Tomáš Bárta tbarta@med.muni.cz The role of the extracellular matrix in development Contents * Introduction ECM function Role in development Components Remodelace Receptors Anoikis 2 Introduction - Extracellular matrix (ECM) Cell Tissue Organ Individual ? ECM – Extra Celluar Matrix 3 •What holds cells together to form tissue? How do cells and tissues interact with each other? •How do they coordinate to form the whole individual? What is an extracellular matrix (ECM)? •It is a three-dimensional insoluble network of extracellular macromolecules such as collagen, enzymes and glycoproteins, etc., which provide structural and biochemical support to surrounding cells. It mediates cell adhesion, communication between cells and differentiation. It instructs cells "when to grow, when to divide, when to produce different molecules, when to die." It consists of about 300 proteins, mainly: collagen, proteoglycans, elastin, glycoproteins (fibronectin and laminin). Each component has certain physical and biochemical properties. 4 5 •There is a certain analogy between cytoskeleton and ECM Just as the cytoskeleton determines the internal structure of the cell, participates in movement, transport, etc., so the ECM has a similar function but outside the cell and at the level of tissues and organs. ECM is composed of many types of macromolecules, fibers, proteins, glycoproteins, but most collagen (about 30% of all proteins). Introduction - Extracellular matrix (ECM) 6 •All ECM components help to attach cells and structure cells into tissues. But they also give cells a signal of when to grow, when to divide, when to produce certain molecules, and even when to die. Obsah obrázku laserová, scéna Popis byl vytvořen automaticky Introduction - Extracellular matrix (ECM) Extracellular matrix (ECM) •Glycoproteins Proteoglycans Fibrous proteins Polysaccharides Collagen is the most represented (~30% of all proteins in mammals) 7 Integrins "connect" the cytoskeleton with the ECM Link to the previous lecture – what you already know 8 What is extracellular matrix (ECM) - Features 9 What is extracellular matrix (ECM) - Features 10 Jak buňká „vnímá“ ECM 11 Components of ECM – how to understand it? Kusindarta, 2018 Proteoglycans Components of the ECM Glycoproteins Heparan sulfate Chondroitin sulfate Fibronectins Laminin Polysaccharides Hyaluronic acid. Proteins Collagens 12 Components of ECM - Proteoglycans * A key role in the transport of paracrine signals. Large molecules that consist of a central protein and a covalently bound polysaccharide chain. Examples include: Heparan Sulfate, Chondroitin Sulfate. 13 Components of ECM – Proteoglycans – Heparan sulfate (HS) * Linear polysaccharide, which occurs in the form of proteoglycan (a protein with covalently bound HS chains) – in almost all animal tissues A key role in ligand binding to -> receptors does not have to take place without it! Mutations that block the synthesis of Heparan sulfate cause various defects in cell migration, morphogenesis and differentiation. Different organs and tissues during development produce different compositions of HS HS changes during development => important for proper development. These findings led to the "sugar code" hypothesis – specific HS - modification controls individual events during development through interaction with signal pathways. many organs, including hematopoietic, skeletal systems and liver, the lungs and kidneys do not form properly if HS is absent. Poulain, 2015 Heparan sulfate (HS) 14 Components of ECM – Proteoglycans – Heparan sulfate Poulain, 2015 General structure •Proteins have a different number of places for carbohydrate binding Some may also contain chondroitin sulfate HS is synthesized in the Golgi apparatus 15 Components of ECM – Proteoglycans – Heparan sulfate – features Function 16 Components of ECM – Proteoglycans – Heparan sulfate – features Function •As receptors/co-receptors (Wnt, FGF, Shh) Increase in the concentration of the ligand, or receptor on the surface of cells in lipid rafts Regulation of receptor signaling Control of ligand secretion Directly as signaling molecules Checking the distribution of signal gradients. ECM component 17 Components of ECM – Proteoglycans – Heparan sulfate – features Ext1 -/- Exostosin Glycosyltransferase 1 This defect is due to a failure in the production of heparan sulfate