Melioidosis, caused by the environmental Gram negative bacillus Burkholderia pseudomallei, is an emerging infectious disease affecting both animals and humans. B. pseudomallei has the ability to enter the host cell and escape from the phagosome. Once in the cytoplasm, the pathogen proliferates and expresses a virulence-associated protein known as BimA which polymerises cellular actin at the pole of the bacterium to promote its movement inter- and intracellularly, a process known as actin-based motility. This actin-based motility is also used as a strategy to evade host immune responses and survive intracellularly. In the first part of the thesis, we demonstrate that a B. pseudomallei ΔbimA mutant displays impaired intracellular survival compared to the isogenic parent strain in BALB/C bone-marrow derived macrophages (BMDMs), notably at later time points post-infection. Macrophages are the key innate immune cells that control B. pseudomallei in vivo and in vitro, and BALB/C mice provide an excellent model of acute human melioidosis. We also have determined that in BMDMs, the ΔbimA mutant is able to escape from the phagosome and enters the cytosol where it is unable to form actin tails. We used targeted, hypothesis-driven experiments to identify potential cell-autonomous innate mechanism/s of killing the mutant. First, we speculated that BimA mediates escape from autophagy. However our studies, including LC3-conversion assays, and bacterial co-localisation studies, failed to demonstrate a role for autophagy in clearance of the ΔbimA mutant from infected BMDMs. In the second part of this thesis, we investigated the role of Toll-like Receptors (TLR) in recognition and elimination of B. pseudomallei. MyD88 (Myeloid differentiation primary-response gene 88) and TRIF (TIR-domain-containing adaptor protein inducing IFNβ) are the main adaptor proteins involved in TLR signalling. We utilised the gene silencing technique using short interfering RNAs (siRNAs) to knockdown MyD88 transcript, and in a separate experiment used MyD88- or TRIF-blocking peptides. In addition, we investigated the involvement of canonical and non-canonical inflammasome pathways in cell-autonomous immunity of the BMDMs. However, none of these pathways were shown to be involved in clearance of the ΔbimA mutant from infected BMDMs. Finally we took an unbiased approach by microarray to characterise the global host transcriptome in BALB/C BMDMs upon B. pseudomallei infection, and to identify specific responses to the ΔbimA mutant. Analyses performed at the gene level revealed that several interferon signalling-related pathways are activated in cells infected with either the WT or ΔbimA mutant strains. A number of other pro-inflammatory mediators that are commonly seen in general inflammatory infections, such as IL-1α, IL-1β, IL-12β, and IL-6, were also upregulated. Interestingly, the cytoplasmic RNA sensors RIG-1 and MDA-5, thought primarily to be involved in the detection of RNA viruses, were also induced upon B. pseudomallei infection. Very few pathways were associated with a specific macrophage response to the ΔbimA mutant, indicating that an as yet undescribed pathway may play a role in sensing and eliminating the ΔbimA mutant. We conclude that actin-based motility mediates escape of B. pseudomallei from macrophage intracellular killing through a novel pathway which has yet to be unravelled.

Le transport intracellulaire met en jeu des vésicules et nécessite ainsi des modifications de la membrane plasmique. En particulier, des nanotubes de membrane de quelques dizaines de nanomètres peuvent se former. Nous avons mis en place un système biomimétique à base de liposomes pour décrypter les mécanismes de changement de forme membranaire, en particulier sous l’action du cytosquelette d’actine. La physique des tubes de membrane est bien connue, notamment la force nécessaire au maintien de ce type de tube, qui dépend de l’élasticité de courbure du liposome et de sa tension de membrane imposée par l’aspiration d’une micropipette. En utilisant une diode quatre quadrants, nous avons atteint une résolution temporelle de l’ordre de 4 µs, et une résolution en termes de force plus précise que le pN. Ce montage permet pour la première fois d’étudier les fluctuations de tels tubes. Cette thèse ouvre la voie à l’étude des effets de la polymérisation d’actine sur ces nanotubes / Intracellular transport involves membrane compartments and thus requires dynamic changes in the morphology of cell membranes. In this case, membrane tubes are formed whose radius is of the order of several tens of nanometers. We develop biomimetic systems based on model lipid membranes to decipher the mechanisms of membrane remodelling in particular under the action of the actin cytoskeleton. The mechanics of membrane nanotubes, especially the force needed to form and maintain a nanotube, are now well understood. The force depends on the curvature elasticity of the membrane and on its mechanical tension that is controlled in our experiment by micropipette aspiration. By using a four-quadrant diode, we obtain an unprecedented temporal resolution, in the order of 4 µs, and a force resolution under pN. This setup allows us to access unrivaled membrane nanotube properties.This thesis paves the way for studying the effect of actin dynamics on membrane nanotubes

Actin isoform specific mutations have been identified as causes for various human diseases. These include twelve missense mutations in γ-nonmuscle actin leading to early onset autosomal dominate nonsyndromic hearing loss and twenty two missense mutations in α-smooth muscle actin leading to thoracic aortic aneurysms and dissections (TAAD). The molecular mechanisms leading to these human pathologies are mainly unknown, principally due to the inability to isolate pure mutant γ-nonmuscle actin and α-smooth muscle actin in quantities required for biochemical analysis. To begin to address these problems, I have individually expressed the human nonmuscle actin isoforms, β– and γ– nonmuscle actin, in a baculovirus expression system and characterized their biochemical properties. Surprisingly, despite a conserved amino acid difference of only 4 residues at or near the N-terminus, Ca-γ-actin exhibits slower monomeric and filamentous biochemical properties than β-actin. In the Mg-form, the difference between the two is smaller. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. Calcium bound in the high affinity binding site of γ-actin may cause a selective energy barrier relative to β-actin that retards the equilibration between G– and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the γ-actin-rich cochlear hair cell stereocilium where local mM calcium concentrations may exist. In hair cells γ-nonmuscle actin seems to play a central role in stereocilia maintenance. To determine how the deafness causing D51N-γ-mutant actin mutation leads to deafness, I expressed and characterized it in the γ-actin background. The D51N mutation, lethal when cloned into yeast, displayed decreased filament stability and polymerization kinetics of an actin more dynamic than γ-actin. This result suggests that the hearing effects of the γ-actin mutations on the hearing apparatus are not simply caused by an inability to polymerize. The observed increased polymerization rates and decreased filament stability may have major implications for the human disease, as the mutation may alter the ability of the γ-actin to fulfill its maintenance functions. To address the basis by which TAAD mutations cause vascular dysfunction I introduced two of the know human mutations, N115T and R116Q, into yeast actin, 86% identical to human α-smooth muscle actin. I then generated yeast strains expressing each of these mutations as the sole actin in the cell to assess their effect on actin function in vivo and in vitro. Both mutant strains exhibited reduced ability to grow under a variety of stress conditions, although the N115T cells were more severely affected. In vitro the mutations caused exhibited altered thermostability and nucleotide exchange rates indicating effects on monomer conformation with R116Q the most severely affected. The N115T actin demonstrated a biphasic elongation phase during polymerization, while R116Q actin demonstrated a markedly extended nucleation phase. Allele-specific effects were also seen on critical concentration, rate of depolymerization and filament treadmilling. R116Q filaments were hypersensitive to severing by the actin-binding protein cofilin. In contrast, N115T filaments were hyposensitive to cofilin, despite near normal binding affinities of actin for cofilin. The mutant specific effects on actin behavior suggest that individual mechanisms may contribute to TAAD. Understanding the mechanisms of actin dependent human diseases requires elucidation of the effects of the mutations on the behavior of actin per se, its regulation, and the impact on actin mediated processes within the cell. The work provided in this thesis and future studies will provide the information required to understand the pathways involved in these diseases and form innovative treatments for deafness and TAAD.

The parallel actin filament bundling protein Fascin is a critical protein in both disease and development. Overexpression of Fascin is linked to increased aggressiveness in a number of cancer types, including breast and colon carcinomas. Importantly, Fascin is not normally expressed in adult epithelial cells from which many of these cancers arise. Therefore, Fascin is increasingly cited as both a potential biomarker and therapeutic target in many types of cancer. Fascin is most commonly associated with the formation of filopodia and invadapodia (parallel actin filament bundle structures) to drive migration and invasion. However, Fascin activity and regulation remain poorly understood. In order for Fascin to be an effective target for cancer therapeutics, a better understanding of the mechanisms regulating Fascin activities in the cell is necessary. Prostaglandins (PGs) are short-lived lipid signaling molecules that mediate a wide range of biological activities. PGs act through G protein-coupled receptors to initiate signaling cascades that affect downstream targets, including actin cytoskeletal remodeling. Importantly, the key enzymes in the synthesis of PGs, cyclooxygenase (COX) 1 and 2, are the targets of non-steroidal anti-inflammatory drugs like aspirin. Interestingly, like Fascin, PGs have been independently implicated in cancer development and metastasis and aspirin may reduce the risk of aggressive cancer. However, the exact mechanisms by which PGs mediate cancer development