1. Cell-to-Cell and Cell-to-Matrix Adhesions
Test Your Knowledge:
Where would you find the basal lamina? Proteoglycans? Fibronectin?
Name one type of cell-to-cell connection.
Name one type of cell to extracellular matrix connection
Name one type of cell membrane to cytoskeleton connection.
Name one function of the extracellular matrix
True/False Specific extracellular matrix components can directly activate cytosolic signal transduction pathways.

2. Cell-to-cell adhesion
Cells combine to form tissues. This requires that cells “adhere” to one another to form a functional unit.
Types of adhesion:
Cell-to-cell adhesion
Cell-to-extracellular matrix adhesion;
Epithelial (examples: skin, digestive tract lining, glands, etc.) – Function is to separate compartments (barriers); selective barriers (blood vessel linings); glandular secretion
Cells tend to have polarity (direction and function) – apical, basal, lateral, or basolateral
Can have specialized structures at the apical surface
All are connected to the underlying tissue by basal membrane (extracellular matrix material)
MOST IMPORTANT TYPE OF CELL ADHESIONS: 1) cell to cell and 2) cell to extracellular matrix
Connective tissue (connects one tissue type to another; includes tendons, ligaments, tissues underlying skin and other epithelial tissues
Characterized by cells that secrete proteins and compounds that make up an extensive extracellular matrix. Matrix:cell ratio is high
Mobile cells from other compartments or the same compartment can move through the extracellular matrix (ie. WBC)
NEED CELL TO EXTRACELLULAR MATRIX TYPES OF ADHESIONS

3. Adapter proteins and cytoskeleton
Basal lamina and ECM proteins such as:;

4. Types of interactions between adhesion proteins:
Homophilic – adhesion created by interaction between two similar adhesion molecules
Heterophilic – adhesion created by interaction between two different adhesion molecules or between adhesion molecules and cytoskeleton or extracellular matrix proteins
Homotypic – adhesion between similar molecules
Heterotypic – adhesion between different molecules
5 classes of CAMs
Not shown: mucins

5. Cell-to-cell adhesion molecules
1. Calcium dependent adhesion molecules (cadherins)
Evolutionarily ancient; widely expressed; over 12 different types known
Almost all vertebrate cells express one or more
Structure: Single-pass transmembrane glycoprotein composed of about residues
Type of binding:
Types:
Interactive with actin cytoskeleton: Cadherins N; P; R; B; E
Desmosome associated: Desmogleins & Desmocollins
Protocadherins
Location:
Evolutionarily ancient; widely expressed; over 12 different types known
Almost all vertebrate cells express one or more
Structure: Single-pass transmembrane glycoprotein composed of about residues
Extracellular domain: 5 tandem repeats, each comprising sandwich of β sheets; Ca2+ binding
Anchor: Transmembrane segment; Intracellular
Cytoplasmic carboxy-terminal domain binds catenins, catenins then bind to actin cytoskeleton
Type of binding: Homophilic: Via most distal cadherin repeats (His-Ala-Val); requires Ca+
Types
Interactive with actin cytoskeleton: Cadherins N; P; R; B; E
Desmosome associated: Desmogleins & Desmocollins
Interact with intermediate filaments
Location: In tight junctions
Protocadherins
Homology to cadherins: Extracellular, but not intracellular, domains
Diseases & Functions
Cadherin E (1): Reduction correlates with tumor malignancy & invasion
Gynecologic malignancies
Point mutations in tumor cells
Somatic loss of heterozygosity common
Gastric malignancies
Susceptibility to Listeria monocytogenes infection
Cadherin M: Myogenesis
Cadherin N: Role in establishment of left-right asymmetry
Cadherin P (3): Congenital hypotrichosis with juvenile macular dystrophy
Cadhein VE: Expression reduced in human angiosarcomas
Cadherin 23: Deafness, Age-related & non-syndromic; Usher syndrome
Catenin β1 (Cadherin-associated protein): Mutations in malignancies
Colon; Hepatoblastoma; Pilomatricoma; Ovarian (Endometrioid)
Desmoglein 3: Antibody target in pemphigus

