8. Molecular Structure and Function of the Extracellular Matrix_Bradshaw_NOTES.pdf

Molecular Structure of the Extracellular Matrix Name: Amy Bradshaw Office: STB 325 Email: [email protected] Phone: 792-4959 OUTLINE: The Extracellular Matrix aka What Holds our Cells Together I). Introduction: What is the ECM for? II). Connective Tissue ECM vs. Basement Membranes & Tissue Specificit...

Molecular Structure of the Extracellular Matrix Name: Amy Bradshaw Office: STB 325 Email: [email protected] Phone: 792-4959 OUTLINE: The Extracellular Matrix aka What Holds our Cells Together I). Introduction: What is the ECM for? II). Connective Tissue ECM vs. Basement Membranes & Tissue Specificity III). Connective Tissue Organization A) Fibrillar Collagen 1) Fibrils, Fibers, and Tissue Organization 2) Collagens I, III, and V a) Procollagen processing b) Fibril incorporation 3) Proteoglycans and Glycosaminoglycans a) Structure b) Function B) Fibronectin: “Master” ECM Protein C) Basement Membranes 1) Laminin 2) Collagen IV 3) Nidogen and Perlecan 4) Assembly OBJECTIVES: After studying these lectures you should be able to: 1) Name the 2 primary types of extracellular matrix. 2) Describe functional and structural differences between the 2 primary types of extracellular matrix. 3) Identify key structural entities in the procollagen molecule and specify the functional significance of these elements. 4) Describe the primary steps in procollagen processing. 5) What mechanism covalently binds collagen molecules into an insoluble fiber. What enzyme is capable of catalyzing this bond? 6) What is a role of proteoglycans in influencing collagen fibril assembly? 7) What are critical functional roles of proteoglycans and glycosaminoglycans in the connective tissue? 8) What are the four primary types of disaccharides present in glycosaminoglycans and proteoglycans? 9) What role does fibronectin play in ECM assembly? How does force generation via the actin cytoskeleton facilitate fibronectin assembly? 10) Specify the primary extracellular matrix proteins that compose basement membranes. 11) Which proteins self-assemble to form a sheet-like structure? 12) What is the role of nidogen and heparan sulfate proteoglycans in basement membrane assembly? READING REFERENCE: Molecular Biology of the Cell, Alberts, Johnson, Lewis, Raff, Roberts and Walter. Garland Science, 6th edition. Chapter 19. Medical Cell Biology, Goodman, Elsevier, 3rd edition. Chapter 6. 7/25/2022 Molecular Structure and Function of the Extracellular Matrix Amy Bradshaw [email protected] • Images from Medical Cell Biology, Goodman • Molecular Biology of the Cell, Alberts The Extracellular Matrix What does it do?  Provides structural support to tissues to maintain form and function  Sequesters growth factors and enhances formation of chemo‐attractive gradients  Provides substrates for cell migration  Promotes cell differentiation The ECM holds multicellular organisms together but also provides information to guide cell behavior. Cells and ECM have a reciprocal relationship. 1 7/25/2022 Two Primary Types of Extracellular Matrix (ECM) 1) Connective Tissue or Interstitial components of organs Space filling type of ECM. Frequently contain cells within this ECM. Dermis, tendon, ligaments are examples of connective tissue. Primary components: Fibrillar collagens, proteoglycans, glycosaminoglycans. 2) Basement Membranes/Basal Lamina: Thin layer of assembled proteins. Epithelial cells reside on top of basement membranes. Primary components: Laminin, collagen IV, heparan sulfate proteoglycan (perlecan), and nidogen Two types of ECM as viewed by scanning electron microscopy E: Epithelial Cells sitting on top of a Basement membrane BL: Basal Lamina or Basement Membrane: Looks like a blanket by electron microscopy. Thin sheets of polymerized protein C: Connective Tissue: space filling type of structure with fibril polymers Molecular Biology of the Cell, Alberts et al. 2 7/25/2022 Illustration of a connective tissue with an overlying epithelial cell layer separated by a basal lamina Epithelial cell Basal lamina Transmission electron microscopy of area in blue box Connective Tissue: collagen fibrils From: Molecular Biology of the Cell (1994), Connective tissue: lots of fibrillar collagen (ECM) along with cells, blood vessels, nerves etc. Basal lamina: thin sheet of ECM separating polarized epithelial cells from connective tissue. Schematic of a single collagen fibril (red box) Collagen triple helix From: Biology (1990), Campbell. Each Tissue Has Specific, Specialized ECMs to Support Form And Function Examples: Cardiac Skeletal Smooth Examples: Skin Lens Gut MBC, Alberts et al. 3 7/25/2022 Fibroblasts are the Primary Producer of Connective Tissue ECM Proteins Fibroblast surrounded by fibronectin fibers Cells in Connective Tissue are Generally Surrounded by ECM Loose Connective Tissue Dense Connective Tissue 4 7/25/2022 ECM also changes with age and development of tissues Connective Tissue in Embryonic Chick: More cells than matrix, collagen fibrils are loosely packed. MBC, Alberts et al. Adult: More matrix than cells, densely packed fibrils. Adult Mouse skin Tadpole skin How do cells organize and assemble age and tissue specific ECMs? 1). Initiation of ECM assembly generally occurs on cell surfaces or in specialized membrane bound structures. Cells can align and assemble ECM structures to their liking through cytoskeleton‐dependent structures. 2). The composition of the ECM can differ significantly between tissues. Differential incorporation of ECM proteins/proteoglycans can alter the structural properties of the ECM. 3). Cross‐linking of collagen molecules and other ECM proteins can influence structure and function. 5 7/25/2022 Type I collagen is a triple helical structure composed of two 1(I) subunits and one 2(I) subunit N and C‐terminal propeptides are removed prior to ECM incorporation. Telopeptides are sites of collagen cross‐link formation. The primary amino acid sequence of collagens encode extended regions of repeating tripeptides Gly ‐ X ‐ Y required for triple helical assembly 6 7/25/2022 The extended triple helical domain of type I collagen generates the rod‐like structure of this fibril‐forming collagen and is the basic building block of our connective tissues Lodish et al. 5th ed. http://www.bu.edu/histology/p/20802ofa.htm Collagen Fibers are Made up of Collagen Fibrils 7 7/25/2022 Making a Collagen I Monomer 1. mRNA encoding the 3 alpha chains of collagen I are transcribed into the Endoplasmic Reticulum (Step 1, see next slide) 2. The chains are modified and assembled in the Endoplasmic Reticulum and the Golgi Apparatus with the assistance of many chaperone proteins that catalyze the specific modifications of collagen subunits including hydroxylation of proteins and addition of sugar moieties. (Steps 2‐3, next slide) 3. C‐propeptides cue chain recognition and bring 3 subunits together to assemble into triple helical collagen which is then secreted as procollagen in specialized secretory vesicles for export to the extracellular space. (Steps 4‐6, next slide) 4. In the extracellular space, procollagens propeptides are removed by specific proteases. The collagen monomer is then ready for incorporation into a collagen fibril and become part of the insoluble ECM stabilized by covalent cross‐link formation. (Steps 7‐9, next slide) Making a Collagen I Fibril Prolyl Hydroxylase 9 9 6 8 6 8 From: Molecular Biology of the Cell From Birk et al. Annals 8 7/25/2022 Fibroblasts are the primary cell type that produce and secrete fibrillar collagens in Connective Tissue CF, collagen fiber FB, fibroblasts cell bodies ICC, intracellular collagen in specialized vessicle Procollagen I is processed to collagen I monomers that are incorporated into collagen fibrils that mature to form collagen fibers 9 7/25/2022 The Enzyme Lysyl Oxidase modifies Lysine Residues in the Telopeptide Region The Oxidized Residues then Form Covalent Links in the Extracellular Matrix Generating Insoluble Stabilized Collagen Cross Links Assembly of single Collagen molecules into fibrils: Role of Proteoglycans Small Leucine Rich Proteoglycans (SLRPs) facilitate tip to tip fibril fusion: to generate length by reducing lateral interaction. Differential expression of SLRPs during development allows for discrete phases: fibrils growing in length, then growing in thickness. 10 7/25/2022 General types of ECM Proteins Proteoglycans and Glycosaminoglycans (GAGs) are Prevalent in Connective Tissues These water‐loving (hydrophilic) molecules provide connective tissues with viscoelasticity and turgor. These molecules also help to diffusion of regulate cations and small molecules in the extracellular space. Essential for the “space filling” capacity of connective tissues. 11 7/25/2022 Proteoglycans and GAGs are Space‐filling components of Connective Tissues Proteoglycans & GAGs are Large Space‐Filling Molecules That Hold Water Alberts et al. 4th ed. Lodish et al. 5th ed. 12 7/25/2022 Glycosaminoglycans (GAGs) Unbranched polysaccharide chains composed of repeating disaccharide units. Hyaluronan (Hyaluronic Acid or Hyaluronate) Sometimes associated with a protein core: Proteoglycan 4 Main types of GAG’s Hyaluronan (Hyaluronic Acid or Hyaluronate): –No protein core –No sulfation –Spun on the cell surface (i.e. not secreted) by specific enzymes. Chondroitin/dermatan sulfates: e.g. proteoglycans: SLRP family members: Decorin and Biglycan Heparan Sulfates: e.g. proteoglycan: perlecan Keratan Sulfates: e.g. proteoglycans: SLRP family members: fibromodulin and lumican 13 7/25/2022 Glycosaminoglycans are composed of characteristic disaccharide repeats. Lodish et al. 5th ed. Proteoglycans and GAGs form Multimeric Structures in Connective Tissues 14 7/25/2022 Fibronectin “Master” ECM protein as multiple types of ECM assemblies are thought to depend on Fn including fibrillar collagen and fibrillin/elastin. Integrin receptor engagement of ECM proteins can be used to generate force 15 7/25/2022 Ribbon Diagram of Fibronectin Unfolding Fibronectin matrix assembly: Schematic representation of the basic structure of FN dimers and the stages of FN assembly into a mature ECM network. ©2015 by Portland Press Ltd Katarzyna I. Wolanska, and Mark R. Morgan Biochm. Soc. Trans. 2015;43:122-128 16 7/25/2022 Basement Membranes/Basal Lamina Lodish Basement Membranes have tissue specific properties that represent different functions 17 7/25/2022 Components of Basement Membranes These basic building blocks are amongst the most highly conserved proteins in multicellular life. Essential components for tissue organization and function. Collagen IV Nidogen Laminin Heparan Sulfate Proteoglycan: e.g. Perlecan and Agrin Laminin A family of proteins that all take the form a cross‐like structure. Like collagens, also a trimeric protein composed of 3 subunits but not rod‐like. These trimers assemble into a polymerized sheet of protein on cell surfaces. Lodish 18 7/25/2022 Laminin A purified laminin molecule (trimer) viewed by rotary shadowing technology Lodish Collagen IV A collagen family member so it has 3 subunits that have triple helical regions with interrupted nonhelical sequences. The interrupted helical regions generate a collagen molecule that is not rod‐shaped but flexible. These trimers also self‐assemble into a polymerized sheet of protein by forming dimers, then tetramers that are held together by covalent cross‐links. Lodish 19 7/25/2022 Collagen IV A purified collagen IV network viewed by rotary shadowing technology Lodish Primary Components Organize into a Sheet‐like Basement Membrane Bound to Cell Surface Receptors Lodish Laminin (blue) assembles on cell surfaces through interaction with cell surface receptors (integrins: green) and forms a network. Collagen IV (red) forms an adjacent, independent network overlying the laminin network. Nidogen (yellow) and perlecan (green) tether the networks together. 20 7/25/2022 Conclusions: Connective tissue: • A space‐filling type of ECM, rich in fibrillar collagens but can also contain cells, blood vessels, nerves etc. • Fibrillar collagens are the major protein component whereas glycosaminoglycans and proteoglycans provide the water‐loving space filling component. • Each tissue has a specialized ECM to support form and function. • Factors that influence ECM structure and function include, ECM composition, cellular organization, and cross‐linking. Basement membranes/basal lamina: • The most ancient type of ECM ‐ required for multi‐cellular life. • Although these ECMs can be tailored in a tissue‐specific manner and include a list of proteins, each basal lamina contains the essential 4 protein components: laminin, collagen IV, nidogen, and heparan sulfate proteoglycan (e.g. perlecan). • Laminin and collagen IV form independent networks that form a sheet like structure tethered together by nidogen and perlecan. • Most basement membranes are too thin to be visible at the level of the light microscope. 21