are unknown. The work presented in this thesis focused on novel PG-dependent regulation and activity of Fascin. The research presented here utilized Drosophila oogenesis as a model system to analyze PG-dependent Fascin activity. Drosophila oogenesis is an ideal model in which to study the activity and regulation of actin binding proteins like Fascin. Oogenesis consists of 14 morphologically defined stages, which are observable many times over within a single isolated pair of ovaries. A developing follicle consists of 16 germline cells – 15 nurse, or support cells, and a single oocyte. The nurse cells are of particular interest because they are the sites of dynamic actin remodeling during mid-late oogenesis. During stage 10B, an array of radially-aligned actin filament bundles form at the nurse cell membranes and extend inwards towards the nucleus. A network of cortical actin is also strengthened during this stage. These actin structures are essential for the completion of oogenesis, and ultimately female fertility. Importantly, PGs and Fascin are required for this actin remodeling; genetic loss of Fascin or the Drosophila COX-like enzyme Pxt (Peroxinectin-like) leads to disruption of cytoplasmic actin remodeling, and ultimately, female sterility. Using this model system, work presented here describes the discovery of Fascin as a downstream target of PGs to promote actin bundle formation, described in Chapter 2. Additionally, Fascin is required for strengthening of the cortical actin network downstream of PGs. This observation is one of the first to describe a role for Fascin in a branched actin network. Additionally, Fascin is regulated by a specific PG – PGF2α – during S10B to promote follicle development. Finally, Chapter 2 shows that PGs target specific actin binding proteins to promote cytoskeletal remodeling; Villin, another actin bundling protein, does not interact with PGs. Chapter 3 describes the novel observation that Fascin localizes to the nucleus and the nuclear periphery in Drosophila nurse cells. This finding is significant, as it is the first to describe Fascin in a context other than cytoplasmic. Fascin localization in and around the nucleus is specific and dynamic, and changes throughout late stage oogenesis, suggesting regulated functions at these sites. Fascin localization is regulated by PGs, and loss of Pxt leads to reduced nuclear Fascin localization and failure to localize to the nuclear periphery. Additionally, Fascin has novel potential functions in the nucleus and at the nuclear periphery. Loss of Fascin leads to disruption of nucleolar morphology in the nurse cell nuclei. Additionally, loss of PGs, which cause reduced nuclear Fascin levels, also causes abnormal nucleolar morphology. These data suggest that PGs regulate Fascin to control nucleolar organization. At the nuclear periphery, Fascin localization requires components of the protein complex that links the nucleoplasm to the cytoplasm, termed the LINC complex. Loss of an essential LINC complex protein, Koi, leads to a loss of nuclear periphery Fascin localization. These data suggest that Fascin may be a novel component of the LINC complex. Finally, Chapter 4 describes regulation of Fascin by phosphorylation at conserved serine residues. PGs affect Fascin phosphorylation, and loss of PGs leads to more heavily phosphorylated Fascin. Additionally, phosphorylation of Fascin alters localization to the nucleus and to the nuclear periphery. These data suggest that one mechanism by which PGs regulate Fascin is to control its phosphorylation status to affect subcellular distribution. In summary, the work presented in this thesis has demonstrated novel regulation and function of the actin bundling protein Fascin using Drosophila oogenesis as a model. Importantly, these functions and regulation of Fascin are likely conserved in mammals, and may have implications in human health and disease. Continued study of the activity and regulation of actin binding proteins like Fascin in Drosophila will likely have great effect on our understanding of many human diseases.

Prostaglandins (PGs) are small, lipid signaling molecules produced downstream of cyclooxygenase (COX) enzymes. PG signaling regulates many processes including pain, inflammation, fertility, cardiovascular function and disease, and cancer. One mechanism by which PG signaling exerts its function is by regulating the dynamics of the actin cytoskeleton; however, the exact mechanisms remain largely undefined. Drosophila oogenesis provides an ideal system to determine how PG signaling regulates the actin cytoskeleton. Drosophila follicles, or eggs, pass through 14 well- characterized, morphologically defined stages of development. Each developing follicle is comprised of 16 interconnected germline-derived cells (15 nurse cells and 1 oocyte) that are surrounded by a layer of somatically derived epithelial cells. During Stage 10B (S10B), the nurse cells form a cage-like network of parallel actin filament bundles that extend from the nurse cell membranes inward, toward the nurse cell nuclei. During Stage 11 (S11), the nurse cells rapidly transfer their cytoplasmic contents into the oocyte in an actomysoin-dependent contraction termed nurse cell dumping. Previous work uncovered that the Drosophila COX-like enzyme, Peroxinectin-like (Pxt), and