BACKGROUND:Transglutaminase-4 (TGase-4), also known as the Prostate Transglutaminase, is an enzyme found to be expressed predominately in the prostate gland. The protein has been recently reported to influence the migration and invasiveness of prostate cancer cells. The present study aimed to investigate the influence of TGase-4 on cell-matrix adhesion and search for the candidate active domains] within the protein.METHODS:Human prostate cancer cell lines and prostate tissues were used. Plasmids that encoded different domains and full length of TGase-4 were constructed and used to generate sublines that expressed different domains. The impact of TGase-4 on in vitro cell-matrix adhesion, cell migration, growth and in vivo growth were investigated. Interactions between TGase-4 and focal adhesion complex proteins were investigated using immunoprecipitation, immunofluorescence and phosphospecific antibodies.RESULTS:TGase-4 markedly increased cell-matrix adhesion and cellular migration, and resulted in a rapid growth of prostate tumours in vivo. This effect resided in the Core-domain of the TGase-4 protein. TGase-4 was found to co-precipitate and co-localise with focal adhesion kinase (FAK) and paxillin, in cells, human prostate tissues and tumour xenografts. FAK small inhibitor was able to block the action mediated by TGase-4 and TGase-4 core domain.CONCLUSION:TGase-4 is an important regulator of cell-matrix adhesion of prostate cancer cells. This effect is predominately mediated by its core domain and requires the participation of focal adhesion complex proteins.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20701号 / 工博第4398号 / 新制||工||1683(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 小寺 秀俊, 教授 中部 主敬, 教授 安達 泰治, 准教授 横川 隆司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Cells adhere to each other and to the extracellular matrix (ECM) through protein molecules on the surface of the cells. The breaking and forming of adhesive bonds, a process critical in cancer invasion and metastasis, can be influenced by the mutation of cancer cells. Several molecules have been reported to play a crucial role in cellular adhesion and proliferation, and eventually in cancer progression, with TGF-β being one of the most important. In this thesis, we propose a general framework to model cancer cells movement and aggregation, in response to nonlocal social interactions (that is, attraction towards neighbours that are far away, repulsion from those that are near by, and alignment with neighbours at intermediate distances), as well as other molecules' effect, e.g., TGF-β. We develop nonlocal mathematical models describing cancer invasion and metastasis as a result of integrin-controlled cell-cell adhesion and cell-matrix adhesion, for two cancer cell populations with different levels of mutation. The models consist of nonlinear partial differential equations, describing the dynamics of cancer cells and TGF-β dynamics, coupled with nonlinear ordinary differential equations describing the ECM and integrins dynamics. We study our models analytically and numerically, and we demostrate a wide range of spatiotemporal patterns. We investigate the effect of mutation and TGF-β concentration on the speed on cancer spread, as well as the effect of nonlocal interactions on cancer cells' speed and turning behaviour.

Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands. The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins. We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

La morphogenèse des tissus dans les organismes multicellulaires est très importante pour le développement et certaines pathologies. La morphogenèse tissulaire est dirigée par des forces bio-mécaniques générées par des moteurs moléculaires tels que la myosine et transmis via le cytosquelette et les structures d'adhésion à l'intérieur et entre les cellules. La contractilité de la myosine, souvent en mode oscillatoire, a été étudiée principalement au niveau du domaine apical des cellules épithéliales au cours du développement mais très peu au niveau de leur domaine basal. L'oscillation de la myosine basale est importante pour le contrôle de l'élongation du tissu durant l'oogenèse chez la Drosophile. Bien que la voie Rho1-ROCK-myosin-MBS soit connue pour contrôler l'activité de la myosine, le mécanisme précis de ce contrôle n'a pas été élucidé. Le but de mon projet de thèse est de répondre à deux questions : Quels sont les facteurs en amont de cette voie ? Comment cette voie de signalisation crée et maintient l'oscillation de la myosine ? 1) Contrairement à ce qui est déjà connu, Je me suis intéressé à l'effet des adhésions cellule-cellule et cellule-matrice dans le contrôle des voies de signalisation gouvernant l'oscillation de la myosine basale. Les adhésions cellule-matrice, mais pas les adhésions cellule-cellule, sont positivement corrélées avec l'intensité et la polarité dorso-ventrale de la myosine, indiquant que les adhésions cellule-matrice pourraient être les facteurs en amont de la voie Rho1-myosine. Les adhésions cellule-matrice régulent positivement l'activité de Rho1 près des jonctions et gouvernent les flux de ROCK et myosine à l'intérieur du domaine median, contrôlant ainsi l'élongation du tissu. D'une autre manière, les adhésions cellule-cellule affectent indirectement les flux de ROCK and myosine en contrôlant la distribution subcellulaire de ROCK et du réseau d'actomyosine. L'inhibition des adhésions cellule-cellule, qui a un effet mineur sur l'élongation du tissu, provoque la redistribution des adhésions cellule-matrice et des filaments F-actin entrainant le chargement de la myosine à différentes positions. 