6. E-Cadherin Domains 1 and 2 In Complex With Calcium
Two of the 5 tandem repeats of extracelluar region
Extracellular domain: 5 tandem repeats, each comprising sandwich of β sheets; Ca2+ binding
Anchor: Transmembrane segment; Intracellular
Cytoplasmic carboxy-terminal domain binds catenins, catenins then bind to actin cytoskeleton

7. Non calcium dependent adhesion molecules
(NCAMs nerve cell adhesion molecules, ICAMs, and L1)
Evolutionarily ancient; widely expressed
Belong to the immunoglobulin (Ig) superfamily
Structure: single pass, transmembrane proteins which may bind to the cytoskeleton inside cells
Type of adhesion:
Can have both homophilic and heterophilic interactions;
homo – neural specific Ig Cell Adhesion molecules (IgCAMs);
hetero systemic IgCAMs
Functions:
neurite outgrowth, myelination, and
firm adhesion of leukocytes
Structure: 1 or more repeats of Ig fold of aa. Form sites of adhesion.
Ig domain + transmembrane domain + cytoplasmic tail
Can have both homophilic and heterophilic interactions; homo – neural specific Ig Cell Adhesion molecules (IgCAMs); hetero systemic IgCAMs
Functions: neurite outgrowth, myelination, and firm adhesion of leukocytes

8. Selectins extravasation
Expressed only in vertebrates; in circulatory cells (endothelium and blood cells)
Transient transmembrane binding proteins (lectins)
In the presence of calcium, bind to specific oligosaccharides on another cell
Structure: single transmembrane polypeptide
Type of adhesion:
Function:
Structure
N-terminal: Homologous to Ca++-dependent lectins
EGF motif
62 amino acid repeats: Homology to complement regulatory proteins
Transmembrane region
Cytoplasmic tail
Induced, then rapidly downregulated
Adhesion
Transient
Architecture
Binding site: Amino-terminal domain
Connecting arm: Contains EGF-like domain & peptide repeats
Ca++ dependent
Ligands: Sialated glycans (Similar pattern of binding to sialoadhesins)
Functions
Slow intravascular leukocytes before transendothelial migration
E-selectin: Mediates initial PMN adhesion to endothelial cells
Adhesion is rolling, not firm
Firm adhesion via LFA/ICAM-1 & VLA-4/VCAM-1
The smallest of the vascular selectins, a kDa molecule, is constitutively expressed at the tips of microfolds on granulocytes, monocytes, and a vast array of circulating lymphocytes. L-selectin is also known as LECAM-1, LAM-1, Mel-14 antigen, gp90 mel, and Leu8/TQ-1 antigen. L-selectin is important for lymphocyte homing and adhesion to high endothelial cells of post capillary venules of peripheral lymph nodes. Moreover, this adhesion molecule contributes greatly to the capture of leukocytes during the early phases of the adhesion cascade. Following capture, L-selectin is shed from the leukocyte surface after chemoattractant stimulation. L-selectin interacts with three known counter receptors or ligands, MAdCAM-1, GlyCAM-1, and CD34. In conjunction with other molecules, L-selectin's function and influence in the adhesion cascade has been under scrutiny in many experiments using gene-targeted mice.
In L-selectin deficient mice, trauma-induced leukocyte rolling in mesentary or cremaster muscle veules is normal initially, but declines over time. Using an intravenous L-selectin antibody, this phenotype can be reproduced, thus blocking the function of L-selectin. This indicates that trauma-inducted rolling in these mice is P-selectin dependent with a velocity highly comparable to that observed in wild-type mice. L-selectin is critical in mediating rolling after surgical trauma and is necessary for neutrophil recruitment after inflammation. Nevertheless, basal neutrophil trafficking appears to remain unaffected by absence of L-selectin since peripheral leukocyte and neutrophil counts in these mice were normal.
In TNF-α, tumor necrosis factor-alpha, treated mice deficient in L-selectin, data suggests that leukocyte rolling is P-selectin dependent. The lack of L-selectin in these mice reduces the efficiency of E-selectin mediated rolling is shown by the sensitivity of rolling to P-selectin antibodies. These experiments consistently show that L- and P-selectin mediate leukocyte rolling, however, L-selectin alone cannot assume this task at normal velocities in vivo. L- and P- selectin cooperate in such a way that in the absence of P-selectin, L-selectin must initiate leukocyte interactions to allow slow rolling on E-selectin. In addition, L- or P-selectin must be present to mediate capture before the commencement of leukocyte rolling. Without the presence of either L- or P-selectin, rolling cannot occur.
extravasation

