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SPARC is also concentrated circurnferentially at the apicolateral PDF

142 Pages·2003·5.46 MB·English
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Preview SPARC is also concentrated circurnferentially at the apicolateral

SPARC is also concentrated circurnferentially at the apicolateral plasma membranes of surface ectodermal cells. Injection of anti-sense Xenopus SPAPC morp;!olinos Ieads to the dissociation of surface ectoderm by mid-tailbud. A similar dissociation is observed with animal cap exp!ants taken fiom morpholino injected embryos. These studies indicate that SPARC promotes epithelial cell-cell adhesion and is required for iaaintaining the integrity of the embryonic epidermis of Xenopus embryos. This represents the first evidence of an adhesive tiinction for SPARC. Collectively, my studies indicate that SPARC tiinctions intracellularly as a calcium- dependent regulator of ciliary movement and that SPARC has adhesive activity in epithelial tissues, in contrast to an anti-adhesive activity in rnesenchymaI tissues. Since the associations of SPARC with cilia and circumferentia! enrichment are observed in amphibians and mammals, it is likely that these intracellular and extracellular fùnctions of this glycoprotein are evoluticnarily conserved. Table of Contenis Table of Corztenîs iv List of figures and Tables vii Thesis Introduction Review ùf ECM molecules Review of SPARC structure and fùnction Review of early Xenopus development Xenoprrs as a mode1 organism Objectives und Approach 18 Chapter 1 :A calcium-binding motif in SPARC/osteonectin inhibits chordomesoderm ce11 migration during Xenopus laevis gastrularion: eviùènce of counter-adhesive activity in vivo Abstract 2 1 Introduction 23 Materials and Methodr Embryo Rearing Microinjection Histolo~y Scanning Electron Microscopy Whclemount In Situ Hybridization Results Peptide 4.2 is associated with an inhibition cf prospective head 3 1 mesoderm involution during gastrulation Peptide 4.2 inhibits spreading of involuting prospective head 3 9 mesodermal and endodermal cells Peptide 4.2 inhibits chordin expression in the anterior and 42 postenor region of tailbud embryos Discussion 49 Chapter 2: Associalialionof SPARC (Osleonectin, BM-40)w ith extmcellrrlm and infracellular components of the ciliated suflace ectoderm of Xenopus embryos Abstract Introduction Materials and Methods Embryos Antibodies Western Blot Analysis Wholemount Immunocytochemistry Animal Cap Assays and Northem Blot Analysis Immunogold Electron Microscopy Results Mammalian anti-SPARC antibodies recognize epitopes on Xet~opusS PARC SPARC is present in the cilia of surface ciliated epidermal cells Cell auton9mous activation of SPARC in the surface ectoderm SPARC accumulates at the interface of surface epidermal cells Sensorial layer cells express higher levels of SPARC mRNA than the surface epidermal cells SPARC mRNA expression in animal caps occurs in the absence of exogenous factors SPARC is associated with the ciliary microtubules Discussion Chapter 3: SPARC is associated with tubulin and promotes epithelial cell-cell adhesion Abstract Introduction Materials and Methods htibodies Co-Immunoprecipitations Whole-mount irnmunocytochemistry Microinjection and Animal Cap Assays Results The CO-localizationo f SPARC and tubulin in cilia and neurons Imrnunoprecipitation indicates a physiological interdction between SPARC and tubu!in SPARC CO-localizesw ith components of tight junctions Decreases in SPARC translation lead to developmental anomalies and dissociation of epidermal cells Resctie of the morpholino phenotype with mouse and Xmopus SPARC mRNA Inhibition of SPARC mRNA translation by mcpholinos Discussion Thesis Discussion ûverall Summary References Appendir A List of figures and Tables Figure 1. Schematic representation of the molecular organization 33 and biological activities of SPARC Figure 2. Injection of low doses of peptide 4.2 and LiCl into the blastocoel 3 7 cavity of stage 8/9 embryos result in different rates of blastopore ring closure and failure in development of anterior structures Figure 3. Injection of peptide 4.2 results in incomplete gastrulation, with 4 1 embryos lacking anterior structures Figure 4. Scanning electron microscopy indicates that peptide 4.2 inhibits 44 the spreading of cells at the leading edge of the involuting chordomesoderm Figure 5. Peptide 4.2-injected embryos show decreased chordin RNA 48 expression during gastrulation and in the chordoneural region of tailbud embryos Figure 6. Immunoblot of Xenoptcs and mouse tissues (whole lysates) 63 using a monoclonal antibody raised against human SPARC (AON-503 1) Figure 7. Scanning electron micrograph of the surface epidermis at 65 mid-tailbud (stage 35) showing evenly distributed ciliated cells in the anterior region Figure 8. Distribution of SPARC