ECM and Cell Adhesion PDF

ECM and Cell Adhesion Gerald Karp, Chapter 7 Alberts et al., Chapter 19 1  Specific Aims of this Section l Specific Aim #1: Why are we interested in cell- extracellular matrix (ECM) & cell- cell adhesions? l Specific Aim #2: Identify...

ECM and Cell Adhesion Gerald Karp, Chapter 7 Alberts et al., Chapter 19 1  Specific Aims of this Section l Specific Aim #1: Why are we interested in cell- extracellular matrix (ECM) & cell- cell adhesions? l Specific Aim #2: Identify the components of the ECM l Specific Aim #3: Understand the basic structure of cell-adhesion junction, types of cell-binding & underlying principles in cell-adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 2 …Because cell-ECM and cell-cell adhesions are greatly involved in tissue formation 3 G. Karp, Cell and Molecular Biology, 4th Ed.  Cells influence ECM and vice versa Endothelial cells on sq. pattern Alberts, et al., Molecular Biology of the Cell, 4th ed. Embryonic chick heart on collagen gel, 4 days Green: actin cytoskeleton; Blue: nuclei G. Karp, Cell and Molecular Biology, 4th Ed. l Because most cells are anchorage dependent, i.e. cell growth, proliferation and survival are dependent on attachment to a substratum l ECM can influence organization of cell’s cytoskeleton. This is mediated mainly by integrins and the intracellular signals they generate l ECM can also affect cell function through physical as well as chemical effects 4  … Because cells influences ECM and vice versa l Cells influence ECM: l Through the specific components cells secrete l By orienting the components of ECM l ECM influences cells: Intracellular actin fibers of cell align with Alberts, et al., Molecular Biology of the Cell, 4th ed. l ECM (e.g. fibronectin) l Cell cytoskeleton pulls on the fibronectin and can uncover more cell binding sites l Create larger scale order in tissues l Cells often show ‘anchorage dependence’ – the need to be attached to surface to survive 5 … Because cells influences ECM and vice versa – Regulation of ECM by cells l ECM degraded by proteases secreted locally by cells (e.g. matrix metalloproteases, MMPs) l Degradation is regulated by: l Proteases secreted as inactive precursors and are activated locally l Proteases are localized by being bound to cell surface receptors l Cells secrete protease inhibitors l Local ECM degradation is important for localized cells migration (e.g. at the growing tips of axons – neurite outgrowth movie clip) 6  Specific Aims of this Section l Specific Aim #1: Why are we interested in cell- ECM & cell- cell adhesions? l Specific Aim #2: Identify the components of the ECM l Specific Aim #3: Understand the basic structure of cell-adhesion junction, types of cell-binding & underlying principles in cell-adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 7 ECM fills space around cells l Volume filling l Hydrated gel with embedded fibrous proteins l Components primarily produced and secreted locally by cells, esp. fibroblasts Alberts, et al., Molecular Biology of the Cell, 4th ed. The ECM influences cell migration, cell shape, gene expression and cell differentiation. ECM provides mechanical support for tissues, it is a substrate on 8 which cells migrate and a place to locate signals for communication Extracellular matrix fills space around cells Figure 7.5. An overview of the macromolecular organization of the extracellular matrix 9 G. Karp, Cell and Molecular Biology, 4th Ed. Extracellular matrix components: l Glycosaminoglycans (GAGs) / proteoglycans – Gel l Collagen l Elastin Fibrous proteins l Laminin l Fibronectin 10 ECM components: Glycosaminoglycans (GAGs) l Unbranched chains of repeating disaccharides l GAGs are synthesized in cells and released into ECM l Carry net negative charge \ hydrophilic l Cloud of cations (such as Na+) forms around the chain and attracts water l The most abundant GAGs are hyaluronic acid (hyaluronan), chondroitin sulfate and heparin Lodish, et al., Molecular Cell Bioloty, 5th Ed. 11 GAGs are Found Covalently Linked to a Core Protein forming proteoglycans l GAGs/proteoglycans fill a large volume of space and can assemble into even larger molecules Proteoglycan: Core protein + Tetrasaccharide link + GAG 12 Alberts, et al., Molecular Biology of the Cell, 4th ed. GAGs & proteoglycans 13 Role of GAGs / proteoglycans in ECM l Form a space-filling gel matrix l Resists compression l Allows