Extracellular matrix (ECM) microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. The focus of this PhD thesis is the study of two microfibrillar assemblies, fibrillin-1 and collagen VI. Fibrillin is a large ECM glycoprotein which facilitates the deposition of elastin in elastic tissues such aorta, skin and lung and sequesters growth factors in the matrix. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the ECM allowing for the transduction of biochemical and mechanical signals. The structures of some individual domains and short fragments of both fibrillin and collagen VI have been solved, but it is not fully understood how they are arranged into microfibrils and how these microfibrils are arranged into tissues. Therefore the aim of this project has been to determine the hierarchical organisation of fibrillin and collagen VI across multiple length scales. The nanoscale structure of the fibrillin microfibril was determined using negative stain TEM and single particle reconstruction. Microfibrils had a hollow tube-like structure with well-defined bead, arm, interbead and shoulder regions. To overcome flexibility observed in the microfibril, separate sub-models of the different fibrillin regions were modelled. The bead region had a complex double layered structure with an interwoven core and ring structures. The arm region had four separate densities which are potentially formed from dimers of fibrillin molecules. Serial block face scanning electron microscopy (SBF-SEM) and electron tomography allowed for the in situ 3D imaging of individual fibrillin microfibrils in ciliary zonule tissue. Microfibrils in ciliary zonule fibres were held together by cross bridges between microfibrils. These ciliary zonule fibres were then organised into larger fascicle-like structures which were stabilised by circumferentially arranged ciliary zonule fibres. The frozen hydrated structure of the collagen VI half-bead was reconstructed using cryo-TEM. The half-bead region had a compact hollow head, and flexible tail regions, the tail regions were linked together by the collagenous interbead region. SBF-SEM and electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM had a meshwork-like organisation formed from globular densities ~30 nm in diameter. Together a combinatorial approach to image ECM microfibrils from the sub-molecular level to intact tissue structures spanning nanometre to millimetre length scales is presented. This provides a better understanding of how fibrillin and collagen VI microfibrils are organised in tissues.

Human embryonic stem cells are pluripotent cells that have indefinite replicative potential and ability to differentiate into derivatives of three germ layers. HESCs are conventionally derived and grown on mitotically inactivated mouse embryonic fibroblasts and there are some alternative feeder types of human origin that have been used to replenish hESCs while trying to prevent cross-species contamination. The trophic factors that are secreted by the feeders are found to be important for long-term pluripotency but there are also supportive culture systems for hESCs lacking feeder cells which might suggest that not only the interactions with the feeders affect the behaviour of hESCs but also the components of the niche may take part in the decision of self-renewal or differentiation. Extracellular matrix components are known to exert their stimulatory or inhibitory effects by localising cells into a specific microenvironment in natural niches but have been relatively little investigated for hESCs. The aim of this study was to investigate ECM components which might have a role in the maintenance of hESCs. I have first investigated human placental stromal fibroblasts and immortalised human placental stromal fibroblasts for the support hESC pluripotency as an anlternative feeder type to conventional mouse embryonic fibroblasts. Secondly, the matrices derived from hPSFs and ihPSFs were assessed for their ability to support hESC pluripotency. Tandem mass spectrometry was used to identify ECM components released by human feeders in order to characterise the range of extracellular matrix proteins that support the growth of self-renewing hESCs. The majority of the molecules was shared between the cell types irrespective of hPSF cell derived matrix was not being supportive for hESC pluripotency, with some ECM components being unique ihPSFs. Collagen VI, tenascin C and versican were tested for hESC attachment and as substrates for feeder-free culture system in order to develop an optimised feeder-free system. Furthermore, integrin receptor profile of different hESC lines was also determined in order to identify the mechanisms of substrate attachment. Integrin attachment was shown to be vital for hESC engagement to fibronectin and vitronectin in feeder-free systems. The components of the integrin signalling machinery were identified in hESCs and the significance of integrin-mediated signalling in hESC self-renewal was demonstrated by blocking integrin β1 on fibronectin and integrin aVβ5 on vitronectin. Moreover, intracellular signalling mediator c-Src was shown to involve in ECMregulated signalling by affecting the phosphorylation of Focal Adhesion Kinase. Inhibition of Src led to a decrease in the expression of pluripotency-associated markers. Finally, the effects of growth factor supplementation on the maintenance of pluripotency in defined feeder-free conditions were studied by withdrawal of growth factors and blocking FGF Receptors. FGF-2 was shown to be essential for long-term self-renewal while the effects on pluripotency deteriorated in the absence of both FGF-2 and Activin A. Taken together this project highlighted the importance of substrate attachment and growth factors on the regulation of hESC self-renewal.

No description available.

Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23770号 / 医博第4816号 / 新制||医||1056(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 安達 泰治, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM