The Extracellular Matrix November 30, 2017

Jeff Miner, Ph.D. Division of Nephrology

7717 Wohl Clinic 362-8235

minerj@wustl.edu

General Organization of Tissues

There are

Wall of the Stomach

Why do all multicellular animals have ECM?

•  Acts as structural support to maintain cell organization and integrity (endothelial tubes of the vascular system; mucosal lining of gut; skeletal muscle fiber integrity)

•  Compartmentalizes tissues (pancreas: islets vs. exocrine component; skin: epidermis vs. dermis)

•  Provides hardness to bone and teeth (collagen fibrils become mineralized/calcified)

•  Presents information to adjacent cells: –  Inherent signals (e.g., RGD motif in fibronectin) –  Bound signals (BMP7, TGFβ, FGF, SHH, etc.)

•  Serves as highways for cell migration during development (neural crest migration), in normal tissue maintenance (intestinal mucosa), and in injury or disease (wound healing and cancer)

Mechanisms of ECM function

Pengfei Lu et al. J Cell Biol 2012;196:395-406

© 2012 Lu et al.

Types of ECMs

•  Basement membrane (basal lamina) – Epithelia, endothelia, muscle, fat, nerves

•  Elastic fibers – Skin, lung, large blood vessels

•  Stromal or interstitial matrix •  Bone, tooth, and cartilage •  Tendon and ligament

Cells Need Receptors to Recognize and Respond to ECM

•  Integrins •  Dystroglycan •  Syndecans •  Muscle-Specific kinase (MuSK) •  Discoidin domain receptors (DDRs) 1 and 2 •  Others

Types of ECM Components •  Collagens •  Proteoglycans

–  Perlecan, aggrecan, agrin, collagen XVIII •  Hyaluronan (no protein core) •  Large Modular Glycoproteins

–  Laminins, nidogens, fibronectin, vitronectin •  Fibrillins, elastin, LTBPs, MAGPs, fibulins •  “Matricellular” Proteins

–  SPARC, Thrombospondins, Osteopontin, tenascins

Basement Membranes

•  Specialized layers of extracellular matrix surrounding or adjacent to all epithelia, endothelia, peripheral nerves, muscle cells, and fat cells

•  Originally defined by electron microscopy as ribbon-like extracellular structures beneath epithelial cells

Primary Components of All Basement Membranes

•  Collagen IV 6 chains form α chain heterotrimers •  Laminin 12 chains form ~15 α-β-γ heterotrimers •  Entactin/Nidogen 2 isoforms •  Sulfated proteoglycans Perlecan and Agrin are the

major ones; Collagen XVIII is another

History: The Engelbreth-Holm-Swarm (EHS) tumor:A blessing with a caveat.

•  Cell proliferation, differentiation, and migration

•  Cell polarization and organization, as well as maintenance of tissue structure

•  Separation of epithelia from the underlying stroma/mesenchyme/interstitium, which contains a non-basement membrane matrix

•  Kidney glomerular filtration (barrier between the bloodstream and the urinary space)

Basement Membranes are Involved in a Multitude of Biological Processes

Laminin Heterotrimers are composed of one α, one β, and one γ chain. •  400 to 800 kDa cruciform, Y, or rod-

shaped macromolecules. •  Major glycoprotein of basement

membranes—it’s required. •  Chains are evolutionarily related. •  5 alpha, 4 beta, and 3 gamma chains are

known. They assemble with each other non-randomly.

•  15 heterotrimers described to date.

LM-521

Laminin Trimers Polymerize

•  Laminin chains assemble into trimers in the ER and are secreted as trimers into the extracellular space.

•  Full-sized laminin trimers can self-polymerize into a macromolecular network through short arm-short arm interactions.

•  The α chain LG domain on the long arm is left free for interactions with cellular receptors.