2) J'ai montré que l'oscillation de la myosine basale dépend peu de la tension corticale de l'actomyosine : l'inhibition du chargement de la myosine sur les filaments d'actine n'affecte pas le flux de myosine alors qu'il bloque fortement le cycle périodique des contractions/relaxations de la cellule indiquant que l'oscillation est principalement due à une réaction biochimique plutôt qu'à une tension corticale. Au cours de l'oscillation de la myosine, les protéines Rho1 et leur activité sont principalement distribuées et enrichies au niveau et près des jonctions basales, et le contrôle majeur de cette oscillation est le flux des signaux ROCK qui diffusent des jonctions basales au cortex medio-basal. Ce mouvement de ROCK est initié grâce à une interaction transitoire entre ROCK et Rho1 actif au niveau et près des jonctions basales, conduisant ainsi à l'ouverture et activation de la kinase ROCK. Au cours de ce mouvement, l'activation de ROCK permet l'accumulation et l'amplification des signaux ROCK; Cette amplification entraîne la phosphorylation de la myosine, qui ensuite génère la redistribution dynamique de la phosphatase MBS. Enfin, l'enrichissement des signaux MBS arrête les signaux ROCK et myosine. Dans ces deux études, nous avons construit un outil optogénétique confirmant les différentes étapes de l'oscillation de la myosine basale. L'ensemble de ces résultats démontrent que le mécanisme contrôlant l'oscillation de la myosine basale nécessite une réaction biochimique, et met en évidence deux contrôles diffèrent de cette oscillation par les adhésions cellule-cellule et les adhésions cellule-matrice. / Tissue morphogenesis in multicellular organisms is very important in both development and human disease. Tissue morphogenesis is driven by bio-mechanic force that is normally generated by molecular motors such as myosin and transmitted via cytoskeleton and adhesion structures within and between cells. Myosin contractility, often as an oscillatory pattern, has been studied mainly in apical but less in basal domains of epithelial cells during development. Basal myosin oscillation is important in control of tissue elongation during Drosophila oogenesis. Although a signal cascade (Rho1-ROCK-myosin-MBS) has been known to regulate myosin activity, the detailed controlling mechanism is unclear. My project is aimed to address two questions: first, what is the upstream factor of this signal cascade? Second, how does this signal cascade create and maintain basal myosin oscillation? For this first question, I am interested in the effect of cell-cell and cell-matrix adhesion in control of this signal cascade governing basal myosin oscillation. Cell-matrix adhesion (Integrin and Talin), but not cell-cell adhesion (E-cadherin), is positively correlated with the intensity and Dorsal-ventral (DV) axis polarity of basal myosin oscillation, indicating that cell-matrix adhesion might be the upstream control of Rho1-myosin signal cascade. Cell-matrix adhesion positively regulates the Rho1 activity near junction and governs the pulsed ROCK and myosin signals within basal-medial domain, thus strongly controlling tissue elongation. Differently, cell-cell adhesion indirectly affects the ROCK and myosin pulses through controlling the subcellular distribution of ROCK and actomyosin network. Inhibition of cell-cell adhesion results in the redistribution of cell-matrix adhesion and F-actin filaments leading to different position of myosin loading, which plays minor effect on tissue elongation. For the second question, I unraveled that basal myosin oscillation is barely dependent on actomyosin cortical tension: inhibition of myosin loading to F-actin filament seems not to affect basal pulsatile myosin flows, while it strongly blocks the periodic cycle of cell contraction and relaxation at basal surface, thus indicating that oscillation is mainly from biochemical reaction rather than cortical tension. This observation highlighted that biochemical reaction is the main control of oscillation occurrence. During basal myosin oscillation, Rho1 proteins and Rho1 activity are mainly distributed and enriched at and near basal junction and the major control of basal myosin oscillation is the flow movement of oscillatory ROCK signals from basal junction to medio-basal cortex. This ROCK flow movement is initiated from the transient interaction of ROCK with active Rho1 at and near basal junction, thus leading to the opening and activation of ROCK kinase capability. During the membrane-medial flow movement, ROCK kinase activity mediates the accumulation and thus the amplification of ROCK signals; this positive signal amplification turns on the phosphorylation of myosin regulatory light chain (MRLC), which governs the dynamic redistribution of MBS. Finally, enriched MBS signals shut off both ROCK and myosin signals. In both study, an optogenetic tool named as LARIAT was built up in vivo to confirm the various status of basal myosin oscillation. Altogether, these results demonstrated two different controls of basal actomyosin signals by cell-matrix adhesion and cell-cell adhesion, and further demonstrated the underlying mechanism of basal myosin oscillation at the biochemical levels.