as revealed by imrnunocytochemistry 67 and in situ hyoridization Figure 9. SPARC mRNA expression is activated in animal caps 7 1 Figure 10. Irnmunoelectron rnicroscopic analysis indicates that SPARC is 74 associated with the microtubules of axonemes Figure 1 1. Co-localization of SPARC and tubulin in surface cilia and 9 1 neural tube Figure 12. Tubulin is irnmunoprecipitated by anti-SPARC antibodies that 94 cross-react with Xenoptrs SPARC Figure 13. Confocal serial sectioning indicates that SPARC is CO-localized 98 with zonula occludin- 1 at apicolateral plasma membranes of epithelial cells Figure 14. Inhibition of ectodermal ceIl adhesion by anti-sense SPARC IO 1 Morpholinos Figure 15. Animal cap dissociation is rescued by SPARC sense cRNAs 1 04 Figure 16. Anti-sense Xenopus SPARC morpholinos inhibit SPARC 10 7 mRNA translation TABLE 1: Defects in Xenopus Iaevis embryos associated with the injection of SPARC peptide 4.2 in comparison tgLiC1.' TABLE II: Rescue of XSMO-induced animal cap dissociation by CO-injectiono f Xerroptcs or mouse SPARC sense cRNAs. First and foremost, 1 am indebted to my supervisor Dr. Maurice Ringuette for his intellectual guidance, support, enthusiasm, and genuine concem for my well-being and scientific growth throughout my years in the laboratory. Reaching the destination was the ultimate goal, but I owe him great thanks for making the journey such an important a.nd enjoyable part of the trip. 1 would also Iike to thank members ofthe faculty: Drs. Ian Brown, Theodore Brown, UIli Tepass, and Jaro Sodek for their technical and intellectual guidance. 1 also owe a great deal of gratitude to former and present members of the 6" floor. Firstly, 1 would like to thank Sashko Damjanovski for providing me with the basic molecular tools and a foundation on which to start my graduate studies, and for his advice and suggestions throughout my degree. To Vernadeth Alarcon, for her constant support and encouragement during the writing of this thesis, and for providing an understanding ear and sound advice when 1 was in dire need of it. To Yusuke Marikawa, for discussions and advice on experiments, and for being a mode1 scientist. 1 have also been incredibly fortunate to have had the companionship of many fellow students on the ' 6 floor, who provided the necessary distractions and countless laughs and good times over the years. To CJ, Yvonne, Liz, Nat, Magda, Derek. Michelle, Lea, and countless others over the years; you've made the lab a second home, and at times in this degree, a tirst home. 1 appreciate your fnendship more than words can adequately express, and will remember Our time together with great fondness always. 1 also would like to acknowledge other fiiends who have provided stress-relief over the last several years: Al, Nancy, Janani. Makeda, Michael, and Erica. Your understanding, words of support, and willingness to listen when 1 needed it are much appreciated. 1 would like to thank rny family for allowing me the tieedom to pursue my goals, and for understanding al1 that it involves. To my niece Vanessa, for coming over to play and for reminding me of what's important in life. Lastly, I'd Iike to thank my "old" fi-iends, Yin- Ling, Keren, Hoa, Kelly, Binh, Erika, and Sue. Tme friends are hard to find, and even harder to hold onto, and 1 thank you for being there with me al1 these years. Introduction The extracellular matrix (ECM) of metazoans is composed of a complex network of macromolecules with diverse morphoregulatory tiinctions. in addition to contributing to the unique physiochernical properties and architecturai design of tissues, ECM molecules act as potent regulators of many cellular activities. For exarnple, cell proliferation, differentiation, migration, signal transduction, and survivai are dependent upon a dynamic reciprocal dialogue between cells and the surroundhg ECM. Mutations or misexpression of ECM rnolecules is the underlying cause of a broad range of morphological defects and diseases (Lukashev and Werb, 1998). This introduction will begin with a brief review of the four classes of ECM molecules, and their structurai and regulatory contributions to development, pattern formation and tissue remodeling. This is followed by a review of the properties and putative activities of SPARC, the focus of my thesis. Lastly, a description of the key morphological events and developmental stages of eariy Xenopus laevis d lbe given, providing criticai background into the experimental design of my experiments and interpretation of my data. Special emphasis is placed on unique aspects of Xetiopirs somitogenesis and embryonic skin development. Structural and functional diversitv of ECM molecules The €CM of animal cells is comprised of four major classes of macromolecules: collagens, proteoglycans, elastins, and glycoproteins. Different combinations of ECM rnacromolecules