Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

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.

The motivation for this PhD thesis was to understand the molecular basis of how cells regulate the formation of an organised and mechanically strong extracellular matrix (ECM). In tendon this process begins during embryogenesis with the appearance of bundles of narrow-diameter (~30 nm) collagen fibrils that are parallel to the tendon long axis. At the onset of collagen fibrillogenesis, the cells elongate, the fibrils are constrained within plasma membrane channels with their ends contained in tension-sensitive actin-stabilised plasma membrane protrusions. The mechanism by which actin is reorganised during cell elongation and the formation of tension-sensitive plasma membrane protrusions is poorly understood. The small GTPase RhoA is the major regulator of actin reorganisation into stress fibres, which have been implicated in mechanotransduction, ECM assembly and remodelling. The hypothesis tested by this PhD thesis was that the organisation and tensioning of extracellular collagen fibrils is generated on a blueprint of tensioned actin filaments within the cell. Rho activity is regulated specifically by Rho GTPase activating proteins (RhoGAPs). By comparing the global gene expression of tendon tissues at different developmental stages, Arhgap28, a novel RhoGAP, which is expressed during tendon development but not during postnatal maturation, was identified.Arhgap28 belongs to a large family of RhoGAPs containing the closely related members, Arhgap6 and Arhgap18, which have previously been shown to regulate RhoA and stress fibre formation. Arhgap28 expression was upregulated in embryonic fibroblasts cultured in a 3D, tensioned embryonic tendon-like construct compared to monolayer culture. Arhgap28 expression was further enhanced during the development of mechanical strength and stiffness of the tendon constructs, but downregulated when the tension in tendon constructs was released. Overexpression of a C-terminal V5-tagged Arhgap28 protein caused a reduction in RhoA activation and disruption of stress fibre assembly. Modulation of Rho signalling using lysophosphatidic acid and Y27632 showed that collagen remodelling by cells in collagen gels and tendon constructs is regulated by RhoA signalling. A tissue-wide qPCR analysis identified Arhgap28 in several tissues including tendon, bone, and skin. An Arhgap28 reporter mouse (Arhgap28gt) and an Arhgap28 knockout mouse (Arhgap28del) were also studied to investigate the role of Arhgap28 in tissue organisation in vivo. Arhgap28gt mice showed Arhgap28 expression in bones at E18.5. Homozygous Arhgap28del mice were viable, appeared normal but expressed a truncated Arhgap28 transcript, which if translated, would produce a protein lacking the RhoGAP domain. Therefore, it was hypothesised that knockout mice were normal due to compensation from another RhoGAP. Overexpression of Arhgap6 in Arhgap28-null bone tissues was confirmed. Upregulation in RhoA expression was also detected, further suggesting that Arhgap28 regulates RhoA. Interestingly, a microarray comparison of bone tissues from wild type and Arhgap28-null mice showed that genes linked to bone dysplasia are downregulated in Arhgap28-null bone. Together, these results suggest that formation of a strong and organised collagen ECM is mediated by RhoA-generated cellular tension and that Arhgap28 and Arhgap6 might be co-regulators of this process.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18880号 / 医博第3991号 / 新制||医||1008(附属図書館) / 31831 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 篠原 隆司, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Magíster en fisiopatología / La hipertrofia cardiaca es una respuesta adaptativa del corazón frente a situaciones de sobrecarga de trabajo, la cual funciona inicialmente como un mecanismo compensatorio. Sin embargo, la hipertrofia sostenida en el tiempo puede conducir a la miocardiopatía dilatada, insuficiencia cardiaca y muerte súbita. Los receptores adrenérgicos juegan un papel fundamental en la regulación de la función cardiaca bajo condiciones normales y patológicas. Las mitocondrias son responsables del 90% de la producción de ATP en el cardiomiocito y su función está regulada dinámicamente por procesos de fusión y fisión. Cambios en la dinámica y energética mitocondrial constituyen una característica distintiva de los corazones hipertrofiados. La estimulación de cardiomiocitos con agonistas adrenérgicos genera hipertrofia y aumento de la fisión mitocondrial, lo que se asocia a una disminución de la producción de ATP. Miro1 es una proteína de la membrana mitocondrial externa involucrada en la dinámica y el transporte de este organelo, cuya función ha sido estudiada principalmente a nivel neuronal. Es ampliamente conocido que alteraciones en proteínas mitocondriales están relacionadas directamente con cambios en el funcionamiento mitocondrial, y por ende con patologías como la hipertrofia del cardiomiocito. Es por ello que en base a estos antecedentes nos propusimos la siguiente hipótesis: “Miro1 regula negativamente la hipertrofia inducida por fenilefrina en cardiomiocitos de rata neonata” Para contestar esta hipótesis los objetivos específicos de este trabajo se enfocaron en evaluar tanto en cardiomiocitos control como en aquellos tratados con fenilefrina 50 μM el contenido de Miro1 y los efectos del silenciamiento y sobrexpresión de esta proteína sobre el área celular y los marcadores de hipertrofia. Resultados Cardiomiocitos de rata neonata tratados con fenilefrina 50 μM aumentaron en un 50% el área celular, así como también la expresión de los marcadores hipertróficos de β-MHC, ANP y BNP. El silenciamiento de Miro1 indujo un aumento significativo en los niveles de ARNm de ANP y BNP cuando los cardiomiocitos fueron estimulados con fenilefrina, no observándose cambios en el área celular ni en los niveles de β-MHC. Por el contrario, la sobre expresión de Miro1 en los cardiomiocitos evitó tanto el aumento del área celular como el aumento en la expresión de marcadores de hipertrofia. Estos resultados sugieren la participación de Miro1 como proteína reguladora de la hipertrofia en cardiomiocitos. / Cardiac hypertrophy is an adaptive response to manage the excessive cardiac workload of the heart to maintain normal cardiac function. However, sustained hypertrophy leads to cardiomyopathy, cardiac failure and death. Adrenergic receptors play a key role in the regulation of cardiac function under normal and pathological conditions. Mitochondria are responsible for 90% of ATP production in the cardiomyocyte and their function is dynamically regulated by fusion and fission processes. Changes in mitochondrial dynamics and metabolism are central issues in hypertrophied hearts. The stimulation of cardiomyocytes with adrenergic agonists generate hypertrophy and an increase in mitochondrial fission, which in turn is associated with a decrease in ATP synthesis. Miro1 is a mitochondrial outer membrane protein which is involved in the mitochondrial dynamics and transport at neuronal level. It is known that alterations in mitochondrial dynamics proteins are directly associated with mitochondrial dysfunction and with cardiac pathologies such as cardiomyocyte hypertrophy. Based on these asseverations we hypothesized that: "Miro1 negatively regulates phenylephrine-induced hypertrophy in neonatal rat cardiomyocytes" In order to answer this hypothesis the specific aims were focused on to evaluate Miro1 content, and the effect of silencing or overexpressing Miro1 on surface cellular area and hypertrophic gene markers expression in control cardiomyocytes and phenylephrine treated cardiomyocytes. Results Neonatal rat cardiomyocytes treated with 50 μM phenylephrine 50% increased the surface cell area, as well as the expression of the hypertrophic gene markers: β-MHC, ANP and BNP. Miro1 silencing induced a significant increase in ANP and BNP mRNA levels when cardiomyocytes were stimulated with phenylephrine, with no changes in surface cell area or β-MHC mRNA levels. In contrast, Miro1 overexpression in cardiomyocytes prevented both surface cell area increase and mRNA levels of hypertrophic gene markers. These results suggest the involvement of Miro1 as a regulatory protein of hypertrophy in cardiomyocytes.

