Cell Junctions, Cell Adhesion & Extracellular Matrix September 4, 2018 Mitch Denning, Ph.D.

Epithelial Tissue Connective Tissue Scanty ECM Many cells Cells are tightly bound together in sheets Cells bear most of the mechanical stress through junctional complexes that attach to the cytoskeleton. Plentiful ECM Sparse distribution of cells in ECM Matrix is rich in fibrous polymers (e.g., collagen) Matrix bears most mechanical stress

Long-Term Connections Require Cell Junctions

Four Functional Classes of Cell Junctions

Tight/Occluding Junctions (Zonula Occludens) Functions Seal adjacent cells to create a permeability barrier Intestinal Epithelium Endothelial cells: Blood-Brain Barrier (Inhibits drug delivery to brain) Regulates Paracellular Transport Leakage between cells Apical vs. Basolateral sorting “Fence” function

Paracellular Transport Transcellular Transport of Glucose Paracellular Transport Transcellular Transport: Active transport of glucose from gut lumen into cells via Na-driven glucose symporter (active transport). Sodium and glucose are transported in the same direction. Passive diffusion of glucose to blood via GLUT4? Channel (facilitated diffusion). Sealing and fence functions required. Paracellular Transport: Transient breach in barrier to allow solutes and water around cells.

Tight/Occluding Junctions Claudins are major tight junction proteins. “Zip lock bag seal”

Anchoring Junctions Stabilize cells against mechanical stress Mechanically attach cells and their cytoskeleton to their neighbors or to the extracellular matrix

Types of Anchoring Junctions

Adherens Junctions Join an actin bundle in one cell to actin bundle in adjacent cell Form adhesive belt just below tight junction Cadherins: Ca2+-dependent transmembrane adhesion proteins Anchor proteins connect cadherins to actin

Cadherins Mediate Calcium-Dependent Binding Between Cells Homophilic/Homotypic Adhesion adaptor proteins (catenins)

Desmosomes Spot welds that anchor cells together. Attached to Intermediate Filaments (IF) Desmosomal Cadherins are Desmogleins and Desmocollins Plaque proteins attach desmosomal cadherins to IF. Pemphigus: Auto-antibodies to desmosomal cadherins cause blistering, loss of skin integrity/barrier function Tonofilaments: Bundles of cytokeratin intermediate filaments

Desmosomes: Cell-Cell “Rivets”

Focal Contacts (Focal Adhesions) Link Extracellular Matrix (ECM) to actin filaments Allows cells to hang on to surroundings Integrins: Transmembrane ECM binding proteins Bind to actin indirectly via anchor proteins

Hemidesmosomes “Half-Desmosomes” Distribute forces on an epithelium to the basal lamina (ECM) Integrins mediate basal lamina adhesion Anchor proteins link integrins to intermediate filaments

Cadherins Mediate Calcium- Dependent Cell-Cell Adhesion Single-pass transmembrane proteins Calcium-dependent adhesion

Cadherin-Dependent Cell Sorting Homophilic Binding E Cadherin: Epithelial N Cadherin: Neurons, heart, fibroblasts, skeletal muscle P Cadherin: Placenta, epidermis, breast epithelium VE Cadherin: Vascular endothelium Early Amphibian Embryo Epidermal: Red Mesoderm: Green Neural Crest: Blue

Movie 19.1 GFP Cadherin

Selectins and Integrins Mediate Transient Cell-Cell Adhesion in the Bloodstream Selectins are Lectins: Carbohydrate binding proteins Transient, calcium-dependent adhesion Heterophilic. Cooperate with integrins E Selectin: Activated endothelial cells Inside-out signaling: Leukocyte integrins become activated Integrins bind adhesion molecules on endothelial cells

19.2 Movie Leukocyte Rolling

Communicating Gap Junctions Sugars (mono/disaccharides) Nucleotides Function Electrically connect cells Averages small molecules throughout tissue More specialized cells uncouple Connexins 4 pass transmembrane proteins 6 connexins form a functional pore: Connexon Permeability varies with connexin composition

Signal-Relaying Junctions: Synapse

The Extracellular Matrix Hydrated network of protein and carbohydrate containing molecules Abundant in connective tissues Secreted by fibroblasts, chondroblasts, osteoblasts, epithelium ECM bears most of mechanical stress

Glycosaminoglycans High negative charge attracts Na+ ions and water • Unbranched polysaccharide chains composed of repeating disaccharide units. • One amino sugar is usually sulfated and the other is a uronic acid • Absorb large amounts of water: Occupy space Heparin Sulfate Uronic Acids: Sugar acids where terminal carbon (#6) hydroxyl (OH) group is oxidized to carboxylic acid. [Glucuronic acid] High negative charge attracts Na+ ions and water

