Dissertations / Theses on the topic 'Actin regulators'

Dendritic cells (DC) are key cells of the innate immune system required to prime adaptive immunity. Migration is central to their function to enable immune surveillance of the whole body and for prompt activation of the adaptive immune system. Immature DC assemble specialised actin structures called podosomes which are thought to be critical for efficient adhesion-mediated migration. Podosomes are, therefore, considered to be essential for DC function. Despite the great increase in literature regarding podosomes and related structures over recent years, still much is unknown about critical components, regulation and function of these structures in DC. Cytoskeletal studies of DC have been complicated by the fact that tools commonly employed for biological manipulation may constitute activation stimuli for DC and dramatically alter the DC cytoarchitecture. A panel of human THP1DC knock-down cell lines was generated using RNAi technology targeting factors suspected or known to be important for podosome formation and/or function such as the integrins CD18 and CD29 and the actin regulators HS1, WASp and WIP. Results obtained from functional analysis of the knock-down cell lines confirm CD18 to be specifically recruited to the DC podosomes and to be essential for their assembly. On the contrary, CD29 knock-down did not attenuate podosome assembly, even when reduced to levels that resulted in a defect in static adhesion. As previously reported, WASp and WIP expression was demonstrated to be necessary for podosome formation. Furthermore, a role for the cortactin homolog HS1 in CD18 activation in myeloid cells is suggested, as HS1 knock-down resulted in defective CD18-dependent adhesion and reduced podosome formation when cells were plated on ICAM but not on fibronectin. The results presented here define a robust method for manipulating immature DC for cytoskeletal studies and advance our current understanding of the regulation of podosome assembly in human DC.

"Cell and process migration are critical to the establishment of neural circuitry. The study of these processes is facilitated through use of model organisms with simple nervous systems, such as C. elegans. Research in this nematode has defined the cytoplasmic adaptor MIG-10 as a key regulator of these processes. Mutation of mig-10 disrupts neuronal and axonal migration and outgrowth of the ‘canals’, or processes, of the excretory cell. MIG-10 directs the localization of UNC-34, which remodels actin filaments at the leading edge of a migrating cell or process to modify the direction or rate of its protrusion. An interactor of MIG-10 identified in a yeast two- hybrid analysis, ABI-1, has several roles in actin remodeling, such as targeting Ena/VASP members for phosphorylation by Abl kinase. Mutation of abi-1 in the nematode produces phenotypes that resemble those of mig-10 mutants, including disrupted outgrowth of the excretory canals, a developmental process in which ABI-1 is known to function cell autonomously. To test the hypothesis that the ABI-1/MIG-10 interaction contributes to cell migration and outgrowth, both in vivo and in vitro analyses were performed. Expression of either MIG-10A or MIG-10B exclusively in the excretory cell partially rescued the canal truncation characteristic of mig-10 mutants, suggesting MIG-10 functions autonomously in this cell during canal outgrowth. Physical interaction between MIG-10 and ABI-1 was confirmed using a co-immunoprecipitation system. Both MIG-10A and MIG-10B interact with ABI-1 through a mechanism that likely involves the SH3 domain of ABI-1 and sites in either the central region or C-terminus of MIG-10. These results suggest that MIG-10 and ABI-1 function together in a cell autonomous manner to promote cell or process migration. A possible consequence of this interaction is modulation of the MIG-10 binding to UNC-34 through Abl-mediated phosphorylation of MIG-10."

Macrophages are one of the three main human cell types infected by HIV-1. They are highly plastic cells requiring a dynamic actin cytoskeleton for their role in development, homeostasis, tissue repair and immunity. For HIV-1, disrupting actin in macrophages is detrimental in that it leads to a complete block of viral uptake and reduces reverse transcription but, significantly, not fusion. Rho GTPases (Rac1, RhoA and Cdc42) regulate many aspects of actin dynamics including those required for endocytosis. Using a pharmacological approach, it was shown that Rac1 along with Rho GTPase effectors Pak1 and N-WASP are important for productive HIV-1 entry in macrophages. However, pharmacological inhibitors aren’t available for many host factors and may have off-target effects. To overcome this, expression of dominant negative (DN) Rho GTPases was attempted in human stem cell-derived macrophages (esMDMs). While DN Rac1 expressing esMDMs were successfully generated, this was not possible for the other two. DN Rac1 expressing esMDMs, as expected, had less filamentous actin and reduced dextran uptake compared to control esMDMs. In contrast to the pharmacological studies, HIV-1 infection studies in Rac1 DN esMDMs revealed a significant increase in HIV-1 fusion, reverse transcription and nuclear import, which could be due to reduced filamentous actin leading to a slower rate of endocytosis thereby allowing more time for viral fusion within endocytic vesicles. Surprisingly, reduced HIV-1 gene expression was observed in Rac1 DN esMDMs. This was corroborated by transfection studies implicating Rho GTPases in LTR driven gene expression. To overcome the ineffectiveness of RhoA and Cdc42 DN constitutive gene expression in esMDMs, an inducible lentiviral gene expression system based on the use of a constitutive promoter and a FLEx switch mediating irreversible DNA inversions was generated. The novel FLEx vector was the first system shown to induce transgene expression in esMDMs albeit at a very low efficiency.

Our immune system protects us from infection and abnormal cell growth. But what happens when the function of immune cells is perturbed by the absence of key cytoskeletal regulators? In the immunodeficiency disorder, Wiskott-Aldrich syndrome, patients express dysfunctional variants of WASp, a haematopoietically restricted regulator of the actin cytoskeleton. The disease manifests in recurrent bacterial infections, thrombocytopenia and a severely defective inflammatory response. In this thesis I have utilised a TILLed zebrafish WASp mutant to characterise the effect of WASp knockout on innate immune cell function using a number of assays and live imaging techniques in transparent larvae. I observe defective neutrophil and macrophage migration to a fin wound in the WASp mutant, and increased larval death from systemic bacterial infection as a result of defective phagocytosis and clearance. I have gone on to use the WASp mutant as a null background to screen an allelic range of human WASp patient mutant genes expressed specifically in neutrophils. I show that expression of wild-type hWASp protein can rescue the neutrophil recruitment defect in WASp mutant larvae. Likewise, expression of two point mutants reveals that Cdc42 binding is not essential for hWASp rescue capability, whereas tyrosine phosphorylation of the protein is required. Moreover, expression of a constitutively active hWASp mutant results in larval neutropenia, that mirrors the WASp-related disorder, X-linked neutropenia. Alongside my WASp mutant study, I have also characterised the role of zebrafish Myosin IXb, a haematopoietically restricted atypical RhoGAP, through use of targeted morpholino. I observe decreased neutrophil recruitment to a fin wound in Myosin IXb morphant larvae, whilst live imaging studies demonstrate a contracted morphology and failure to form a polarised leading edge in morphant cells. In summary, I report a live imaging zebrafish study to assess the role of two key actin regulators in vivo, and describe the first instance whereby an allelic range of human mutations are tested in zebrafish to gain a unique insight into immunodeficiency disorders at both a cellular and whole organism level.