Spore formation is an essential step in the fungal life cycle that contributes to the dispersal of the organism and also to survival under harsh environmental conditions. The morphology of spores shows an astonishing diversity in the fungal kingdom and varies from very simple round and small spores to very complex multi-armed or sigmoid structures. With exception of the regulation of ascospore formation in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are well-characterized model organisms for spore development in fungi, little is currently known about the regulation of more complex spore morphologies. In this study, the filamentous ascomycete Ashbya gossypii is used as a model system for the investigation of a complex and composite spore morphology. A. gossypii produces linear, needle-shaped spores possessing a length of 30 µm, which can be divided into three major segments: a rigid tip segment, a more fragile membrane compartment and a stable tail-cap. Furthermore, the different compartments were shown to correlate with distinct materials. While the tip segment and the tail-cap of the spores consist of stabilizing materials like chitin and chitosan, these materials are absent from the compartment in the middle. The actin cytoskeleton plays an essential role in several steps of spore formation in A. gossypii. Different regions of actin accumulation were identified that directly correlate with the developing spores. Especially the developing tip segment is characterized by heavy-bundled linear actin structures. Furthermore, proteins of the formin family, a class of actin organizing proteins, were identified to be directly involved in spore formation in A. gossypii. The formin AgBnr2 fulfills an actin-related key function during spore development by linking actin to the spindle pole body during sporulation. Downregulation of AgBNR2 leads to severe sporulation defects, indicating a central function in spore development. Moreover, AgBni1, another representative of the formin family, also has a regulatory function in size determination of the typical needle-shaped spores of A. gossypii. Using a modified yeast two-hybrid approach, four potential activators of the formin AgBni1 were identified: the Rho-type GTPases AgRho1a, AgRho1b, AgRho3 and AgRho4. The interaction of AgBni1 with the two Rho1 GTPases plays an important role during spore development. In this study, the Rho binding domain of AgBni1 was further examined to identify amino acids that are essential for the interaction with the Rho-type GTPases. Using random mutagenesis combined with a two-hybrid screen, the point mutation S250P in the Rho binding domain of AgBni1 was identified to reduce the interaction of the formin with the Rho1 GTPases. Integration of AgBni1 S250P causes an increase in spore length, suggesting a direct effect of this signaling pathway in spore length determination. An actin-regulating protein network that includes the formin AgBni1, the Rho-type GTPases AgRho1a and AgRho1b and the paxillin-like protein AgPxl1 was identified to be mainly involved in the regulation of the spore length. Thereby, this network seems to be involved in the arrangement of the different spore compartments via the actin cytoskeleton.