thus PG signaling, is required to promote both actin filament bundle formation during S10B and subsequent nurse cell dumping. This finding established Drosophila oogenesis as a genetically tractable model in which to elucidate the conserved mechanisms underlying PG- dependent actin remodeling. The research presented in this dissertation focused on identifying actin-binding proteins that are regulated by PG signaling during Drosophila oogenesis. To identify these downstream effectors, we performed a dominant modifier screen to uncover factors that could suppress or enhance the ability of COX inhibitors to block nurse cell dumping in vitro. This screen revealed a number of actin-binding proteins that enhance the dumping defects caused by COX-inhibition, including the actin bundling protein, Fascin (Drosophila Singed, Sn); the actin filament elongation factor, Enabled (Ena); and the actin filament capper, Capping protein (Drosophila Capping protein alpha, Cpa, and beta, Cpb). Through a collaborative effort between Christopher Groen and myself, Fascin was shown to mediate PG-dependent cortical actin integrity and actin bundle formation during Drosophila ooogenesis. Ena and Capping protein regulate actin filament elongation through opposing actions: Ena promotes their elongation, while Capping protein binds to, or caps, the growing end of actin filaments to prevent their further elongation. However, genetic reduction of either Ena or Capping protein enhance the nurse cell dumping defects caused by COX inhibition. These findings suggest that Ena activity must be balanced to promote proper actin remodeling during S10B. Ena localization to the growing ends of actin filament bundles is reduced in pxt mutants during S10B, suggesting that PG signaling is required to promote Ena localization at this stage. Together, these data support a model in which PG signaling promotes actin remodeling during S10B, at least in part, by modulating Ena-dependent actin remodeling. While PG signaling promotes parallel actin filament bundle formation during S10B, PGs also restrict actin remodeling during Stage 9 (S9). Loss of Pxt results in early actin remodeling, including the formation of extensive actin filaments and actin aggregate structures within the posterior nurse cells of S9 follicles. Wild-type follicles exhibit similar structures at a low frequency. Ena preferentially localizes to the early actin structures observed in pxt mutants and reduced Ena levels strongly suppress early actin remodeling in pxt mutants. These data indicate that PG signaling temporally restricts actin remodeling during Drosophila oogenesis, at least in part, through negative regulation of Ena localization or activity during S9. The data presented here support a model in which PG signaling coordinates the concerted activity of a number of actin-binding proteins to regulate actin remodeling during Drosophila oogenesis. Specifically, PG signaling temporally restricts actin remodeling during S9 of Drosophila oogenesis, but promotes parallel actin filament bundle formation during S10B. PG signaling achieves this temporal regulation, at least in part, through differential regulation of Ena-dependent actin remodeling. Based on prior pharmacologic studies, we hypothesize that PGE2 is required to restrict Ena-dependent actin remodeling during S9, while PGF2Α; is required to promote Ena-dependent actin remodeling during S10B. Determining how these signaling cascades achieve differential regulation of Ena throughout Drosophila oogenesis is an important area for future investigation. As both the actin cytoskeletal machinery and PG signaling are conserved across species, the data presented here provide new and significant insights into the likely conserved mechanisms by which PG signaling regulates actin remodeling across species.

Orientador: Ana Lia Anbinder / Coorientadora: Noala Vicensoto Moreira Milhan / Banca: Fábio Luiz Coracin / Banca: Monica Ghislaine Oliveira Alves / Resumo: Granuloma piogênico (GP) é uma lesão de origem inflamatória que ocorre frequentemente em pele e cavidade oral. Existem dois subtipos histológicos: o tipo não lobular (GPNL), que é caracterizado por proliferação vascular semelhante a tecido de granulação, sem padrão de organização; e o tipo lobular (GPL) que se caracteriza pela organização dos vasos em agregados lobulares, separados por feixes de tecido conjuntivo. O objetivo deste estudo foi revisar todos os casos de granulomas piogênicos do nosso serviço de patologia bucal, a partir do ano 2000, reclassificar e correlacionar as características clínicas, microscópicas e imuno-histoquímicas com os subtipos da lesão. No levantamento foram encontrados no arquivo 197 casos diagnosticados como granuloma piogênico e hemangioma lobular capilar. Após revisão das lâminas, 9 casos foram reclassificados, e 19 foram excluídos, restando 169 casos, sendo 62 de GPL e 107 de GPNL. Foram coletados ainda dados como sexo, idade, local da lesão, tipo de nódulo, ocorrência de trauma prévio e hipótese diagnóstica clínica. Reações imuno-histoquímicas (GLUT-1, CD34, D2-40, AML e Mast cell) de 22 casos, sendo 11 lobulados e 11 não lobulados. A média de idade de acometimento foi de 38,59±16,96 anos, com 55,62% dos casos ocorrendo em pacientes do sexo feminino (10,12% durante gravidez), com maior acometimento em gengiva (39,64%), 44,97% dos nódulos eram do tipo pediculado e 13,02% relataram trauma mecânico prévio. O GPNL ocorre mais em gengiva, enquant... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pyogenic granuloma (GP) is an inflammatory lesion that occurs frequently in the skin and oral cavity. There are two histological subtypes: the non-lobular type (NLCH), which is characterized by vascular proliferation similar to granulation tissue, without organization pattern; and lobular capillary hemangioma (LCH) characterized by the organization of vessels in lobular aggregates, separated by bundles of connective tissue. The purpose of this study was to review all cases of pyogenic granulomas of our oral pathology service, from the year 2000, to reclassify and correlate the clinical, microscopic and immunohistochemical characteristics with the subtypes of the lesion. In the survey, 197 cases diagnosed as pyogenic granuloma and lobular capillary hemangioma were found in our files. After review, 9 cases were reclassified, and 19 were excluded, remaining 169 cases, being 62 LCH and 107 NLCH. Data such as sex, age, site of lesion, type of nodule, previous trauma and clinical diagnostic hypothesis were also collected. Immunohistochemical reactions (GLUT-1, CD34, D2-40, SMA and Mast cell) of 24 cases, 11 LCH and 11 NLCH. Mean age was 38.59±16.96 years, with 55.62% of cases occurring in female patients (10.12% during pregnancy), with a greater involvement in gingiva (39.64%), 44.97% of the nodules were pedunculated and 13.02% reported previous mechanical trauma. The NLCH occurs more in gingiva, while LCH affects more lips (p<0,05). The number of microvessels (CD34 positive), SMA ... (Complete abstract click electronic access below) / Mestre

Adherens junctions are essential for embryogenesis and tissue homeostasis. The major transmembrane adhesion receptors in adherens junctions are the cadherins, which mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one cytoplasmic component of the cadherin adhesion complex, but unlike other junction components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cellmatrix adhesions has commanded the most attention, while little is known about its role in cell-cell adhesions. To define the role of vinculin in adherens junctions, I established a short hairpin RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell surface. I investigated the mechanism for this effect and found that vinculin must bind to beta-catenin to regulate E-cadherin surface expression. Having established a role for vinculin in cell-cell adhesions, the critical question became how vinculin recruitment to and activation at cell-cell junctions are regulated. I found that á-catenin triggers activating vinculin conformational changes. Unlike all of the known vinculin activators in cell-matrix adhesions, alpha-catenin binds and activates vinculin independently of an A50I substitution. Thus, adherens junction activators and cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822. Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially recruited to adherens junctions and cell-matrix adhesions and describes the first known role for vinculin at cell-cell adhesions.

Das Aktin-Zytoskelett ist für viele zelluläre Funktionen unerlässlich, dazu gehören der strukturelle Aufbau von Zellen, die Zellwanderung und Vesikeltransportprozesse. Die funktionelle Vielfalt der Aktinstrukturen spiegelt sich in einer Vielzahl verschiedener molekularer Mechanismen wieder, welche die Polymerisierung von Aktinfilamenten regulieren. Die sponante Aktinpolymerisierung wird jedoch verhindert aufgrund der Instabilität von kleinen Aktin Oligomeren und durch Aktin Monomer bindende Proteine, welche die Bildung solcher Oligomere unterbinden. Aktinnukleationsfaktoren helfen diese kinetische Barriere der Filamentbildung zu überwinden und sind wesentlich für die Erzeugung von neuen Aktinfilamenten an bestimmten subzellulären Kompartimenten. Spir Proteine sind die ersten beschriebenen Mitglieder der neuen Klasse von WH2 Domänen Aktinnukleationsfaktoren. Sie leiten die Polymerisierung von Aktin ein, indem sie Aktinmonomere an die vier WH2 Domänen im Zentrum des Proteins binden. Trotz ihrer Eigenschaft Aktinpolymerisation in vitro selber zu nukleieren, bilden Spir Proteine einen regulatorischen Komplex mit anderen Aktinnukleatoren der formin Untergruppe von forminen. Spir hat eine Funktion bei der Regulierung von vesikulär erzeugten filamentösen Aktinstrukturen, Vesikeltransportprozessen und der Bildung der Teilungsfurche während der asymmetrischen meiotischen Zellteilung. Das Säugetiergenom kodiert zwei spir Gene, spir-1 und spir-2. Die entsprechenden Proteine haben einen identischen strukturellen Aufbau und sind zu einem großen Teil homolog zueinander. Um die Spir Funktion im sich entwickelnden und adulten Nervensystem zu untersuchen, wurde die bisher unbekannte Expression des Maus spir-2 Gens analysiert. Real-time PCR Analysen haben ergeben, dass spir-2 in adulten Mäusen in Oozyten, dem Gehirn, im Gastrointestinaltrakt, den Hoden und der Niere exprimiert wird. In situ Hybridisierungen wurden durchgeführt um die zelluläre Natur der spir Expression nachzuweisen. Während der Embryogenese haben in situ Hybridisierungen gezeigt, dass spir-2 im sich entwickelnden Nervensytem und Darmtrakt exprimiert