Intellectual disability (ID) is a common phenotype of brain-development disorders and is heterogeneous in etiology with numerous genetic causes. PAK3 is one gene with multiple mutations causing ID. Affected individuals have microcephaly, and other brain-structure defects have been reported. Additionally, PAK3 is in a genetic network with eighteen other genes whose mutations cause ID, suggesting the molecular mechanisms by which PAK3 regulates of cognitive function may be shared by other genetic ID disorders. Studies in rodent models have shown that the orthologs of PAK3 are important for regulating dendrite spine morphology and postnatal brain size. In Drosophila melanogaster, the morphological processes of oogenesis, dorsal closure during embryogenesis, and salivary gland-lumen formation require Pak, the Drosophila ortholog of PAK3. Additionally, Pak is important for development of the subsynaptic reticulum of the neuromuscular junction, sensory axon pathfinding and terminal arborization in the Drosophila central nervous system (CNS). However, the role of Pak in mushroom body (MB) structure and intrinsic neurite arbor morphogenesis, as well as details of the underlying cellular and molecular mechanisms are unknown. To address this gap, I used Drosophila models of PAK3 gene mutations, Pak, and a combination of immunostaining, primary cell culture, and genetic interaction studies to elucidate these mechanisms. I performed a detailed characterization of the previously reported adult Pak phenotypes of decreased survival as well as leg and wing morphology. I found that decreased survival is a low-penetrance phenotype that is enhanced by chromosomes from the same mutagenesis. Defects of the adult wing include folding and misalignment between the layers, blisters, and missing or partial cross veins. The Pak-mutant legs are short and often misdirected in the pupal case with morphological defects in the shape of the leg segments themselves. The mushroom bodies are important insect learning and memory brain structures whose lobes are composed of axon bundles with individual axons bifurcating to form the α and β lobes. Mutations in Pak cause defects in the length, thickness, and direction of the MB α and β lobes. These defects increase in severity during metamorphosis, when neurogenesis and differentiation of these structures occur, suggesting that Pak stabilizes the branches of the α/β mushroom body neurons. Pak-mutant cultured neurons have reduced neurite arbor size with defects in neurite caliber. Initial outgrowth was normal, followed by a decrease in neurite branch number, again supporting the role of Pak in neurite-branch stability. There are defects in the cytoskeleton in growth cones at six hours post-plating as well as in neurons after three days in vitro. The Pak-mutant phenotype severity depends on the phosphorylation status of myosin regulatory light chain, supporting the mechanistic hypothesis that Pak regulates neurite-branch stability by inhibiting myosin light chain kinase. The neuronal phenotype of decreased branch stability suggests a mechanism of excessive retraction as the cellular pathogenesis underlying PAK3 mutation-associated brain disorders. I used western blotting to characterize the protein products of four nonsense mutations in Drosophila Pak to interpret genotype-phenotype relationships. Each allele has molecularly unique consequences: Pak¹¹, stop-codon read through and truncated protein; Pak¹⁶, no read through, but truncated protein; Pak⁶, read through with no truncated protein; Pak ¹⁴, neither readthrough nor truncated protein. Truncated proteins produced by Pak¹¹ and Pak¹⁶ alleles retained partial function for survival, wing blistering, leg morphology, and neurite length. Conversely, truncated protein increased the severity of the mushroom body defects. Truncated proteins have no effect on neuron branch number, wing folding, or vein defects. Together, these results demonstrate a role of Pak in regulating epithelial morphology, brain structure, and neurite arbor size and complexity. These closely resemble features of the human disorder, providing evidence that this is a good genetic model for this cause of ID.

