation de PLK-1 à l’enveloppe nucléaire avant sa rupture et j’ai identifié un complexe de nucléoporines impliqué dans ce processus. En effet, NPP-1, NPP-4 et NPP-11 dont la fonction est de réguler le transport nucléo-cytoplasmique, ont également un second rôle dans le recrutement de PLK-1 aux pores nucléaires. PLK-1 interagit avec ses substrats phosphorylés par deux types de mécanismes d’amorçage Plk1-dépendant et indépendant, impliquant une autre kinase en amont comme Cdk1-Cycline B par exemple. J’ai montré que le recrutement de PLK-1 aux pores dépend des deux mécanismes, nécessitant donc une coordination entre Cdk1-Cycline B et PLK-1. Une fois que PLK-1 est au centre du pore nucléaire, elle peut alors probablement phosphoryler de nombreuses nucléoporines et participer au désassemblage des pores, conduisant à la rupture de l’enveloppe nucléaire
During cell division, a mother cell duplicates (interphase) and then segregate its genetic material equally between the two daughter cells (mitosis). Between these two stages, the cell undergoes a drastic reorganization governed by the major actor Cdk1-Cyclin B, leading to mitotic entry. The activation of this kinase is regulated by an auto-amplification loop where the first molecules of Cdk1-Cyclin B stimulate activation of the following. Plk1 kinase has been shown to initiate this self-amplification loop by stimulating activators and repressing upstream Cdk1-Cyclin B inhibitors. For this kinase to be fully active, it must itself be activated by Aurora A, in the presence of its coactivator Bora. It is crucial to understand how all these actors coordinate in space and time to trigger mitotic entry because a disruption could lead to a segregation of anarchic DNA, leading to the formation of tumors and the appearance of cancers. During my thesis, I first contributed to demonstrate a conserved mechanism of Plk1 activation in human cells and in C. elegans (PLK-1), involving the coactivator Bora or SPAT-1 in C. elegans. We have shown that the phosphorylation of SPAT-1 by Cdk1-Cyclin B induces its interaction with PLK-1, which promotes the phosphorylation of PLK-1 by Aurora A and thus its activation in vitro. This phosphory-dependent mechanism of SPAT-1 is important in vivo for controlling the entry into mitosis over time. In addition, activation of Plk1 in vitro with human proteins strongly suggests conservation of the mechanism. We then showed that the phosphorylation of Bora and SPAT-1 by Cdk1 on residues S41, S112, S137 and S119, S190, T229 respectively, is necessary for their interaction with Plk1 / PLK-1, then triggering the activation of Plk1 / PLK-1 and mitotic entry. These results demonstrate that phosphorylated Bora / SPAT-1 is part of the self-amplification loop of Cdk1-Cyclin B via the activation of Plk1, ultimately enabling irreversible activation of the actors of mitotic entry. Subsequently, I focused on the role of PLK-1 in nuclear envelope breakdown using the C. elegans early embryo as a model system. After demonstrating that PLK-1 is crucial for the nuclear envelope breakdown in embryos, I observed a localization of PLK-1 to the nuclear envelope before its rupture and I identified a nucleoporin complex involved in this process. Indeed, NPP-1, NPP-4 and NPP-11 whose function is to regulate nucleo-cytoplasmic transport, also have a second role in the recruitment of PLK-1 to nuclear pores. PLK-1 interacts with its phosphorylated substrates by two types of Plk1-dependent and independent priming mechanisms, involving another upstream kinase such as Cdk1-Cyclin B for example. I have shown that the recruitment of PLK-1 to the pores depends on both mechanisms, thus requiring coordination between Cdk1-Cyclin B and PLK-1. Once PLK-1 is at the center of the nuclear pore, it can probably phosphorylate many nucleoporins and participate in the disassembly of pores, leading to tnuclear envelope breakdown

Une etude en immunofluorescence sur coupe au cryostat, grace a des anticorps anti-tubuline, anti-proteine associees aux microtubules et anti centre organisateur des microtubles, de la distribution des proprietes et du role des microtubules au cours de la reprise de la meiose de l'ovcyte de souris

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2010
