THE EXTRACELLULAR MATRIX OF CONNECTIVE TISSUE

SAA

Introduction
Tissues are not made up solely of cells A substantial part of their volume is extracellular space which is largely filled by an intricate network of macromolecules constituting the extracellular matrix The extracellular matrix in connective tissue is frequently more plentiful than the cells it surrounds, and it determines the tissue's physical properties

COMPOSITION
This matrix is composed of a variety of proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surface of the cell that produced them. The proteoglycan molecules in connective tissue form a highly hydrated, gel-like ground substance in which the fibrous proteins are embedded. The polysaccharide gel resists compressive forces on the matrix while permitting the rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells. The collagen fibers both strengthen and help organize the matrix, and rubberlike elastin fibers give it resilience. Finally, many matrix proteins help cells attach in the appropriate locations Consist of ground substance, fiber and cell

Two main macromolecule

Two main classes of extracellular macromolecules make up the matrix: (1) polysaccharide chains of the class called glycosaminoglycans (GAGs), which are usually found covalently linked to protein in the form of proteoglycans, (2) fibrous proteins, including collagen, elastin, fibronectin, and laminin, which have both structural and adhesive functions.

The Extracellular Matrix of Animals

Glucosaminoglycan (GAG)-usually linked to protein in the form of proteoglycans, resists compressive force Fibrous proteins - Structural: collagen and elastin, tensile strength - Adhesive: fibronectin and laminin

Figure 19-37. The relative dimensions and volumes occupied by various macromolecules. Several proteins, a glycogen granule, and a single hydrated molecule of hyaluronan are shown. 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

Figure 19-59. The comparative shapes and sizes of some of the major extracellular matrix macromolecules. Protein is shown in green, and glycosaminoglycan in red.

Function
Connective tissues form the framework of the vertebrate body Attachment Strengthen Organization Resist compressive force Resilience Permitting rapid diffusion Tissue repair

Variation
Variations in the relative amounts of the different types of matrix macromolecules and the way in which they are organized in the extracellular matrix give rise to an amazing diversity of forms, each adapted to the functional requirements of the particular tissue

ECM is made by cell within It

Fibroblast Chondroblasts Osteoblast

Scanning electron micrograph of fibroblasts in connective tissue

GAG Chains Occupy Large amount of space

Unbranched polyscharide chain Repeating dissaccharide unit Consist of ;
Hyaluronan Chondroitin sulfate and dermatan sulfate Heparan sulfate Keratan sulfate

Glycosaminoglycan
hyaluronan, 25,000 Nonsulfated discccharide units ; Space filler; wound repair; lubricant; chondroitin sulfate and dermatan sulfate, heparan sulfate and heparin, keratan sulfate
Negative Charge Na+ H2O withstand compressive forces Less than 10% by weight; Porous hydrated gels Classified by sugar residues, type of linkage, the number and location of sulfate

Schematic structures of GAGs. Heparin/HS and hyaluronic acid (HA) are glycosaminoglycans; chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S) and dermatan sulfate (DS) are galactosaminoglycans; keratan sulfate (KS) is a sulfated polylactosamine. Since heparin/HS structures are highly heterogeneous, only their most abundant disaccharide unit IdoA(2OSO3)-GlcNSO3(6-OSO3) is shown here.

 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.

Figure 19-40. Examples of a small (decorin) and a large (aggrecan) proteoglycan found in the extracellular matrix. These two proteoglycans are compared with a typical secreted glycoprotein molecule, pancreatic ribonuclease B. All three are drawn to scale. The core proteins of both aggrecan and decorin contain oligosaccharide chains as well as the GAG chains, but these are not shown. Aggrecan typically consists of about 100 chondroitin sulfate chains and about 30 keratan sulfate chains linked to a serine-rich core protein of almost 3000 amino acids. Decorin decorates the surface of collagen fibrils, hence its name.

An aggrecan aggregate from fetal bovine cartilage

The repeating disaccharide sequence of a dermatan sulfate glycosaminoglycan (GAG) chain

Dwarf: prematurely aged appearance, generalized defects in skin , joints, muscles, and bones.