La motilité cellulaire est impliquée dans plusieurs fonctions biologiques critiques, et sa dérégulation peut conduire à des maladies graves comme le cancer. Il est donc essentiel d'étudier les mécanismes moléculaires qui régissent la formation des structures impliquées dans la motilité cellulaire. La première étape de la migration cellulaire de type mésenchymateuse est la formation du lamellipode, protrusion membranaire supportée par un réseau de filaments d’actine et propulsée vers l’avant par la polymérisation de l'actine. La coordination spatio-temporelle des régulateurs de l’actine dans le lamellipode détermine la polarité, l'architecture et les mouvements du réseau de filaments d’actine. Ces régulateurs sont notamment le complexe WAVE (WRC) et le complexe Arp2/3. L'activation du WRC est l'événement moléculaire qui déclenche l'activation du complexe Arp2/3 et donc l'initiation de la formation du réseau de filaments d’actine dans le lamellipode. L'activation du WRC repose sur la libération du domaine WCA activateur du complexe Arp2/3, situé à l'extrémité C-terminale de la sous-unité WAVE. Des études in vitro montrent que deux domaines WCA sont nécessaires pour activer efficacement le complexe Arp2/3.Mais on ne sait pas comment l'organisation spatiale du WRC à l’échelle moléculaire est traduite en activation du complexe Arp2/3 déclenchant la formation du lamellipode. En d'autres termes, la stœchiométrie et la distribution spatiale requise pour traduire l'activation conformationnelle du WRC en une activation efficace du complexe Arp2/3 sont des informations essentielles manquantes.L'utilisation récente de la microscopie de super-résolution (SRM) et du suivi de particules uniques (SRM) a permis de repenser radicalement les assemblages macromoléculaires, en particulier les structures impliquées dans la migration cellulaire telle que le lamellipode. En suivant les protéines individuelles et en fournissant des images avec une résolution spatiale inférieure à la limite de diffraction de la lumière, ces techniques donnent accès à l'organisation et à la dynamique à l'échelle nanométrique des complexes protéiques dans les cellules.Pour révéler l’organisation moléculaire du WRC, nous avons utilisé le DNA-PAINT, une technique de SRM avec une résolution spatiale inférieure à 10 nm. Le DNA-PAINT est basé sur l'hybridation de brins d'ADN complémentaires, l'un situé sur la protéine cible (brin d'amarrage) et l'autre sur le fluorophore (brin d’imagerie). Le DNA-PAINT permet le comptage moléculaire des protéines (qPAINT) et l'imagerie de super-résolution multicouleurs (Ex-PAINT). Cela nous a permis d'évaluer la stœchiométrie, les colocalisations et la composition des complexes protéiques dans le lamellipode.Des expériences de qPAINT ont montré que la stœchiométrie du WRC à l'extrémité du lamellipode est de une ; tandis que l'imagerie en direct par nanoscopie RESOLFT a révélé que ces WRCs uniques forment des clusters isolés à l'extrémité du lamellipode. La SRM multicouleur du corps du WRC et de son domaine WCA a montré que l’activation conformationnelle du WRC induit une libération du domaine WCA dans un rayon de 40 nm. En utilisant des protrusions stéréotypées en forme de vague, nous avons corrélé l'organisation moléculaire du WRC avec leur vitesse de déplacement. Nous avons montré que la distance entre les WRCs individuels est inférieure au rayon de leur dépliage conformationnel dans des régions de protrusion rapide du lamellipode, augmentant la possibilité de rencontre des domaines WCA et donc l’activation du complexe Arp2/3. Ainsi, le WRC, fonctionnant comme un complexe isolé, doit être espacé d'une distance inférieure à son dépliage conformationnel pour activer efficacement le complexe Arp2/3 dans le lamellipode. Dans l'ensemble, nos résultats montrent qu'en plus de l'activation biochimique des circuits de signalisation, l'organisation spatiale des protéines est cruciale pour le contrôle de leur fonction dans les cellules
Cell motility is involved in critical biological functions, and dysregulation of adhesion, migration and of the actin cytoskeletal can lead to severe disease like cancer. Therefore, it is essential to study the molecular mechanism driving the formation of sub-cellular structures involved in cell motility. The first step in mesenchymal cell migration is the forward protrusion of the lamellipodium which is a thin sheet of membrane-enclosed actin filaments (F-Actin) networks propelled by actin polymerization. The spatiotemporal coordination of F-actin regulators in the lamellipodium determines the polarity, architecture and movements of branched F-actin networks. This includes two interacting nanomachines, the WAVE regulatory complex (WRC) and the Arp2/3 complex. WRC activation is the central molecular event triggering Arp2/3 complex activation and thus, the initiation and formation of a branched F-actin network in the lamellipodium. WRC activation relies on the exposure of the cryptic Arp2/3-activating WCA domain located at the C-terminal extremity of the WAVE subunit tail. In vitro studies showed that two WCA domains are needed to efficiently activate the Arp2/3 complex.But how the local spatial organization of WRC at the molecular level translate into activation of the Arp2/3 complex triggering the morphogenesis of the lamellipodium is unknown. In other words, the stoichiometry and the spatial distribution required to translate WRC conformational activation to an efficient activation of Arp2/3 complex are essential missing information.The recent application of super-resolution microscopy (SRM) and single particle tracking (SRM) lead to a drastic rethinking of macromolecular assemblies, in particular structures involved in cell migration, including the lamellipodium. By tracking individual proteins and delivering images with spatial resolutions below the diffraction limit of light, these techniques give access to the nanoscale organization and dynamics of protein complexes in live cells.To reveal the molecular organization of WRC, we use DNA-PAINT, a SRM technique which allows spatial resolution below 10 nm. DNA-PAINT is based on hybridization of complementary DNA strands, one located on the target protein (docking strand) and the other on the dye (imager strand). DNA-PAINT enable absolute molecular counting in protein complexes (Quantitative-PAINT) and multi-color super-resolution imaging (Exchange-PAINT). This allowed us to assess stoichiometries, colocalizations and composition of nanomachines in the lamellipodium.Using Quantitative-PAINT, we showed that the stoichiometry of WRC at the lamellipodium tip of migrating mouse melanoma cell (B16) is one; while live super-resolution imaging based on RESOLFT nanoscopy revealed that these single WRC form discrete foci at the lamellipodium tip. Multicolor Exchange-PAINT super-resolution microscopy of the WRC core and its WCA domain, showed that its conformational activation induces the release of the WCA domain in a radius of 40 nm away from its core. Using stereotyped waveform protrusions, we correlated WRC molecular organization with the rate of membrane protrusions. We showed that the spatial distribution of individual WRC is below the radius of its conformational unfolding in regions of faster lamellipodial protrusion, increasing the possibility of WCA domain dimerization and thus activation of the Arp2/3 complex. This way, the WRC, functioning as an isolated complex, must be spaced at a distance less than its conformational unfolding to activate efficiently the Arp2/3 complex in the lamellipodium. Altogether, our results show that besides biochemical activation of signaling circuitry, the spatial organization of proteins is crucial for controlling their function in cells

The binding of antigen (Ag) to the B cell receptor (BCR) induces the activation of intracellular signaling and the reorganization of the actin cytoskeleton. However, the function of actin reorganization and the mechanisms by which BCR signaling and actin reorganization is coupled have not been well studied. This thesis has investigated how BCR signaling regulates actin reorganization and how actin remodeling in turn influences BCR signalig. My studies show that the key stimulatory signaling molecule of the BCR, Bruton's tyrosine kinase (Btk), is critical for actin polymerization at the activation surface and BCR clustering and B cell spreading, events that are essential for signaling initiation and amplification. The key inhibitory signaling molecule, SH2-containing phosphatidylinositol-5 phasphatase (SHIP-1), is important for removal of F-actin from the activation surface, and actin-mediated B cell contraction and the formation of BCR central clusters. SHIP-1 suppresses actin polymerization by inhibiting Btk-dependent activation of Wiskott-Aldrich syndrome protein (WASP). These results suggest that BCR signaling can regulate B cell morphology and surface BCR clustering via modulationg actin dynamics. To understand the roles of actin reorganization in BCR signaling, I investigated the effects of gene knockout of the two actin regulators, WASP and its homolog, neuronal (N)-WASP. My results show that both WASP and N-WASP are required for optimal BCR clustering, B cell spreading, and BCR signaling, but they play distinct roles. WASP promotes actin polymerization, B cell spreading, BCR clustering, and signaling amplification, and N-WASP inhibits actin polymerization at the activation surface and promotes B cell contraction, BCR central cluster formation, and signaling attenuation. Importantly, B cell-specific N-WASP knockout causes increases in the levels of autoantibody. In addition, WASP and N-WASP negatively regulate each other, compete for Arp2/3, and are inversely regulated by Btk and SHIP-1. Taken together, these results demonstrate that the balance of stimulatory and inhibitory BCR signaling controls actin dynamics and organization through regulating the activities of WASP and N-WASP. Actin remodeling in turn amplifies BCR signaling activation or down regulation by modulating B cell morphlogy and the organization of surface BCRs.This research reveals a bidrectional feedback loop between BCR signaling and the actin cytoskeleton.

