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extra cellular matrix

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  2. 2. ECM Many types of animal cells are surrounded by an extra cellular matrix (ECM)- An Organized network of extra cellular materials. It often plays a key regulatory role in determining the shape and activities of the cell. One of the best defined ECM is the basement membrane (basal lamina ) a continuousSheet 50 to 200 nm thickness. It surrounds nerve fibers, muscles, and fat cells, underlies the basal surfaces of epithelialTissues such as epidermis of the skin, lining of digestive and respiratory tracts. Basement membrane provides mechanical support for attached cells, generate signals, That serve as a substratum for cell migration-it act as a barrier for the passage of macromolecules
  3. 3. Composition of Extracellular Matrix (ECM) • Cells (mesenchymal origin) - fibroblasts - smooth muscle cells - chondroblasts - osteoblasts and epitelial cells • Organic fibrilar matrix • Organic nonfibrilar matrix • Water
  5. 5. Structure of ECM • collagen – the main ECM component, forms the main fibres • elastin • proteoglycans - heteropolysacharides • structural glycoproteins - fibronectin, laminin
  6. 6. Functions of ECM • Provides support anchorage and for cells. • Regulates and determine cells dynamic behaviour : - polarity of cells - cell differentiation - adhesion - migration • Provides mechanical support for tissues and organ architecture. - growth - regenerative and healing processes - determination and maintenance of the structure • Place for active exchange of different metabolites, ions, water.
  7. 7. Cells Need Receptors to Recognize and Respond to ECM • Integrins • Dystroglycan • Syndecans • Muscle-Specific kinase (MuSK) • Others
  8. 8. 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
  9. 9. Why do all multicellular animals have ECM? • Act as structural support to maintain cell organization and integrity (epithelial tubes; mucosal lining of gut; skeletal muscle fiber integrity) • Compartmentalize tissues (pancreas: islets vs. exocrine component; skin: epidermis vs. dermis) • Provide hardness to bone and teeth (collagen fibrils become mineralized) • Present information to adjacent cells: – Inherent signals (e.g., RGD motif in fibronectin) – Bound signals (BMP7, TGFb, FGF, SHH) • Serve as a highway for cell migration during development (neural crest migration), in normal tissue maintenance (intestinal mucosa), and in injury or disease (wound healing; cancer)
  10. 10. Types of ECM Components • Collagens • Proteoglycans – Perlecan, aggrecan, agrin, collagen XVIII • Hyaluronan (no protein core) • Large Glycoproteins – Laminins, nidogens, fibronectin, vitronectin • Fibrillins, elastin, LTBPs, MAGPs, fibulins • “Matricellular” Proteins – SPARC, Thrombospondins, Osteopontin, tenascins
  11. 11. 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
  12. 12. Generic Tissue Structure Stratified, pseudostratified, or monolayer (aka stroma) “Tube within a Tube” concept
  13. 13. Basement Membrane M. Loots, Univ. of Pretoria, S.A. J. Schwarzbauer, Curr. Biol. 1999
  14. 14. Basement Membranes • In general, basement membranes appear very similar to each other by EM. • But all are not alike! • There is a wealth of molecular and functional heterogeneity among basement membranes, due primarily to isoform variations of basement membrane components.