diffusion of molecules and cell migration l Regulate movement of cells and molecules l Enhance or inhibit activity of biomolecules, e.g. growth factors l Protect biomolecules from degradation l Concentrate biomolecules l Present the signaling molecule to cells l Depending on the specific GAGs in the matrix – can have different properties – different charge density, 14 different pore sizes – different interaction with cells ECM Components: Collagen l Fibrous structure protein embedded in the proteoglycan gel l Most abundant protein in mammals (25% of total protein) l Primary role – provide tensile strength to the matrix Alberts, et al., Molecular Biology of the Cell, 4th ed. l Hierarchical structure: 3 rope-like polypeptide chains (alpha chains) form triple helix l Primary repeating amino acid sequence is Gly-Pro-X l Cross links form between lysine residues l Self-assemble into fibrils (10-300 nm diameter, hundreds µm long) l Fibrils assemble into fibers (several µm15 diameter) Collagen Type I: Higher Structure Incorrect collagen assembly making skin hyperextensible ~1890 l Every 3 repeating units: Gly l Pro & Hydroxy proline: 35% 16 Collagen fibers under EM: Banding patterns observed 17  ECM Components: Elastin l Structural protein embedded in the proteoglycan gel l Provide elasticity / resilience l Main component of elastic fibers l Stabilized by cross links between lysine residues l Hydrophobic domains and alpha helices (where the cross links form) l Very abundant in tissues Alberts, et al., Molecular Biology of the Cell, 4th ed. such as aorta 18 ECM Components: Fibronectin l Dimer: two subunits that each have several binding domains l Subunits linked by disulfide bonds l Involved in binding to other ECM components (Collagen, heparin, etc) and also to cell surface receptors (integrins) l E.g. RGDS sequence (arginine-glycine-aspartic acid-serine) binds to integrins 19 l Helps to guide cell migration Alberts, et al., Molecular Biology of the Cell, 4th ed.  ECM Components: Laminin l Three chains form a cross shape l Found mainly in the basal lamina l Provides strength and flexibility Lodish, et al., Molecular Cell Biology l Has multiple binding domains – both to other ECM 20 components and to cells  Specific Aims of this Section l Specific Aim #1: Understand why we are interested in cell- ECM & cell- cell adhesions l Specific Aim #2: Identify the components of the ECM l Specific Aim #3: To understand the basic structure of cell-adhesion junction, the types of cell-binding & the underlying principles in cell-adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 21  Cell-ECM (& cell-cell) Binding is mediated by Cell Adhesion Molecules l Basic structure of Cell Anchoring Junction includes: l Transmembrane protein: l Extracellular domain binds to molecules in ECM or on another cell l Transmembrane domain in plasma membrane stabilized by hydrophobic forces l Intracellular domain connects to adapter proteins l Intracellular adapter proteins link to components of cell cytoskeleton l Anchoring prevents lateral diffusion of 22 adhesion molecules in the membrane Lodish, et al., Molecular Cell Bioloty, 5th Ed. Cell adhesion occurs through binding of membrane proteins (e.g. integrins) & adhesion molecules, (e.g. RGD seq.) l Attachment of mammalian blood cells and tissue cells to other cells, extracellular matrices and biomaterial surfaces is controlled by members of various families of membrane proteins and adhesion molecules l Cell-matrix adhesion is generally mediated by small sequences of the cell binding region on certain extracellular proteins such as collagen, fibronectin, etc.. (e.g. RGD seq) (arginine-glycine-aspartic acid) 23  Long term tight junction adhesion: Cell Interactions includes short term non- junctional adhesions & long- term tight junction adhesions Short-term non-junctional adhesion: 24 Cell Interactions includes short term non-junctional adhesion & long-term tight junction adhesion l Initially, cells interact by non-junctional adhesion l Adhesion proteins allow cells to attach quickly but not too tightly. They allow reversible changes in adhesion that aid in migration and intercalation (e.g. tissue development) l Cell attachments are stabilized by the formation of long-term tight cell junctions l Cytoskeleton help stabilize the clustering of adhesion molecules for multipoint binding and also for force transmission l Each cell has an assortment of both number and type of cell adhesion molecules that allow it to bind appropriately to other cells and ECM 25  Specific Aims of this Section l Specific Aim #1: To understand why we are interested in cell- ECM & cell- cell adhesions l Specific Aim #2: To Identify the components of the ECM l Specific Aim #3: To understand the basic structure of cell-adhesion junction, the types of cell-binding & the underlying principles in cell- adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 26  Cell-ECM Anchoring Junctions l Types of Cell-ECM Junction: l Focal Adhesions l Hemidesmosomes 27 G. Karp, Cell and Molecular Biology, 4th ed. Lodish, et al., Molecular Cell Biology  Vinculin (indicating Integrin clusters) Focal Adhesions Actin filament Nucleus l Transmembrane protein is Integrin l Connect ECM components (e.g. fibronectin, collagen) to actin cytoskeleton l Matrix binding promotes integrin clustering and association with the cytoskeleton. This in turn promotes further integrin clustering and matrix organization linker proteins: in a positive feedback system l Mainly seen in vitro 28  Specific Aims of this Section l Specific Aim #1: To understand why we are interested in cell- ECM & cell- cell adhesions l Specific Aim #2: To Identify the components of the ECM l Specific Aim #3: To understand the basic structure of cell-adhesion junction, the types of cell-binding & the underlying principles in cell- adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 29 Integrins are the major players involved in cell-ECM adhesion l Family of transmembrane glycoprotein l Composed of 2 chains, a and b l Combinations: at least 18 types of a chains and 8 types of b chains l Cytoplasmic tail of integrins binds to linker proteins that connects to the cytoskeleton l Ca2+ and Mg2+ which are needed for integrins to adhere l Forms a bridge from ECM to cytoskeleton Cytoplasmic tail l Can activate intracellular signal pathways & transmit signals to nucleus thus altering cell phenotype (motility, growth, survival, etc.) 30 Integrins 18 alpha-subunits and 8 beta-subunits have been identified 24 different heterodimer combinations l a-subunit conveys ECM specificity and is involved in regulation of b-subunit’s extracellular specificity and interactions of cytoplasmic b tail with cytoskeletal and signaling molecules 31  Inactive Active Copyright © 2016 John Wiley & Sons, Inc. All rights reserved. Fig_7-13  Specific Aims of this Section l Specific Aim #1: To understand why we are interested in cell- ECM & cell- cell adhesions l Specific Aim #2: To Identify the components of the ECM l Specific Aim #3: To understand the basic structure of cell-adhesion junction, the types of cell-binding & the underlying principles in cell- adhesion l Specific Aim #4: Identify types of cell-ECM anchoring junctions l Specific Aim #5: Identify types of cell-ECM adhesion molecules – integrins l Specific Aim #6: Understand the difference in binding strength of adhesion molecules and its effect on cellular migration in vivo (neutrophil migration) 33  Example of Cell-cell adhesion under Flow: Leukocyte Movement into Tissue PAF: platelet activating factor http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.6507 Lodish, et al., Molecular Cell Biology Selectins mediate transient, Ca2+ dependent cell-cell adhesion in bloodstream. P-selectin on endothelial cell bind to white blood cell (WBC) – weak binding, rolling along surface Integrins become activated on WBC. Integrins on WBC binds to ICAM on EC – strong binding, attachment and migration of 34 WBC into tissue Rolling leukocytes Specific Aims - Summary l Specific Aim #1: Cell- ECM & cell- cell adhesions are greatly involved in tissue formation and can also affect cellular behavior (differentiation, proliferation, etc.) l Specific Aim #2: ECM comprises mainly of GAGs, proteoglycans, collagen, elastin, fibronectin, laminin l Specific Aim #3: Cell-adhesion junctions comprise of cell adhesion molecules that link cell to ECM proteins or to neighboring cells. Cell adhesion may be long- term or short-term. l Specific Aim #4: Cell-ECM anchoring junctions include focal adhesions (in vitro mostly) and hemidesmosomes (in vivo). l Specific Aim #5: Integrins are the major cell-ECM adhesion molecules. l Specific Aim #6: Selectin binding results in transient cell binding. Integrin binding provides strong cell binding – Neutrophil migration example 36