La motilité cellulaire est un processus intégré essentiel à de nombreux phénomènes physiologiques tels que la formation du cône de croissance et la plasticité synaptique. Des dérégulations de la motilité cellulaire peuvent être à l’origine de la formation de métastases ou de pathologies neuropsychiatriques comme la schizophrénie et l'autisme. La compréhension des mécanismes régulant la migration cellulaire est donc un enjeu majeur. La motilité cellulaire repose sur la formation de diverses structures constituées de réseaux d’actine branchés telles que le lamellipode. La formation du lamellipode nécessite l’intervention de protéines régulatrices de l’actine telles que Rac1 et les complexes Wave et Arp2/3. Grâce à l’utilisation de suivi de protéine unique, nous avons pu comprendre comment la coordination spatio-temporelle de ces régulateurs contrôle la formation et la morphologie des lamellipodes de cellules migrantes. Nous avons ainsi découvert que l’activation et la localisation du complexe Wave étaient régulées de manière enzymatique mais également mécanique. Dans une première étude, nous avons montré que la RhoGTPase Rac1 active le complexe Wave spécifiquement à l’extrémité du lamellipode. Dans une seconde étude, nous avons révélé que la localisation du complexe Wave est régulée par la dynamique des filaments des réseaux branchés d’actine. Ces données soulignent l’importance du complexe Wave dans la formation du lamellipode et révèlent l’existence d’une régulation mécanique de la localisation du complexe Wave
Cell motility is an integrated process involved in critical phenomena such as axonal pathfinding and synaptic plasticity. Dysregulation of cell motility can induce metastasis and abnormal spine shapes observed in neuropsychiatric disorders like autism and schizophrenia. Therefore it is essential to understand how cell motility is regulated. Cell motility requires the formation of branched actin networks propelled by actin polymerization that lead to the formation of membrane protrusions such as the lamellipodium. Several actin regulatory proteins are involved in this process, such as Rac1 and the WAVE and ARP2/3 complexes. Using single protein tracking, we revealed key phenomena concerning the spatio-temporal regulation of lamellipodium formation by actin regulatory proteins. We found that the localization and activation of the WAVE complex was enzymatically regulated, but also mechanically. First, we showed that the Rac1 RhoGTPase activates the WAVE complex specifically at the tip of the lamellipodium. We also showed that WAVE complex localization is regulated by the dynamics of branched-network actin filaments. This study confirms the crucial role of the WAVE complex in lamellipodium formation and reveals the existence of a mechanical regulation of the localization of this complex in the cell

Thesis (LLD)--Stellenbosch University, 2015
ENGLISH ABSTRACT : Section 25 of the South African Constitution authorises and sets the limits for two forms of legitimate regulatory interference with property, namely deprivation and expropriation. The focus of this dissertation is on the requirement in section 25(1) that no law may authorise arbitrary deprivation of property. According to the Constitutional Court, deprivation is arbitrary when there is insufficient reason for it. The Court listed a number of factors to consider in determining whether there is a sufficient relationship between the purpose to be achieved by deprivation and the regulatory method chosen to achieve it. The outcome of the arbitrariness question depends on the level of scrutiny applied in a particular case. The level of scrutiny ranges from rationality review to proportionality review. Deprivation that results in an excessively harsh regulatory burden for one or a small group of property owners will probably be substantively arbitrary and in conflict with section 25(1). Courts generally declare unconstitutional regulatory interferences with property rights invalid. However, invalidating legitimate regulatory measures that are otherwise lawful purely because they impose a harsh and excessive burden on some property owners may not always be justified if the regulatory measure fulfils an important regulatory purpose. Invalidating excessive regulatory measures may in some instances also be meaningless and may not constitute appropriate relief in vindicating the affected rights. The purpose of this dissertation is to investigate the appropriateness of alternative solutions to invalidating otherwise lawful and legitimate but excessive regulatory deprivations of property. The goal is to identify remedies that allow courts to uphold the regulatory measure and simultaneously balance out the excessive regulatory burden it imposes on property owners. One alternative solution is to transform the excessive regulatory measure into expropriation and require the state to pay compensation to the affected owner. This approach is referred to as constructive expropriation. However, in view of the Constitutional Court’s approach to and the wording of section 25 it seems unlikely that it will adopt constructive expropriation as a solution. Another alternative solution is for the legislature to include a statutory provision for compensation in the authorising statute. Examples from German, French, Dutch and Belgian law show that this approach balances out the excessive regulatory burden and allows courts to uphold the otherwise lawful and legitimate but excessive regulatory statute without judicially transforming the deprivation into expropriation. An overview of South African law indicates that there is legislation that includes non-expropriatory compensation provisions. In cases where the regulatory statute does not contain a compensation provision, the courts might consider reading such a duty to pay compensation into the legislation or awarding constitutional damages. In conclusion, it is possible for the state to deprive owners of property in a manner that may result in an excessive regulatory burden being suffered by one or a small group of property owners if the regulatory purpose is necessary in the public interest, provided that the legislature explicitly or implicitly provides for non-expropriatory compensation in the regulatory statute.
AFRIKAANSE OPSOMMING : Artikel 25 van die Suid Afrikaanse Grondwet magtig en stel grense daar vir twee regmatige vorme van regulerende staatsinmenging met eiendom, naamlik ontneming en onteiening. Die fokus van hierdie proefskrif is op die vereiste in artikel 25(1) dat geen wet arbitrêre ontneming van eiendom mag toelaat nie. Volgens die Grondwetlike Hof is ʼn ontneming arbitrêr as daar nie ʼn voldoende rede daarvoor is nie. Die Hof het faktore gelys wat oorweeg moet word om te bepaal of daar ʼn voldoende verhouding bestaan tussen die doel wat die staat met ontneming van eiendom nastreef en die regulerende maatreël wat vir die doel gebruik word. Die uitkoms van die toets vir arbitrêre ontneming hang af van die hersieningsstandaard wat die howe in ʼn spesifieke geval gebruik. Die standaard wissel van ʼn redelikheidstoets tot ʼn proporsionaliteitstoets. ʼn Ontneming wat ʼn oormatige swaar las op een of ʼn beperkte groep eienaars plaas sal waarskynlik arbitrêr en teenstrydig met artikel 25(1) wees. Die howe se benadering is om ongrondwetlike ontnemings van eiendom ongeldig te verklaar, maar dit is nie altyd geregverdig om toelaatbare en andersins regmatige ontnemings wat ʼn oormatige las op sommige eienaars plaas ongeldig te verklaar nie. Die ongeldigverklaring van wetgewing wat ʼn oormatige ontneming magtig mag soms ook nutteloos wees en nie ʼn gepaste remedie wees om die eienaar se regte te herstel nie. Die doel van hierdie proefskrif is om die geskiktheid van alternatiewe oplossings tot die ongeldigverklaring van andersins regmatige maar oormatige ontnemings van eiendom te ondersoek Die doel is om remedies te identifiseer wat die howe toelaat om regulerende ontnemings in stand te hou en terselfdertyd die oormatige las op enkele eienaars uit te balanseer. Een alternatiewe oplossing is om die oormatige ontneming te omskep in onteiening en die staat sodoende te verplig om aan die eienaar vergoeding te betaal. Hierdie benadering staan bekend as konstruktiewe onteiening. Gegewe die Grondwetlike Hof se benadering tot en die bewoording van artikel 25 is dit onwaarskynlik dat die howe konstruktiewe ontneming as ʼn oplossing sal aanvaar. ʼn Ander alternatiewe oplossing is vir die wetgewer om ʼn statutêre bepaling vir vergoeding in die magtigende wetgewing in te voeg. Voorbeelde uit die Duitse, Franse, Nederlandse en Belgiese reg toon aan dat hierdie benadering ʼn oormatige las kan uitbalanseer en die howe toelaat om die andersins geldige en regmatige ontneming in stand te hou sonder om dit in onteiening te omskep. ʼn Oorsig van Suid Afrikaanse reg dui aan dat daar wetgewing bestaan wat wel voorsiening maak vir sodanige vergoeding. In gevalle waar die magtigende wetgewing nie vergoeding voorsien nie kan die howe oorweeg om ʼn vergoedingsplig in die wet in te lees of om grondwetlike vergoeding toe te ken. Hierdie proefskrif kom tot die gevolgtrekking dat dit grondwetlik moontlik is vir die staat om eienaars van eiendom te ontneem op ʼn wyse wat soms daartoe kan lei dat enkele eienaars ʼn oormatige swaar las moet dra, mits die ontneming ʼn belangrike openbare doel dien en die wetgewer uitdruklik of implisiet voorsiening maak vir vergoeding.