  15. 15. Basement Membranes are Involved in a Multitude of Biological Processes • Influence cell proliferation, differentiation, and migration • Maintain cell polarization and organization, as well as tissue structure • Act as a filtration barrier in the kidney between the vasculature and the urinary space • Separate epithelia from the underlying stroma/mesenchyme/interstitium, which contains a non-basement membrane matrix
  16. 16. Lamina Densa + Lamina Lucidae Kidney Glomerular Basement Membrane
  17. 17. Kidney Basement Membranes Laminin b1 Laminin b2
  18. 18. Primary Components of All Basement Membranes • Collagen IV 6 chains form α chain heterotrimers • Laminin 12 chains form several α-β-γ heterotrimers • Entactin/Nidogen 2 isoforms • Sulfated proteoglycans Perlecan and Agrin are the major ones; Collagen XVIII is another
  19. 19. Collagen • The most abundant protein in the body, making 25%-35% of all the whole-body proteins. • Collagen contributes to the stability of tissues and organs. • It maintains their structural integrity. • It has great tensile strenght. • The main component of fascia, cartilage, ligaments, tendons, bone and skin. • Plays an important role in cell differentiation, polarity, movement • Plays an important role in tissue and organ development
  20. 20. Collagen structure Collagen is insoluble glycoprotein (protein + carbohydrate) Collagen polypeptide structure: - G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – A – – A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – – A – A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G - glycine, X - proline or hydroxyproline, Y – lysin or hydroxylysine, A – amino acid • Proline and hydroxyproline constitute about 1/6 of the total sequence, provide the stifness of the polypeptide chain. • Carbohydrates : glucose, galactose
  21. 21. Structure of collagen1
  22. 22. Collagen provides structural support to tissues • The principal function of collagens is to provide structural support to tissues. • Collagens are a family of over 20 different extracellular matrix proteins. – Together they are the most abundant proteins in the animal kingdom.
  23. 23. 15.3 Collagen provides structural support to tissues • All collagens are organized into triple helical, coiled-coil “collagen subunits.” – They are composed of three separate collagen polypeptides. • Collagen subunits are: – secreted from cells – then assembled into larger fibrils and fibers in the extracellular space
  24. 24. Cells Need Receptors to Recognize and Respond to ECM • Integrins • Dystroglycan • Syndecans • Muscle-Specific kinase (MuSK) • Others
  25. 25. Three helical polypeptide units twist to form a triple-helical collagen molecule: a molecular "rope" which has some bending stiffness and does not undergo rotation.
  26. 26. 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)
  27. 27. Collagen IV: Network or Sheet Forming • Six genetically distinct a chains: α1- α6, ~180 kDa each • Chains form three types of heterotrimers: – (a1)2(α2), α3α4α5, (α5)2(α6) • Like all Collagens, comprised mainly of Gly-x-y repeats, y is frequently proline • Gly-x-y pattern has multiple interruptions – Provides flexibility to the collagen network and to the basement membrane Hudson et al., NEJM 2003
  28. 28. Collagen IV Trimer • 7S domain at N-terminus • Interrupted Gly-x-y triple helical domain • C-terminal non-collagenous domain--NC1
  29. 29. Collagen IV Network Trimers (aka protomers) associate with each other, four at the N-terminus and two at the C-terminus (hexamer), to form a chicken wire-like network that provides strength and flexibility to the basement membrane.
  30. 30. What Directs Chain-Chain-Chain Recognition and Hexamer Assembly?
  31. 31. Sulfilimine: The Bond that Crosslinks Type IV Collagen NC1 Domains Vanacore et al., Science 2009
  32. 32. Fibrillar Collagens (I, II, III, V)
  33. 33. Fibrillar Collagens (I, II, III, V) • Connective tissue proteins that provide tensile strength • Triple helix, composed of three a 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
  34. 34. CCoollllaaggeenn CCrroosssslliinnkkiinngg • Once formed, collagen fibrils are greatly strengthened by covalent crosslinks that form between the constituent collagen molecules. • The first step in crosslink formation is the deamination by the enzyme lysyl oxidase of specific lysine and hydroxylysine side chains to form reactive aldehyde groups. • The aldehydes then form covalent bonds with each other or with other lysine or hydroxylysine residues.
  35. 35. CCoollllaaggeenn CCrroosssslliinnkkiinngg • If crosslinking is inhibited (Lysyl hydroxylase mutations; vitamin C deficiency), collagenous tissues become fragile, and structures such as skin, tendons, and blood vessels tend to tear. There are also many bone manifestations of under-crosslinked collagen. • Hydroxylation of specific lysines governs the nature of the cross-link formed, which affects the biomechanical properties of the tissue. Collagen is especially highly crosslinked in the Achilles tendon, where tensile strength is crucial.
