Dissertations / Theses on the topic 'Drebrin A'

Des pathologies lourdes, telles que les dystrophies de la rétine et certains cancers, impliquent une désorganisation de l'épithélium et la famille Crb, dont la protéine apicale Crumbs3 (isoformes Crb3A et Crb3B) fait partie. Les protéines transmembranaires Crb possèdent un domaine intracellulaire fortement conservé et des partenaires communs. Il est donc essentiel de comprendre comment ces protéines Crb sont régulées afin de mieux appréhender ces pathologies. Pour cela, j'ai étudié le complexe de polarité apical Crb3A (Crb3A, Pals1, PATJ) impliqué dans l'établissement et le maintien de la polarité apico-basale. Je me suis, tout d'abord, intéressée à la régulation des isoformes de Crb3 par leurs partenaires (Pals1 et PATJ), puis, à la régulation des protéines de la membrane apicale, dont Crb3A, par la Drebrine E2, un nouveau partenaire de Crb3A impliqué dans l'organisation du cytosquelette d'actine et la morphogenèse apicale. Mon travail a permis de mettre en évidence: 1) la régulation de la dynamique membranaire des isoformes de Crb3 par PATJ dans les cellules Caco-2/TC7, une lignée épithéliale intestinale humaine, mais aussi, 2) d'identifier une nouvelle fonction de la Drebrine E2 dans la régulation du trafic de plusieurs protéines de la membrane apicale dans ces cellules, dont, par exemple, la DPPIV (DiPeptidyl Peptidase IV). Dans les cellules déplétées en Drebrine E2, l'expression des protéines apicales est diminuée et leur endocytose est augmentée, puis, elles sont relocalisées dans le compartiment majeur de dégradation, le lysosome
Some serious diseases like retinal dystrophies and some cancers involve epithelial cells disorganization and the Crumbs (Crb) proteins family. The apical Crb3 (Crb3A and Cr3B isoforms) protein belongs to Crb family. The transmembrane proteins Crb have a conserved intracellular domain with common partners. It is unclear how Crb proteins are regulated by their partners and this information is required to better understand these pathologies. Here, we decided to study the apical polarity Crb3A complex (Crb3A, Pals1, PATJ) which is involved in apico-basal polarity establishment and maintenance. First, I investigated Crb3 isoforms regulation by their partners (Pals1 and PATJ). Then, I studied the regulation of apical membrane proteins, such as Crb3A, by Drebrin E2, a new partner of Crb3A which is involved in actin cytoskeleton remodeling and apical morphogenesis. During my thesis, I demonstrated: 1) the regulation of Crb3 isoforms dynamics by PATJ in Caco-2/TC7 human intestinal epithelial cells, but also, 2) a new function for Drebrin E2 in regulating the trafficking of apical membrane proteins, like DPPIV (DiPeptidyl Peptidase IV). In Drebrin E2 KD cells, apical membrane proteins expression is decreased and we observe an increased endocytosis. This leads to relocalization of the apical membrane proteins to the main degradative compartment, the lysosome. These new datas suggest a role for Drebrin E2 in the regulation of apical membrane proteins recycling pathway. The Drebrin E2 KD cells phenotype is reminiscent of the microvillar inclusions disease (MVID). Now, I am trying to investigate the link between theses pathways

Die vorliegende Arbeit erforscht die Regulation der Expression von Drebrin; DBN (Developmentally Regulated Brain Protein) in Neuronen. DBN ist ein Protein das Actin bindet und Actin-Filamente bündeln kann. Änderungen der Morphologie der Spines verändern die synaptische Aktivität und Plastizität – wichtigen Prozessen bei der Gedächtnisbildung und Alterung des Gehirns, sowie bei geistigen Störungen bzw. Behinderungen. DBN-Expression im Alter und in einigen neurodegenerativen Krankheiten reduziert ist. Eine schwächere Expression von DBN in Spines geht außerdem mit einem Verlust an synaptischen Verbindungen einher, einem gemeinsamen Merkmal von Alterung und neurologischen Störungen wie der Alzheimer Krankheit. Diese Befunde bildeten die Motivation und Grundlage für meine Erforschung der Produktion und Lokalisierung von DBN. In meinem Projekt, habe ich den Effekt der sequenzspezifischen S647-Phosphorylierung von DBN untersucht. Die Arbeit zeigt, dass diese post-translatorische Modifikation die Stabilität von DBN reguliert. Ich habe FUNCAT-PLA und Puro-PLA für die Visualisierung von de novo synthetisierten Proteinen in situ benutzt. Mittels hochauflösender Fluoreszenz-Hybridisierung konnte ich zeigen, dass DBN nicht nur im Zellkörper sondern auch lokal in den Spines translatiert wird. Meine Resultate bieten eine Grundlage für das Verständnis der Regulierung de DBN-Konzentration in Zellen und ermöglichen die weitere Erforschung der Rolle der S647-Phosphorylierung von DBN für die Morphologie von Spines. Die Arbeit bildet außerdem eine experimentelle Plattform für weitere Studien der Rolle von DBN für Spines, sowohl in Bezug auf Stabilität als auch der synaptischen Funktion und Stabilität.