10. Cell to extracellular matrix adhesion molecules
Integrins (examples: laminin, fibronectin, fibrinogen)
A family of transmembrane adhesion molecules (usually glycoproteins) that exist in variable activation states
Extracellular matrix receptors on integrins have selective affinity for certain matrix proteins; allows cells to explore their environment
Structure: have an alpha and a beta subunit (heterodimer); alternative splicing has led to 16 different a chains and 8 different b chains
Type of adhesion:
Function: WBC binding to endothelium;

11. Epithelial tissues – what do they need adhesion molecules for?
tight junctions
adherens junctions
desmosome
hemidesmosome

12. Tight Junctions Function: Protein composition: occludin claudin
junction adhesion molecules (JAMs)
Cytosolic face

14. Adherens junctions (adhesion belt); attach to actin
Desmosomes; attach to intermediate filaments
Focal adhesions
Hemidesmosomes

15. Adherens Junctions Composition: cadherens
Function: can contract (with help of myosin)
Folding of sheets into tubes during morphogenesis, other folding processes during morphogenesis
Binding partners:
catenins, and via catenins to cytoskeleton (actin)

16. desmosome
pemphigus

17. Focal Adhesions
Examples: myotendinous junction
fibroblast migration in connective tissue

19. Basal Lamina – extracellular matrix; a sheetlike meshwork underlying or surrounding groups of cells
Function is to organize cells into tissues, also works in tissue repair; provides a guide for migrating cells during tissue formation (I.e. neural cells)
Function :

20. Entactin (nidogen and laminin) Perlecan
Components of the basal lamina; produced by cells that rest on it (mainly fibroblasts); sometimes called the basement membrane
Type IV collagen
Laminins
Entactin (nidogen and laminin)
Perlecan
Collagens are
insoluble, extracellular glycoproteins
found in all animals
the most abundant proteins in the human body
They are essential structural components of all connective tissues, such as
cartilage
bone
tendons
ligaments
fascia
skin
19 types of collagens have been found (so far) in humans. The major ones are:
Type I. The chief component of tendons, ligaments, and bones.
Type II. Represents more than 50% of the protein in cartilage. It is also used to build the notochord of vertebrate embryos.
Type III. Strengthens the walls of hollow structures like arteries, the intestine, and the uterus.
Type IV. Forms the basal lamina of epithelia. (The basal lamina is often called the basement membrane, but is not related to lipid bilayer membranes.) A meshwork of Type IV collagens provides the filter for the blood capillaries and the glomeruli of the kidneys.
The other 15 types are probably equally important but they are much less abundant.
Collagen Type IV (Col-IV) forms one network. Laminin (Lm) forms another. And entactin (En) and perlican (Perl) interact with the two networks. HS is the heparan sulfate glycosaminoglycans linked to the perlican core protein.