Made available in DSpace on 2014-06-11T19:30:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-19Bitstream added on 2014-06-13T18:47:26Z : No. of bitstreams: 1 andreotti_pf_dr_arafcf.pdf: 3700497 bytes, checksum: 44fed3a7c2ab7db1a4fba80fbe6c2c51 (MD5) / Universidade Estadual Paulista (UNESP) / Os mecanismos de virulência de Paracoccidioides brasiliensis não estão completamente esclarecidos. Há consenso que subcultivos sucessivos deste fungo acarretam a perda de sua patogenicidade que pode ser revertida pelo reisolamento do agente após passagem em animal, recuperando assim alguns fatores de virulência. No presente estudo, tivemos como objetivo clonar e sequenciar uma banda polimórfica de 300pb do DNA genômico do isolado de P. brasiliensis com alta virulência (após reisolamento de animal), e assim caracterizar um provável fator de virulência deste fungo. Os resultados mostraram que as adesinas são capazes de induzir apoptose nas células estudadas por diferentes mecanismos. Assim podemos concluir que P. brasiliensis dispõe, como outros patógenos, de adesinas que participam, não só da adesão deste fungo às células do hospedeiro, mas também de outros importantes mecanismos que possa estar contribuindo para a sua patogênese. / The Paracoccidioides brasiliensis virulence mechanisms are not completely clarified. There is a consensus that successive subculturing of this fungus causes the loss of its pathogenicity, which can be reversed by passage in animals, with recovery of some factors of virulence. In the present study, we cloned and sequenced a polymorphic band of 300bp from P. brasiliensis genomic DNA of the one isolate with high virulence (after reisolation of animal). Thus, we can conclude that P. brasiliensis use, as many other pathogens, the adhesins as virulence factors to participate, not only of the adhesion of this fungus to the host cells, but in other important mechanisms to its pathogenesis.

Prions are infectious protein entities capable of self-replication. Prions are the causal agents behind the transmissible spongiform encephalopathies causing neurodegeneration and death in affected organisms. Prions have been identified in yeast with the best-characterized prions being [PSI+] and [PIN+], whose respective native proteins are the Sup35 translation termination factor and Rnq1 (function unknown). Autophagy is a cellular housekeeping mechanism mediating the degradation of damaged proteins and superfluous organelles. It is a highly sequential process regulated by autophagy related genes (ATGs). Autophagy has also been implicated in the clearance of amyloidogenic proteins including prions. However, the mechanistic basis underlying this activity is poorly understood, and a key objective of this project was to characterize how autophagy prevents spontaneous prion formation. Our study found that the deletion of core ATGs correlated with an increase in de novo [PSI+] and [PIN+] formation as well as Sup35 aggregation. Enhancement of autophagic flux through spermidine treatment attenuated the increased levels of de novo [PSI+] formation in mutants that normally show elevated levels of [PSI+] formation. Defective autophagy correlated with increased oxidatively damaged Sup35 in an atg1 mutant whereas anaerobic growth abrogated the increased [PSI+] formation in the atg1 mutant to wild-type levels. Our data suggest that autophagy serves a protective role in the clearance of oxidatively damaged Sup35 proteins that otherwise has a higher propensity towards [PSI+] prion formation. We also investigated the role of prion formation and autophagy during yeast chronological ageing which is the time that non-dividing cells remain viable. Prion diseases are associated with advanced age which correlates with a decline in cellular protective mechanisms including autophagy. Our study found an age dependent increase in the frequency of de novo [PSI+] formation with chronological age of yeast cells, more so in an atg1 mutant relative to the wild-type. Autophagy competent cells carrying the [PSI+] and [PIN+] prions also had improved chronological lifespan relative to prion free cells and atg1 cells. Cells carrying the [PSI+] prion elicited elevated autophagic flux that may promote improved lifespan thus suggesting a beneficial role of the [PSI+] prion during chronological ageing. The actin cytoskeleton provides the structural framework essential for a multitude of cellular processes to occur. We investigated the role of the Arp2/3 complex responsible for branching of actin filaments towards prion formation. Knockout mutants of the nucleation promoting factors of the Arp2/3 complex, in particular the abp1 mutant, showed reduced de novo [PSI+] formation and Sup35 aggregation under basal and oxidative stress conditions. Similarly, treatment with latrunclin A, an actin monomer-sequestering drug also abrogated de novo [PSI+] formation. Colocalization studies revealed that Sup35 often does not colocalize with Rnq1, a marker for the insoluble protein deposit (IPOD) in an abp1 mutant. This suggests a role for the Abp1 protein in the efficient transport of Sup35 molecules to the IPOD that may facilitate de novo [PSI+] prion formation under vegetative states and oxidant challenges.