  36. 36. Bone is Composed of Mineralized Type I Collagen Fibrils Bone is 70% mineral and 30% protein, mostly collagen Mineral is Dahllite, similar to hydroxyapatite (contains calcium, phosphate, carbonate)
  37. 37. 15.3 Collagen provides structural support to tissues • Mutations of collagen genes can lead to a wide range of diseases, from mild wrinkling to brittle bones to fatal blistering of the skin.
  38. 38. 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
  39. 39. Scurvy • Liver spots on skin, spongy gums, bleeding from mucous membranes, depression, immobility • 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)
  40. 40. Some Genetic Diseases of Collagen • Collagen I – Osteogenesis imperfecta – Ehlers-Danlos syndrome type VII • Collagen II – Multiple diseases of cartilage • Collagen III – Ehlers-Danlos syndrome type IV • Collagen IV – Alport syndrome, stroke, hemorrhage, porencephaly • Collagen VII – Dystrophic epidermolysis bullosa (skin blistering)
  41. 41. Different Types of Mutations in Collagen I a Chain Genes Cause Different Disease Severities Gene location mutation Syndrome COL1A1 17q22 Null alleles OI type I Partial deletions; C-terminal substitutions OI type II N-terminal substitutions OI types I, III or IV Deletion of exon 6 EDS type VII COL1A2 7q22.1 Splice mutations; exon deletions OI type I C-terminal mutations OI type II, IV N-terminal substitutions OI type III Deletion of exon 6 EDS type VII
  42. 42. 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: 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)
  43. 43. Generalizations • Most ECM proteins are large, modular, multidomain glycosylated or glycanated proteins • Some domains recur in different ECM proteins – Fibronectin type III repeats – Immunoglobulin repeats – EGF-like repeats – Laminin Globular (G) domain – von Willebrand factor Perlecan
  44. 44. Endostatin: Noncollagenous Tail of Collagen XVIII • Exhibits anti-angiogenic activity • Targets tumor vasculature
  45. 45. Type IV Collagen NC1 Domains • Exhibit anti-angiogenic activity • Target tumor vasculature
  46. 46. 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: 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)
  47. 47. Dominant Negative COL1 Mutations * Gly subst. in COL4A2 * Gly subst. in COL4A1 Byers P. Connective Tissue and Its Inheritable Disorders 1993, pp317-50. ½ of the trimers are abnormal ¾ of the trimers are abnormal
  48. 48. Elastin aanndd EEllaassttiicc FFiibbeerrss EExxhhiibbiitt RRuubbbbeerr--LLiikkee PPrrooppeerrttiieess • Physiological importance lies in the unique elastomeric properties of elastin. Found in tissues in which reversible extensibility or deformability are crucial, such as the major arterial vessels (esp. aorta), the lung and the skin. • Elastin is characterized by a high index of hydrophobicity (90% of all the amino acid residues are nonpolar). One-third of the amino acid residues are glycine with a preponderance of the nonpolar amino acids Ala, Val, Leu, and Ile. As in collagen, one-ninth of the residues are proline (but with very little hydroxylation). • Early in development, the elastic fibers consists of microfibrils, which define fiber location and morphology. Over time, tropoelastin accumulates within the bed of microfibrils.
  49. 49. Elastic Fiber Biogenesis • Elastic fibers are very complex, difficult to repair structures • There are two morphologically distinguishable components – Microfibrils – Elastin • Assembly follows a well-defined sequence of events: 1. Assembly of microfibrils 2. Association of tropoelastin aggregates with microfibrils 3. Crosslinking of tropoelastins with each other by lysyl oxidase to form polymers Shifren and Mecham, 2006
  50. 50. Major steps underlying the assembly of Ramirez, F. et al. Physiol. Genomics 19: 151-154 2004; doi:10.1152/physiolgenomics.00092.2004 Copyright ©2004 American Physiological Society microfibrils and elastic fibers
  51. 51. Microfibril Components: ~30 • Fibrillin--three forms • Microfibril-associated glycoproteins (MAGPs)--two forms • Latent TGFb Binding Proteins (LTBPs)-- four forms • Proteoglycans, MFAPs, Fibulins, Emilins, Collagens, Decorin, et al.