are co-assembled in different tissues, providing tremendous structural and tiinctional diversity. Collagens comprise the most abundant class of ECM molecules, accounting for up to 25% of the total protein content of some mammais. Collagen molecules include variable amounts of a characteristic triple helicai structure comprised of three a chains wound around each other into a right-handed superhelix. The a chains are constmcted of a repeating Glycine- X-Y sequence motif, where X and Y can be any amino acid, but is often proline and hydroxyproline respectively (Hay, 1991). Glycine plays a critical role in the tight folding of the triple-heiix whereas hydroxyproiine-rnediates interchain H-bonding and ensures the assernbly of a thermaily stable superheiii. Once secreted, collagens can form a complex variety of supramolecular structures. Based on their design, collagens are divided into six classes: fibril- forming (types 1, II, III, V, M), FACIT: fibril-associated with intempted triple helices (types TX. XII), network-forming (IV), flamentous (VI), short chain (VIII, X), long chah (VII). Fibril forming collagens are secreted as procollagens that require N- and C-terminal proteolytic processing before they can self-assemble into striated fibrils. During fibrillogenesis, collagens are stabilized by intramolecular and intermolecular covalent cross-links by lysyl oxidase at terminai lysine residues (Kagan, 2000). The cross-linking generates fibrils with tremendous tensile strength. In contrast to fibrillar collagens, network-forming collagen type IV molecules are secreted as mature molecules and seiI-assemble in extended cornplex polygonal sheets. Type IV collagen assernbly is stabilized by N-terminal tetramerization, C-terminal dimerization and lateral zssociations. Lateral associations are possible because, unlike in type 1 collagen, the collagenous dornains (triple-helical domains) of type IV collagen are intempted by numerotis non-collagenous dornains. These two major classes illustrate how different a-chains lead to the formation of radically different supramolecular structures. Moreover, different cornbinations of collagens are ofien CO-assernbledw ith one another, playing a key role in determining the biophysical properties of fibrils. For exarnple, the ratio of type 1 to type III collagen in tissues is a prime determinant of tissue flexibility, such as in arteries (Intengan and Schifin, 2000). SPARC has a strong affinity for both fibrillar collagens and type IV collagen (Yan and Sage, 1999). However, its role in the assernbly and fùnction of these collagens remains to be detennined. Profeo~Ivcars - Proteoglycans (PGs) are composed of glycosaminoglycan (GAG) chains attached to a core protein. GAGs are long linear polymers of repeated disaccharides. Usually one sugar is D- glucoronic acid or L-iduronic acids and the second sugar is either N-acetylglucosamine or N- acetylgalactoseamine. GAGs faIl into three groups or types: chondroitiddermatan sulfate (CSDS), keratan sulfate (KS), and heparan sulfàtc: (HS). Two features add to ttic tremendous variety of PGs found in tissues. First, core proteins Vary in sequence and size, ranging From 10 KDa to 400,000 KDa (Lander AD, 1999). Moreover, the number, length, and type of GAG chains associated with individual proteins Vary immensely. The tissue and cellular distribution of individual PGs also Vary. While the majority of PGs are secreted, some are membrane- spaming, linked to plasma membranes via phospholipids. or as in the case of serglycin, located in storage vesicles inside cells (Kolset and Gallagher. 1990). Through their high negative charge densities, the GAG chains associate with large amounts of water molecules, aiid are prirnarily responsible for the viscoelastic properties of tissues. In addition, PGs also regulate cell-cell and cell-matrix interactions. For example, syndecan promotes the binding of integrins to matrix glycoproteins such as fibronectin (Woods et al., 2000). Moreover, PGs can also act as reservoirs for growth factors and can present growth factors to their cognate cell surface receptors. In some case, surh as fibroblast growth factor, the growth factor cannot interact with its cognate receptor unless bound to a proteogly~m( Botta et al., 2000). Elastin Elastin is the major protein of elastic fibres, and is responsible for imparting extensibility and resiliency to tissues such as the dermis, ligaments, lung and major blood vessels (Mecham,

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Chapter 3: SPARC is associated with tubulin and promotes epithelial Decreases in SPARC translation lead to developmental ûverall Summary . Collagens comprise the most abundant class of ECM molecules, accounting for up to hedgehog were gifis frorn Drs. Chns Kintner, Eddy De Robertis, and Rand
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