This thesis studies the abundance of the protein Drebrin; DBN (Developmentally Regulated Brain Protein) in neurons, which is an actin-binding protein capable of bundling actin filaments. Synapses in the mammalian brain are formed on tiny protrusions, called dendritic spines. Changes in spine morphology affect synaptic activity and plasticity, which are processes underlying memory formation. DBN abundance plays an important role in regulating dendritic spine morphology. Cognitive decline and neurodegenerative conditions have been shown to be linked with a decrease in DBN levels. A weakening in the expression of this protein in spines is associated with the loss of synaptic connections, a common feature of ageing and neurological disorders such as Alzheimer''s disease. This evidence was the underlying motivation for studying the localization and turnover of DBN. I studied the effect of the site-specific S647 phosphorylation of DBN and found that such post-translational modification regulates protein stability. For the project, I established several novel techniques in our laboratory, including state-of-the-art methods such as FUNCAT-PLA and Puro-PLA for the visualization of de novo synthesized proteins in situ. My results show that DBN translation occurs not only in somata but also locally in the dendrites and spines. The same observation is true for DBN transcripts, which are present both in the soma and dendrites of neurons. These observations suggest that DBN could play an important role during synaptic plasticity. My results allow the future investigation of the potential role of site-specific phosphorylation of DBN in spine morphology. This PhD thesis represents a contribution to better understanding the regulation of DBN abundance. It also provides an experimental platform for additional investigation about the role of DBN in spine morphology, regarding its stability and its correlation with synaptic maintenance and function.

The brain is a highly complex structure; neurons extend axons which follow precise paths to make connections with their targets. This extension is guided by a specialised and highly motile structure at the axon tip -the growth cone- which integrates guidance cues to steer the axon through the environment. Aberrant pathfinding is likely to result in developmental impairments causing disruption to brain functions underlying emotion learning and memory. Furthermore, pre-existing connections are constantly remodelled, the ability to do so declines with age, and can have huge impacts on quality of life and well-being. Examining how changes in growth cone behaviour triggered by external cues occurs is crucial for understanding processes in both development and disease. Controlled reorganisation of growth cone cytoskeletal components, such as actin filaments, generate membrane protrusions forming lamellipodia and filopodia. Filopodium formation is commonly associated with sensing the mechanical and chemical environment of the cell. Despite our understanding of the guidance choices that can be made, how filopodia transmit information at a molecular level leading to profound changes in morphology, motility and directionality remains largely unknown. Various actin-binding proteins regulate the number, stability and branching of filopodia. They may therefore have a key role in priming or abrogating the ability of the growth cone to respond to a given guidance cue. I have shown that the actin binding proteins drebrin and cofilin, whilst displaying opposing molecular activities on actin filaments, work synergistically in a temporally regulated manner. A fluorescent membrane marker combined with tagged cofilin and drebrin enabled accurate correlation of cofilin and drebrin dynamics with growth cone morphology and filopodial turnover in live neurons. In contrast to previous in vitro experiments, cofilin was found to enhance the effect of drebrin to promote filopodia formation in intact neurons, and that growth cone spread was significantly constrained when cofilin was knocked down. Importantly, this adds to our understanding of how the two actin binding proteins contribute to directed motility in neuronal growth cone filopodia during guidance. Furthermore, following acute treatment with low concentrations of the repulsive guidance cue semaphorin-3A, neuronal growth cones expressing cofilin displayed increased morphological complexity and filopodial stability. This suggests that traditional collapse signals may serve as pause signals allowing neurons to increase the surface area to sense the environment adequately and enable precise wiring decisions. Remodeling of the cytoskeleton is perturbed in a number of degenerative diseases including Alzheimer's, Huntington's, and Amyotrophic Lateral Sclerosis. These conditions are associated with widespread synaptic loss, resulting in memory loss, cognitive impairment, and movement disorders which leads to severe deterioration in quality of life for those afflicted in addition to wider negative socioeconomic impacts. How widespread synaptic loss occurs is poorly understood. One common characteristic is neuronal stress which can be initiated through different conditions such as neuroinflammation, energetic stress, glutamate excitotoxicity, and accumulation of misfolded proteins, all of which have been associated with perturbation of the actin cytoskeleton and the initiation of the cofilin-actin rod stress response. Dysfunction of the cytoskeleton can lead to the disruption of synaptic activity by blocking the delivery of elements such as organelles and proteins required for maintenance of the synapse. Modulating this stress response offers an approach to protecting the integrity of normal synaptic function. Actin interacting protein-1 is a conserved actin binding protein that enhances the filament disassembly activity of cofilin. I have discovered that AIP-1 has a potent ability to prevent the formation of cofilin rods which are thought to contribute to the neuronal dysfunction in several neurodegenerative disorders, even when they are treated with amyloid-β or subjected to metabolic stress. This is the first study to demonstrate a molecular mechanism for preventing rod formation in the presence of a neuronal stressor and has the potential to protect against rod formation by other stressors associated with disease such as inflammation and excitotoxicity. AIP-1 offers the exciting possibility of a means to reverse cofilin rod formation and the subsequent cytoskeletal pathology associated with dementia and has potential for therapeutic exploitation in human disease. Furthermore, it is the first study to demonstrate that AIP-1 localises to areas of rapid actin remodeling in neuronal growth cones. Exploiting the action of AIP-1 therefore represents an exciting and novel therapeutic avenue to tackle neurodegeneration.

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along he rostral migratory stream (RMS) to become inerneurons in the olfactory bulb (OB). This migration is a fundamental event controlling the proper integration of new neurons in a pre-existing synaptic network. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. We have investigated the function of fascin and drebin, two actin-binding proteins highly expressed by RMS neuroblasts in the postnatal mammalian brain. We show that fascin-1 ko mice display an abnormal RMS and a smaller OB. Bromodeoxyuridine labeling experiments show that lasck of fascin significantly impairs neuroblast migration, but does not affect cell proliferation. Moreover, fascin depletion alters the polarized morphology of rat neuroblasts. Protein kinase C (PKC)-dependent posphoylation of fascin on Ser39 regulates its actin-bundling function. Postnatal electroporation of phosphomimetic (S39D) or nonphosphorylatable (S39A) fascin variants followed by time-lapse imaging microscopy (FLIM) studies in rat neuroblasts reveal that the interaction between fascin and PKC can be modulated by cannabinoid signaling, which controls neuroblast migration in vivo. These findings identify fascin as a crucial regulator of neuroblast motility. We propose that a tightly regulated phospho/dephosphofascin cycle modulated by extracellular signals is required for the polarized migration of stem cell-derived neuroblasts. Depletion of drebrin using different RNAi approaches affects neuroblast morphology and impairs neuroblast migration both in vitro and in vivo. Drebrin phosphorylation on Ser142 by Cdk5 promotes actin bundling and microtubule binding. Electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin followed by time-lapse imaging shows decreased neuroblast migration compared to control. Our findings demonstrate that drebrin is necessary for efficient neuroblast migration and suggest that its phosphorylation on Ser142 plays an important role in regulating neuroblast orientation along the RMS.