  52. 52. Marfan Syndrome • Caused by dominant Fibrillin-1 (FBN1) mutations – Haploinsufficiency is the culprit • Skeletal, ocular, and cardiovascular defects • Deficiency of elastin-associated microfibrils • Syndrome may result from alterations in TGFb signaling, rather than purely structural changes in microfibrils
  53. 53. Evidence for FBN/BMP7 Interactions Fbn2+/-; Bmp7+/- trans-heterozygous animals show limb patterning defects. Artaga-Solis et al., J. Cell Biol. 2001 Specific fragments of Fibrillin 1, but not LTBP1, bind to BMP7 Gregory et al., JBC 2005
  54. 54. Elastic Fiber Biogenesis • Elastic fibers are very complex, difficult to repair structures • There are two morphologically distinguishable components – Microfibrils – Elastin • Assembly follows a well-defined sequence of events: 1. Assembly of microfibrils 2. Association of tropoelastin aggregates with microfibrils 3. Crosslinking of tropoelastins with each other by lysyl oxidase to form polymers Shifren and Mecham, 2006
  55. 55. Structure of a cartlage type proteoglycan complex
  56. 56. Sulfated Proteoglycans • Have protein cores with large glycosaminoglycan (GAG) side chains (from 1 to >100) attached to serines • Some PGs contain heparan sulfate – Perlecan, Agrin, Collagen XVIII (endostatin) • Others contain chondroitin, keratan or dermatan sulfate • GAG chains are responsible for most of the biological properties of proteoglycans and provide charge to basement membranes Heparan sulfate: Composed of D-glucuronate-2-sulfate + N-sulfo-D-glucosamine-6-sulfate
  57. 57. Some Major Proteoglycan Family Members From: Iozzo, R.V. (1998) Ann. Rev. Biochem. 67:609 From: Iozzo, R.V. (2001) J. Clinic. Invest. 108:165
  58. 58. 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
  59. 59. Endorepellin: Domain V of Perlecan • Exhibits anti-angiogenic activity • Targets tumor vasculature
  60. 60. Glycosaminoglycan Classification Proteoglycans can be categorised depending upon the nature of their glycosaminoglycan chains. • Hyaluronic acid (does not contain any sulfate) - non-covalent link complex with proteoglycans • Chondroitin sulfate cartilage, bone • Dermatan sulfate skin, blood vessels • Heparan sulfate basement membrane, component of cells surface • Keratan sulfate cornea, bone, cartilage often aggregated with chondroitin sulfate
  61. 61. Function of Proteoglycans • organize water molecules - resistant to compression - return to original shape - repel negative molecules • occupy space between cells and collagen • high viscosity - lubricating fluid in the joints • specific binding to other macromolecules • link to collagen fibers - form network - in bone combine with calcium salts (calcium carbonate, hydroxyapatite) • cell migration and adhesion - passageways between cells • anchoring cells to matrix fibers
  62. 62. Structural Glycoproteins • Direct linkage to collagen or proteoglycans - anchoring collagen fibers to cell membrane - covalent attachment to membrane lipid • Major adhesive structural glycoproteins - fibronectin - laminin
  63. 63. Fibronectin Structure • Dimer connected at C-terminal by S-S linkage • Rigid and flexible domains • Cell binding domain RGDS (arg, gly, asp, ser) - binding receptor in cell membranes • Domain is binding to - collagen type I, II and III - heparin sulfate - hyaluronic acid - fibrin
  64. 64. Fibronectin Function • cell adhesion • cell differentiation • anchoring basal laminae to other ECM • blood clothing - clothing process, link to fibrin
  65. 65. Laminin Heterotrimers are composed of one a, one b, and one g 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
  66. 66. Laminin Structure and Function • cross-shaped glycoprotein • 3 polypeptide chains • domain bind - collagen type IV - heparin - heparin sulfate • cell surface receptor • cell adhesion • cell differentiation • anchoring the glycoprotein to basal laminae
  67. 67. The Laminin Trimers Miner and Yurchenco, 2004
  68. 68. Laminin • All laminin chains share structural homology • Contain globular, rod (EGF-like repeats), and coiled-coil domains • Alpha chains are unique, contain a C-terminal laminin globular “LG” domain, ~100 kDa (New nomenclature)
  69. 69. 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 a chain LG domain is left free for interactions with cellular receptors.