Centrosome reorientation is defined as the positioning of the centrosome in a region between the nucleus and the leading edge and is important for cell polarization and directional cell migration. Cdc42 is the key regulator on this process and two main pathways are involved in centrosome reorientation; on one side, centrosome centration by a mechanism dependent of Par complex and dynein/dynactin, and for other side, a rearward nuclear movement dependent on Cdc42-effector MRCK and actin-myosin retrograde flow. Recently, the LINC complex was found to be involved in the rearward nuclear movement pathway. This complex spans the nuclear envelope and involves a SUN domain-containing protein which interacts with a KASH domain-containing protein, localized in the inner and in the outer nuclear membrane, respectively. SUN proteins bind to lamins and KASH proteins to actin filaments; in this way, the LINC complex makes the connection between actin retrograde flow and the nucleus. In fibroblasts, Sun2 and Nesprin-2 co-localize with dorsal actin cables on TAN lines; actin dorsal cables move back by actin retrograde flow and the nucleus moves with them. Other proteins can be involved in the nuclear movement. In a siRNA screen for nuclear envelope proteins, a putative role for Tmem201 in nuclear movement was identified. In S.pombe, Tmem201 homolog connects the heterochromatin with the LINC complex. A connection with LINC complex in mammalians was never reported so far. Tmem201is a nuclear envelope protein and is localized in TAN lines in fibroblasts. The TMEM201depletion by RNA interference inhibited centrosome reorientation. Tmem201 is involved in nuclear movement, probably by the stabilization of the LINC complex in the nuclear membrane. However, Tmem201 might also be involved in centrosome positioning and could act as a key regulator of both pathways.
Durante muito tempo, o papel do invólucro nuclear foi desvalorizado, sendo visto como um mero compartimento de armazenamento de cromossomas. Hoje em dia, após a descoberta de uma nova categoria de proteínas que permitiram criar o elo entre os componentes nucleares e o citosqueleto, o seu papel como organizador essencial é lhe amplamente reconhecido. De facto, a dinâmica entre citosqueleto e invólucro nuclear é fundamental para um correcto posicionamento do núcleo, que depende, por sua vez, de uma correcta migração e ancoragem do núcleo. O posicionamento nuclear é importante em fenómenos tão diversificados como a fertilização, a formação de fibras musculares, a oogénese e a migração celular. A reorientação do centrossoma é um processo que ocorre em fibroblastos, células endoteliais, células epiteliais, astrócitos, células T e neurónios e que leva à polarização celular. Considera-se que o centrossoma está orientado quando este se encontra posicionado entre o núcleo e a frente condutora da célula na migração. Pensa-se que a polarização do centrossoma é um fenómeno prévio à migração celular, importante para a orientação do Golgi, possibilitando a secreção polarizada de precursores membranares e de factores importantes para a frente da célula, possibilitando a migração. Durante muito tempo, pensou-se que seria o centrossoma a mover-se para uma posição anterior ao núcleo. Hoje em dia, sabe-se que é o núcleo que se move, adquirindo uma posição posterior ao centrossoma. A proteína Cdc42 tem um papel fundamental neste processo, conduzindo à activação dos dois mecanismos necessários à reorientação do centrossoma: por um lado, o centrossoma é mantido no centro da célula. Este mecanismo é dependente do complexo Par (Par6, Par3 e aPKC), da dineína e de dinactina. Por outro lado, MRCK, um efector de Cdc42, activa o movimento retrógrado do núcleo. Este movimento aparece aliado ao movimento retrógrado da actina, dependendo da contracção da miosina. Recentemente, um complexo de proteínas transmembranares do invólucro nuclear foi identificado como fundamental para este movimento nuclear. Este complexo é constituído por duas proteínas, uma com um domínio SUN e a outra com um domínio KASH, ambos localizados na extremidade C-terminal da proteína. Este domínio C-terminal é altamente conservado em todos os Metazoa e em leveduras. A proteína com domínio SUN localiza-se na membrana interna e interage com a lamina nuclear pela sua extremidade N-terminal. A proteína com domínio KASH localiza-se na membrana externa; algumas proteínas que contêm este domínio podem atingir dimensões de mais de 800kDa e podem interagir com a actina pelo seu domínio N-terminal. A localização da proteína com o domínio KASH na membrana externa é absolutamente dependente da proteína com o domínio SUN. Uma grande especulação existe ainda em torno da retenção da proteína com o domínio SUN na membrana nuclear. Em mamíferos, por exemplo, a localização de Sun2 parece ser parcialmente dependente da lamina nuclear, enquanto que Sun1 não depende da lamina para se localizar na membrana nuclear interna. Este complexo atravessa assim todo a membrana nuclear e estabelece assim a ligação entre o núcleo e a actina. Este par proteico é assim chamado de Complexo “LINC” – LIgação entre o Núcleo e o Citosqueleto (LInkers of Nucleoskeleton and Cytoskeleton). Um estudo recente reporta a implicação directa do complexo “LINC” na reorientação do centrossoma em fibroblastos de ratinho. Neste caso, o par de proteínas que está envolvido no movimento nuclear é Sun2/Nesprin-2. Este estudo descreve pela primeira vez