to which Hedgehog binds and uses as part of its diffusion mechanism Lin et al., 2000 18 Inhibition of HS synthesis - > disruption of gastrulation Components of ECM – Proteoglycans – Heparan sulfate * Link to the previous lecture * => Heparan sulfate is key for FGF signaling 19 * Izme = mutation in the gene Ugdh – UDP-glucose dehydrogenase, which is needed for the synthesis of lateral glycosaminoglycan (GAG) chains of proteoglycans. Ciruna and Rossant, 2001 Garcia-Garcia and Anderson, 2003 Phenotypes of mutations in proteoglycan genes often mimic mutations in growth factor receptors Without Heparan sulfate or FGF signaling, the mesoderm does not migrate Components of ECM – Proteoglycans – Heparan sulfate 20 Components of ECM – Proteoglycans – Heparan sulfate 21 Components of ECM – Proteoglycans – Heparan sulfate Right-left (a)symmetry Kupffer's vesicle - is a ciliated organ that controls left-right (LR) patterning Ferreira et al., 2017 22 Components of ECM – Proteoglycans – Heparan sulfate Right-left symmetry – Sdc2 MO Arrington et al., 2013 23 Components of ECM – Proteoglycans – Heparan sulfate 24 Components of ECM – how to understand it? Kusindarta, 2018 Proteoglycans Components of the ECM Glycoproteins Heparan sulfate Chondroitin sulfate Fibronectins Laminin Polysaccharides Hyaluronic acid. Proteins Collagens 25 COMPONENTS OF ECM – Glycoproteins •Fibronectin, Laminin Responsible for the organization of the ECM and cells into ordered structures 26 Components of ECM – Glycoproteins - Fibronectins •Large glycoproteins (ca. 460 kDa) – heterodimers composed of 2 subjenotes connected by disulfide bond Two groups of fibronectins: •Soluble fibronectin (plasma – 300 μg/ml) – produced by hepatocytes Insoluble, cellular fibronectin •It binds to: collagen, fibrin, heparin, DNA, actin, integrin. "Link between cell (integrin) and ECM components" Function: cell adhesion (cell-to-cell contact and also mediates cell-collagen contact, proteoglycan), maintaining the shape of cells => development and morphogenesis It has several distinct binding sites and their interaction with the respective molecules leads to the correct arrangement of cells within the ECM. 27 Components of ECM – Glycoproteins - Fibronectins •Important role in cell migration Fibronectin refers to the pathways through which cells migrate – e.g. migration of germ cells into the gonads, migration of heart cells to the middle part of the embryo. Linask, Lash, 1988 28 Components of ECM – Glycoproteins - Fibronectins •Important role in cell migration Fibronectin paves the ways through which cells migrate – during gastrulation it is crucial for the migration of mesoderm cells. 29 30 M. Marsden and D. W. DeSimone, 2001 Components of ECM – Glycoproteins - Fibronectins 31 Intra-blastocoelar injection of anti-FN mAbs disrupts gastrulation. (A-C,G) Embryos injected at stage 9.5 with mAb 4H2 show normal development at stage 10.5, as indicated by dorsal lip formation (A, arrowhead). (B) At stage 11.5 the blastopore is almost closed (arrowhead) and embryos go onto develop into tadpoles (C,G). mAb 1F7-injected embryos (D-F,H) develop a normal blastopore at stage 10.5 (D, arrowhead). (E) By stage 11.5, there is a significant delay in blastopore closure and little movement of the blastopore lip is apparent (arrowhead). (F) mAb 1F7-injected embryos are truncated along the AP axis and bent ventrally. The blastocoel is retained and displaced ventrally (arrowhead). (H) Blastulae injected with mAb 1F7 develop into tadpoles that have small eyes, display head edema, and lack gut (arrowheads), heart, blood vessels and blood. M. Marsden and D. W. DeSimone, 2001 Components of ECM – Glycoproteins - Fibronectins Components of ECM – Glycoproteins - Fibronectins 32 Components of ECM – Glycoproteins - Laminin •Together with collagen, it represents an essential component of the basal lamina Binding to other laminin and ECM components Vazba na buňku Přes integriny, dystroglykan 33 Components of ECM – Glycoproteins - Laminin •Nomenclature: Laminin-521 contains α5, β2, γ1 chains. Binding to other laminin and ECM components Cell binding Through integrins, dystroglycan 34 Components of ECM – how to understand it? Kusindarta, 2018 Proteoglycans Components of the ECM Glycoproteins Heparan sulfate Chondroitin sulfate Fibronectins Laminin Polysaccharides Hyaluronic acid. Proteins Collagens 35 Components of ECM – Proteins - Collagens •The main structural protein of ECM (about 30% of all proteins in mammals) Produced mainly by fibroblasts Gelatin is essentially hydrolyzed collagen. 