The repeating disaccharide sequence in hyaluronan, a relatively simple GAG

Hyaluronan acts as a space filler and cell facilitator during repair

Lubricant Need hialuronidase enzyme Not from inside the cell

Proteoglycans
Can regulate the activities of secreted proteins Composed of GAG chains covalently linked to core protein Acts as co-receptors

The linkage between a GAG chain and its core protein in a proteoglycan molecule

Glycosul transferases Sulfation and epimerization reaction 95 % carbohydrate by weight

Proteoglycans Can Regulate the Activities of Secreted Signaling Molecules

Fibroblast growth factor (FGF) binds to heparan sulfate chains of proteoglycans which is a required step for FGF to activate its cell-surface receptor TGF- binds to the core proteins of several matrix proteoglycans, ex. Decorin; binding to decorin inhibits the activity

Cell-surface proteoglycans act as co-receptors

Syndecans - extracellular domain: carries chondroitin sufate and heparan sulfate GAG chains - intracellular domain interact with actin cytoskeleton - Serve along with integrins as receptors for collagen, fibronectin, and also binds to fibroblast growth factor and present it to FGF receptor

Collagens
Major proteins of ECM Fibrous protein, triple stranded-helical structure, alfa chain (Gly-X-Y) Rich proline and glycine Human genome contain 2 distinct gene coding for different collagen alfa chain Less than 40 types of collagen molecules have been found Divide into collagen fibrils, fibrils associated collagen, network-forming collagen, anchoring fibrils

Type Collagen
Note that types I, IV, V, IX, and XI are each composed of two or three types of chains, whereas types II, III, VII, XII, XVII, and XVIII are composed of only one type of chain each. Only 11 types of collagen are shown, but about 20 types of collagen and about 25 types of chains have been identified so far.

Electron micrograph of fibroblasts surrounded by collagen fibrils in the connective tissue of embryonic chick skin

Figure 19-43. The structure of a typical collagen molecule. (A) A model of part of a single collagen chain in which each amino acid is represented by a sphere. The chain is about 1000 amino acids long. It is arranged as a left-handed helix, with three amino acids per turn and with glycine as every third amino acid. Therefore, an chain is composed of a series of triplet Gly-X-Y sequences, in which X and Y can be any amino acid (although X is commonly proline and Y is commonly hydroxyproline). (B) A model of part of a collagen molecule in which three chains, each shown in a different color, are wrapped around one another to form a triple-stranded helical rod. Glycine is the only amino acid small enough to occupy the crowded interior of the triple helix. Only a short length of the molecule is shown; the entire molecule is 300 nm long.(by B.L. Trus.)

Figure 19-50. The shaping of the extracellular matrix by cells. This micrograph shows a region between two pieces of embryonic chick heart (rich in fibroblasts as well as heart muscle cells) that were cultured on a collagen gel for 4 days. A dense tract of aligned collagen fibers has formed between the explants, presumably as a result of the fibroblasts in the explants tugging on the collagen. (From D. Stopak and A.K. Harris, Dev. Biol. 90:383398, 1982. Academic Press.)

Figure 19-46. Cross-links formed between modified lysine side chains within a collagen fibril. Covalent intramolecular and intermolecular crosslinks are formed in several steps. First, certain lysines and hydroxylysines are deaminated by the extracellular enzyme lysyl oxidase to yield highly reactive aldehyde groups. The aldehydes then react spontaneously to form covalent bonds with each other or with other lysines or hydroxylysines. Most of the cross-links form between the short nonhelical segments at each end of the collagen molecules.

Series of post translational modification

Pro alfa chain Propeptide Hydroxylation and glycosylated procollagen

Figure 19-47. The intracellular and extracellular events in the formation of a collagen fibril. (A) Note that collagen fibrils are shown assembling in the extracellular space contained within a large infolding in the plasma membrane. As one example of how collagen fibrils can form ordered arrays in the extracellular space, they are shown further assembling into large collagen fibers, which are visible in the light microscope. The covalent cross-links that stabilize the extracellular assemblies are not shown. (B) Electron micrograph of a negatively stained collagen fibril reveals its typical striated appearance. (B, courtesy of Robert

Elastin
The main component of elastic fiber and covered by micofibril (fibrillin) To recoil after transient stretch (lung, skin and blood vessel) Five times more extensible than rubber band Interwoven collagen and elastic : to prevent tissue from tearing Not rich proline and glycine, some hydroxyproline tropoelastin

Figure 19-49. Type IX collagen. (A) Type IX collagen molecules binding in a periodic pattern to the surface of a fibril containing type II collagen. (B) Electron micrograph of a rotary-shadowed type-II-collagen-containing fibril in cartilage, sheathed in type IX collagen molecules. (C) An individual type IX collagen molecule. (B and C, from L. Vaughan et al., J. Cell Biol. 106:991997, 1988. The Rockefeller Unive

Figure 19-52. Stretching a network of elastin molecules. The molecules are joined together by covalent bonds (red) to generate a cross-linked network. In this model, each elastin molecule in the network can expand and contract as a random coil, so that the entire assembly can stretch and recoil like a rubber band

Figure 19-51. Elastic fibers. These scanning electron micrographs show (A) a low-power view of a segment of a dog's aorta and (B) a high-power view of the dense network of longitudinally oriented elastic fibers in the outer layer of the same blood vessel. All the other components have been digested away with enzymes and formic acid. (From K.S. Haas, S.J. Phillips, A.J. Comerota, and J.W. White, Anat. Rec. 230:8696, 1991. Wiley-Liss, Inc.)