a existência de cabos actínicos, organizados numa posição dorsal em relação ao núcleo, que se movem retrogradamente ao mesmo tempo do que o núcleo. Estes cabos, e logo também o movimento retrógrado de actina a eles associados, parecem ser o motor para o movimento nuclear. O envolvimento de Sun2 e Nesprin-2 é reportado, quando se observa que ambas as proteínas se co-localizam com estes cabos de actina e que a depleção destas proteínas inibe o movimento nuclear. Esta associação das proteínas do complexo LINC com os cabos dorsais de actina define uma nova estrutura nuclear denominada de “linhas TAN” – linhas Nucleares Transmembranares associadas a Actina (Transmembrane Actin-associated Nuclear). A força gerada pelo movimento retrógrado de actina é assim transmitida ao núcleo através destas estruturas, conduzindo ao movimento. Sabe-se que Sun2 e Nesprin-2 interagem no espaço perinuclear, mas pouco ainda se sabe sobre esta interacção. Para além do mais, a localização de Sun2 no invólucro nuclear continua mal elucidada. Para além disso, as forças aplicadas sobre estas proteínas durante o movimento nuclear, sugerem o envolvimento de outras proteína na estabilização, organização e interacção deste complexo. Num screen de siRNA para proteínas do invólucro nuclear, algumas proteínas revelaram um potencial papel no movimento nuclear, entre estas a proteína Tmem201. Esta proteína é conservada evolutivamente, estando presente em todos os Metazoa e ainda em S.pombe. Pouco se sabe ainda sobre esta proteína, havendo apenas dois estudos realizados até ao momento, um em S.pombe e outro em células humanas. Em S.pombe, o homólogo de Tmem201, Ima1, participa na associação da heterocromatina com o complexo LINC. Ima1 é importante para a estabilização do complexo, e de facto, quando é eliminada do sistema, observam-se deformações do invólucro nuclear e quebras no complexo LINC. Ima1 funcionará assim como estabilizador do complexo LINC à membrana, função ainda não atribuída a nenhuma outra proteína em mamíferos. Em células humanas, a proteína homóloga Samp1 aparece associada com estruturas membranares que se sobrepõem ao fuso mitótico. É também sugerido um possível papel desta proteína na ligação entre centrossoma e núcleo. Este trabalho propõe-se elucidar o envolvimento da proteína Tmem201 no movimento nuclear e na reorientação do centrossoma. Infelizmente, um anticorpo eficaz para a marcação de Tmem201 não está disponível no mercado e assim, um dos passos fundamentais deste trabalho passou pela produção de um anticorpo capaz de reconhecer eficazmente as três isoformas da proteína. Tal foi conseguido com sucesso, sendo possível visualizar perfeitamente uma marcação nuclear da proteína. Uma marcação a nível dos centrossomas, provavelmente não específica, foi também observada. Esta proteína também foi observada em associação aos cabos dorsais de actina que fazem parte das linhas TAN. Este fenómeno é particularmente interessante, se considerarmos que Sun2 e Nesprin-2 são as duas únicas proteínas que foram identificadas até ao momento com localização nestas estruturas. Tendo em conta que as laminas não se encontram nas linhas TAN, Tmem201 poderia funcionar como estabilizador do complexo LINC a este nível, num papel evolutivamente conservado ao papel de Ima1 em S.pombe. O movimento nuclear e da reorientação do centrossoma pode ser estudado facilmente através de um ensaio experimental em fibroblastos em cultura. Neste ensaio, é efectuado uma lesão linear (através de uma ponta de pipeta) numa monocamda confluente de células aderentes (em meio desprovido de soro). A reorientação do centrossoma é depois estimulada por adição de um factor específico (LPA). Através deste ensaio, procurou-se estudar os efeitos da depleção da proteína, por siRNA, na reorientação do centrossoma. Verificou-se uma inibição da reorientação do centrossoma quando a proteína não está presente, o que corrobora o resultado inicial do screen efectuado. Analisando a posição do centrossoma e do núcleo, uma forte inibição do movimento nuclear é visualizada, enquanto que a posição do centrossoma não é afectada. Os efeitos de depleção podem ser parcialmente recuperados aquando da microinjecção de um plasmídeo que codifica para a mais pequena isoforma de Tmem201 (Tmem201 B-GFP). Os resultados obtidos implicam o envolvimento de Tmem201 no movimento nuclear. Contudo, um resultado inesperado foi obtido, aquando da microinjecção do primeiro domínio da proteína: uma deslocalização do centrossoma. Este resultado sugere um possível envolvimento também no posicionamento do centrossoma. Através dos ensaios de microinjecção, foi também possível concluir que o primeiro domínio da proteína parece estar envolvido na localização nuclear da proteína (visto que Tmem201 628-GFP tem uma localização nuclear), enquanto que o segundo domínio da proteína deverá ter um papel