wird. In adulten Mausgeweben, wurde die höchste Expression von spir-2 in Epithelzellen des Verdauungstraktes, in neuronalen Zellen des Nervensystems und in Spermatocyten gefunden. Im Gegensatz zur eher begrenzten Expression des Maus spir-1 Gens, welches überwiegend im Nervensystem, den Oozyten und Hoden zu finden ist, zeigen die hier aufgeführten Daten ein breiteres Expressionsmuster des spir-2 Gens und unterstützen damit eine allgemeinere zellbiologische Funktion der neuen Aktinnukleatoren. Um die Funktion des Spir Proteins im sich entwickelnden und adulten Nervensystem zu untersuchen, wurden Spir-1 defiziente Mäuse mit Hilfe der gene trap Methode generiert. Spir-1 defiziente Mäuse sind lebensfähig und eignen sich daher perfekt um die Neurobiologie des Spir-1 Aktinnukleators zu untersuchen. Die Analyse von primären kortikalen Neuronen von Spir-1 defizienten Mäusen zeigte eine Reduktion dendritischer Verzweigungen und ist die erste Beschreibung einer neuronalen Funktion von Spir-1. Desweiteren wurde eine transgene Mauslinie (thy1-GFP-M) eingesetzt, die das grüne Fluoreszenzprotein (GFP) unter der Kontrolle von Neuronen-spezifischen Elementen des thy1 Promoters exprimiert. GFP ist dabei nur in einer Teilmenge von Neuronen exprimiert, färbt diese Neuronen jedoch in ihrer Gesamtheit an. Spir-1 defiziente Mäuse, die das GFP Transgen exprimieren wurden generiert und analysiert. Es wurde herausgefunden, dass Spir-1 defiziente Mäuse eine reduzierte Anzahl an dendritischen Dornen im entorhinalen Kortex im Vergleich zu Wildtyp- Geschwistertieren aufweisen. Zusammengefasst gibt diese Studie neue Erkenntnisse über die zellbiologische Funktion von Spir und liefert Einsichten wie das neuronale Netzwerk sturkturiert wird. / The actin cytoskeleton is essential for many cellular functions, such as the regulation of cell morphology, cell migration and vesicle transport processes. The functional diversity of actin structures is reflected in a variety of distinct molecular mechanisms regulating the polymerization of actin filaments. The spontaneous polymerization of actin however is inhibited, by both the instability of small actin oligomers and by actin monomer binding proteins, which prevent the formation of such oligomers. Actin nucleation factors help to overcome this kinetic barrier of filament initiation and are essential for the generation of novel actin filaments at specified subcellular compartments. Spir proteins are the founding members of the novel class of WH2 domain containing actin nucleation factors. They initiate actin polymerization by binding of actin monomers to four WH2 domains in the central part of the protein. Despite their ability to nucleate actin polymerization in vitro by themselves, Spir proteins form a regulatory complex with the distinct actin nucleators of the formin subgroup of formins. Spir functions in the regulation of vesicular originated filamentous actin structures, vesicle transport processes and the assembly of the cleavage furrow during asymmetric meiotic cell divisions. The mammalian genome encodes two spir genes, spir-1 and spir-2. The corresponding proteins have an identical structural array and share a high degree of homology. In order to elucidate the Spir function in developing and adult mouse tissues, the yet unknown expression of the mouse spir-2 gene was addressed. Real-time PCR analysis revealed highest expression of spir-2 in oocytes, the brain, throughout the gastrointestinal tract, testis and kidney of adult mice. In situ hybridizations were performed to substantiate the cellular nature of spir gene expression. During embryogenesis in situ hybridizations show spir-2 to be expressed in the developing nervous system and intestine. In adult mouse tissues highest expression of spir-2 was detected in the epithelial cells of the digestive tract, in neuronal cells of the nervous system and in spermatocytes. In contrast to the more restricted expression of the mouse spir-1 gene, which is mainly found in the nervous system, oocytes and testis, the data presented here show a distinct and broader expression pattern of the spir-2 gene and by this support a more general cell biological function of the novel actin nucleators. In order to address the function of Spir proteins in the developing and adult nervous system, Spir-1 deficient mice were generated by a gene trap method. Spir-1 deficient mice are viable and provide a perfect tool to address the neurobiological function of the Spir-1 protein. Analyses of primary cortical neurons from Spir-1 deficient mice revealed a specific reduction of dendritic branchpoints and are the first description of a neuronal Spir-1 function. Further, a transgenic mouse line (thy1-GFP-M) was employed that expresses the green fluorescent protein (GFP) under the control of neuron specific elements from the thy1 promoter. GFP is thereby expressed in only a subset of neurons and labels the neurons in their entirety. Spir-1 deficient mice carrying the GFP transgene were generated and analyzed. It was found that Spir-1 deficient mice exhibit a reduced number of dendritic spines in the entorhinal cortex compared to wildtype littermates. All together this study gives novel information about the cell biological function of Spir and provides insights how cytoskeletal functions structure the mammalian neuronal network.