Laminin Mutations in Mice (M) and Humans (H) Have Consequences

Lama1, Lamb1, Lamc1: Peri-implantation lethality (M) Lama2: Congenital muscular dystrophy (M, H) Lama3, Lamb3, Lamc2: Junctional epidermolysis bullosa (skin blistering) (M, H) Lama4: Mild bleeding disorder, moto-nerve terminal defects (M); cardiac and endothelial defects (H) Lama5: Neural tube closure, placenta, digit septation, lung, kidney, tooth, salivary gland defects (M); kidney disease? (H) Lamb2: Neuromuscular junction and kidney filtration defects (M); Iris muscle, neuromuscular, kidney filtration defects (H; Pierson syndrome) Lamc3: Brain malformations, autism spectrum disorder? (H)

Perlecan •  Found widely in basement membranes and in

cartilage. •  Contains domains similar to LDL receptor, laminin,

and N-CAM •  Binds to Collagen IV and to Entactin/Nidogen

The Collagens •  The most ubiquitous structural protein.

A triple helical protein containing peptide chains with repeating Gly-Xaa-Yaa (usually Pro) triplets.

•  The triple helix forms through the association of three related polypeptides (α-chains) forming a coiled coil, with the side chain of every third residue directed towards the center of the superhelix. Steric constraints dictate that the center of the helix be occupied only by Glycine residues.

•  Many Proline and Lysine residues are enzymatically converted to hydroxyproline and hydroxylysine.

•  ~28 distinct collagen types; each is assigned a Roman numeral that generally delineates the chronological order in which the collagens were isolated/characterized.

Diversity of Collagens Type I fibrils Skin, tendon, bone, ligaments, dentin,

interstitium

Type II Fibrils Cartilage, vitreous humor

Type III Fibrils Skin, muscle, bv

Type IV 2D sheets All basement membranes

Type V Fibrils with globular end

Cornea, teeth, bone, placenta, skin, smooth muscle

Type VI Fibril-assoc. (I) Most interstitial tissues

Type VII Long anchoring fibril

Skin--connects epidermal basement membrane/hemidesmosome to dermis

Type IX Fibril-assoc. (II) Cartilage, vitreous humor

Type XIII Transmembrane Hemidesmosomes in skin

Type XV HSPG Widespread; near basement membranes in muscle

Type XVII Transmembrane Hemidesmosomes in skin (aka BPAG2 or BP180)

Type IV Collagen Mutations and Human Disease

•  COL4A1 mutations –  Small vessel disease/retinal vascular

tortuosity –  Hemorrhagic stroke –  Porencephaly –  HANAC syndrome

•  COL4A3/A4/A5 mutations –  Alport syndrome/hereditary

glomerulonephritis

Kidney Glomerular BM

Fibrillar Collagens (I, II, III, V)

Fibrillar Collagens (I, II, III, V) •  Connective tissue proteins that

provide tensile strength •  Triple helix, composed of three α chains

•  Glycine at every third position (Gly-X-Y)

•  High proline content –  Hydroxylation required for proper

folding and secretion •  Found in bone, skin, tendons,

cartilage, arteries

Scurvy

•  Liver spots on skin, spongy gums, bleeding from mucous membranes, immobility, depression

•  Caused by Vitamin C deficiency •  Ascorbate is required for prolyl

hydroxylase and lysyl hydroxylase activities

•  Acquired disease of fibrillar collagen

Illustration from Man-of-War by Stephen Biesty (Dorling-Kindersley, NY, 1993)

Osteogenesis Imperfecta (brittle bone disease)

Clinical: Ranges in severity from mild to perinatal lethal

bone fragility, short stature, bone deformities, teeth abnormalities, gray-blue sclerae, hearing loss

Biochemical: Reduced and/or abnormal Type I collagen

Molecular Genetics: Mutations in either Type I collagen gene, COL1A1 or COL1A2, resulting in haploinsufficiency or disruption of the triple helical domain (dominant negative: glycine substitutions most common)

Marfan Syndrome •  Caused by dominant

Fibrillin-1 (FBN1) mutations –  Haploinsufficiency is the culprit

•  Skeletal, ocular, and cardiovascular defects

•  Deficiency of elastin-associated microfibrils

•  Initially was thought to be caused by a structural defect in microfibrils

Cell-Matrix Interactions December 5, 2017

Jeff H. Miner, Ph.D. Division of Nephrology

7717 Wohl Clinic (Next year, McDonnell Sciences Bldg)

362-8235 minerj@wustl.edu

Twitter: @JeffMinerPhD

Fibronectin (FN)