no movimento do núcleo (visto que apenas a Tmem201 B-GFP é capaz de recuperar a reorientação do centrossoma). A inibição do movimento nuclear pode dever-se a diversos factores: se Tmem201 for importante na localização nuclear de proteínas envolvidas no movimento do núcleo, ou se por acaso levar à inibição do movimento retrógrado de actina. Verificou-se que a depleção de Tmem201 não afecta a retenção de Sun2, Nesprin-2, lamin A/C, lamin B e Emerin. Quanto à actina, não parece haver uma alteração do citosqueleto actínico. Em suma, Tmem201 aparece implicada na reorientação do centrossoma, mais precisamente no movimento nuclear. Tendo em conta que não afecta a localização de outras proteínas na membrana nuclear e não altera o citosqueleto actínico, Tmem201 poderá ter um papel como estabilizador do complexo LINC na membrana nuclear, sendo importante para o movimento nuclear. Contudo, poderá também estar envolvida no posicionamento do centrossoma. Tmem201 poderá assim actuar como proteína reguladora das duas vias.

No
Ran is a small ras-related GTPase that controls the nucleocytoplasmic exchange of macromolecules across the nuclear envelope. It binds to chromatin early during nuclear formation and has important roles during the eukaryotic cell cycle, where it regulates mitotic spindle assembly, nuclear envelope formation and cell cycle checkpoint control. Like other GTPases, Ran relies on the cycling between GTP-bound and GDP-bound conformations to interact with effector proteins and regulate these processes. In nucleocytoplasmic transport, Ran shuttles across the nuclear envelope through nuclear pores. It is concentrated in the nucleus by an active import mechanism where it generates a high concentration of RanGTP by nucleotide exchange. It controls the assembly and disassembly of a range of complexes that are formed between Ran-binding proteins and cellular cargo to maintain rapid nuclear transport. Ran also has been identified as an essential protein in nuclear envelope formation in eukaryotes. This mechanism is dependent on importin-β, which regulates the assembly of further complexes important in this process, such as Nup107–Nup160. A strong body of evidence is emerging implicating Ran as a key protein in the metastatic progression of cancer. Ran is overexpressed in a range of tumors, such as breast and renal, and these perturbed levels are associated with local invasion, metastasis and reduced patient survival. Furthermore, tumors with oncogenic KRAS or PIK3CA mutations are addicted to Ran expression, which yields exciting future therapeutic opportunities.

Tese de doutoramento em Medicina
The multi-subunit protein cytoplasmic dynein 1 (dynein) is the major retrograde microtubule motor in the cell. Dynein has multiple roles during brain development and human mutations in dynein-related genes lead to severe neurodevelopmental disorders. In the inner proliferative zones of the neocortex, division of neural stem cells depends on dynein recruitment to the nucleus during interkinetic nuclear migration. Moreover, dynein is required for the multipolar-to-bipolar transition of post-mitotic neurons, and subsequent neuronal migration requires dynein transport of both the nucleus and centrosome to form the layered neocortex. The mechanisms for dynein functional diversity in brain development and in general are unclear. In this work, we used in utero electroporation to deliver cDNAs and shRNAs into the developing rat brain. Analysis was performed by fixed and live imaging 4 days post injection. We found that dynein containing the Light Intermediate Chain 1 subunit is required for neural stem cell proliferation, multipolar-to-bipolar transition and glial-guided neuronal migration. We found no role in the previous mechanisms for Light Intermediate Chain 2-containing dynein, but this dynein population was required for terminal somal translocation of neurons, which was unknown to depend on microtubule motors. Further, our in vitro and in vivo evidence demonstrates that Nesprin-2, a resident protein at the nuclear envelope, recruits dynein via its adaptor BicD2, to mediate nuclear transport during glial-guided neuronal migration. Disruption of Nesprin-2 or BicD2 dynein recruitment caused a severe block in migration, as cells were arrested before reaching the cortical plate. Centrosomal movement appeared intact, but nuclear transport was impaired which led to an increase by more than 50 fold in nucleus-centrosome distance. Overall, our data define discrete dynein populations that contribute differentially to brain development. These data also elucidate the mechanisms for nuclear movement during glial-guided neuronal migration and terminal somal translocation. Ultimately, these advances might help to understand the neurodevelopmental pathologies arising from human mutations in dynein related genes.