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.

 

This study utilized large-scale gene expression data to define the regulatory networks of genes expressing in the hippocampus to which multiple disease pathologies may be associated. Specific aims were: ident i fy key regulatory transcription factors (TFs) responsible for observed gene expression patterns, reconstruct transcription regulatory networks, and prioritize likely TFs responsible for anatomically restricted gene expression. Most of the analysis was restricted to the CA3 sub-region of Ammon&rsquo
s horn within the hippocampus. We identified 155 core genes expressing throughout the CA3 sub-region and predicted corresponding TF binding site (TFBS) distributions. Our analysis shows plausible transcription regulatory networks for twelve clusters of co-expressed genes. We demonstrate the validity of the predictions by re-clustering genes based on TFBS distributions and found that genes tend to be correctly assigned to groups of previously identified co-expressing genes with sensitivity of 67.74% and positive predictive value of 100%. Taken together, this study represents one of the first to merge anatomical architecture, expression profiles and transcription regulatory potential on such a large scale in hippocampal sub-anatomy.

Clathrin mediated endocytosis (CME) in S. cerevisiae requires the dynamic interplay between many proteins at the plasma membrane. Actin polymerisation provides force to drive membrane invagination and vesicle scission. The WASp homologue in yeast, Las17 plays a major role in stimulating actin filament assembly during endocytosis. The actin nucleation ability of WASP family members is attributed to their WCA domain [WH2 (WASP homology2) domain, C central, and A (acidic) domains] which provides binding sites for both actin monomers and the Arp2/3 complex. In addition, the central poly-proline repeat region of Las17 is able to bind and nucleate actin filaments independently of the Arp2/3 complex. While Las17 is a key regulator of endocytic progression and has been found to be phosphorylated in global studies, the mechanism behind regulation of Las17 actin-based function is unclear. Therefore, the aims of this study were to investigate the post-translation modification of Las17 by phosphorylation, and to determine how this modification impacts on Las17 function both in vivo and in vitro.

To what extent is regulatory forbearance an issue in the regulation of banking In Nigeria? If it is indeed a problem, what impact has it had on domestic banking crises, and how have they occurred? And could the problems be adequately addressed through the application of mandatory early resolution rules based on the regime applied under the Prompt Corrective Action system In the United Sates? This thesis begins by analysing regulatory forbearance generally, focusing on its causes and problems. As a background to the introduction of PCA rues, it examines the impact of forbearance on the US thrifts crisis. It focuses rules because they were developed specifically to address regulatory forbearance; their purpose was to curb discretion, and to compel swift resolution of failing banks in order to minimise costs. The PCA rules have been successfully applied in the US since 1991, during which time the banking system has remained largely stable.

Exonic mutations can result in altered protein function by affecting exon recognition during splicing. To understand this mechanism I have extensively evaluated the exonic regulatory elements affected by the disease G to T mutation at position +6 of BRCAl exon 18. This substitution induces the exclusion of the exon and it has been suggested that it disrupts an ASF/SF2-dependent enhancer. Using a pulldown assay with an internal standard, I show that WT and T6 sequences bind ASF/SF2 with similar efficiency, which is significantly lower compared to the binding to a typical enhancer derived from the fibronectin EDA exon. Consistent with the absence of an ASF/SF2 enhancing effect, siRNA depletion of ASF/SF2 did not induce exon WT exclusion indicating that ASF/SF2 is not essential for BRCAl exon 18 splicing.

Ovarian cancer is a highly metastatic disease having a poor prognosis (<25%). The factors and underlying mechanisms that regulate ovarian cancer metastasis, however, are still incompletely understood. p70 S6 kinase (p70S6K), a serine/threonine kinase, is frequently activated in high-grade malignant human ovarian cancer. The aim of this study is to investigate the molecular mechanisms by which p70S6K may promote ovarian cancer metastasis. The results show that p70S6K is a critical regulator of the actin cytoskeleton, peritoneal adhesion and dissemination, and multicellular aggregates/spheroids formation in the acquisition of the metastatic phenotype. The regulation of p70S6K on the actin cytoskeleton is through two important functions: as an actin cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70S6K induced a marked reorganization of the actin cytoskeleton and directional migration of ovarian cancer cells. Actin binding and immunofluorescence studies showed that p70S6K had a direct interaction with the actin filaments with no other proteins involved. This interaction did not affect actin polymerization kinetics but cross-linked the actin filaments to inhibit cofilin-induced actin depolymerization. In addition, p70S6K mediated the activation of Rac1 and Cdc42 GTPases and their downstream effector p21-activated kinase 1, but not RhoA. Peritoneal adhesion and dissemination is regulated by p70S6K through integrin expression. Expression of p70S6K siRNA efficiently inhibited ovarian cancer cell adhesion to fibronectin and laminin among different peritoneal extracellular matrix components, as well as to human primary peritoneal mesothelial cells. These effects were associated with the expression of alpha5 and beta1 integrin. Studies into the mechanisms suggest that p70S6K may upregulate alpha5 integrin by a transcriptional mechanism whereas beta1 integrin is regulated at a post-transcriptional level. Enhanced expression of alpha5 and beta1 integrin by active p70S6K mediated the subsequent peritoneal adhesion. In ovarian cancer xenografts, p70S6K and beta1 integrin interference significantly inhibited peritoneal dissemination through reduction in the number and weight of tumors. Multicellular spheroids are present in the malignant ascites of ovarian cancer patients. Using a 3-dimensional culture system, expression of p70S6K siRNA resulted in inhibition of multicellular spheroid formation, which was mediated by N-cadherin but not E- or P-cadherin. In addition to spheroid formation, inhibition of p70S6K was associated with reduced growth of spheroids and disaggregation capabilities on different extracellular matrix components. Taken together, these findings indicate that p70S6K plays an important role in the biology of ovarian cancer metastasis through regulation of several critical steps in dissemination and migration, suggesting that p70S6K could be explored as a potential therapeutic target in ovarian cancer.
published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy

Proper contraction of striated muscle requires the assembly of actin filaments with precise spacing, polarity and lengths, however the mechanisms by which the cell accomplishes this remain unclear. In one model, the giant protein nebulin is proposed to function as a "molecular ruler" specifying the final lengths of the actin filaments. This dissertation focuses on determining the mechanisms by which nebulin regulates thin filament assembly. We found that nebulin physically interacts with CapZ, a protein that caps the barbed end of the actin filament within the Z-disc. Reduction of nebulin levels in chick skeletal myocytes via siRNA results in a reduction of assembled CapZ, and a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin-filament barbed ends to the Z-disc via a direct interaction with CapZ. Unexpectedly, the CapZ binding site was mapped to a site on nebulin that was previously predicted to localize outside of the Z-disc. Thus, we also propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres. To determine the mechanism by which nebulin regulates thin filament length and directly test the molecular ruler hypothesis, a unique small nebulin molecule ("mini-nebulin") was constructed. The introduction of mini-nebulin into chick skeletal myocytes, with endogenous nebulin knocked down, does not result in corresponding shorter actin filaments; an observation that is inconsistent with a strict ruler function. Treatment of these cells, however, with the actin depolymerizing agent Latrunculin A produces filaments that match the length of the mini-nebulin molecule, indicating mini-nebulin stabilizes the actin filaments. Furthermore, knockdown of nebulin results in more dynamic populations of the thin filament components actin, tropomyosin and tropomodulin. Strikingly, introduction of mini-nebulin is able to restore the normal stability of the actin filaments. Taken together, these data indicate that nebulin is responsible for proper actin organization within the Z-disc and contributes to actin filament length regulation by stabilizing the filament, preventing actin depolymerization.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 137-154).
Because there is little knowledge in the areas of stereocilia development, maintenance, and function in the hearing system, I decided to pursue a proteomics-based approach to discover proteins that play a role in stereocilia function. I employed a modified "twist-off" technique to isolate hair bundle proteins, and I developed a method to purify proteins and to process them for analysis using multi-dimensional protein identification technology (MudPIT). The MudPIT analysis yielded a substantial list of proteins. I verified the presence of 21 out of 34 (62%) existing proteins known to be present in stereocilia. This provided strong evidence that my proteomics approach was efficient in identifying hair bundle proteins. Next, I selected three proteins and localized them to murine cochlear stereocilia. StarD10, a putative phospholipid binding protein, was detectable along the shaft of stereocilia. Nebulin, a putative F-actin regulator, was located toward the base of stereocilia. Finally, twinfilin 2, a putative modulator of actin polymerization, was found at the tips of stereocilia. In order to determine the function of twinfilin 2, I localized the protein predominately to the tips of shorter stereocilia where it is up-regulated during the final phase of elongation. When overexpressed, I found that twinfilin 2 causes a shortening of microvilli in LLC-PK1/CL4 cells and in native cochlear stereocilia. The main result of this thesis was determining the sub-cellular localization of three interesting proteins and functionally characterizing one protein. My thesis also confirmed the proteomics screen I developed as an efficient method for identifying proteins in stereocilia.
by Anthony Wei Peng.
Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Vertebrate nervous system development requires the careful interpretation of many attractive and repulsive guidance molecules. For the incredibly complicated wiring diagram comprising the vertebrate nervous system to elaborate properly, the highly motile "growth cone" at the tip of an axon must sense extracellular embryonic cues and respond through a number of intracellular interactions leading ultimately to coordinated changes in cytoskeletal morphology and modulation of the axonal path. Here I describe axon pathfinding defects displayed by mice genetically deficient for all three vertebrate Ena/VASP homologues: Mena, VASP, and EVL. As has been reported previously in invertebrates, these defects share phenotypic overlap with those seen in mice genetically deficient for the repulsive guidance molecules Slit and Robo. I find that the pathfinding errors observed in Ena/VASP deficient mice are likely a result of failure to respond to Slit/Robo. Furthermore, based on my findings, I propose a "four-step" model of growth cone responses to repulsive cues. Finally I find that the direct binding of Ena/VASP proteins to Robo seen in invertebrates is conserved and expanded in vertebrates. These interactions appear to be tunable by phosphorylation, suggesting a model by which context dictates the Ena/VASP:Robo interaction, potentially leading to changes in growth cone responsiveness to guidance cues.
by John Edward van Veen.
Ph.D.

Several lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also
trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins.

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

Macrophage migration, an essential component of many biological processes and pathologic conditions, is mediated by integrated cellular signaling processes and cytoskeleton rearrangement. Recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. Furthermore, our lab has provided evidence demonstrating the involvement of a key innate immunity signaling kinase, IRAK-1, as a critical modulator of murine macrophage migration. Macrophage migration induced by a potent PKC activator, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) was significantly decreased in IRAK-1-/- murine macrophages compared with wild type cells. Mechanistically, we first demonstrated that IRAK-1 works downstream of PKCε and directly binds to VASP, a cytoskeleton regulatory protein, to regulate PMA-induced macrophage migration. Secondly, we proved that IRAK-1 is required for LPS-induced macrophage migration and expression of MCP-1, a chemotactic cytokine for macrophages, via transcription factor C/EBPδ instead of NFκB. IRAK-1 binds directly to IKKε and inhibition or knock-down of IKKε results in a significant decrease in C/EBPδ expression and MCP-1 mRNA expression. Lastly, we identified the direct association between IRAK-1 and Rac1, a member of the Rac subfamily in the Rho family of GTPases. These finding further confirmed the essential role of IRAK-1 during macrophage migration. Our research provides a novel facet regarding the molecular signaling processes regulating macrophage migration.
Ph. D.

The Class Action Fairness Act of 2005 (CAFA) was signed into law on February 18, 2005. Prior to CAFA, plaintiffs found it easier for class action lawsuits to be tried in their preferred venue—state courts. Changes introduced by CAFA practically removed the majority of class action jurisdiction from state to federal courts. Since law and regulation might serve as an external corporate governance mechanism, an interesting question is whether CAFA has strengthened or weakened corporate governance. If CAFA improves corporate governance, associated marginal benefits would outweigh marginal costs. The opposite would be true if CAFA weakens corporate governance. This issue was hotly debated in the US Congress. The proponents argued that CAFA would reduce costs for the affected firms, while opponents argued the opposite. The main purpose of this paper is to examine which side of the debate is reflected in market reactions to various events that either enhanced or reduced the chances of the passage of CAFA. We identify the firms that are most likely to be affected by CAFA and find that the overall market reaction for these firms is positive when the likelihood of CAFA passage increases, while the reaction has been negative when the chance of its passage diminishes. We also hypothesize that firms that are more likely to be exposed to product liability litigation would experience a significantly higher (positive or negative) abnormal return than firms that are more likely to be involved in contract liability law suits. The results support this hypothesis. We also examine potential factors that might explain cross-sectional variations in abnormal returns and find that duality of Chairmanship and CEO has negative impact, while the

Limited oxygen availability (hypoxia) influences cell migration and invasion, but the underlying mechanisms are poorly understood. Much of the cellular response to hypoxia is regulated by a family of Hypoxia Inducible Factor (HIF) prolyl hydroxylases (PHD1-3), each of which is thought to regulate specific pathways.Their activity is dependent on the availability of oxygen and alpha-ketoglutarate but despite intensive studies their activity in vivo and their substrates are poorly defined. In this study we performed a quantitative proteomic screen to identify new substrates of PHDs. Co-immunoprecipitations using FLAG-tagged PHDs were performed under hypoxia to trap the enzyme-substrate interactions, and binding partners were identified by mass spectrometry. Actin was identified to interact with PHD3 specifically under hypoxia. Subsequently two defined prolyl residues in beta-actin were shown to be hydroxylated. Hypoxia-induced rearrangement of the actin cytoskeleton was shown to be dependent on PHD3 activity as a knockdown of PHD3 was sufficient to increase the intracellular G- to F-actin ratio. An increase in cell migration and invasion was also found to be dependent on PHD3 activity. Mutation of both hydroxylated prolyl residues led to a similar phenotype regarding actin rearrangement and cell migration. Using constantly active HIF-mutants, we could show that these PHD3-dependent pathways are independent of HIF. All together, this study shows a pro-invasive pathway linking HIF-independent oxygen-sensing pathways and actin signalling. However, the mechanism of how hypoxia-induced actin rearrangement leads to increased migration and invasion remains to be elucidated.

Stress responsive genes play a key role in shaping the manner in which plants process and respond to environmental stress. Their gene products are linked to DNA transcription and its consequent translation into a response product. However, whilst these genes play a significant role in manufacturing responses to stressful stimuli, transcription factors coordinate access to these genes, specifically by accessing a gene&rsquo
s promoter region which houses transcription factor binding sites. Here transcriptional elements play a key role in mediating responses to environmental stress where each transcription factor binding site may constitute a potential response to a stress signal. Arabidopsis thaliana, a model organism, can be used to identify the mechanism of how transcription factors shape a plant&rsquo
s survival in a stressful environment. Whilst there are numerous plant stress research groups, globally there is a shortage of publicly available stress responsive gene databases. In addition a number of previous databases such as the Generation Challenge Programme&rsquo
s comparative plant stressresponsive gene catalogue, Stresslink and DRASTIC have become defunct whilst others have stagnated. There is currently a single Arabidopsis thaliana stress response database called STIFDB which was launched in 2008 and only covers abiotic stresses as handled by major abiotic stress responsive transcription factor families. Its data was sourced from microarray expression databases, contains numerous omissions as well as numerous erroneous entries and has not been updated since its inception.The Dragon Arabidopsis Stress Transcription Factor database (DASTF) was developed in response to the current lack of stress response gene resources. A total of 2333 entries were downloaded from SWISSPROT, manually curated and imported into DASTF. The entries represent 424 transcription factor families. Each entry has a corresponding SWISSPROT, ENTREZ GENBANK and TAIR accession number. The 5&rsquo
untranslated regions (UTR) of 417 families were scanned against TRANSFAC&rsquo
s binding site catalogue to identify binding sites. The relational database consists of two tables, namely a transcription factor table and a transcription factor family table called DASTF_TF and TF_Family respectively. Using a two-tier client-server architecture, a webserver was built with PHP, APACHE and MYSQL and the data was loaded into these tables with a PYTHON script. The DASTF database contains 60 entries which correspond to biotic stress and 167 correspond to abiotic stress while 2106 respond to biotic and/or abiotic stress. Users can search the database using text, family, chromosome and stress type search options. Online tools have been integrated into the DASTF 
database, such as HMMER, CLUSTALW, BLAST and HYDROCALCULATOR. User&rsquo
s can upload sequences to identify which transcription factor family their sequences belong to by using HMMER. The website can be accessed at http://apps.sanbi.ac.za/dastf/ and two updates per year are envisaged.