23. Hormones: estrogens, glucocorticoids,
The regulatory factors which impact on matrix synthesis, degradation and function are many and include 'growth factors', cytokines, hormones, vitamins, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).
Vitamins: C, D
Hormones: estrogens, glucocorticoids,
Matrix Metalloproteinases MMP: zinc containing enzymes that degrade most molecules of the ECM
Tissue Inhibitors of Metalloproteinases TIMP: zinc binding endopeptidases
Growth Factors: TGFß promotes cellular movement through matrix, and is involved in imflammation and repair
Vitamins: A list of matrix regulators might historically start with the recognition in the 18th century that Vitamin C was an essential nutrient, required for hydroxylation of proline to hydroxyproline and consequent stability of the collagen triple helix and vitamin D plays a major role in the dynamic reactions of the matrix of bone.
Hormones: The effects of hormones on matrix production, stability and degradation have been applied clinically for decades for a variety of conditions ranging from estrogens for osteoporosis to glucocorticoids for inflammatory processes which often have fibrotic sequelae. Growth factors and/or cytokines may mediate some or many of these effects while others may act directly on matrix components and/or their receptors.
Matrix Metalloproteinases MMP: Matrix metalloproteinases (MMP) are a family of enzymes which contain zinc at their active site and can degrade most of the matrix macromolecules found in connective tissues. The activation of some of the MMPs is linked to integrin receptors and is promoted by SPARC. MMPs are secreted by both matrix cells and infiltrating leukocytes in response to inflammaory mediators. MMPs are the major class of proteinases responsible for degradation of cartilage in rheumatoid arthritis.
Tissue Inhibitors of Metalloproteinases TIMP: TIMPs are natural inhibitors of the matrix metalloproteinases, collagenases, stromelysins, and gelatinases, a group of zinc-binding endopeptidases involved in the degradation of the extracellular matrix. At least three different TIMPs have been characterized, each inhibiting all known eukaryotic metalloproteinases. TIMPs affect activation of prometalloproteinases and modulate proteolysis of extracellular matrix components. TIMPs are particularly active in development during tissue remodeling and in pathologic disorders associated with inflammatory processes and tumor metastasis. Because of the intimate relationship between solid phase matrix components and growth regulatory agents, TIMPs play an important role in matrix cell regulation.
Growth Factors: TGF-beta plays a major role in matrix production and degradation . It also induces leukocyte margination and accumulation both through direct chemotaxis and by inducing cell surface integrin expression on monocytes. As a consequence, TGF-ß promotes monocyte adhesion to type IV collagen, laminin, and fibronectin, facilitating cellular movement through the matrix. In that TGF-beta also stimulates gelatinase/type IV collagenase it plays a major role in inflammation and repair. Most growth factors, directly or indirectly affect the matrix, a subject which is beyond the scope of this discussion.

24. Proteoglycans – glycoproteins containing covalently linked polysaccharide chains called glycosaminoglycans (GAGs); high viscosity and low compressibility
Proteoglycans are molecules that contain carbohydrate structures called glycosaminoglycans covalently attached to the protein core. Besides glycosaminoglycans they usually contain oligosaccharides, too. There are seven different types of glycosaminoglycans found. Exceptionally, hyaluronan is always present as a free carbohydrate chain.
The 7 different types are: chondroitin or dermatan sulfate, keratan sulfate, heparan sulfate/heparin, hyaluronon
GAGs are highly negatively charged molecules, with extended conformation that imparts high viscosity to the solution. GAGs are located primarily on the surface of cells or in the extracellular matrix (ECM). Along with the high viscosity of GAGs comes low compressibility, which makes these molecules ideal for a lubricating fluid in the joints. At the same time, their rigidity provides structural integrity to cells and provides passageways between cells, allowing for cell migration.
hyaluronan
Also: chondroitin or dermatan sulfate, keratan sulfate, heparan sulfate/heparin-

25. Common structural make-up of GAGs attachment to proteins; proteoglycans or mucopolysaccharides
95% carbohydrate by weight
Possible functions:
Selective sieve; regulate movement of molecules and cells
Chemical signaling between cells; bind certain growth factors (FGF) to stimulate proliferation in the area; TGFb binds to several core proteins of the proteoglycan group
Bind and regulate proteases and protease inhibitors (may restrict range of action, sterically block activity, provide a reservoir for later release, prolong action, or alter concentration)
Biosynthesis of glycosaminoglycans is initiated (with some exceptions) by adding the reducing end of xylose to the serine residue of the core protein [Grebner et al, 1966; Kjellen & Lindahl, 1991]. Sometimes also threonine may accept the xylose. The reaction is catalyzed by a soluble enzyme xylosyl-transferase.
Elongation of glycosaminoglycans is performed by the action of specific enzymes that add one monosaccharide at a time to the end of growingchains. N-acetylgalactosaminyl- transferase and glururonyl-transferase are involved in the biosynthesis of chondroitin and dermatan sulphates. Sulphate is added simultaenously with carbohydrate chain polymerization [de Luca et al, 1973]. Inorganic sulphate is activated with ATP into phosphoadenosylphosphosulphate (PAPS), that donates the sulphate group to glycosaminoglycans. The donation is catalyzed by specific sulphotransferases that donate the sulphate into carbon 4 or 6 of the GalNAc.