La morphogenèse épithéliale est un processus complexe qui résulte en une organisation particulière des épithélia, leur permettant ainsi d’assurer leurs fonctions physiologiques. Les cellules épithéliales sont polarisées et présentent un domaine apical en contact avec le milieu extérieur et un domaine basal en contact avec la lame basale. Ces deux domaines sont séparés par un complexe jonctionnel qui permet de maintenir une cohésion du tissu, et qui joue un rôle de barrière physique entre l’extérieur et l’intérieur du corps. Du côté apical, ces cellules présentent une bordure en brosse composée de microvillosités hautement organisées, qui leur permet ainsi d’augmenter leur surface d’échange avec le milieu extérieur. Dans la cellule, ces microvillosités vont s’ancrer au niveau d’un réseau dense d’actine, le terminal web. Au cours de ma thèse, je me suis intéressée à la morphogenèse des cellules épithéliales intestinales en utilisant comme modèle la lignée cellulaire Caco2 qui en culture est capable de se différencier en entérocytes. L’acquisition de leur polarité ainsi que de leur forme colonnaire nécessite des remodelages du cytosquelette d’actine et la régulation du trafic intracellulaire des composants de la membrane plasmique. Dans cette étude, j’ai identifié la Drebrine, qui est une protéine de liaison à l’actine, comme une protéine nécessaire à la formation du terminal web et de la bordure en brosse. Parallèlement, j’ai montré que la Drebrine est impliquée dans la redistribution de différentes vésicules d’endocytose, et/ou de recyclage, lors de la morphogenèse épithéliale
Epithelial morphogenesis is a complex process that provides a unique organization to epithelial cells in order to perform their physiological functions. Epithelial cells are highly polarized cells with an apical domain facing the outside environment and a basolateral domain contacting the underlying basal lamina. These two cell surfaces are delimited by a set of junctions providing tissue integrity and a controlled physical barrier between the outside and the inside of the body. In particular, in single layered columnar epithelia, cells are highly organized along the apico-basal axis with an apical surface that is often covered by microvilli developed to enlarge the apical surface and the exchanges with the outside medium. These microvilli in the cells are anchored in a dense actin-based network called terminal web. This cell polarity relies on the local organization of the cell cytoskeleton and the regulation of intracellular trafficking of plasma membrane components. Here we showed that Drebrin, which is an actin-binding protein, is required for the formation of the terminal web and thus for the brush border organization. In parallel, we also demonstrate a role for Drebrin in the correct distribution of endocytic and recycling vesicle during the process of cell morphogenesis

p83 é uma proteína com massa molecular aparente de 83 kDa, supostamente ainda não descrita, específica de sistema nervoso, e desenvolvimento regulada. p83 interage fortemente com laminina, Tau, tubulina e heat shock protein 90. p83 foi inicialmente detectada por imunohistoquímica e western blot usando-se um anticorpo anti-lectina KM+ purificado por afinidade. Sua purificação a partir de cérebro de rato está em progresso. Identificar o envolvimento de p83 em processos do Sistema Nervoso Central humano é um passo necessário em direção à compreensão de sua função biológica. Uma biblioteca de expressão de cDNA de cerebelo de rato (Lambda ZAP II, Stratagene) foi submetida ao screening, usando-se um anticorpo específico para isolar o cDNA de p83. O anticorpo anti-KM+ foi pré-adsorvido contra proteínas de E. coli XL1 Blue MRF, antes de ser usado no screening. As membranas foram reveladas por imunodetecção cromogênica (fosfatase alcalina e NBT/BCIP). A análise de todos os clones Lambda ZAP II foi feita por excisão in vivo do fagomídeo pBluescript, subclonagem em E. coli XL1 Blue MRF, purificação do DNA plasmidial e digestão com Eco RI. A seqüência correspondente ao clone isolado foi analisada usando-se ferramentas e bancos de dados do NCBI. A seqüência nucleotídica mostrou identidade com as isoformas A e E de drebrina. As isoformas A e E de drebrina foram detectadas em adulto e embrião, respectivamente. Drebrina A é uma proteína sistema nervoso-específica, desenvolvimento regulada e associa-se com F-actina. Embora drebrina e p83 compartilhem propriedades em comum, nossos dados de western blot indicaram que parecem não se tratar da mesma proteína. Nós investigamos a expressão de drebrina em Displasia Cortical Focal tipo IIB, comparando com córtex normal. As secções de tecido foram coradas com hematoxilina-eosina e prata (Bielchowsky). Secções foram processadas por imunohistoquímica usando-se os anticorpos anti-drebrina M2F6 e o DAS2, e recuperação antigênica. A detecção foi feita usando-se um anticorpo biotinilado, e DAB como cromógeno. Os tecidos displásicos (13 casos) foram obtidos cirurgicamente de tecidos exibindo epilepsia droga-resistente. Os controles foram obtidos de necrópsia de 15 pacientes sem história prévia de doenças neurológicas ou alterações patológicas. Nossos resultados sugerem uma associação entre drebrina e DCF IIB, um distúrbio do desenvolvimento cortical.