A dineína citoplasmática-1 (dineína) é um complexo proteico composto por várias subunidades que desempenha a maioria do transporte retrógrado dependente de microtúbulos na célula. A dineína tem múltiplas funções durante o desenvolvimento cerebral, e no humano mutações em genes que codificam proteínas do complexo causam patologias graves associadas ao neurodesenvolvimento. Nas zonas mais internas do neocórtex embrionário, a proliferação de células estaminais neurais depende do recrutamento de dineína para o núcleo, durante a oscilação nuclear característica destas células. Além disso, a dineína é necessária para a transição morfológica de neurónios multipolares para bipolares, e a subsequente migração neuronal para o córtex requer o transporte do núcleo e do centrossoma pela dineína. Globalmente, os mecanismos da diversidade funcional da dineína não são bem entendidos. Neste trabalho, usamos eletroporação in utero para injetar plasmídeos no cérebro de embriões de ratos, que foram analisados por microscopia confocal 4 dias após a cirurgia. Com isto, descobrimos que a dineína que contém a subunidade Light Intermediate Chain 1 é necessária para a proliferação de células estaminais neurais, para a transição de neurónios multipolares para bipolares, e para a migração neuronal. Não encontramos nenhuma função relevante nos mecanismos anteriores para a subunidade Light Intermediate Chain 2, mas esta população de dineína é fundamental para a terminal somal translocation de neurónios, um processo em que se desconhecia o envolvimento de motores de microtúbulos. Encontramos também através de experiências com imunofluorescência em células e co-imunoprecipitações que a Nesprina-2, uma proteína do invólucro nuclear, recruta dineína através do seu adaptador BicD2. Esta cadeia de interações é importante para a migração neuronal, e quando afetada, os neurónios não conseguem entrar para a Placa Cortical, o precursor embrionário do neocórtex. A disrupção da função da Nesprina-2 ou da BicD2 interrompe o movimento nuclear, mas curiosamente não afeta o transporte do centrossoma. Então as células afectadas apresentam uma separação anormal do núcleo em relação ao centrossoma. Em suma, os nossos dados definem populações específicas de dineína que contribuem de diferente forma para o desenvolvimento cerebral, e ajudam a perceber os mecanimos envolvidos na migração neuronal. Isto é importante para ajudar a entender as patologias do neurodesenvolvimento decorrentes de mutações humanas em genes relacionados com a dineína.
Financial support for this PhD work was provided by FCT (Fundação para Ciência e Tecnologia) fellowship PD/BD/113782/2015, awarded through the University of Minho MD/PhD program.

Loss of regulation of cell cycle events mediated by changes in cytosolic Ca$\sp{2+}$ ion activity has been implicated in the progression of normal cells to neoplasia. In this study, the Ca$\sp{2+}$ buffer 5,5$\sp\prime$-difluoro 1,2-bis(2-aminophenoxy) ethane-N,N,N$\sp\prime N\sp\prime$-tetra-acetic acid (5,5$\sp\prime$-dfBAPTA, abbreviated "dfB") has been used to modulate cell division in transformed and primary mouse keratinocytes. Exogenous application, via the tetra(acetoxymethyl) ester ("AM"), of 18-20 $\mu$M dfB/AM to the growth media of transformed cells inhibits cell division and DNA synthesis, without compromising the cells' viability, as shown by $\sp3$H-thymidine incorporation and flow cytometry. Bulk fluorimetry shows that cells treated with dfB/AM are able to buffer Ca$\sp{2+}$ in proportion to the concentration of dfB/AM applied. Primary cultured cells treated with 18-20 $\mu$M dfB/AM die within 3 hours of treatment. Viable dfB/AM treated cultures of transformed cells have a higher proportion of cells in the G$\sb2$ phase of the cell cycle than do controls, as shown by flow cytometry. This result, in combination with that showing reduced $\sp3$H-thymidine incorporation, suggests that 18-20 $\mu$M dfB/AM inhibits a pre- or mid-mitotic step. Light, electron, and confocal microscopies show 18-20 $\mu$M dfB/AM-treated cells to have prominent, thickened nuclear envelopes along with actin cytoskeletons that are distinguishable from controls. Upon return to medium that does not contain dfB/AM, treated transformed cells gradually resume their pre-treatment growth and division patterns.

Nuclear positioning occurs in different cellular contexts: from dividing yeast to more specialized cells like neuronal glial progenitor and skeletal muscle cells. Interestingly, abnormal nuclear positioning is associated with diseases such as muscular dystrophy where nuclei occupy a central rather than peripheral location. Moreover, rearward nuclear positioning is typical of migratory cells. Active nuclear movement in most cases involves coupling of cytoskeletal components with the nucleus by a group of transmembrane proteins in the nuclear envelope called the LINC (linker of nucleoskeleton and cytoskeleton) complex. It is composed of the inner nuclear membrane SUN (Sad1p, UNC-84) proteins associated with nuclear lamins and the outer nuclear membrane KASH (Klarsicht, ANC-1, Syne Homology) proteins, which interact with the cytoskeleton. In my thesis, the murine fibroblast cell line NIH3T3 was used as a model system to study nuclear positioning in states of active movement and static homeostatic positioning. Nuclear positioning and centrosome reorientation are hallmarks of cell polarity in migrating fibroblasts. The Gundersen lab has established that the nucleus moves rearward to orient the centrosome in serum starved fibroblast monolayers stimulated by the serum-derived factor lysophosphatidic acid (LPA). LPA stimulates the GTPase Cdc42, which in turn activates the Cdc42 effector MRCK to phosphorylate myosin II and activate actin retrograde flow to move the nucleus to the rear. A second Cdc42 effector, Par6 functions with Par3 and dynein to maintain the centrosome in the cell centroid. The nucleus is moved rearward by the attachment of retrograde dorsal actin cables to the nucleus through transmembrane actin-associated nuclear (TAN) lines. TAN lines are composed linear arrays of the LINC complex proteins nesprin-2G (N2G) and SUN2 and dorsal actin cables. Disrupting TAN lines components blocks nuclear movement and efficient cell migration. Interestingly, TAN lines are analogous to other membrane adhesions, such as focal adhesions, in that they are transmembrane structures linked to the actin cytoskeleton and transmit force. Given the large number of proteins composing structures such as focal adhesions, we predicted there would be additional components in TAN lines necessary for their formation and function. Thus, I set out to identify and study cytoplasmic factors required for TAN line formation and/or function during active nuclear positioning in fibroblast. A collaborator detected N2G as a hit in a yeast two-hybrid screen for FHOD1 interactors. FHOD1 is an actin regulator and belongs to the formin family. Like other formin family members, it has an FH2 actin binding domain, an FH1 domain and DID and DAD domains that interact to autoinhibit FHOD1. Unlike other formins, FHOD1 is not activated by GTPase binding and contains a second actin binding domain (ABS domain), giving it actin bundling activity. We show that spectrin repeats (SRs) 10-13 of N2G and the N-terminus of FHOD1 interacts with each other directly by biochemical assays with purified proteins. SiRNA against FHOD1 and overexpression of either FHOD1 or N2G interacting domains prevented LPA-stimulated nuclear movement in wounded monolayers of NIH3T3 fibroblasts, suggesting that the interaction between FHOD1 and N2G is required for nuclear movement and centrosome reorientation. FHOD1 was required for TAN line formation, but was dispensable for the formation of dorsal actin cables and retrograde actin flow. By re-expressing an artificial construct containing the N2G-binding domain of FHOD1 and the actin-binding domain of α–actinin in FHOD1 depleted cells, we show that the FHOD1 ABS domain provides N2G with an additional contact to actin filaments required for nuclear movement. This study thus identifies FHOD1 as a new TAN line component and suggests that the interaction of FHOD1 with N2G may reinforce TAN lines so that they can resist the force necessary to move the nucleus. The above study identifies a new component in a pathway that actively moves the nucleus. We have far less knowledge about the mechanism that maintains the nucleus in position when it is not moving. For example, it is unknown whether the static nuclear positioning is an active process or simply an inactivation of mechanisms that actively move nuclei. To answer this question, I developed a novel method to artificially displace the nucleus in adherent cells by centrifugation and used this