Les cèl·lules eucariotes internalitzen i redistribueixen les molècules de la superfície a través de la ruta endocítica. Aquest és un procés clau per a l’adquisició de nutrients i el catabolisme que també controla l’exposició de receptors i complexos d’adhesió cel·lular, entre d’altres. Treballs anteriors del nostre laboratori van identificar la kazrina C com una proteïna que bloquejava l’endocitosi depenent de clatrina (CME) quan es sobreexpressava. El treball exposat a aquesta tesi confirma el paper de la kazrina en CME, ja que les cèl·lules KO de kazrina generades pel sistema de CRISPR-cas9 no van internalitzar correctament la transferrina (Tfn), un marcador endocític. La kazrina C va co-localitzar amb marcadors d’unions adherents com la N-cadherina a la membrana plasmàtica i en estructures intracel·lulars. De fet, un fraccionament subcel·lular va demostrar la presència de la kazrina als early endosomes (EEs). En concordança amb un paper de la kazrina a EEs, la seva depleció va provocar una acumulació de EEs que contenien N-cadherina, que a més tenien una distribució més perifèrica que a les cèl·lules control. Les cèl·lules KO de kazrina no transportaven Tfn cap al compartiment de reciclatge endocític (ERC) i tenien el conseqüent defecte al reciclatge de Tfn. Tots els fenotips en les cèl·lules KO de kazrina es van recuperar amb la re-expressió de GFP-kazrina C però no amb la de GFP. Aquestes evidències apunten cap a un paper de la kazrina C al reciclatge endosomal i al transport de EEs cap al ERC. En consonància amb aquesta hipòtesi, la depleció de kazrina va causar defectes en processos cel·lulars que depenen del reciclatge a través del ERC, com ara la migració cel·lular i la citoquinesi. Aquest estudi també va analitzar els mecanismes moleculars de la funció de la kazrina C en el tràfic endocític. Es va demostrar que la kazrina C interacciona amb els motors associats a microtúbuls kinesina-1 i dineïna, i que s’uneix directament a la cadena intermitja lleugera de la dineïna, LIC1. De fet, la kazrina C té un domini coiled-coil similar als dels adaptadors de la dineïna. La kazrina C també té en comú amb aquests adaptadors la localització a la regió pericentriolar, on semblava atrapar EEs. Per tant, proposem que la kazrina C promou el transport de EEs a través de microtúbuls, probablement com un adaptador de EEs i la dineïna. En la mateixa línia, aquest i anteriors estudis del laboratori van mostrar una interacció directa i co-localitzacions parcials de la kazrina C amb components de EEs, com l’adaptador de clatrina AP-1 i les GTPases EHD1/3. A més, la kazrina C va interaccionar amb PI3P i amb la PI3K de classe III, i la seva depleció va causar un augment als nivells endosomals de la sonda de PI3P GFP-FYVE. Finalment, establim una relació entre la kazrina C i un altre element clau del trànsit endocític: la maquinària de polimerització d’actina associada a Arp2/3. Es van observar interaccions directes amb la cortactina i el N-WASP, així com la co-localització de la GFP-kazrina C i la cortactina a la membrana plasmàtica i estructures intracel·lulars. A més, la depleció de la kazrina va provocar una reducció en l’actina ramificada cortical i un augment en l’endosomal. En conjunt, hem provat una funció de la kazrina C al reciclatge endosomal i proposem que aquesta funció ocorre a través de la regulació del transport a través de microtúbuls, la polimerització d’actina i el metabolisme de PI3P.
Las células eucariotas internalizan y redistribuyen las moléculas de su superficie a través de la ruta endocítica. Este es un proceso clave para la adquisición de nutrientes y el catabolismo que también controla la exposición de receptores y complejos de adhesión celular, entre otros. Trabajos anteriores de nuestro laboratorio identificaron la kazrina C como una proteína que bloqueaba la endocitosis dependiente de clatrina (CME) cuando se sobreexpresaba. El trabajo expuesto en esta tesis confirma el papel de la kazrina en la CME, al demostrar que células KO de kazrina generadas por el sistema de CRISPR-cas9 no internalizaban correctamente la transferrina (Tfn), un marcador endocítico. La kazrina C co-localizó con marcadores de uniones adherentes como la N-cadherina en la membrana plasmática y en estructuras intracelulares. De hecho, un fraccionamiento subcelular demostró la presencia de la kazrina en endosomas tempranos (EEs). Además, la depleción de la kazrina provocó una acumulación de EEs con N-cadherina, y estos tenían una distribución más periférica que en las células control, lo cual es coherente con un papel de la kazrina en EEs. Las células KO de kazrina incubadas con Tfn no transportaban el cargo hacia el compartimento de reciclaje endocítico (ERC) y tenían el consiguiente defecto en el reciclaje de Tfn. Todos los fenotipos en las células KO de kazrina se recuperaron con la re-expresión de GFP-kazrina C pero no con la de GFP. Estas evidencias apuntan hacia un papel de la kazrina C en el reciclaje endosomal y el transporte de EEs hacia el ERC. De acuerdo con esta hipótesis, la depleción de la kazrina causó defectos en procesos celulares que dependen del reciclaje a través del ERC, tales como la migración celular y la citoquinesis. Este estudio también analiza los mecanismos moleculares de la función de la kazrina C en el tráfico endocítico. Se demostró que la kazrina C interacciona con los motores asociados a microtúbulos kinesina-1 y dineína, y que se une directamente a la cadena intermedia ligera de la dineína, LIC1. De hecho, la kazrina C tiene un dominio coiled-coil similar a los de los adaptadores de la dineína. La kazrina C tiene también en común con estos adaptadores su localización en la región pericentriolar, donde parecía atrapar EEs. Por lo tanto, proponemos que la kazrina C promueve el transporte de EEs a través de microtúbulos, probablemente como un adaptador de EEs y la dineína. En consonancia, este y anteriores estudios del laboratorio mostraron una interacción directa y co-localizaciones parciales de la kazrina C con componentes de EEs, tales como el adaptador de clatrina AP-1 y las GTPasas EHD1/3. Además, la kazrina C interaccionó con PI3P y con la PI3K de clase III, y su depleción causó un aumento en los niveles endosomales de la sonda de PI3P GFP-FYVE. Por último, hemos establecido una relación entre la kazrina C y otro elemento clave del tráfico endocítico: la maquinaria de polimerización de actina asociada a Arp2/3. Se observaron interacciones directas con la cortactina y el N-WASP, así como la co-localización de la GFP-kazrina C y la cortactina en la membrana plasmática y estructuras intracelulares. La depleción de la kazrina causó una reducción en la actina ramificada cortical y un aumento en la endosomal. En conjunto, probamos una función de la kazrina C en el reciclaje endosomal y proponemos que esta función está mediada por la regulación del transporte a través de microtúbulos, la polimerización de actina y el metabolismo de PI3P.
Eukaryotic cells internalise and redistribute the molecules from their surface through the endocytic pathway. This process is key to nutrient uptake and catabolism, and controls the surface exposure of signalling receptors and cell adhesion complexes, among others. Previous work in the laboratory identified kazrin C as a protein that blocked Clathrin-Mediated Endocytosis (CME) when overexpressed. The work presented in this thesis further supported the role of kazrin in CME, as kazrin KO cells generated with the CRISPR-cas9 system were defective in the uptake of the endocytic marker Transferrin (Tfn). Kazrin C co-localised with markers of adherence junctions, such as N-cadherin, at the plasma membrane and on intracellular structures. Indeed, subcellular fractionation analysis showed the localisation of kazrin in Early Endosomes (EEs). Consistent with a role of kazrin in EEs, kazrin depletion caused an accumulation of N-cadherin-loaded EEs, which showed a more peripheral distribution as compared to WT cells. Kazrin KO cells loaded with Tfn were unable to transport the cargo towards the Endocytic Recycling Compartment (ERC) and had a concomitant defect in Tfn recycling. All phenotypes on KO cells were recovered by the re-expression of GFP-kazrin C but not GFP. These evidences indicated a role of kazrin C in endosomal recycling and the transport of EEs towards the ERC. In agreement with this hypothesis, kazrin depletion caused defects in cellular processes that strongly depend on recycling through the ERC, such as cell migration and cytokinesis. This study also analysed the molecular mechanisms of kazrin C function in endocytic traffic. Kazrin C was found to interact with the microtubule motors kinesin-1 and dynein, and directly bind to the dynein Light Intermediate Chain LIC1. In fact, kazrin C contains a coiled-coil domain similar to those found in dynein adaptors. Also similar to those, GFP-kazrin C localised to the pericentriolar region, where it seemed to trap EEs. Therefore, we proposed that kazrin C promoted microtubule-dependent transport of EEs, possibly as an EE dynein adaptor. Accordingly, this and previous studies in the laboratory showed direct interactions and partial co-localisations of kazrin C with EE components, such as the AP-1 clathrin adaptor complex and EHD1/3 GTPases. In addition, kazrin C interacted with PI3P and the class III PI3K, and its depletion caused an increase in the endosomal levels of the PI3P probe GFP-FYVE. Finally, we linked kazrin C with another player in endocytic traffic: the Arp2/3-associated machinery for actin polymerisation. Direct interactions were observed with cortactin and N-WASP, as well as co-localisation of GFP-kazrin C with cortactin at the plasma membrane and intracellular structures. Moreover, kazrin depletion caused a reduction in cortical and an increase in endosomal branched actin. Altogether, we proved that kazrin C functions in endosomal recycling and propose that this function is mediated by the regulation of microtubule-dependent transport, actin polymerisation and PI3P metabolism.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2003.
Includes bibliographical references (p. 149-164).
Calponins and transgelins are members of a conserved family of actin-associated proteins widely expressed from yeast to humans. While a role for calponin in muscle cells has been described, the biochemical activities and in vivo functions of non-muscle calponins and transgelins are largely unknown. I have used genetic and biochemical analyses to characterize the budding yeast member of this family, Scpl, which most closely resembles transgelin and contains one calponin homology (CH) domain. I showed that Scpl is a novel component of yeast cortical actin patches and shares in vivo functions and biochemical activities with Sac6/fimbrin, the one other actin patch component that contains CH domains. Similar to Sac6, purified Scpl binds directly to actin, cross-links actin filaments, and stabilizes filaments against disassembly. Furthermore, Scpl competes with Sac6 for binding to actin filaments and may share an overlapping binding site on actin. Overexpression of SCP1 suppresses sac6defects and deletion of SCP1 enhances sac6 defects. Together, these data show that Scpl and Sac6/fimbrin function together to stabilize and organize the yeast actin cytoskeleton. I used the genetic interactions between SCP1 and SAC6 to develop the first in vivo assay for function of any transgelin-like protein and established that actin binding is important for at least some Scpl functions. Sequences necessary and sufficient for actin cross-linking were identified in the carboxyl terminus of Scpl, outside the CH domain. Scpl may regulate actin cytoskeleton not only via direct binding to actin filaments, but also via its interaction with another actin binding protein, Abpl. Scpl and Abpl physically interact in a yeast two hybrid and co-immunoprecipitation assays. In vivo patch localization of Scp1 mutant defective for binding to actin filaments requires src-homology 3 (SH3) domain of Abpl. In vitro, Scpl specifically modulates Abpl-dependent activation of the Arp2/3 complex. In summary, Scpl may function in complex with Abpl to regulate actin nucleation by the Arp2/3 complex.
by Anya L. Goodman.
Ph.D.