26. synovial fluid, vitreous humor, ECM of loose connective tissue
GAG
Localization
Comments
Hyaluronate
synovial fluid, vitreous humor, ECM of loose connective tissue
large polymers, shock absorbing
Chondroitin sulfate
cartilage, bone, heart valves
most abundant GAG
Heparan sulfate
basement membranes, components of cell surfaces
contains higher acetylated glucosamine than heparin
Heparin
component of intracellular granules of mast cells lining the arteries of the lungs, liver and skin
more sulfated than heparan sulfates
Dermatan sulfate
skin, blood vessels, heart valves
Keratan sulfate
cornea, bone, cartilage aggregated with chondroitin sulfates
The arrangement of sugar residues in GAG chains and the modification of specific sugars in the chains can determine their function and that of the proteoglycans contaiing them. Groupings of modified sugars can control the binding of growth factors to certain receptors, the activities of proteins in the blood clotting cascade, and the activity of lipoprotein lipase.
Pentasaccharride sequences found in a subset of heparin GAGTs that control the activity of antithrombinIII, and inhibitor of the key blood clotting protease thrombin. When these sequences are sulfated at two specific positions, heparin can activate ATIII, inhibiting clot formation.

28. ECM proteins in connective tissue
Collagen
Proteoglycans
Adhesion proteins
Hyaluronan
Elastic fibers
Tendon – dense connective tissue

29. Type IV collagen
Repeating sequence
(glycine (Gly) - X - Y)n
where X is often proline (Pro) and Y is often hydroxyproline (proline to which an -OH group is added after synthesis of the polypeptide).
The resulting molecule twists into an elongated, left-handed helix (NOT an alpha helix). When synthesized, the N- terminal and C- terminal of the polypeptide have globular domains, which keep the molecule soluble.
As they pass through the endoplasmic reticulum (ER) and Golgi apparatus,
The molecules are glycosylated.
Hydroxyl (-OH) groups are added to the "Y" amino acid.
S-S bonds link three chains covalently.
The three molecules twist together to form a triple helix.
In some collagens (e.g., Type II), the three molecules are identical (the product of a single gene). In other collagens (e.g., Type I), two polypeptides of one kind (gene product) assemble with a second, quite similar, polypeptide, that is the product of a second gene.
When the triple helix is secreted from the cell (usually by a fibroblast), the globular ends are cleaved off. The resulting linear, insoluble molecules assemble into collagen fibers. They assemble in a staggered pattern that gives rise to the striations seen in this electron micrograph (courtesy of Dr. Jerome Gross). (Type IV collagens are an exception; they form a meshwork rather than striated fibers.)
Goodpasture's Syndrome
Some people develop antibodies against an epitope on their Type IV collagen molecules. These
attach to the basal lamina of epithelial cells;
"fix" complement which damages the basal lamina.
So Goodpasture's syndrome is an example of an autoimmune disorder.
The basal laminae of the lung epithelia and the glomeruli of the kidney are especially likely to be affected. In this photo (courtesy of Dr. Frank J. Dixon), a fluorescent antibody against human IgG shows the autoantibodies coating the basement membranes of the glomeruli in a patient with Goodpasture's syndrome.
Repeating sequence (Glycine – X –Y)n X = proline Y= hydroxyproline
Left handed helix. N-terminal and C-terminal ends have globular domains (solubility; cleaved when secreted from cell - insoluble)
In ER and Golgi they are glycosylated, OH groups added, S-S links three chains
End result a triple helix

30. Collagen is a highly abundant and varied protein with more than 14 types discovered. All variations are formed from tropocollagen molecules which are inelastic. The tropocollagen molecule is formed from three peptide chains. Each chain is formed from 1/3 glycine, 1/3 proline and hydroxyproline and 1/3 other amino acids. The different collagen types are formed by variations in the sequence and amount of amino acids found in the alpha-chains. The three chains are principally stabilised by Van de Waals bonds and some occasional covalent bonds forming a right-handed helix. The helix is 300nm in length and 1.5nm in diameter with a screw pitch of 0.27nm.
The tropocollagen molecules are arranged in a quarter stagger array, with a 26.5nm overlap and a 37.5nm gap between each end of the molecule. A sequence of tropocollagen molecules is a microfibril. These microfibrils are organised in parallel to form a collagen fibril with a 50nm diameter. The collagen fibrils then twist together to form a single collagen strand [Black, 1988]. It is the cross-linking between the molecules at each structural level that gives collagen its high tensile strength.