system to identify active mechanisms of homeostatic nuclear positioning. By subjecting wounded monolayers of starved NIH3T3 fibroblast on coverslips to centrifugal force perpendicular to the wound, I find that nuclei are displaced towards the direction of centrifugal force, so that on one wound edge, the nuclei are in the cell rear while on the other, in the cell front. After returning centrifuged cells to the incubator, I used fixed and live cell recordings to show that the displaced nuclei actively re-center within one hour, although nuclei moving rearward did so faster than those moving forward. Treating centrifuged cells with cytoskeletal drugs, revealed an actin/myosin II-dependent rearward recentration and a microtubule (MT)/dynein-dependent forward recentration. I knocked down LINC complex components to test their involvement in these movements. N2G was required for both rearward and forward movement while SUN1 and SUN2 were required for forward and rearward movement, respectively. Overexpression of different N2G constructs in N2G-depleted cells showed that different regions of N2G were necessary for each direction of movement: N-terminal constructs rescued rearward nuclear recentration whereas C-terminal constructs rescued forward recentration. Based on the minimal N2G construct that rescued forward (MT dependent) nuclear recentration, I identified a dynein and dynactin site in the C terminus of N2G. To test whether the homeostatic nuclear positioning mechanisms were active in uncentrifuged cells, I depleted cells of nesprin-2 and then re-expressed nesprin-2 constructs capable of interacting with actin, MTs or both cytoskeletal elements. Nuclei in nesprin-2-depleted cells were no longer maintained at the cell centroid and only re-expression of a construct that contained sites for interaction with both actin and MTs rescued this defect. Thus, both actin- and MT- interaction domains of N2G are required for homeostatic nuclear positioning. To test whether the actin and MT activities of N2G were important for cell migration, I depleted NIH3T3 fibroblasts of nesprin-2 and re-expressed N2G constructs capable of interaction with actin, MTs or both and tested these cells in single and collective cell migration assays. I found that only the MT-dependent activity of N2G is required for the directionality of single cell migration while both N- and C- terminal (actin- and MT- dependent) N2G are required for the velocity of collective cell migration. These results show that different cytoskeletal linkages are used in different modes of cell migration. My thesis studies identify the first cytoplasmic factor required for TAN lines structure, establish a novel method to artificially displace the nucleus in adherent cells, and reveal different mechanisms of LINC complex coupling cytoskeletons during active and homeostatic nuclear positioning, as well as specific cytoskeleton-dependent contributions of nuclear envelope protein N2G during cell migration.

碩士
長庚大學
生物醫學研究所
103
Epstein-Barr nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-associated tumors, including nasopharyngeal carcinoma (NPC) which is a common malignant disease in Taiwan. However, the role of EBNA1 in cell tumorigenesis has not been well-established. EBNA1 in prototype B95.8 (B-EBNA1) is P-ala subtype. Examination of various Taiwanese EBNA1 clones isolated from NPC (N-EBNA1) and peripheral blood lymphocytes of healthy individuals (P-EBNA1) has demonstrated that they all belonged to V-val subtype but had different sizes in the middle domain (Glycine/Alanine repeat region). The role of different clones of EBNA1 in the NPC oncogenesis is thereby an interesting question remains to be investigated. The preliminary data in the lab indicated that cell migration was promoted by N-EBNA1, but not by P-EBNA1. The migration ability of B-EBNA1 was 2-fold lower than that of N-EBNA1. To further investigate the molecular domains responsible for the migration activity of EBNA1, B- and N-EBNA1 chimeras have been constructed and the results suggested that both of the N- and C-terminus of EBNA1 might play roles in the cell migration. Furthermore, cell migration ability of N-EBNA1 was inhibited by the ROCK inhibitor, Y27632. Collectively, these data suggest that both the N- and C-terminal sequences and sizes of middle domain of EBNA1 are important for the cell migration activity of EBNA1, which might be regulated through the activation of the Rho-ROCK signaling pathway.