Fibronectin
Non collagen protein having multiple domain as receptor or binding site Organizing the matrix and helping cell attach As repellent that keep cells out of forbidden area Guide cell movement by serving as track along which cells can migrate Glycoprotein for many cell-matrix interaction Composed by dimer of two large subunit joined by disulfide bond Type III fibronectin

Fibronectin
Can exist both in a soluble form, circulating in the blood and insoluble fibronectin fibrils Binds to integrin through an RGD Motif, as type III repeat Other RGD sequence : blood clotting, anticlotting drug Disintegrins from snake

Figure 19-53. The structure of a fibronectin dimer. (A) Electron micrographs of individual fibronectin dimer molecules shadowed with platinum; red arrows mark the C-termini. (B) The two polypeptide chains are similar but generally not identical (being made from the same gene but from differently spliced mRNAs). They are joined by two disulfide bonds near the C-termini. Each chain is almost 2500 amino acids long and is folded into five or six domains connected by flexible polypeptide segments. Individual domains are specialized for binding to a particular molecule or to a cell, as indicated for five of the domains. For simplicity, not all of the known binding sites are shown (there are other cell-binding sites, for example). (C) The three-dimensional structure of two type III fibronectin repeats as determined by x-ray crystallography. The type III repeat is the main repeating module in fibronectin. Both the Arg-Gly-Asp (RGD) and the synergy sequences shown in red form part of the major cell-binding site (shown blue in B). (A, from J. Engel et al., J. Mol. Biol. 150:97120, 1981. Academic Press; C, from Daniel J. Leahy, Annu. Rev. Cell Dev. Biol. 13:363393, 1997. Annual Reviews.)

Figure 19-54. Coalignment of extracellular fibronectin fibrils and intracellular actin filament bundles. (A) The fibronectin is revealed in two rat fibroblasts in culture by the binding of rhodamine-coupled anti-fibronectin antibodies. (B) The actin is revealed by the binding of fluorescein-coupled antiactin antibodies. (From R.O. Hynes and A.T. Destree, Cell 15:875886, 1978. Elsevier.)

Basal Laminae Are Composed Mainly of Type IV Collagen, Heparan Sulfate Proteoglycan, Laminin, and Entactin

How type IV collagen molecules are thought to assemble into a multilayered network

Three ways in which basal laminae (yellow lines) are organized

A current model of the molecular structure of a basal lamina

Regeneration experiments indicating the special character of the junctional basal lamina at a neuromuscular junction

Laminin

Figure 19-57. The structure of laminin. (A) The subunits of a laminin-1 molecule. This multidomain glycoprotein is composed of three polypeptides (, , and ) that are disulfide-bonded into an asymmetric crosslike structure. Each of the polypeptide chains is more than 1500 amino acids long. Five types of chains, three types of chains, and three types of chains are known; in principle, they can assemble to form 45 (5 3 3) laminin isoforms. Several such isoforms have been found, each with a characteristic tissue distribution. (B) Electron micrographs of laminin molecules shadowed with platinum. (B, from J. Engel et al., J. Mol. Biol. 150:97120, 1981. Academic Press.)

Matrix Degradation
Important for tissue repair In remodelling so as to adapt to the stresses Enable to divide and to travel Need ability to cut through matrix because embedded and rounded, unable to extend (impermeable) To escape from confinement by basal lamina need localized degradation Like cancer cell can spread and proliferate

MD is localized to the vicinity of cells

Need proteoses
Matrix metalloproteases; bound Ca and Zn Serine proteases

Degrade collagen, laminin, fibronectin Others : collagenases Three basic mechanism : local activation (plasminogen), confinement by receptor and secretion of inhibitor (TIMP and Serpin)

How the extracellular matrix could propagate order from cell to cell within a tissue

Figure 19-63. The importance of proteases bound to cellsurface receptors. (A) Human prostate cancer cells make and secrete the serine protease uPA, which binds to cell-surface uPA receptor proteins. (B) The same cells have been transfected with DNA that encodes an excess of an inactive form of uPA, which binds to the uPA receptors but has no protease activity. By occupying most of the uPA receptors, the inactive uPA prevents the active protease from binding to the cell surface. Both types of cells secrete active uPA, grow rapidly, and produce tumors when injected into experimental animals. But the cells in (A) metastasize widely, whereas the cells in (B) do not. To metastasize via the blood, tumor cells have to crawl through basal laminae and other extracellular matrices on the way into and out of the bloodstream. This experiment suggests that proteases must be cell-surface bound to mediate migration through the matrix.

Penutup
Connective tissue contain matrix extracellular