Rozprawy doktorskie na temat „Myosin IIs”

Thesis (M.S.)--West Virginia University, 2010.
Title from document title page. Document formatted into pages; contains viii, 37 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 33-37).

Les mécanismes de formation des plaquettes sanguines à partir des mégacaryocytes ne sont pas totalement compris, mais l’environnement médullaire semble y avoir une influence cruciale. Dans ce travail nous montrons que i) les intégrines β3, récepteurs de protéines de matrice extracellulaire, semblent impliquées dans la mégacaryopoïèse et la formation des plaquettes, ii) la différenciation des cellules hématopoïétiques dans un environnement 3D de rigidité comparable à la moelle osseuse améliore la maturation des mégacaryocytes différenciés in vitro et iii) la myosine IIA est impliquée dans la distribution des organelles dans les mégacaryocytes. Parallèlement, Nous avons caractérisé la spécificité d’expression du transgène Pf4-cre pour valider son utilisation dans nos approches expérimentales. Ce travail apporte un éclairage nouveau sur le rôle de la myosine IIA et des intégrines dans les mégacaryocytes et souligne l’influence de la rigidité de l’environnement dans la mégacaryopoïèse
Megakaryocytes differentiation (megakaryopoiesis) and platelet formation mechanisms are not entirely understood, but the bone marrow environment seems to be crucial in these processes. In this thesis, we show i) that integrin β3, the extracellular matrix protein receptors, are involved in megakaryopoiesis and platelet formation, ii) that recreating a 3D environment of stiffness in the range of that of bone marrow improves the maturation of in vitro differentiated megakaryocytes and iii) a new role for myosin IIA in the cytoplasmic distribution of organelles within the megakaryocyte. As a side-project, we characterized the specificity of expression of the Pf4-cre transgene to validate its use in our experimental approaches. This work enlightens new roles for myosin IIA and integrins in megakaryocytes and indicates that stiffness of the environment influences megakaryopoiesis

Plusieurs méthodes de capture antigénique ont été décrites dans l’intestin grêle, surtout en cas d’infection: échantillonnage direct par les cellules dendritiques (DC), capture par les macrophages qui délivrent ensuite l’antigène aux DC du stroma, passage des antigènes à travers les cellules caliciformes. Des travaux antérieurs in vitro dans le laboratoire ont montré l’importance de la myosine IIA dans la coordination de la migration des DC avec la capture et de l’apprêtement antigénique. L’objectif de ma thèse était de combiner plusieurs méthodes d’imagerie telle que la microscopie intravitale, la microscopie confocale ex vivo et l’immunofluorescence sur tissus à la cytométrie en flux pour déterminer l’impact de la myosine IIA sur la capture antigénique in vivo. Cette étude montre que les DC patrouillent en permanence dans l’épithélium de l’intestin grêle, y compris hors conditions infectieuses. Elles sont recrutées dans la lamina propria (LP) et pénètrent dans l’épithélium par transmigration à travers la membrane basale qui sépare ces deux compartiments. La myosine IIA est indispensable à la transmigration de CD103+CD11b+DC. Ces événements de transmigration surviennent plus fréquemment dans les parties proximales de l’intestin grêle, duodénum and jéjunum, que dans l’iléon. Chez les souris adultes, ces DC ne sont pas recrutées sous l’influence du microbiote mais sont sensibles au rétinal, un métabolite de la vitamine A qu’elles transforment en une molécule active l’acide trans-rétinoïque (AtRA). D’après notre analyse transcriptomique, les DC intra-épithéliales constituent une population homogène dont le profil est distinct de celui de leurs homologues de la LP. Elles sont enrichies en ARN des voies liées à l’apprêtement antigénique, l’autophagie et les lysosomes. Ces résultats suggèrent qu’elles ont une fonction différente des CD103+CD11b+DC de la LP: elles n’agissent pas sur la prolifération ni la différenciation des lymphocytes T mais contrôlent spécifiquement l’effectif des lymphocytes intra-épithéliaux CD8+αβ. Ces découvertes reflètent l’importance de l’épithélium comme première ligne de défense contre les pathogènes. Elles soulèvent également de nouvelles questions concernant la régulation de la réponse immune dans l’épithélium et les interactions mutuelles entre la lumière intestinale, l’épithélium et le stroma des villosités
Several routes for antigen capture have been described in the small intestine, mainly upon pathogenic infection: direct sampling by Dendritic Cells (DCs), sampling by macrophages that deliver antigens to DCs in the stroma, antigenic passage through goblet cells. Previous in vitro work in the lab showed that myosin IIA is essential to coordinate antigen uptake and processing with DC migration. The objective of my thesis was to combine several imaging methods including intravital microscopy, ex vivo confocal microscopy and immunofluorescence on gut tissue to flow cytometry in order to unravel the impact of myosin IIA on DC physiology in vivo. My work shows that CD103+CD11b+ DCs, which are unique to the gut, constantly patrol the epithelium of the small intestine at steady state: they are recruited from the lamina propria (LP) and penetrate into the epithelium by transmigrating through the basal membrane that separates these two compartments. DC transmigration requires myosin IIA in vivo. Remarkably, we found that DC transmigration into the epithelium occurs mainly in the upper parts of the small intestine, the duodenum and the jejunum, but is not observed in the ileum. DC transmigration does not require the gut microbiota but relies on retinal, a vitamin A metabolite of that they convert into its active form all-trans retinoic acid (AtRA). Strikingly, single cell RNA-seq showed that intra-epithelial CD103+CD11b+ DCs constitute a homogenous cell population with a distinct transcriptomic signature from their LP counterpart. They are enriched with RNA related to antigen presentation, autophagy and lysosome pathways. Our results further suggest that these cells have a different function from LP CD103+CD11b+ DCs, as they do not significantly impact proliferation or differentiation of T helper lymphocytes but control the CD8+αβ intraepithelial lymphocytes (IELs) pool. These findings highlight the importance of the epithelial tissue as a first line of defense against pathogens in the upper parts of the small intestine. They also raise new questions about the regulation of the immune response in the epithelium and the mutual influences between lumen, epithelium and intestinal lamina propria