90% of collagen consists of Collagen I, a total of about 30 types of collagen Collagen is glycosiled through prolyl-4-hydroxylase and lysyl hydroxylase – I need Vitamin C as a cofactor. Vitamin C deficiency then leads to Scurvy 36 37 ECM - remodeling •The splitting of ECM components is a key process during ECM remodeling. •ECM quantity control ECM composition regulation Structure regulation Release of growth factors (and other regulatory molecules) •Matrix metalloproteinase (MMP) •The main enzymes that degrade ECM Free (secreted) or bound to the cell membrane. Secreted in the form of zymogene – activation up to ECM (other MMP or oxidation of thiol. group). •Adamalysins (ADAMTS), Meprins 38 ECM - remodeling 39 ECM - remodelace 40 ECM – remodelling - apoptosis 41 ADAMTS - a disintegrin and metalloproteinase with thrombospondin motifs •Degradation of interdigital (between the fingers) tissue involves intensive remodeling of the ECM. Remodeling using ADAMTS5 and its substrate – a component of ECM versican. Versican cleavage product induces apoptosis In the absence of ADAMTS5, syndactyly occurs. ECM – remodelling - apoptosis 42 In the absence of ADAMTS5, syndactyly occurs... ....Because there is no apoptosis ECM – remodelling - apoptosis 43 Even the presence of the cleaved form of Versican is capable of inducing apoptosis ECM - remodeling 44 ECM - remodeling 45 Components of the ECM — Conclusion 46 •Know the basic components of ECM, composition, functions + know examples Know the principles of ECM remodeling + know examples Components of ECM – Receptors A cell must somehow "perceive" if and how it is anchored in the ECM = > it is an important source of signals and information for it. 47 Components of ECM – Receptors - Integrins •The ability of the cell to bind the adhesive glycoproteins ECM (laminin, fibronectin) depends on the expression of membrane receptors. Integrins = integrate the extracellular and intracellular matrix so that they work together. In the ECM, they bind to the arginine-glycine-aspartate (RGD) motif – present in the adhesive proteins of ecM – fibronectin, laminin, vitronectin. Inside the cell, it always binds to the cytoskeleton. 48 Components of ECM – Receptors - Integrins •They have the ability to signal from the outside of the cell inward, which leads to a change in gene expression. Perceive the physical properties (pressure, stiffness) of the ECM - they activate/deactivate various signal pathways. Key to control gene expression during tissue development (mammary gland - able to signal the expression of estrogen receptor and casein protein genes) 49 Obsah obrázku text, barevné Popis byl vytvořen automaticky Components of ECM – Receptors - Integrins •Activation of integrins can: •Stimulate proliferation Inhibit apoptosis Activate the processes required to change cell shape or polarity Change cell motility 50 Components of ECM – Receptors – Integrins 51 •Mouse embryos lacking the 1 integrin subunit arrest in development, shortly after implantation. •The first observable defect is the inability of the inner cellmass to generate primitive endoderm and ectoderm layers. Further proliferation of the inner cellmass is also impaired. Components of ECM – Receptors - Integrins •Example: Which genes are regulated? 52 Components of ECM – Receptors - Integrins 53 Components of ECM – Receptors - Integrins 54 Anoikis •If the cell separates from the epithelium (or ECM), it is potentially dangerous – it loses an important regulatory component (basal lamina/ECM). Therefore, there is an anoikis = programmed cell death, if the cell separates from the ECM, a signaling pathway is triggered that leads to programmed cell death. The binding of integrins to ECM components prevents Anoikis – a still unknown mechanism. 55 Integrins - Signalling 56 ECM receptors - conclusion 57 •Know the basic components of ECM •What happens when ECM components is missing. To know what Integrins are, their structure and function. How the cell "perceives" contact with the ECM. ANOIKIS – even apoptosis is important in the course of development. ECM – Dotazy? 58 Děkuji za pozornost 59 Tomáš Bárta tbarta@med.muni.cz