31. the case of the interstitial collagens the characteristic banding pattern is accounted for by an ordered staggered arrangement of the collagen molecules within the collagen fibrils and the collagen fibers. The manner of molecular packing within the fibril in turn is determined by the amino acid sequence. The way the molecules are staggered in the fibril, however, gives rise to regions in which the molecules overlap and others in which there is no overlap
Some tissues are characterized by a marked predominance of one typee.g., types II, IX, X, and XI in cartilage and type I collagen in bone. It is likely that each of these collagen types is largely responsible for the functional and morphologic properties of each connective tissue, although it has not yet been possible to relate function to a particular chemical modification.

32. Type I. The chief component of tendons, ligaments, and bones.
Type II. Represents more than 50% of the protein in cartilage. It is also used to build the notochord of vertebrate embryos.
Type III. Strengthens the walls of hollow structures like arteries, the intestine, and the uterus.
Type IV. Forms the basal lamina of epithelia. (The basal lamina is often called the basement membrane, but is not related to lipid bilayer membranes.) A meshwork of Type IV collagens provides the filter for the blood capillaries and the glomeruli of the kidneys.
osteogenesis imperfecta
Collagen type II is found predominantly, but not exclusively, in hyaline cartilage where it accounts for 90% of the total collagen. Type II exists in two splice variants (IIA and IIB), in IIB, the dominant form found in mature cartilage, exon 2 is spliced out (encodes a 69 a.a cys-rich domain in the N-terminal propeptide). In IIA, a transient embryonic form found in prechondrogenic mesenchyme, perichondrium and vertebrae, this domain is retained [Ryan and Sandell, 1990, Sandell et al., 1994, Sandell et al., 1991].
Collagen II InteractionsThe Col1 domain interacts with integrin receptors, principally a2b1, and the non-integrin binding protein anchorin CII [Turnay et al ., 1995]. The fibrillar form of type II collagen is cross-linked to type IX collagen via lysine/hydroxylysine residues in the N- and C-telopeptides of type II collagen and in the helical Col2 domain of type IX. The fibrillar form of type II collagen also interacts with the protein cores of fibromodulin and decorin [Hedbom and Heinegård, 1993]. Interacting proteins: Decorin, Fibromodulin, Lumican, Chondroadherin, COMP, Fibronectin and Aggrecan (KS-rich region).
Different types of collagen can co-assemble to form large fibers. Type VI +type I in tendons, form in direction of stress. Type II and Type IX oriented randomly and are in cartilage for strength and compressibility.

33. Perlecan
PERLECAN is a large heparan sulfate proteoglycan with a wide tissue distribution and multiple potential functions (see Iozzo ). The glycosaminoglycan chains, located in the NH2-terminal domain of the core protein, bind basic FGF-2 and have been shown to promote the mitogenic and angiogenic activities of FGF-2. They also interact with the basement membrane components, laminin-1 and collagen IV, and are thought to represent a barrier to the passage of cationic macromolecules across glomerular basement membranes in the kidney. The 400–450-kD core protein, composed of several protein modules arranged in five distinct domains, binds to a variety of small and large molecules, including FGF-7, fibronectin, heparin, laminin-1, PDGF-BB, and integrins (Fig 1). The physiological significance of such interactions is illustrated by the demonstration that lowering of perlecan levels by stable expression of antisense constructs in colon carcinoma cells depresses their FGF-7–dependent growth, and reduces tumor growth and tumor-induced angiogenesis in nude mice (Sharma et al ). Also, perlecan is a strong inducer of FGF-2–dependent neovascularization (Aviezer et al ). These same interactions of perlecan may well explain the finding that perlecan produced by vascular endothelial cells can inhibit proliferation and binding of FGF-2 in smooth muscle cells (Forsten et al ).
In homozygous knockout embryos, no abnormalities are observed before embryonic day 10 (E10)1, but between E10 and E12, most of the embryos die with evidence of bleeding into the pericardial sac. A few animals survive, but die around birth with severe defects in the brain and in the skeleton. The lack of any defects before E10 is surprising because perlecan is first expressed in two-cell embryos and increases on the external surface of trophoectodermal cells of blastocysts. Thus, it has been thought to play a role in the initial attachment of the embryo to the uterine wall (Smith et al ). The absolutely normal Mendelian ratio of wild-type, heterozygous, and homozygous embryos at E9.5 rules out a limiting role for perlecan during implantation. Also, the normal appearance of most basement membranes in homozygous embryos suggests that perlecan does not have a critical role in the assembly of basement membranes (Timpl and Brown ). Perhaps the proteoglycan agrin, which shares homology with perlecan, can substitute for the absence of perlecan. Another possibility is that collagen XVIII, also a heparan sulfate proteoglycan component of most basement membranes, can substitute for the loss of perlecan function (Muragaki et al ; Halfter et al ).