La formation et le remodelage des jonctions intercellulaires sont essentiels pour de nombreux processus biologiques tels que la compaction et la morphogenèse de l’embryon, la formation et la cicatrisation des tissus, le maintien de l’homéostasie tissulaire. Il est maintenant bien décrit que la myosine II non musculaire (NMII) agit comme un générateur de force et un support mécanique pour les jonctions adherens (E-cadhérine-dépendantes) lors de la migration collective et de la morphogenèse. Cependant, la contribution de NMII pendant les premières étapes de la formation de jonctions adherens reste mal connue, probablement en raison de la difficulté technique à capter un tel évènement transitoire mais complexe. Dans ce travail, nous avons étudié le rôle des isoformes non musculaires de la myosine II (NMIIA et NMIIB) au cours de la biogenèse des jonctions adherens dans les cellules MDCK, en utilisant une approche réductionniste in vitro. Cette approche, basée sur l’utilisation de substrats de culture micropatternés, chimiquement activables, mais permit un contrôle spatio-temporel de la formation des contacts intercellulaires. Mes travaux montrent que les cellules forment des contacts irréversibles base de E-cadhérine. L’élongation de ces contacts est accompagnée de la repolarisation du cytosquelette d’actine et de l’axe noyau-centrosome. En utilisant des shRNA spécifiques aux isoformes NMIIA et IIB, j’ai montré que ces deux isoformes ont contributions distinctes la formation et la dynamique des jonctions. NMIIA et NMIIB régulent différemment la biogenèse des jonctions par association avec des réseaux d'actine distincts. L'analyse de la dynamique des jonctions, de l'organisation de l'actine et des forces mécaniques a révélé que NMIIA fournit la force de traction mécanique nécessaire au renforcement et la maintenance des jonctions cellulaires. Le NMIIB est impliquée dans le clustering de la E-cadhérine, le maintien d'une couche d'actine branchée reliant les complexes de cadhérine et les fibres d'actine péri-jonctionnelles conduisant la création d'un stress mécanique anisotrope. Ces données révèlent des fonctions complémentaires imprévues de NMIIA et NMIIB dans la biogenèse et l'intégrité des jonctions adherens
Adherens junction formation and remodeling is essential for many biological processes like embryo compaction, tissue morphogenesis and wound healing. It is now well described that non-muscle myosin II (NMII) acts as a mechanical support and force-generator for E-cadherin junctions during collective migration and morphogenesis. However, the contribution of NMII during early steps of junction formation remains obscure, probably because of the technical difficulty to catch such a transient event. In this work, we investigate the role of non-muscle myosin II isoforms (NMIIA and NMIIB) during adherens junction biogenesis in MDCK cells, using an in vitro reductionist approach. This system, based on chemically switchable micropatterns allows a spatio-temporal control of adherens junction formation. Our observations on MDCK cells show that the cells form irreversible E-cadherin based contacts, junction elongation is accompanied by the repolarization of actin cytoskeleton and nucleus-centrosome axis. Using isoform-specific ShRNA for NMIIA and IIB, we show that they have distinct contributions to junction formation and dynamics. NMIIA and NMIIB differentially regulate biogenesis of AJ through association with distinct actin networks. Analysis of junction dynamics, actin organization, and mechanical forces of control and knockdown cells for myosins revealed that NMIIA provides the mechanical tugging force necessary for cell-cell junction reinforcement and maintenance. NMIIB is involved in E-cadherin clustering, maintenance of a branched actin layer connecting E-cadherin complexes and perijunctional actin fibres leading to the building-up of anisotropic stress. These data reveal unanticipated complementary functions of NMIIA and NMIIB in the biogenesis and integrity of AJ