Development is punctuated by morphogenetic rearrangements of epithelial tissues, including complete detachment of individual motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling; yet, neither the precise series of actin rearrangements at cell-cell junctions that accompany EMT, nor the machinery that controls actin rearrangement during EMT, have been identified. This work represents a detailed study of junctional actin reorganization in cells undergoing EMT, identifies actin regulatory proteins that control this actin reorganization, and defines the specific function of one regulatory protein, zyxin, in EMT. Using immunofluorescence and live cell imaging of HGF induced scattering of MDCK cells, dynamic actin rearrangement events occurring during EMT are characterized. Junctional actin characteristic of cell-cell adherent cells is rearranged into contractile medial actin networks linked to the junctional membrane in the initial steps of cell scattering. This actin rearrangement is accompanied by dynamic redistribution of specific actin regulatory proteins, namely α-actinin and zyxin-VASP complexes. α-Actinin mediates higher order structure of junctional actin. Zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction membranes. Zyxin regulation of actin-membrane linkage controls whether cell migration during EMT occurs independently in solitary cells or is coordinated through tissues. The functional analysis employed here uses novel, quantitative methods that define specific cellular EMT ‘phenotypes’ to reveal the precise role of zyxin in EMT. Constitutive active zyxin mutants exhibit persistent actin-membrane linkages and a scattering phenotype in which cells migrate without loss of cell-cell adhesion. Zyxin is proposed to regulate EMT progression by regulating disruption or maintenance of actin membrane linkages at cell-cell junctions. Zyxin alters the ability of cells to fully detach and migrate independently during EMT and may be an important regulator of morphogenetic plasticity.

In recent years, a global trend of increasing woody vegetation densities in semi-arid savanna habitats has been recorded, commonly described in South Africa as 'bush encroachment'. The shrubs and trees that do this (Increasers) have wrought significant economic and ecological impacts upon carrying capacities of large areas of savannas. This occurs, as suitable grazing areas are incrementally engulfed in shrubs and trees establishing new equilibria, from open savannas (essentially grasslands with scattered trees) into closed woodlands (treelands with scattered grasses). This thesis demonstrated a link between grass biomass, small mammal abundance and diversity, and their potential increaser seed/seedling predatory activities in the semi-arid Lowveld Savannas of South Africa.

Genetics is among the most rewarding fields of biology for the theoretically inclined, offering both room and need for modeling approaches in the light of an abundance of experimental data of different kinds. Many aspects of the field are today understood in terms of physical and chemical models, joined by information theoretical descriptions. This thesis discusses different mechanisms and phenomena related to genetics, employing tools from statistical physics along with experimental biomolecular methods. Five articles support this work. Two articles deal with interactions between proteins and DNA. The first one reports on the properties of non-specific binding of transcription factors proteins in the yeast Saccharomyces cerevisiae, due to an effective background free energy which describes the affinity of a single protein for random locations on DNA. We argue that a background pool of non-specific binding sites is filled up before specific binding sites can be occupied with high probability, thus presenting a natural filter for genetic responses to spurious transcription factor productions. The second article describes an algorithm for the inference of transcription factor binding sites for proteins using a realistic physical model. The functionality of the method is verified on a set of known binding sequences for Escherichia coli transcription factors. The third article describes a possible genetic feedback mechanism between human cells and the ubiquitous Epstein-Barr virus (EBV). 40 binding regions for the major EBV transcription factor EBNA1 are identified in human DNA. Several of these are located nearby genes of particular relevance in the context of EBV infection and the most interesting ones are discussed. The fourth article describes results obtained from a positional autocorrelation analysis of the human genome, a simple technique to visualize and classify sequence repeats, constituting large parts of eukaryotic genomes. Applying this analysis to genome sequences in which previously known repeats have been removed gives rise to signals corroborating the existence of yet unclassified repeats of surprisingly long periods. The fifth article combines computational predictions with a novel molecular biological method based on the rapid amplification of cDNA ends (RACE), coined 5’tagRACE. The first search for non-coding RNAs encoded in the genome of the opportunistic bacterium Enterococcus faecalis is performed here. Applying 5’tagRACE allows us to discover and map 29 novel ncRNAs, 10 putative novelm RNAs and 16 antisense transcriptional organizations. Further studies, which are not included as articles, on the monitoring of secondary structure formation of nucleic acids during thermal renaturation and the inference of genetic couplings of various kinds from massive gene expression data and computational predictions, are outlined in the central chapters.
QC 20110316