Pemphigus vulgaris (PV) ist eine blasenbildende Autoimmunerkrankung der Haut. Ein wesentliches Charakteristikum der Erkrankung sind Autoantikörper, welche gegen die humanen Zell-Adhäsionsmoleküle Desmoglein (Dsg) 3 und 1 gerichtet sind und zu zunehmender Zell-Dissoziation der Keratinozyten führen (Akantholyse). Neben der Dsg3-Reorganisation sind zytoskelettale Veränderungen in Form einer ZK-Retraktion und einer Reorganisation des Actin-Zytoskeletts als ein wichtiges Merkmal akantholytischer Zellen beschrieben worden. Dennoch ist der zeitliche Verlauf und die funktionelle Relevanz dieser zytoskelettalen Veränderungen im Vergleich zu anderen Prozessen, wie der Dsg3-Reorganisation oder der Zell-Dissoziation, unklar. In dieser Arbeit wurde daher die Rolle der ZK-Filamente und der Actinfilamente für die PV-Pathogenese untersucht. Inkubation von kultivierten Keratinozyten mit PV-IgG resultierte in einer ZK-Retraktion, welche eng mit dem Beginn der Dsg3-Reorganisation und der Zell-Dissoziation korrelierte. Weiterhin fand sich eine Abhängigkeit der PV-IgG-induzierten ZK-Retraktion und der Zell-Dissoziation von der p38MAPK-Signalkaskade, während die Beteiligung der p38MAPK an der Dsg3-Reorganisation von untergeordneter Rolle zu sein scheint. Übereinstimmend dazu führte eine Überexpression von E-Cadherin zu einer Hemmung der p38MAPK-Aktivierung, der ZK-Retraktion und der Zell-Dissoziation, so dass den Cadherinen eine übergeordnete Rolle in der Vermittlung der PV-Pathogenese zuzukommen scheint. Neben einer ZK-Retraktion zeigten die Zellen als Reaktion auf eine Inkubation mit PV-IgG auch wesentliche Reorganisationen der Actinfilamente, welche ebenfalls eng mit der Dsg3-Reorganisation und der Zell-Dissoziation korrelierten. Darüber hinaus interferierte die pharmakologische Modulation des Actin-Zytoskeletts mit den PV-IgG-Effekten. So führte eine Stabilisierung der Actinfilamente zu einer Reduktion sowohl der Dsg3-Reorganisation als auch der Zell-Dissoziation, während eine Zerstörung der Filamente die Effekte verstärkte. Zur Unterstützung dieser Ergebnisse wurde die Rolle des Actins für die durch Rho-GTPasen vermittelte Hemmung von PV-IgG-Effekten untersucht. Eine Aktivierung der Rho-GTPasen führte neben einer Hemmung PV-IgG-vermittelter Effekte auch zu einer Verstärkung des kortikalen Actin-Rings, während eine Hemmung der Actin-Polymerisation die protektiven Effekte der Rho-GTPasen-Aktivierung aufheben konnte. Zusammenfassend lässt sich sagen, dass die Ergebnisse dieser Arbeit eine übergeordnete Rolle sowohl der desmosomalen als auch der klassischen Cadherine für die PV-Pathogenese zeigen. Daneben scheint auch der Actin-Reorganisation eine wesentliche Position zuzukommen. Die ZK-Retraktion hingegen scheint, zumindest im Bezug auf die Dsg3-Reorganisation, sekundär zu sein, trägt aber möglicherweise im Anschluss an eine p38MAPK-Aktivierung wesentlich zum Verlust der Zell-Zell-Adhäsion bei. / In human autoimmune blistering skin disease pemphigus vulgaris (PV) autoantibodies are mainly directed against keratinocyte cell adhesion molecules desmoglein (Dsg) 3 and 1 and cause keratinocyte cell dissociation (acantholysis). Early ultrastructural work revealed cytokeratin (CK) retraction to be a characteristic hallmark of acantholytic keratinocytes and recent studies reported profound alterations of the actin cytoskeleton. Nevertheless, the temporal sequence and relevance of these cytoskeletal phenomena in pemphigus pathogenesis compared to other events such as Dsg3 reorganisation or keratinocyte dissociation are only poorly understood. We examined roles of CK and actin filaments in PV-IgG-mediated keratinocyte dissociation. Incubation of cells with PV-IgG resulted in a CK retraction which closely correlated with the onset of cell dissociation and Dsg3 reorganisation. Both, PV-IgG-induced CK retraction and cell dissociation were found to be p38MAPK-dependent whereas the contribution of p38MAPK activation for Dsg3 reorganisation seemed to be secondary. According to this, overexpression of E-cadherin prevented PV-IgG-induced p38MAPK activation, cell dissociation and CK retraction. Therefore cadherins seem to have a primary role for PV pathogenesis. Parallel to CK retraction, PV-IgG treatment resulted in striking changes in actin cytoskeleton organization which also closely correlated with cell dissociation and Dsg3 reorganisation.Therefore, we investigated whether pharmacologic manipulation of actin polymerization modulates pathogenic effects of PV-IgG. Pharmacological stabilization of actin filaments significantly blocked cell dissociation and Dsg3 fragmentation whereas actin depolymerisation strongly enhanced pathogenic effects of PV-IgG. To substantiate these findings, we studied whether the protective effects of Rho GTPases, which are potent regulators of the actin cytoskeleton and were shown to be involved in pemphigus pathogenesis, were dependent on modulation of actin dynamics. Activation of Rho-GTPases enhanced the cortical junction-associated actin belt and blunted PV-IgG-induced cell dissociation. However, when actin polymerization was blocked under these conditions the protective effects of Rho-GTPase activation were abrogated. Taken together, these experiments indicate a primary role of both desmosomal and classical cadherins for PV pathogenesis. Furthermore actin reorganization seems to be critical for PV-IgG-induced acantholysis. CK retraction may contribute to p38MAPK-dependent keratinocyte dissociation in pemphigus but appears to be secondary, at least to Dsg3 reorganisation.