(Hyaluronic Acid, Hyaluronate) Hyaluronan (Hyaluronic Acid, Hyaluronate) Simplest GAG Very long chain length: up to 25,000 disulfide units No sulfated sugars Synthesized from the basal side of an epithelial sheet Creates a cell-free space into which cells can migrate: Embryogenesis Diffusion of nutrients, metabolites, growth factors… Resists compressive forces - Cartilage Wound Healing Promotes cell migration and proliferation Wound Healing: Inflammation, granulation tissue, cell migration, angiogenesis, reepithelialization

Proteoglycans Glycoproteins Glycosaminoglycans covalently attached to a core protein in golgi At least 1 GAG Very high carbohydrate content Long, unbranched side chains Regulate movement of molecules and cells Chemical signaling between cells Glycoproteins Few, short, branched carbohydrate chains Low carbohydrate content

Structural Proteins Collagens The most abundant proteins of the extracellular matrix 25% of protein mass in mammal Responsible for the strength of the ECM Bones strength Connective tissue integrity

Collagen Structure Triple-stranded, a-chains ~1000 amino acids long Gly-X-Y repeat Glycine – smallest aa X: Proline Y: Hydroxyproline Vitamin C (Scurvy) OH-Pro, OH-Lys H-bonding Lysine aldehydes covalent intra-molecule and inter-fibril cross-linking >40 different alpha chains can assemble into different collagen molecules. Interchain OH-bonding. Gelatin is hydrolyzed collagen

Synthesis of Collagen Fibrils and Fibers

42 α chain genes assemble into ~40 types of collagen

Ehlers-Danlos Syndrome Collagen Genetic Disorders Osteogenesis Imperfecta Defect in Type I collagen Movie :”Unbreakable” Ehlers-Danlos Syndrome Mutations in collagen Types I, III, V Collagen maturation/secretion Both are Autosomal Dominant OI: Decreased collagen synthesis, mutations, rearrangements Samuel L. Jackson had OI

Elastin Imparts elasticity to tissues Super rubber band Highly cross-linked Major ECM of arteries Fibrillin covers elastin fibers. Fibrillin is essential for elasticity of elastin Fibrillin Mutation Marfan’s Syndrome: Aorta rupture Mitral valve prolapse Skeletal abnormalities Tall stature

Fibronectin Adhesive ECM Glycoprotein Binds cells to the ECM and guides cellular migration Arg-Gly-Asp (RGD) binds integrins Directs cell migration during development RGD: Arginine-Glycine-Aspartate

Synthesized by cells on both sides. Basal Lamina Flexible, thin extracellular matrix mats that underlie epithelial cell sheets and tubes, surround fat, muscle, and Schwann cells Functions as a molecular filter: Kidney glomerulus Functions to orient/polarize cells: Hemidesmosome/Integrins Important for cell survival: Anchorage dependence Barrier to cell migration: Cancer Guide for cell migration: Neuromuscular Junction Around Under Between Synthesized by cells on both sides. Schwann Cells: Myelin producing cells of peripheral nervous system. Basal lamina serves as a scaffold for axon regeneration.

Composition of the Basal Lamina Laminin Adhesive protein which binds cells, collagen, GAG, PG Fibronectin Type IV Collagen Perlecan (proteoglycan) Nidogen (glycoprotein) Heparin Sulfate (glycosaminoglycan)

Regeneration of Basal Lamina at Neuromuscular Junction

Integrins Focal Contact Extracellular Matrix Receptors (Laminin, Fibronectin, Collagen…) Low Affinity, High copy number Binding is Ca2+ or Mg2+-dependent Heterodimers of a and b glycoproteins Overlapping specificity Many bind RGD tripeptide Cytoplasmic tails of b subunits link to cytoskeleton (usually Actin) Focal Contact

Integrin Binding Specificities INTEGRIN LIGAND DISTRIBUTION a5b1 Fibronectin Ubiquitous a6b1 Laminin Ubiquitous a7b1 Laminin Muscle aLb2 (LFA-1) Ig superfamily White blood cells ICAM-1 a2b3 Fibrinogen Platelets a6b4 Laminin Epithelial hemidesmosomes b1 subunits Partner with at least 12 a subunits

Regulation of Integrin Activity Inside-Out Signaling Integrins don’t bind ECM until activated by cells Platelets activate b3 integrin to bind fibrinogen and aggregate. T lymphocytes activate b2 integrins to bind antigen presenting cells.

Integrins Outside-In Signaling Integrin binding to ECM activates intracellular signaling Clustering of integrins at ECM contact sites Focal Adhesion Kinase recruitment, clustering, activation Similar to growth factor receptors Responsible for survival/growth/polarization signals in epithelia attached to a basement membrane.