Orientador: Maria José Soares Mendes-Giannini / Banca: Christiane Pienna Soares / Banca: Maria Célia Jamur / Banca: Rosely Maria Zancopé Oliviera / Banca: Sônia Rozental / Resumo: Os mecanismos de virulência de Paracoccidioides brasiliensis não estão completamente esclarecidos. Há consenso que subcultivos sucessivos deste fungo acarretam a perda de sua patogenicidade que pode ser revertida pelo reisolamento do agente após passagem em animal, recuperando assim alguns fatores de virulência. No presente estudo, tivemos como objetivo clonar e sequenciar uma banda polimórfica de 300pb do DNA genômico do isolado de P. brasiliensis com alta virulência (após reisolamento de animal), e assim caracterizar um provável fator de virulência deste fungo. Os resultados mostraram que as adesinas são capazes de induzir apoptose nas células estudadas por diferentes mecanismos. Assim podemos concluir que P. brasiliensis dispõe, como outros patógenos, de adesinas que participam, não só da adesão deste fungo às células do hospedeiro, mas também de outros importantes mecanismos que possa estar contribuindo para a sua patogênese. / Abstract: The Paracoccidioides brasiliensis virulence mechanisms are not completely clarified. There is a consensus that successive subculturing of this fungus causes the loss of its pathogenicity, which can be reversed by passage in animals, with recovery of some factors of virulence. In the present study, we cloned and sequenced a polymorphic band of 300bp from P. brasiliensis genomic DNA of the one isolate with high virulence (after reisolation of animal). Thus, we can conclude that P. brasiliensis use, as many other pathogens, the adhesins as virulence factors to participate, not only of the adhesion of this fungus to the host cells, but in other important mechanisms to its pathogenesis. / Doutor

No útero gestante dos animais com placentação do tipo hemocorial, ocorre uma migração e acúmulo transitório de linfócitos natural killer (NK) , cuja atuação na gestação não está totalmente elucidada. Estas células NK do ambiente uterino (uNK) apresentam comportamento distinto daquelas encontradas no sangue circulante (cNK), constituindo uma sub-população das células NK com expressão gênica específica ditada pelo ambiente uterino gestante. De fato, se estas células isoladas do útero de camundongos prenhes forem inoculadas em machos da mesma espécie eram capazes de induzir a resposta imunológica com produção de anticorpos que reagem especificamente com as células uNK. No presente trabalho foi utilizado um destes anticorpos monoclonais denominados de \"mouse anti-mouse uterine natural killer cell clone 1 (mam-uNK1)\" obtidos anteriormente em nosso laboratório para avaliar a especificidade deste anticorpo e a localização da molécula antigênica correspondente. Para tanto, foram utilizados cortes histológicos do útero no 9º dia de gestação, dos órgãos linfóides (baço, timo e linfonodo), do cérebro, do fígado e do coração submetidos à reações imunocitoquímicas com o anticorpo mam-uNK1 em nível da microscopia de luz e, pela imunomicroscopia eletrônica nas células uNK do útero gestante e células estriadas cardíacas do miocárdio. Foram obtidos homogenados teciduais dos mesmos órgãos avaliados pela imunocitoquímica para realização do SDS-PAGE e Western-blot com o intuído de identificar as frações protéicas reativas e homologia entre os diversos órgãos. Os padrões de imunomarcação com o mam-uNK1 foram comparadas com o padrão de reatividade da lectina DBA (Dolichos biflorus) tanto nos cortes histológicos quanto nos homogenados submetidos ao Western-blot. Os resultados demonstraram reação positiva distribuída difusamente no citoplasma, com maior intensidade no perímetro das células uNK, e marcação difusa no citoplasma das células deciduais e das células musculares lisas do miométrio. Reações positivas foram encontradas também no citoplasma das células musculares cardíacas, no citoplasma das células reticulares dos órgãos linfóides, nos feixes de nervos do sistema nervoso central e no citoplasma dos hepatócitos. Pela imunomicroscopia eletrônica foram observadas partículas de ouro coloidal em maior número no citoplasma que preenchem os prolongamentos citoplasmáticos tipo microvilosidades e no citoplasma marginal abaixo da membrana plasmática nas células uNK. Nas células musculares cardíacas as marcações mais intensas foram constatadas no citoplasma da extremidade destas células onde as miofibrilas eram menos organizadas e se ancoravam à membrana plasmática. Pelo Western-blot, foram identificadas duas bandas reativas ao mam-uNK1 com peso molecular de 52 e 54 kDa comuns a todos os órgãos analisados. Estes dados demonstram que a molécula reconhecida pelo anticorpo mam-uNK1 tem ampla distribuição em diversos tipos celulares não sendo específica para as células uNK, porém apresentam uma localização peculiar nestas células e nas células musculares cardíacas. Pelo padrão de localização identificado em imunomicroscopia eletrônica, presume-se que estas moléculas estejam associadas com a modulação do citoesqueleto nas diversas atividades que estes componentes estruturais desempenham nas células, sendo particularmente interessante a relação com a motilidade celular. / In the pregnant uterus of animals developing hemochorial type placentation occurs a transient migration and accumulation of natural killer lymphocytes (NK) which activity in the pregnancy has not been fully elucidated. These NK cells from uterine environment (uNK) present a distinct behavior from those found in the peripheral circulating blood (cNK), composing a subset of NK cells with specific gene expression that is regulated by pregnant uterus. Actually, these cells isolated from pregnant mice uteri were inoculated in the male mice of same strains, they induced immune response with production of antibodies reactivity specifically to uNK cells. In the present work it was used one of these mouse anti-mouse uterine natural killer cells clone 1 (mam-uNK1) monoclonal antibody that was obtained previously in our laboratory, in the aim to evaluate the specificity of this antibody and localization of corresponding antigenic molecule. It was used histological sections of uteri on 9º gestational day, lymphoid organs (spleen, thymus and lymph node), brain, liver and heart processed for immunocytochemistry with mam-uNK antibody at light microscopy and immunoelectron microscopy for uNK cells in pregnant uterus and cardiac muscle cells. Tissue homogenates from the same organs that were evaluated by immunocytochemistry were obtained to perform SDS-PAGE and Western-blot primary to identify the proteins fractions reactive with mam-uNK antibody and possible homology among the tissues. The immunolabeling pattern using mam-uNK both, in histological sections and Western-blot were compared with pattern of Dolichos biflorus (DBA) lectin reactions. The results showed diffuse positive reaction distributed in the cytoplasm with higher intensity on perimeter of uNK cells and diffuse labeling in the cytoplasm of decidual cells and, in smooth muscle cells of miometrium. Positive reaction was also found in the cytoplasm of cardiac muscle cells, reticular cells of lymphoid organs, hepatocytes and in the axon bundles of the brain. By immunelectron microscopy, were observed higher number of gold particles in the cytoplasm of microvillous-like cell processes and in the marginal cytoplasm near the plasma membrane of uNK cells. In the cardiac muscle cells the most conspicuous labeling was seen in the cytoplasm of muscle cells at the end portions, that is, where the myofibrills were less organized and anchoring to plasma membrane. The Western-blot identified two bands with 52 and 54 kDa reactive do mam-uNK constantly found in all organs analyzed. These data show that the molecule that is recognized by mam-uNK1 antibody is widely distributed in several cell types, not specific for mouse uNK cells, but has a very peculiar localization in these cells and in the cardiac muscle cells. To the localization pattern that was identified by immunelectron microscopy it was suggested that, these molecules were associated to the modulation of the cytoskeleton in many activities that these structural component potentially could carry out in the cells, being particularly attractive those related to cell motility.