S100A4 is a member of the S100 family of proteins and increases the motility of many cell types. This is also thought to explain its association with the epithelial-mesenchymal transition (EMT), a developmental program re-activated during tumourigenesis. Mechanistically, S100A4 interacts with a number of targets including Smad3 and liprin-β1; however, the best characterised is non-muscle myosin IIA (NMIIA) which regulates many aspects of the cytoskeleton. There is a large body of in vitro data indicating that S100A4 promotes the monomeric state of NMIIA; however, in vivo evidence for the interaction in cells is lacking. Accordingly, the first aim of this study was to determine if S100A4 interacts with, and promotes the monomeric state of NMIIA in A431 cells undergoing SIP1-induced EMT. Intriguingly, co-localisation analysis of S100A4 and NMIIA in A431-SIP1 cells using immunoelectron microscopy indicated that NMIIA is present in a folded, 10S state, and unfolded 6S state, and S100A4 interacts with both. This represents the first evidence of 10S and 6S states of NMIIA in non-muscle cells. In addition, FRAP analysis demonstrated that cells with attenuated expression of S100A4 turned over NMIIA with a slower rate, consistent with S100A4 promoting the monomeric state. The second part of the study explored the mechanism of the S100A4-NMMIA interaction. In vitro analysis of phosphomimetic S1916D and S1943D NMIIA showed no differences in binding affinity with S100A4 compared to WT NMIIA, contrary to the published literature. Based on the NMR structure of S100A4 and NMIIA, V77 and C81 were identified as key S100A4 residues that mediated the interaction with NMIIA. Mutation of these sites abolished the interaction with NMIIA, an effect reflected in null-phenotypes for both proteins when over-expressed in A431 cells compared to WT S100A4. In conclusion, this study suggests S100A4 is an important regulator of NMIIA dynamics in cells.