  70. 70. Laminins- Primordial germ cell migration
  71. 71. 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) Lamb2: Neuromuscular junction and kidney filtration defects (M); Iris muscle, neuromuscular, kidney filtration defects (H; Pierson syndrome) Lamc3: Brain malformations, autism spectrum disorder? (H)
  72. 72. Proteases degrade extracellular matrix components MMPs – A large family of zinc containing enzymes called MMP that are either secreted into the – Extra cellular space or anchored to plasma membrane • Cells must routinely degrade and replace their extracellular matrix as a normal part of – development (embryonic cell migration) – wound healing – .
  73. 73. MMPS MMPs have been implicated in a number of pathological conditions, including Arthritis, hepatitis,atherosclerosis,tooth and gum diseases and tumor progression Inherited skeletal disorders have been found mutations in MMP genes.
  74. 74. • Extracellular matrix proteins are degraded by specific proteases, which cells secrete in an inactive form. • These proteases are only activated in the tissues where they are needed. • Activation usually occurs by proteolytic cleavage of a propeptide on the protease.
  75. 75. Proteases degrade extracellular matrix components • The matrix metalloproteinase (MMP) family is one of the most abundant classes of these proteases. – It can degrade all of the major classes of extracellular matrix proteins. • MMPs can activate one another by cleaving off their propeptides. – This results in a cascade-like effect of protease activation that can lead to rapid degradation of extracellular matrix proteins.
  76. 76. Proteases degrade extracellular matrix components • Cells secrete inhibitors of these proteases to protect themselves from unnecessary degradation. • Mutations in the matrix metalloproteinase-2 gene give rise to numerous skeletal abnormalities in humans. – This reflects the importance of extracellular matrix remodeling during development.
  77. 77. Interaction of cells with with extra cellular materials The components of ECM such as Fibronectin, laminin, proteoglycan, and collagen Are capable of binding to receptors situated on the cell surface. The most important family of receptors that attach cells to their microenviroinment Is the Integrins Integrins which are found only in animals are a family of membrane proteins that play a key role in integrating the extracellular and intracellular enviroinments. On the One side of the plasma membrane – binds to array molecules (ligands) that are Present in the extra cellular enviroinment. On the intracellular side of the membrane, integrin binds to interact with directly or Indirectly with different proteins. Integrins composed of two membrane spanning polypeptides α ,ß that are Non covalently linked
  78. 78. Integrins can be activated rapidly by events within the cell that alter the conformation of Cytoplasmic domains of the integrin subunits. Integrins have been implicated in two main types of activities Adhesion of cells to their substratum and transmisssion of signals from the external enviroinment to the cell interior. The binding of the extra cellular domain of an integrin to a ligand such as fibronectin, Collagen can induce confirmational changes at the opposite cytoplasmic end of integrins.
  79. 79. Integrin structure
  80. 80. Most integrins are receptors for extracellular matrix proteins • Virtually all animal cells express integrins. – They are the most abundant and widely expressed class of extracellular matrix protein receptors. • Some integrins associate with other transmembrane proteins.
  81. 81. 15.13 Most integrins are receptors for extracellular matrix proteins • Integrins are composed of two distinct subunits, known as α and β chains. • The extracellular portions of both chains bind to extracellular matrix proteins • The cytoplasmic portions bind to cytoskeletal and signaling proteins.