Orientador: Sara Terezinha Olalla Saad / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T12:26:47Z (GMT). No. of bitstreams: 1 Borges_Luciene_D.pdf: 3990737 bytes, checksum: 7c865de830ecbe5f830e992e6c69fb2c (MD5) Previous issue date: 2007 / Resumo: Estímulo mecânico é um dos principais eventos envolvidos na hipertrofia cardíaca que afeta vários componentes de vias de sinalização do miocárdio. Recentemente, um novo transcrito altamente expresso em músculo cardíaco, denominado de ARHGAP21, foi descrito como um membro da família de proteínas Rho GAP, que demonstrou atividade catalítica sobre Cdc42 e interação com ARF1, ARF6 e a-catenina, proteínas importantes do remodelamento do citoesqueleto e junções aderentes. No presente estudo, tivemos como objetivo analisar a expressão da ARHGAP21 em resposta ao estresse mecânico agudo em corações de ratos adultos, e sua associação com FAK e PKC?. Utilizando-se fracionamento subcelular, microscopia confocal e eletrônica, demonstramos que ARHGAP21 relocaliza-se das regiões nucleares e miofilamentos para linhas Z, discos intercalares e costâmeros de cardiomiócitos submetidos à sobrecarga de pressão, sugerindo que esta roteína pode desenvolver uma importante função no remodelamento cardíaco. Além do mais, ensaio de imunoprecipitação mostrou que ARHGAP21 interage com PKC? e FAK em ratos controle, submetidos à coarctação da aorta e espontaneamente hipertensos (SHR). Três diferentes regiões de FAK, contendo cauda de GST acoplada, foram utilizadas em ensaio de ligação in vitro, demonstrando que ARHGAP21 se liga à porção carboxi terminal de FAK. Além disso, ARHGAP21 associa-se à PKC? fosforilada em Thr410 em o-imunoprecipitados de extratos protéicos de ratos controle e SHR. Entretanto, ARHGAP21 associa-se apenas à FAK fosforilada em Tyr925 em SHR. Também foi verificado que PKC? é fosforilada por estímulo mecânico. Estes resultados sugerem que ARHGAP21 pode atuar como uma molécula sinalizadora ou proteína adaptadora das vias de sinalização de FAK e PKC? em cardiomiócitos, provavelmente desempenhando importante função durante estresse cardíaco / Abstract: Mechanical stimulus is one of the major events involved in cardiac hypertrophy, and affects components of essential myocardium signaling pathways. Recently, we described a highly expressed mRNA in the cardiac muscle as a member of the RhoGAP family of proteins, ARHGAP21, which demonstrated GAP activity over Cdc42 and interacted with ARF1, ARF6 and a-catenin important proteins of the cytoskeleton assembly and adherent junctions. In the present work, we aimed to analyze the expression of ARHGAP21 in response to acute mechanical stress in the adult rat heart and its association with FAK and PKC? proteins. By subcellular fractionation, confocal and immunoelectron microscopy, we demonstrated that ARHGAP21 is relocated from the nucleus to the plasma membrane, Z-lines and costameres of cardiomyocytes submitted to pressure overload conditions, suggesting that this protein may develop a role in cardiac remodeling. Furthermore, immunoprecipitation assay showed that ARHGAP21 interacted with PKC? and FAK in control rats, rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Using three different GST-tagged regions of FAK, we found that ARHGAP21 binds to the carboxyl terminal portion of FAK. Moreover, ARHGAP21 binds to PKC? phosphorylated on Thr410 in co-immunoprecipitates of protein extracts from sham control rats and SHR. However, ARHGAP21 only binds to FAK phophorylated on Tyr925 of SHR. We have also shown that PKC? is phosphorylated by mechanical stimuli. Altogether, these results suggest that ARHGAP21 may act as a signaling molecule or scaffold protein of FAK and PKC? signaling pathways in cardiomyocytes, probably developing an important function during cardiac stress / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

De plus en plus d’études soulignent l’importance de la localisation intracellulaire des voies de signalisation. Nous avons développé des méthodes permettant de perturber cette localisation à l’aide de nanoparticules magnétiques. Ces dernières sont fonctionnalisées avec les protéines d’intérêts et deviennent ainsi un vecteur permettant de contrôler la localisation de la signalisation. Nous avons tout d’abord appliqué cette méthode dans un système modèle, des gouttes d’extrait cellulaire de Xénope, dans lesquelles nous avons créé artificiellement un gradient de protéines de signalisation à l’aide de nanoparticules magnétiques. Nous avons mis en évidence l’influence d’une asymétrie biochimique sur la localisation d’asters de microtubules. Dans un deuxième temps nous avons examiné la possibilité d’appliquer cette méthode dans des cellules HeLa adhérentes, pour perturber la localisation d’endosomes de signalisation rendus magnétiques. Nous avons cherché à optimiser les conditions expérimentales nécessaires pour contrôler la position d’endosomes de signalisation magnétiques Enfin, un troisième projet dont les résultats préliminaires sont présentés dans cette thèse, a consisté à utiliser un actuateur, non plus magnétique, mais biologique pour confiner une cascade de signalisation. Plus précisément la contraction d’un réseau d’actine confiné dans des gouttes d’extrait cellulaire est utilisée pour localiser des protéines de signalisation. Ces résultats démontrent l’intérêt de nanoparticules magnétiques pour induire et étudier des phénomènes de brisures de symétries dans des environnements biologiques / An increasing number of studies highlight the importance of signaling localization. We developed methods to perturb this localization using magnetic nanoparticles. Proteins of interest are grafted on magnetic nanoparticles, allowing to magnetically localize them. We first propose a new method to engineer directly a spatial gradient of signaling protein concentration within in cell extract droplets using super-paramagnetic nanoparticles. We observed a link between a spatial asymmetry in biochemical cues and microtubules aster positional information. Our assay provides a bottom-up approach to examine the minimum ingredients generating polarization and symmetry breaking within cells. We then examined the possibility to magnetically perturb endosomes position in HeLa cell. We found the experimental conditions to achieve this goal. Finally, we used directly cytoskeleton elements as actin filament to trigger asymmetrically confined signaling proteins and trigger microtubule assembly, in cell extract droplets. More generally, these results show how symmetry breaking within cells can be induced and studied using magnetic nanoparticles and biophysical tools.

Le cytosquelette se compose de microfilaments (polymères d’actine), de microtubules (polymères de tubuline) et de filaments intermédiaires (polymères de desmine, de lamines …). Le sepsis défini par une infection associée à une réaction inflammatoire systémique est responsable de dysfonctions myocardiques de mauvais pronostique. Cette cardiomyopathie apparait dans les premières heures du sepsis et guérit en moins de deux semaines chez les survivants. Même si certaines études démontrent l’implication d’éléments du cytosquelette dans la cardiomyopathie septique, les rôles des microfilaments et des microtubules ne sont pas clairement établis.Macrophage migration inhibitory factor (MIF) est une cytokine pro-inflammatoire sécrétée en excès dans le sepsis qui serait responsable d’un ralentissement de la récupération myocardique. Dans un premier temps, notre travail a consisté à caractériser l’implication des microtubules dans la dysfonction musculaire cardiaque induite par MIF. Dans un modèle de trabécules auriculaires droites humaines nous avons démontré que MIF induit une hyperpolymérisation des microtubules responsable d’une hyperviscosité intracellulaire, d’une dysfonction mitochondriale et d’une dysfonction contractile. Nos résultats suggèrent qu’une hyperpolymérisation des microtubules induite par MIF pourrait être responsable d’un ralentissement de la récupération myocardique à la phase tardive de la myocardiopathie septique. Dans un second temps, nous avons évalué l’implication des microfilaments dans un modèle murin de dysfonction myocardique inflammatoire induite par l’injection d’une endotoxine bactérienne, le lipopolysaccharide. Nos résultats suggèrent qu’à la phase précoce de la cardiomyopathie inflammatoire il existe une hyperpolymérisation des microfilaments responsable de dysfonctions contractile et mitochondriale.Les connaissances fondamentales acquises au cours de ce travail de thèse suggèrent une implication directe des microtubules et des microfilaments dans la physiopathologie des cardiomyopathies inflammatoires. / The cytoskeleton is composed of intracellular microfilaments (actin polymers), microtubules (tubulin polymers) and intermediate filaments (desmin, lamin … polymers).Sepsis, the association of an infection and a systemic inflammatory response, induces myocardial dysfunction. Septic cardiomyopathy appears in the early phase of sepsis and is associated with fatal outcome. Complete recovery of myocardial function occurs within two weeks following the onset of myocardial dysfunction in surviving patients. Although several studies demonstrate a role of cytoskeleton in septic cardiomyopathy, the involvement of microfilaments and microtubules is not clear.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine secreted during sepsis that is suggested to postpone myocardium recovery. The first aim of the study was to characterize microtubule implications in MIF-induced cardiac muscle dysfunction. In a model of human right atrial trabecule we demonstrated that MIF induces microtubule stabilizations which are responsible for high intracellular viscosity, contractile and mitochondria dysfunctions. Our results suggest that MIF-induced microtubule stabilizations might be responsible for a delay of myocardial recovery during septic cardiomyopathy. The second aim of the study was to characterize microfilament involvements in a murine inflammatory cardiomyopathy induced by a lipopolysaccharid (LPS) injection. Our results suggest that microfilament stabilizations might be responsible for LPS-induced contractile and mitochondria dysfunctions in the early phase of inflammatory cardiomyopathy. Thus, these new fundamental mechanisms suggest a direct involvement of microtubules and microfilaments in the development and evolution of inflammatory cardiomyopathies.