Systemic steroid hormone and intracellular signaling pathways are known to act cooperatively during the development of vertebrate and invertebrate epithelia. However, the mechanism of this interaction is poorly understood. Morphogenesis of Drosophila leg imaginal disc epithelia is regulated both by the steroid hormone 20-hydroxyecdysone (ecdysone) and the RhoA GTPase signaling pathway. Recent evidence suggests that these pathways act cooperatively to control imaginal disc morphogenesis. Thus, leg imaginal disc morphogenesis is an excellent system in which to study the interaction of steroid hormone and intracellular signaling pathways. We have identified mutations in three genes, 12-5, 18-5, and 31-6, with roles in the morphogenesis of leg epithelia. Of particular interest, these mutations interact genetically with each other, mutations in the RhoA signaling pathway, and the ecdysone regulated Sb-sbd (Stubble) transmembrane serine protease. This suggests that the 12-5, 18-5, and 31-6 gene products may link hormone and RhoA signaling responses. The goal of this research was to identify and characterize the 18-5 and 12-5 genes in order to discern the mechanistic relationship between the RhoA pathway and ecdysone hierarchy.18-5 and 12-5 were precisely mapped to molecular locations within the Drosophila genome utilizing a P-element recombination mapping technique. This work narrowed the location of the 18-5 locus to within an interval of 112 kb within the Drosophila genome sequence. This interval contains 17 known and predicted genes. I also mapped the location of the 12-5 locus to a 2.6 Mb interval of the 2nd chromosome. Based on phenotypic analyses and the site of the molecularly mapped interval, a candidate gene for the 18-5 mutation was identified. Sequence analysis of the candidate gene was inconclusive and requires further analysis. Genetic interaction assays indicate that the 18-5 gene product acts upstream or at the level of Rho kinase in the RhoA signaling pathway.

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