Parkin gene is associated with the development of autosomal recessive juvenile parkinsonism (Kitada et al., 1998) which is a common form of familial Parkinson’s disease (Klein and Schlossmacher, 2006). Since Parkin has multiple cell protective effects, increasing the expression level of Parkin in the brain might be able to rescue cells in danger, which in turn might prevent or slow down the development of Parkinson’s disease (Ulusoy and Kirik, 2008). In order to increase Parkin expression, it is important to understand the transcriptional mechanisms regulating Parkin expression (Maston et al., 2006). Since human Parkin is very big (~1.4 Mb) (Asakawa et al., 2001), in this study we use the smaller Fugu parkin gene, which is an ortholog of human Parkin (Yu et al., 2005), to search for the transcriptional factors and signaling pathways regulating Parkin expression. We have cloned vertebrate constructs that allow for the monitoring of an entire genomic Fugu parkin gene tagged with a reporter (eGFP or luciferase) in mammalian cells; and have established cellular model for studying the expression. According to the “TRANSFAC” transcription factor database, as well as “TFBIND” and “TFSEARCH” softwares (Wingender et al., 1996; Heinemeyer et al., 1998; Heinemeyer et al., 1999; Tsunoda and Takagi, 1999; Akiyama 1995), potential Nrf2 binding sites are conserved in the promoters of mammalian parkin (including human Parkin and mouse parkin) and in Fugu parkin. In this study, we could not find a link between the presence of the potential Nrf2 binding site(s) in the parkin promoter and the up-regulation of parkin; and we could not find an association between the Nrf2 pathway activation and the induction of parkin under the specific experimental conditions.

Dosage compensation in female mammals is achieved by XIST/Xist RNA mediated transcriptional silencing of one of the two X chromosomes. Several developmental specific cis-acting regulatory elements for mouse XIST have been demonstrated. One of these regulatory elements is the mouse Tsix locus, which is transcribed antisense to Xist and represses Xist on one of the X chromosomes at the onset of X inactivation. A transcript antisense to human XIST has been shown; however, its functional significance has been repeatedly challenged. My thesis aims to uncover cis-acting regulatory elements for human XIST and determine whether the elements are comparable to those found in mice. Currently, multi-copy integrations of a transgene containing human XIST into male mouse embryonic stem (ES) cells or into male human fibrosarcoma cells are the model systems of choice for studying the initiation of human X inactivation because the transcription antisense to human XIST can be detected by RT-PCR from the transgenes. Using DNase I hypersensitivity (HS) mapping, I found one HS site on the human transgene in mouse ES cells located approximately 11 kb downstream of XIST3’ end. While this HS site does not correspond to the transcription starts of TSIX previously described, it encompasses a small cluster of CTCF binding sites based on in silico search. Besides the downstream HS site, I discovered two HS sites in differentiated cell lines. One of the HS sites is immediately upstream of the XlST transcription start site. The other HS site (HS 101), located approximately 65 kb upstream of human XIST transcription start, resides within a region that shares above 70% sequence identity with cow and dog but not mouse. I analyzed these upstream HS sites and found that HS 101 exhibits bi-directional promoter and enhancer activity. My thesis revealed three previously unidentified HS sites flanking the human XIST locus; the presence of only one ES cell specific HS site downstream of XIST3’ end is in sharp contrast to the seven sites reported in mouse. The results suggest that mouse Xist and human XIST are regulated differently. To account for the differences in regulatory elements, I propose an alternative model for human XIST regulation.

Small GTP-binding proteins are molecular switches that serve as important regulators of numerous cellular processes. In animal and plant cells, the Rho family of small GTPases participate in e.g. organisation of the actin cytoskeleton, production of reactive oxygen species through the NADPH oxidase complex, regulation of gene expression. The three most extensively studied subgroups of the Rho GTPase family are Cdc42, Rho and Rac. One of the mechanisms by which animal Rac and Cdc42 GTPases regulate actin filament organisation is through activation of the ARP2/3 complex, a multimeric protein complex which induces branching and nucleation/elongation/polymerisation of actin filaments. Activation of the ARP2/3 complex by Rac and Cdc42 is mediated through the proteins WAVE and WASP, respectively.

In a search for Ras-like GTPases in Arabidopsis, we identified a family of genes with similarity to Rac GTPases. Screens of cDNA and genomic libraries resulted in the finding of 11 genes named ARACs/AtRACs. Genes encoding Rho, Cdc42 or Ras homologues were not identified. Expression analysis of AtRAC1 to AtRAC5 indicated that AtRAC1, AtRAC3, AtRAC4 and AtRAC5 are expressed in all parts of the plant, whereas AtRAC2 is preferentially expressed in root, hypocotyl and stem.

The AtRAC gene family can be divided into two main groups based on sequence similarity, gene structure and post-translational modification. AtRAC group II genes contain an additional exon, caused by the insertion of an intron which disrupts the C-terminal geranylgeranylation motif. Instead, group II AtRACs contain a putative motif for palmitoylation. Phylogenetic analyses indicated that the division of plant RACs into group I and group II occurred before the split of monocotyledonous and dicotyledonous plants. Analyses of the genes neighbouring AtRAC genes revealed that several of the plant RAC genes have been created through duplications.

The restricted/tissue-specific expression pattern of AtRAC2 led us to do a more detailed expression analysis of this gene. A 1.3 kb fragment of the upstream (regulatory) sequence of AtRAC2 directed expression of GUS or GFP to developing primary xylem in root, hypocotyl, leaves and stem. In root tips, the onset GUS staining or GFP fluorescence regulated by the AtRAC2 promoter slighty preceded the appearance of secondary cell walls. In stems, GUS staining coincided with thickening of xylem cell walls. Transgenic plants expressing constitutively active AtRAC2 displayed defects in the polar growth of leaf epidermal cells, indicating that AtRAC2 may be able to regulate the actin cytoskeleton. Surprisingly, an AtRAC2 T-DNA insertion mutant did not show any observable phenotypes. GFP fusion proteins of wild type and constitutively active AtRAC2 were both localised to the plasma membrane. The data suggest that AtRAC2 is involved in development of xylem vessels, likely through regulation of the actin cytoskeleton or NADPH oxidase.

The role of RAC GTPases in regulation of the actin cytoskeleton in plants is well documented. However, although the ARP2/3 complex had been identified in plants/Arabidopsis, the mechanisms regulating this complex were unknown. Through database searches, we identified three Arabidopsis genes, AtBRK1, AtNAP and AtPIR, which encoded proteins with similarity to subunits of a protein complex shown to regulate the activity of WAVE1 in mammalian cells. T-DNA inactivation mutants of AtNAP and AtPIR displayed morphological defects on epidermal cells undergoing polar expansion, such as trichomes and leaf pavement cells. The phenotypes were similar to those observed for ARP2/3 complex mutants, suggesting that AtNAP and AtPIR act in the same pathway as the ARP2/3 complex in plants. The actin cytoskeleton in atnap and atpir mutants was less branched than in wild type plants; instead, actin filaments aggregated in thick actin bundles.

Finally, we have recently discovered a small gene family encoding putative WAVE homologues. In mammalian cells, Rac activates WAVE1 through binding to PIR121 or Sra1 (the mammalian homologues of AtPIR). The discovery of a putative WAVE regulatory complex as well as putative WAVE homologues in Arabidopsis suggests that plant RAC GTPases regulate organisation of the actin cytoskeleton during polar growth at least partly through the ARP2/3 complex, using an evolutionarily conserved mechanism.

The main objective of this study was to investigate promoter sequences of putative HSR genes for the presence of unique regulatory elements and modules that might be involved in the regulation of HSR. In order to achieve this objective, an in silico promoter analysis strategy was devised, which focused on the identification of promoter sequences and regulatory elements, and modelling of promoter modules by using Genomatix software tools such as MatInspector and ModelInspector. Results showed that two modules (EGRF_SP1F_01 and SP1F_CEBP_01) were conserved in the promoter sequences of three well-known Hsp-genes (Hsp90, Hsp105β and αβ-crystallin). Screening the 60 target gene promoters for the presence of the two modules revealed that 12 genes (20 %) contained both modules. These included Moesin, Proline-4 hydroxylase, Poly(A) binding protein and Formin-binding protein. None of these genes had been previously associated with heat shock response.