  82. 82. Most integrins are receptors for extracellular matrix proteins • In vertebrates, there are many α and β integrin subunits. – These combine to form at least 24 different αβ heterodimeric receptors. • Most cells express more than one type of integrin receptor. – The types of receptor expressed by a cell can change: • over time or • in response to different environmental conditions
  83. 83. Most integrins are receptors for extracellular matrix proteins • Integrin receptors bind to specific amino acid sequences in a variety of extracellular matrix proteins. • All of the known sequences contain at least one acidic amino acid.
  84. 84. Integrin receptors participate in cell signaling • Integrins are signaling receptors that control both: – cell binding to extracellular matrix proteins – intracellular responses following adhesion • Integrins have no enzymatic activity of their own. – Instead, they interact with adaptor proteins that link them to signaling proteins.
  85. 85. Integrin receptors participate in cell signaling • Two processes regulate the strength of integrin binding to extracellular matrix proteins: – affinity modulation • varying the binding strength of individual receptors – avidity modulation • varying the clustering of receptors
  86. 86. Integrin receptors participate in cell signaling • Changes in integrin receptor conformation are central to both types of modulation. • They can result from changes: – at the cytoplasmic tails of the receptor subunits or – in the concentration of extracellular cations
  87. 87. Integrin receptors participate in cell signaling • In inside-out signaling, changes in receptor conformation result from intracellular signals that originate elsewhere in the cell. – For example, at another receptor • In outside-in signaling, signals initiated at a receptor are propagated to other parts of the cell. – For example, upon ligand binding
  88. 88. Integrin receptors participate in cell signaling • The cytoplasmic proteins associated with integrin clusters vary greatly depending on: – the types of integrins and extracellular matrix proteins engaged. • The resulting cellular responses to integrin outside-in signaling vary accordingly. • Many of the integrin signaling pathways overlap with growth factor receptor pathways.
  89. 89. Integrins and extracellular matrix molecules play key roles in development • Gene knockout by homologous recombination has been applied in mice to; – over 40 different extracellular matrix proteins – 21 integrin genes • Some genetic knockouts are lethal, while others have mild phenotypes.
  90. 90. Integrins and extracellular matrix molecules play key roles in development • Targeted disruption of the β1 integrin gene has revealed that it plays a critical role in: – the organization of the skin – red blood cell development
  91. 91. Integrin activation
  92. 92. Selectins Comprise a family of integral membrane glycoproteins that recognize and bind To a particular arrangement of sugars in the oligo saccharides. Selectins possess a small cytoplasmic domain, a single membrane spaning Domain. 3 known selectins – E selectins- present in the endothelial cells. L- Selectins LEUKOCYTE P- Selectins- PLATELETS All three selectins recognize a particular group of sugars that is found at the Ends of the carbohydrate chains of certain complex glycoproteins. The importance of integrins in the inflammatory response is demonstrated by A rare disease called leukocyte adhesion deficiency (LAD), Leukocyte Of these individuals lack the ability to adher to the endothelial layer of Venules. These patients suffer from repeated bacterial infections.
  93. 93. Selectins control adhesion of circulating immune cells • Selectins are cell-cell adhesion receptors expressed exclusively on cells in the vascular system. • Three forms of selectin have been identified: – L-selectin – P-selectin – E-selectin
  94. 94. Selectins control adhesion of circulating immune cells • Selectins function to arrest circulating leukocytes in blood vessels so that they can crawl out into the surrounding tissue. • In a process called discontinuous cell-cell adhesion, selectins on leukocytes bind weakly and transiently to glycoproteins on the endothelial cells. – The leukocytes come to a “rolling stop” along the blood vessel wall.
  95. 95. Tight junctions form selectively permeable barriers between cells • Tight junctions also preserve epithelial cell polarity by serving as a “fence.” – It prevents diffusion of plasma membrane proteins between the apical and basal regions.