TNF-alpha can stimulate a variety of kinases with the ability to activate non-muscle myosin II. As a result, increases in actin filament formation and actomyosin contractility (AMC) have been reported in response to TNF-alpha. These events are thought to play an important role in mediating TNF-alpha induced apoptosis but how they do so is unclear. In this study we prevented non-muscle myosin II activation in response to TNF-alpha by treating cells with the myosin light chain kinase (MLCK) inhibitor ML-7 or through isoform specific siRNA knockdown of myosin IIA and IIB. We found that treatment with ML-7 or knockdown of myosin IIB, but not IIA, impaired the cleavage of caspase 3 and caspase 8 as well as nuclear condensation in response to TNF-alpha. During this cell death process myosin II seemed to function independent of AMC since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF-alpha induced caspase cleavage. Immunoprecipitation studies revealed associations of myosin IIB with clathrin and FADD in response to TNF-alpha suggesting a role for myosin IIB in TNFR1 endocytosis and DISC formation. Taken together these findings suggest that myosin IIB activation promotes TNF-alpha cell death signaling in a manner independent of its force generating property.

Mitochondria are important organelles of eukaryotic cells that provide energy through cellular respiration. Cells have evolved several quality control mechanisms to preserve functional mitochondria and avoid cell damage. Damaged mitochondria are recognized and removed by mitophagy to avoid the production of reactive oxygen species. A major signal for the recognition of damaged mitochondria is the electrical membrane potential depolarization, which leads to the recruitment of PTEN-induced putative kinase 1 (PINK1), followed by the recruitment of the E3 ubiquitin ligase parkin at the outer mitochondrial membrane. Parkin ubiquitinates numerous mitochondrial outer membrane proteins and initiates mitophagy. Mutations in the gene encoding parkin are frequently found in familiar forms of Parkinson’s disease. Although several factors involved in parkin mitochondrial recruitment have been characterized, additional proteins may be involved. The aim of this work was to determine whether other factors may be involved in colocalizing parkin to damaged mitochondria. Following up a SILAC-immunoprecipitation experiment, we hypothesized that an unconventional myosin (myosin IIa) may be involved in the recruitment of parkin to the mitochondria. Myosins are a large family of actin-based cytoskeletal motors that use energy derived from ATP hydrolysis to generate movement. Non-conventional myosins are well studied for their contribution to synaptic function in neuronal cells. MYH9 was identified as potential interactor of parkin by mass spectrometry. This interaction was validated through co-immunoprecipitation and immunofluorescence experiments. In addition, myosin alteration with small chemicals that depolymerize actin filament or inhibit myosin activity, impaired parkin localization to mitochondria upon stress. This study potentially implicates myosin IIa as a modulator of parkin recruitment to the mitochondria, and may thus open the door to new therapeutic strategies for Parkinson’s disease.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

Les isoformes des chaînes lourdes de la myosine (MyHC) sont d'excellents marqueurs du développement et de la physiopathologie du tissu musculaire strié. L'isoforme MyHC IIB, caractéristique des fibres squelettiques à métabolisme glycolytique, est exprimée tardivement au cours du développement. Son expression est soumise à de nombreuses régulations physiologiques telles que l'activité contractile ou le taux circulant d'hormones thyroi͏̈diennes. Nous avons isolé la totalité du gène MyHC IIB de souris ainsi que son promoteur. Pour identifier les séquences déterminantes pour la régulation de ce gène, nous avons cloné en amont d'un gène rapporteur CAT, différents fragments de la région promotrice. Des expériences de co-transfection, avec des vecteurs d'expression pour les facteurs myogéniques, dans des cellules musculaires murines C2 nous ont permis de mettre en évidence une activation du promoteur MyHC IIB, par les facteurs myogéniques. Cette activation en trans est indépendante de l'intégrité d'une boîte E proximale qui est un site de fixation potentiel pour les facteurs de la famille MyoD. Trois motifs nucléotiques sont importants pour le fonctionnement de ce promoteurs dans les cellules C2 : les régions mAT1 et mAT2 (riches en nucléotides A et T et pouvant fixer les facteurs MEF2 et Oct-1) et la boîte TATA. Les techniques classiques de biologie moléculaire, mutagenèse, étude des protéines affines à l'ADN et transfection, nous ont permis de définir les rôles de ces différents éléments dans la régulation de l'expression du gène MyHC IIB.