p83 is 83 kDa protein supposedly not yet described, nervous system specific, and developmentally regulated. p83 strongly interacts with laminin, Tau, tubulin and heat shock protein 90. It was initially detected by immunohistochemistry and western blot using an affinity-purified anti-lectin KM+ antibody. Its purification from rat brain is in progress. Identifying the involvement of p83 in human Central Nervous System processes is a required step towards understanding its biological roles. A premade cDNA rat cerebellum expression library (Lambda ZAP II, Stratagene) has been screened, using a specific antibody to isolate p83 cDNA. Anti-KM+ antibody was pre-adsorbed against E. coli XL1 Blue MRF proteins, before using in screening. Membranes were revealed by cromogenic immunodetection (alcaline fostase and NBT/BCIP). The analysis of all positive Lambda ZAP II clones was carried out by in vivo excision of pBluescript, subcloning in E. coli XL1 Blue MRF, plasmidial DNA purification and Eco RI digestion. The sequence corresponding to the clone isolated was analyzed using the NCBI tools and database. The nucleotide sequence showed identity with drebrin A and E isoforms. Drebrin A and E isoforms were detected in adults and embryos. Drebrin A is a neuron-specific, development-regulated F-actin-binding protein. It participates in growth cone extension and dendritic spine formation. Although have same drebrin and p83 properties in common, they not seem to be the same protein. We have investigated the expression of drebrin in Focal Cortical Dysplasia type IIB (FCD IIB) as compared to normal cortex. Tissue sections were stained with hematoxylin-eosin and silver (Bielchowsky). Sections were processed for immunohistochemistry using anti-drebrin antibodies M2F6 and DAS2, and an antigen retrieval technique. Detection was carried out using a biotinylated antibody, using DAB as chromogen. Dysplastic tissues (13 cases) were obtained at surgery for drug-resistant epilepsy. Controls were obtained at autopsy from 15 patients without history of neurological disorder and gross pathological changes. A specific drebrin labeling in dysplastic tissue was more intense than in controls. Indeed, most control cases exhibited at most a slightly higher staining than the background. Balloon, clear and undetermined cells, and giant, dysmorphic neurons, showed a conspicuous labeling by anti-drebrin. These cells showed a thin rim labeling of the nuclear membrane, and a finely punctate nuclear labeling. In contrast, a coarse nuclear, but a faint cytoplasm labeling was observed in autopsy cases. Our data suggest an association between Drebrin expression and the FCD IIB, a disturbance of cortical development.

Die Arbeit überprüft anhand der Gesetze zu Vorratsdatenspeicherung in Deutschland und Österreich die Erklärkraft des Multiple Streams Ansatz. Die Analyse kommt zu dem Ergebnis, dass der Multiple Streams Ansatz die Entstehung dieser Gesetze besser erklären kann, je weniger die EU den Prozess mitbestimmt. Weitere Ergebnisse der Arbeit sind, dass insbesondere die ideologische Position der Regierungsparteien sowie fokussierende Ereignisse das Zustandekommen einer Vorratsdatenspeicherung begünstigen, während die Verfassungsmäßigkeit der Gesetze keine Rolle zu spielen scheint.