Das invasive Potential maligner Gliome beeinflusst maßgeblich die schlechte Prognose dieser Tumorentität. Migration und Invasion von Tumorzellen werden entscheidend durch die Cofilin-vermittelte Umstrukturierung des Aktin-Zytoskeletts geprägt, die durch die Aktivität antagonistischer Cofilin-Kinasen und -Phosphatasen reguliert wird. Im Rahmen der vorliegenden Arbeit konnte ein progressiver Expressionsverlust der Cofilin-Phosphatase Chronophin mit ansteigendem Malignitätsgrad astrozytärer Gliome aufgezeigt werden, der mit einer Zunahme der Phosphorylierung von Cofilin einhergeht. In den entsprechenden Gewebeproben gelang gleichzeitig der Nachweis einer gesteigerten Expression der Cofilin-Kinase LIMK-2. Genetische und epigenetische Analysen des Chronophin-Locus konnten eine Hypermethylierung im Bereich der Promotorregion der Phosphatase identifizieren, die möglicherweise dem Verlust von Chronophin in Glioblastom-Gewebeproben zugrunde liegt. In Glioblastom-Zelllinien, die unterschiedliche Expressionsmuster von Chronophin aufwiesen, konnten hingegen keine molekularen Alterationen festgestellt werden. Untersuchungen des Einflusses von ROCK- und LIMK-Inhibitoren auf Glioblastomzellen konnten ausgeprägte Veränderungen der Zellmorphologie dokumentieren, wobei erstmals die Induktion eines stellate cell-Phänotyps unter Einfluss des LIMK-Inhibitors BMS-5 beschrieben wird. Während ROCK- und LIMK-Inhibitoren keinen Einfluss auf die 2D-Motilität der Tumorzellen hatten, wiesen die Glioblastomzellen in Abhängigkeit ihrer basalen Cofilin-Aktivität eine verstärkte bzw. verminderte 3D-Invasivität auf. Die Erkenntnisse dieser Arbeit unterstreichen die Bedeutung des Cofilin-Signalweges für die Migration und Invasion von Gliomzellen, zeigen neue Angriffspunkte in der Therapie maligner Gliome auf und warnen zugleich vor einem unkritischen Einsatz neuer Wirkstoffe. / The invasive potential of malignant gliomas is the main reason for the dismal prognosis of this tumor entity. Migration and invasion of tumor cells is crucially determined by the cofilin dependent reorganization of the actin cytoskeleton regulated by the activity of antagonistic cofilin kinases and phosphatases. This study revealed a progressive loss of expression of the cofilin phosphatase chronophin with increasing malignancy grade of astrocytic glioma, accompanied by an increase of cofilin phosphorylation. Moreover, the analyzed glioma specimens were characterized by a simultaneous increase of expression of the cofilin kinase LIMK-2. Integrated genetic and epigenetic analysis of the chronophin locus identified an aberrant promoter methylation as a possible mechanism leading to chronophin down-regulation in glioblastoma tissue samples. In contrast, molecular alterations of the chronophin locus were undetectable in glioblastoma cell lines characterized by different chronophin expression levels. Analysis of glioblastoma cells demonstrated striking effects of ROCK and LIMK inhibitors on cell morphology, providing first evidence of the induction of a stellate cell-phenotype by the LIMK inhibitor BMS-5. While ROCK and LIMK inhibitors had no detectable effect on the 2D motility of the tumor cells, the inhibitors increased or decreased the 3D-invasiveness of the glioma cells depending on their basal cofilin activity. The findings of this study stress the importance of the cofilin signaling pathway as a key regulator of glioma migration and invasion, indicate novel targets for glioma therapy, but also warn against a noncritical use of new drugs.