The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL's tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

L'épithélium sensoriel auditif des mammifères (organe de Corti) détecte et analyse les ondes sonores. Ces ondes mécaniques se propagent au sein de l'organe et stimulent la touffe stéréociliaire mécanosensible située à l'apex des cellules sensorielles (cellules ciliées). La répartition et l'orientation des cellules ciliées parmi les cellules de soutien, ainsi que l'orientation des touffes stéréociliaires sont invariables, et critiques pour l'audition. Au cours de ma thèse, j'ai développé des outils permettant de suivre les étapes précoces de cette organisation, par l'analyse des mouvements cellulaires et de la polarisation du kinocil présent dans les touffes stéréociliaires en développement. Par immunohistochimie sur tissu fixé, et vidéo-microscopie à fluorescence sur explants d'organe de Corti de souris transgéniques, j'ai entrepris une étude de ces phénomènes. De plus, cet organe, grâce à ses jonctions d'adhérence serrées entre les cellules peut résister aux fortes tensions mécaniques qu'il subit, encore accrues par l'électromotilité de ses cellules ciliées externes. Nous avons pu montrer, par une analyse de la circonférence apicale des cellules ciliées externes, que ces jonctions, initialement cylindriques, adoptent une forme non-convexe et qui dépend de la géométrie de la touffe ciliaire. Enfin, cette transition apicale est accompagnée d'un remodelage du cytosquelette d'actine et d'une polarisation de la répartition des myosines II, et Vlla, et de la protéine shroom2. Les travaux contribuent à la compréhension des étapes nécessaires à la formation d'un épithélium spécialisé, dont la fonction s'est affinée au cours de l'évolution
The mammalian auditory organ (organ of Corti) detects and analyzes sound waves. These mechanical waves spread through the organ and stimulate the mechanosensitive stereociliary bundle at the sensory hair cells' apex. The distribution and orientation of hair cells among supporting cells, and stereociliary bundle orientation in the epithelium plane are constant and critical for audition. I have developed tools to follow the first steps of this organization by analyzing cell movements and polarization of the kinocilium that is present in the developing hair bundle. By immunohistochemistry on fixed tissues and time-lapse videomicroscopy on explant cultures of the organ of Corti from transgenic mice, I undertook the study of these phenomena. Moreover, thanks to specific tight-adherens junctions between outer hair cells and their supporting cells, this organ can resist to sound mechanical stress that is locally amplified by electromotility of the outer hair cells. We have shown, by analyzing the apical circumference of outer hair cells, that these junctions, initially cylindric, adopt a non-convex shape that depends on strereociliary bundle geometry. This transition is accompanied by actin cytoskeleton remodeling and myosinll, myosinVIIa and shroom2 protein polarized redistribution. This work contributes to the understanding of the developmental steps leading to a highly specialized epithelium, which function was refined through evolution

Le gene murin codant pour la chaine lourde de la myosine rapide (myhc) iib n'est exprime que dans le muscle squelettique differencie et au stade adulte. Il existe de nombreux motifs de sequence dans son promoteur, dont plusieurs sont egalement retrouves dans les promoteurs d'autres genes de la famille myhc, et qui lient des proteines nucleaires. Il y a une boite e a -940pb qui a une affinite elevee pour des proteines myogeniques bhlh des extraits de myoblastes et de myotubes, mais qui ne conduit pas a une grande augmentation de l'activite transcriptionnelle dans les experiences de transfection. Les proteines srf et ctf se lient autour de -100pb, et leur fixation induit une courbure dans l'adn. L'inclusion de cette region dans les constructions deletees augmente l'activite transcriptionnelle dans les myotubes mais diminue dans les myoblastes. Entre -140 et -190 pb, il existe les sites mat1 et mat2, qui sont riches en a et t. Les deux peuvent fixer mef-2 et une proteine qui parait etre une proteine a homeodomaine, 2bb2, bien qu'il semble que mat1 soit normalement occupe par mef-2 et mat2 par 2bb2 ; la fixation de mef-2 a mat1 est necessaire pour l'activation du promoteur. Des interactions entre des facteurs de transcription specifiques du muscle et ubiquitaires contribuent a l'expression restreinte de ce gene