•  A glycoprotein associated with many extracellular matrices and present in plasma/serum

•  Alternative splicing generates many isoforms that heterodimerize covalently via S-S bonding

•  Fibroblasts make it, assemble it, stick to it, and respond to it

•  FN harbors the “RGD (Arg-Gly-Asp)” motif (in domain III-10) that serves as a ligand for various integrins, especially α5β1 and αvβ3

•  Fn-/- mouse embryos die at E8.5 due to defects in the vasculature and in heart development

Mao and Schwarzbauer, Matrix Biol. 2005

Fibronectin and Branching Morphogenesis

Sakai et al., Nature 2003

Integrins Direct FN Fibril Formation

Mao and Schwarzbauer, Matrix Biol. 2005

Secreted compact soluble FN binds integrin

FN binding induces reorganization of actin and signaling

Cell contractility leads to changes in FN conformation, exposing FN interaction domains and allowing fibril formation

Integrins

•  Large family of transmembrane receptors for extracellular matrix and cell surface proteins.

•  Consist of an α and a β subunit, both with a single-pass transmembrane domain.

•  16 different α chains and 8 different β chains associate to form 22 distinct heterodimers.

•  Cytoplasmic tails of both α and β chains mediate cell signaling events in response to ligand binding.

Integrins

•  Some integrins bind to a specific site on matrix proteins, such as Arg-Gly-Asp (RGD), which is found in fibronectin, vitronectin, tenascin, et al.

•  Ligand binding absolutely requires divalent cation**

•  As mechanotransducers, integrins link the extracellular matrix to the force generating actin-myosin cytoskeleton. This both allows the cell to influence the nature of the extracellular matrix, and allows the ECM to influence cellular architecture and behavior.

Integrins Need to be Activated

•  Integrin adhesiveness can be dynamically regulated through a process termed inside-out signaling.

•  Ligand binding transduces signals from the cellular environment to the interior of the cell through outside-in signaling.

•  Protein structure analyses have provided insights into the mechanisms whereby integrins become activated to bind ligand and how ligand binding translates to changes in intracellular signaling.

Adair and Yeager, Meth. Enzymol. 2007

Focal Adhesions are Organized by Integrins and Form Along Actin Stress Fibers

Do they exist in tissues in vivo?

Anchors Feet Sensors

Anoikis

•  Apoptosis induced by inadequate or inappropriate cell/matrix interactions.

•  Resistance to anoikis can lead to metastasis of epithelium-derived cancer cells.

Receptors for the Basement Membrane

•  Cells are thought to recognize the basement membrane through receptors that interact with specific basement membrane components, primarily with laminin. –  Integrins –  Dystroglycan

•  Binding of receptors to the basement membrane can result in signal transduction and alterations in cell behavior.

Muscular Dystrophy

•  Muscular dystrophy refers to a group of genetic muscle diseases that cause progressive muscle weakness due to defects in muscle proteins, which lead to death of muscle cells and tissue (necrosis).

•  There are many forms of muscular dystrophy that vary in onset and severity based on the protein affected and the nature of the mutation.

•  It has been proposed that the trigger of muscle cell necrosis is the focal breakdown of the plasma membrane due to contraction-induced damage.

•  MD is a disease characterized by defective muscle cell/matrix interactions. Straub et al. J. Cell Biol. 1997

Evans Blue Assay

Mutations in Lama2 Cause Congenital Muscular Dystrophy

•  Laminin-211 (α2β1γ1) is the major laminin in skeletal muscle fiber basement membranes and in peripheral nerve basement membranes.

•  Mutations, some null, affect humans and mice.

•  Skeletal muscle BM breaks down with the severe mutations, due to disrupting linkage of the actin cytoskeleton to the ECM.

ECM and Cell/Matrix Interactions