The microtubule cytoskeleton plays a vital role in the spatial-temporal organization of subcellular cargo required to maintain homeostasis and direct cell division. Cytoplasmic dynein and kinesin are opposite-polarity, microtubule-based motors that transport a wide variety of cargo throughout eukaryotic cells. While much is known about the stepping mechanism of kinesin from decades of study, cytoplasmic dynein's size and complexity has limited our understanding of its underlying motor mechanism. Here, a minimal, artificially-dimerized dynein motor was observed with two-color, near-simultaneous, high-precision, single-molecule imaging, which reveals the stepping pattern of each motor domain as dynein moves along the microtubule. Although the stepping behavior appeared highly irregular and erratic, with large variability in step sizes, side stepping behavior, and back stepping behavior, dynein did show evidence of tension-based, coordinated stepping. Furthermore, advances in DNA nanotechnology enabled us to engineer a synthetic motor-cargo system, referred to as a chassis, to investigate how multiple cytoskeletal motors work in teams to produce the myriad of motile behaviors observed in vivo. Specifically, the mechanisms that coordinate motor ensemble behavior was examined using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing control of motor type, number, spacing, and orientation in vitro. Ensembles of 1-7 identical-polarity motors displayed minimal interference with respect to directional velocity, while ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species. This experimental system allowed us to test directly the tug-of-war proposed to occur during dynein's delivery to the microtubule plus-end by the kinesin Kip2. This work led to the mechanistic understanding that Lis1/Pac1, CLIP170/Bik1, and EB1/Bim1 proteins function to enhance kinesin's processivity, allowing it to win a tug-of-war and transport dynein toward the microtubule plus-end. Overall, this work elucidated mechanisms of ensemble motor function and dynein's stepping mechanism in addition to building significant tools to further pave the way for future studies to elucidate how cytoskeletal motors function to organize cellular cargos.

Cell migration is required for a number of physiological processes and is implicated in pathologies such as tumor metastasis. Cell motility is dependent upon dynamic actin reorganization, and is regulated by the Rho family of small GTPases. Rho GTPases are molecular switches that cycle between their active and inactive conformations. The best-studied members of this family are Rac1, RhoA, and Cdc42. Each is responsible for the formation of specific actin structures. Diacylglycerol kinases (DGKs) act at the membrane to convert diacylglycerol (DAG) and phosphatidic acid (PA), maintaining the balance of these two lipid second messengers. Previous studies from our lab have demonstrated that the ζ isoform of DGK facilitates the release of Rac1 and RhoA from their inhibitor, RhoGDI. Here we studied a closely related isoform, DGKι, using mouse embryonic fibroblasts (MEFS) in which the gene for DGKι had been deleted. Aberrations in cell morphology, spreading, and migration were identified in DGKι-null MEFs. We showed that the activity of Rac1 and RhoC, but not RhoA, was impaired in the absence of DGKι, yet only RhoC protein levels were affected. Reduced activation of these Rho GTPases was accompanied by defects in Rac1- and RhoC- related actin structures. These data demonstrate that DGKι, in addition to DGKζ, contributes to the regulation of GTPase activation and remodeling of the actin cytoskeleton.