A isoforma 3 do trocador Na+ /H+ (NHE3), presente em membrana apical, é a proteína de transporte que medeia a maior parte da reabsorção de NaCl e NaHCO3- em túbulo proximal renal. A fosforilação direta do NHE3 por PKA na serina 552 é um dos mecanismos pelos quais a sua atividade pode ser inibida. A ligação da angiotensina II (Ang II) ao receptor AT1 (AT1R) em túbulo proximal estimula a atividade do NHE3 por diferentes vias de sinalização. Entretanto, não foram ainda bem estabelecidos os efeitos da ativação da via AT1R/Gi, com consequente diminuição nos níveis de cAMP, na regulação do NHE3. A Ang II pode ainda estimular a atividade do NHE3 por promover a sua translocação da base para o corpo das microvilosidades, entretanto, o papel da proteína motora miosina IIA nesta translocação em resposta à Ang II ainda não foi estabelecido. Sendo assim esta tese teve como objetivos: (1) testar a hipótese de que a Ang II diminui os níveis de fosforilação do NHE3 mediados pelo cAMP/PKA na serina 552 aumentando a sua atividade por reduzir os níveis de cAMP e (2) testar a hipótese de que a miosina IIA participa da redistribuição do NHE3 da base para o corpo das microvilosidades em túbulo proximal renal em condições de estímulo da reabsorção de sódio, como ocorre em resposta à Ang II. Visando avaliar os efeitos da ativação da via AT1R/Gi na regulação do NHE3, verificamos, por meio da técnica de recuperação do pH dependente de Na+, que, em condições basais, a Ang II estimulou a atividade do NHE3, mas não alterou a atividade da PKA e nem afetou os níveis de fosforilação do NHE3 na serina 552 em uma linhagem de células de túbulo proximal (OKP). Entretanto, na presença da forskolin (FSK), agente que eleva os níveis intracelulares de cAMP, a Ang II foi capaz de contrapor-se ao efeito inibitório da FSK sobre o NHE3 por promover redução na concentração de cAMP, diminuição da atividade da PKA e, consequentemente, diminuição nos níveis de fosforilação da serina 552. Todos os efeitos da Ang II foram bloqueados quando um pré-tratamento com Losartan, antagonista do receptor AT1, foi feito nas células OKP, destacando a contribuição da via AT1R/proteína Gi no aumento da atividade do NHE3 pela Ang II. Observamos que a inibição da proteína Gi com PTX (toxina pertussis) diminuiu a atividade do NHE3 em células OKP e que a PTX diminuiu a atividade do NHE3 assim como preveniu o efeito estimulatório da Ang II sobre a atividade do NHE3 em túbulo proximal de ratos Wistar. Adicionalmente, com a intenção de avaliar os efeitos da miosina IIA na redistribuição do NHE3, constatamos que a blebistatina, inibidor da miosina IIA, preveniu completamente o aumento de atividade do NHE3 mediado pela Ang II em ratos Wistar e que o uso da blebistatina foi capaz de prevenir o aumento do NHE3 na superfície de células OKP tratadas com Ang II. Em conjunto, nossos resultados sugerem que a Ang II contrapõe-se aos efeitos do cAMP/PKA sobre a fosforilação e a atividade do NHE3 pela ativação da via AT1R/Gi e que a miosina IIA desempenha um papel na mediação da regulação da atividade do NHE3 em túbulo proximal renal de ratos em resposta à Ang II. Sugerem ainda que a desfosforilação do NHE3 na serina 552 pode representar um evento chave na regulação do manuseio de sal tubular proximal pela Ang II na presença de hormônios natriuréticos que promovem o aumento dos níveis de cAMP e da fosforilação do transportador e que a miosina IIA está envolvida na regulação do tráfego do NHE3 em túbulo proximal renal
The Na+/H+ exchanger isoform 3 (NHE3), expressed on the apical membrane, is responsible for most NaCl and NaHCO3 - reabsorption in the renal proximal tubule. Direct phosphorylation of NHE3 by PKA at serine 552 is one of the mechanisms by which its activity is inhibited. Binding of angiotensin II (Ang II) to the AT1 receptor (AT1R) in the proximal tubule stimulates NHE3 activity through multiple signaling pathways. However, the effects of AT1R/Gi activation and subsequent decrease in cAMP accumulation on NHE3 regulation are not well established. Ang II can also stimulate NHE3 activity by promoting its translocations from the base to the body of the microvilli, however, the role of the myosin IIA motor protein in this translocation in response to Ang II is not yet established. Therefore, the aims of this thesis are: (1) to test the hypothesis that Ang II decreases the cAMP/PKA-mediated NHE3 phosphorylation levels at serine 552 increasing its activity by reducing cAMP levels and (2) to test the hypothesis that myosin IIA participates in the NHE3 redistribution from the base to the body of the microvilli in the renal proximal tubule under conditions in which sodium reabsorption is stimulated, such as in response to Ang II. In order to evaluate the effects of AT1R/Gi pathway activation on NHE3 regulation, by means the intracellular pH recovery technique, we verified that under basal conditions, Ang II stimulated NHE3 activity but did not affect PKA-mediated NHE3 phosphorylation at serine 552 in opossum kidney (OKP) cells. However, in the presence of the cAMP-elevating agent forskolin (FSK), Ang II counteracted FSK-induced NHE3 inhibition, reduced intracellular cAMP concentrations, lowered PKA activity, and prevented the FSK-mediated increase in NHE3 serine 552 phosphorylation. All effects of Ang II were blocked by pretreating OKP cells with the AT1R antagonist Losartan, highlighting the contribution of the AT1R/Gi pathway in Ang II-mediated NHE3 upregulation under cAMP-elevating conditions. We also verified that Gi protein inhibition by pertussis toxin treatment decreased NHE3 activity both in vitro and in vivo and, more importantly, prevented the stimulatory effect of Ang II on NHE3 activity in Wistar rat proximal tubules. Additionally, we assessed the effects of myosin IIA on NHE3 redistribution, and found that blebbistatin, a myosin IIA inhibitor, completely prevented the increase of Ang II-mediated NHE3 activity in Wistar rats and that blebbistatin was able to prevent the increase of NHE3 on the Ang II-treated OKP cells surface. Collectively, our results suggest that Ang II counteracts the effects of cAMP/PKA on NHE3 phosphorylation and inhibition by activating the AT1R/Gi pathway and that myosin IIA plays a role in mediating the NHE3 activity regulation in the rat renal proximal tubule in response to Ang II. Furthermore, these findings support the notion that NHE3 dephosphorylation at serine 552 may represent a key event in the regulation of renal proximal tubule sodium handling by Ang II in the presence of natriuretic hormones that promote cAMP accumulation and transporter phosphorylation, and that myosin IIA is involved in NHE3 trafficking regulation in the renal proximal tubule