碩士
長庚大學
醫學生物技術暨檢驗學系
99
N-myc downstream regulated gene 1 (NDRG1) is overexpression in head and neck cancer (HNC) tissues as we reported previously. However, the cellular function of this molecule on tumorigenesis of HNC is still not clear. The three-tandem repeats (3R) of ”GTRSRSHTSE” in the C-terminal region of NDRG1 is a specific feature. NDRG1 is predicted phosphorylated at the serine sites in its 3R motif by glycogen synthase kinase 3 β (GSK3β). In this study, we examined the significance of 3R motif of NDRG1 functions leading to HNCs. Expression of the deletion construct NDRG1ΔC [deletion of 3R motif] suppressed nuclear translocation of NDRG1, and led to significantly reduction of cell migration and invasion in HNC cells. We also observed that SB 216763, an inhibitor of GSK3β decreased the phosphorylation of 3R motif of NDRG1 and attenuated the nuclear translocation, cell migration and invasion. We further introduced mutations at the serine sites at 342 [S342A], 352 [S352A] and 362 [S362A], which are susceptible phosphorylation by GSK3β. Expression of all these mutants repressed NDRG1 nuclear import, cell migration and invasion. Together, these results suggest a novel function by which NDRG1 modulates cell motility and invasion through GSK3β phosphorylated serine sites of 3R motif at serine 342, 352 and 362.

碩士
長庚大學
生物醫學研究所
101
Epstein-Barr virus nuclear antigen 1 (EBNA1) is the only viral protein detected in all EBV-associated malignant tumors, including nasopharyngeal carcinoma (NPC). To elucidate the role of EBNA1 in NPC, EBNA1 genes, isolated from prototype B95.8 (B-EBNA1, P-ala subtype), NPC samples (N-EBNA1, V-val subtype), and peripheral blood lymphocytes (P-EBNA1, V-val subtype) of healthy individuals in Taiwan, have been cloned and expressed in cultured cells. These EBNA1 clones are different in not only sequences but also sizes, which are determined by the middle Gly/Ala repeats. The role of different clones of EBNA1 in the NPC oncogenesis remains to be investigated. The preliminary data in the lab indicated that cells expressing N-EBNA1 had the best ability to undergo cell migration in a trans-well assay. Therefore, the constructed chimeras between cloned B- and N-EBNA1 sequences and a serial of N-EBNA1 deletion clones with different sizes of middle domain were used here to study the molecular domains responsible for the migration activity of EBNA1. The data obtained from the B- and N-EBNA1 chimeras indicated that the C-terminal of EBNA1 ( aa 487~641 ) played an important role in cell migration. Since amino acids (aa) 502 and 524 are significantly different between B- and N-EBNA1, double mutations at these two specific sites have been constructed. The trans-well data have shown that aa 502 and 524 are crucial for cell migration activity promoted by N-EBNA1. Among seven deletion clones of N-EBNA1, only four of them had the ability to support cell migration. Collectively, these data strongly suggest that both sequences and sizes of middle domain of EBNA1 are important for the cell migration activity of EBNA1. Furthermore, treatment with Rho-kinase (ROCK) inhibitor diminished the cell migration ability of N-EBNA1 indicated that signaling pathway of Rho small GTPases might contribute to the cell migration activity of EBNA1. The connection between the activation of Rho-ROCK pathway by N-EBNA1 and the functional domains of EBNA1 affecting cell migration remains to be investigated.

碩士
中山醫學大學
生物醫學科學學系碩士班
101
Transforming Growth Factor (TGF)- β1 may induce epithelial-to-mesenchymal transition (EMT) and cancer invasion via Smad-dependent or -independent signaling pathways. Epigallocatechin-3-gallate (EGCG), the green tea polyphenol, has been reported to block TGF-β1-elicited EMT and invasion of human A549 lung cancer cells in a Smad-dependent manner. Herein, whether EGCG can inhibit TGF-β1-induced EMT and invasion of A549 cells from the modulation of a Smad-independent route was investigated. We found that EGCG suppressed TGF-β1-induced β-catenin nuclear localization and function through inhibiting PI3K/Akt/GSK-3β signaling activation, paralleled with the presence of membrane-bound β-catenin and epithelial marker E-cadherin and the downregulation of aggressive invasion phenotypes of A549 cells. These findings define a new molecular basis that EGCG may block TGF-β1-induced EMT and malignant dissemination of A549 cells through the modulation of a cross-talk between PI3K/Akt/ GSK-3βand Wnt/β-catenin signaling pathways.

One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...