35. Laminins – multiadhesive matrix proteins
Function: organization of basement membrane; have binding sites for itegrin receptors (important in embryonic development and tissue remodeling)
Laminins are tightly associated with entactin or nidogen a 150-kD sulfated glycoprotein, which also binds to type IV collagen. As a result of these multiple interactions, laminin, entactin, type IV collagen, and perlacan form crosslinked networks in the basal lamina.
Laminins are extracellular matrix proteins which consist of alpha, beta and gamma chains with molecular masses of kDa. Chain association occurs through a large triple alpha-helical coiled-coil domain towards the C-terminus of each chain. Eight genetically distinct laminin chains (alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 1, gamma 2) and seven different assembly forms (laminins-1 to -7) are known so far. The most extensively characterized laminin-1 (alpha 1 beta 1 gamma 1) shows calcium-dependent self assembly and heterotypic binding to perlecan, nidogen, fibulin-1 and other matrix components. This binding indicates a crucial role in the supramolecular organization of basement membranes. Laminins also possess binding sites for at least six different integrin receptors and are thus involved in many cell-matrix interactions. Such interactions have been shown to be important during embryonic development and for tissue homeostasis and remodelling.
The globular LG domains at the C terminus mediate Ca2+ dependent binding to specific carbohydrates on certain cell-surface molecules such as syndecan and dystroglycan. LG domains can mediate binding to steroids and proteins as well.
transmembrane protein Dystroglycan is a highly glycosylated central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin (see Drosophila Laminin A), agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Drosophila Dystroglycan (Dg) is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers.

36. Cartilage proteoglycan aggregate
Hyaluronan/hyaluronic acid or hyaluronate – nonsulfated GAG formed as a disaccharide repeat cojmposed of glucuronic acid and N acetylglucosamine. A major component of the EM that surrounds migrating and proliferating cells, especially in embryos. Also forms the backbone of complex proteoglycan aggregates found in cartilage.
Imparts stiffness and resilience, and lubrication
Binds lots of water and acts as a large hydrated sphere. Because of the negative charges, attracts ions, which in turn attract water. This leads to increased turgor pressure, which is how many connective tissues resist compression (I.e. intervertebral discs,
HA is bound to the surface of many migrating cells by adhesion receptors. The hyaluronon coat bound to cells appears to keep cells apart from one another, giving them the freedom to move about and proliferate. As the cells anchor and become stable they lose hA. Thus it appears to be involved in migration of cells during embryogenesis.
The proteoglycans of articular cartilage consist of core proteins of variable length with different oligosaccharide and glycosaminoglycan chains linked to them. The molecular weight of aggrecan in the tissue ranges from 1 to 3.5 MDa, and their size based on electron microscopy varies in the range of nm. They form large aggregates (molecular size up to 800 MDa) with hyaluronan and so-called link-protein. The percentage of aggregating proteoglycans is 50-85% of total weight of proteoglycans. In addition, there is another group of large proteoglycans (10-40% of the total amount), however, unable to form aggregates [Heinegård & Sommarin, 1987; Carney & Muir, 1988; Lohmander, 1988]. These groups derive probably from the aggrecan through partial cleavage by proteinases, instead of being an individual gene product.
Cartilage proteoglycan aggregate

38. Adhesions between non-epithelial, cells and the extracellular matrix – short and long term adhesions that help in motility
Focal adhesions
Focal contacts
Focal complexes,
3D adhesions
Fibrillar adhesions
Podosomes

40. Two conformations of integrins
Change in conformation transferred to other proteins scaffolded to internal signaling pathways