碩士
國立陽明大學
生化暨分子生物研究所
102
Cell migration, a dynamic physical process, mediates most of morphologic processes, and is known to be controlled by the maturation states of a cellular adhesive organelle, focal adhesions (FAs). FAs, an integrin-based adhesive organelle, serve to physically connect cells to, as well as transduce signals to and from, the surrounding extracellular matrix (ECM). It is well established that nascent adhesions (small size) are formed at the leading edge, and then undergo a maturation process to grow into focal complexes (medium size) and mature FAs (large size), in which their composition change simultaneously. However, key regulators that drive FA maturation remain unknown. To identify key molecules that drive the maturation process, we find that non-muscle myosin IIA (NMIIA) and IIB (NMIIB) are recruited into focal complexes, which is signaled by Rac1 activity. Indeed, we found that NMIIA and NMIIB are localized at focal complexes and mature FAs, but not at nascent adhesions. Perturbation of NMIIA and IIB expression significantly inhibited the maturation process, leading to abnormal cell migration. In addition, we further determined whether Rac1-mediated actin polymerization promotes FA recruitment of NMIIA and NMIIB, and unexpecteddly found that actin polymerization acts as a negative regulator on FA recruitment of NMIIA and NMIIB. In summary, I found that NMIIA and NMIIB are able to serve as the key regulators in driving FA maturation, and I will further determine the mechanism of their FA recruitment.