  96. 96. Tight junctions : sealing the extra cellular space Tight junctions are located at the very epical end of the junctional complex between Adjacent epithelial cells.
  97. 97. Septate junctions in invertebrates are similar to tight junctions • The septate junction: – is found only in invertebrates – is similar to the vertebrate tight junction • Septate junctions appear as a series of either straight or folded walls (septa) between the plasma membranes of adjacent epithelial cells.
  98. 98. Septate junctions in invertebrates are similar to tight junctions • Septate junctions function principally as barriers to paracellular diffusion. • Septate junctions perform two functions not associated with tight junctions: – they control cell growth and cell shape during development. • A special set of proteins unique to septate junctions performs these functions.
  99. 99. ADHEREN JUNCTIONS Adherens junctions : are found in a variety of sites within the body.They are particularly Common in epithelia such as the lining of the intestine where they occur as belts Binding cell to its surrounding neighbours. The cadherin clusters of adherin junction – connect the external enviroinment to theActin cytoskeleton and provide a pathway for signals to be transmitted from the Cell exterior to the cytoplasm. Adherin junctions situated between endothelial cells that line walls of blood vessels transmit signals that ensure the survival of the cells.
  100. 100. Adherens junctions link adjacent cells • Adherens junctions are a family of related cell surface domains. – They link neighboring cells together. • Adherens junctions contain transmembrane cadherin receptors.
  101. 101. Adherens junctions link adjacent cells • The best-known adherens junction is the zonula adherens. – It is located within the junctional complex that forms between neighboring epithelial cells in some tissues. • Within the zonula adherens, adaptor proteins called catenins link cadherins to actin filaments.
  102. 102. Desmosomes : are disk-shaped adhesive junctions approximately 1mm in diameter. That are found in a variety of tissues. Desmosomes are particularly numerous in tissues that are subjected to mechanical stress such as cardiac muscle and the epithelial layers of the skin and uterine Cervix.
  103. 103. Desmosomes are intermediate filamentbased cell adhesion complexes • The principal function of desmosomes is to: – provide structural integrity to sheets of epithelial cells by linking the intermediate filament networks of cells.
  104. 104. Desmosomes are intermediate filament-based cell adhesion complexes • Desmosomes are components of the junctional complex. • At least seven proteins have been identified in desmosomes. • The molecular composition of desmosomes varies in different cell and tissue types.
  105. 105. Desmosomes are intermediate filament-based cell adhesion complexes • Desmosomes function as both: – adhesive structures – signal transducing complexes • Mutations in desmosomal components result in fragile epithelial structures. – These mutations can be lethal, especially if they affect the organization of the skin.
  106. 106. Hemidesmosomes attach epithelial cells to the basal lamina • Hemidesmosomes, like desmosomes, provide structural stability to epithelial sheets. • Hemidesmosomes are found on the basal surface of epithelial cells. – There, they link the extracellular matrix to the intermediate filament network via transmembrane receptors.
  107. 107. Hemidesmosomes attach epithelial cells to the basal lamina • Hemidesmosomes are structurally distinct from desmosomes. • They contain at least six unique proteins.
  108. 108. Hemidesmosomes attach epithelial cells to the basal lamina • Mutations in hemidesmosome genes give rise to diseases similar to those associated with desmosomal gene mutations. • The signaling pathways responsible for regulating hemidesmosome assembly are not well understood.
  109. 109. Gap junctions : are sites between animal cells that are specilized for Intercellular communication. Gap junctions have a simple molecular compostion they are composed entirely Of an integral membrane protein called connexin. Connexins are organized Into multi subunit complexes called connexons that are completely span the Membrane. Each connexin is composed of six connexin subunits arranged in a ring around A central opening.
  110. 110. Gap junctions allow direct transfer of molecules between adjacent cells • Gap junctions are protein structures that facilitate direct transfer of small molecules between adjacent cells. • They are found in most animal cells.
  111. 111. Gap junctions allow direct transfer of molecules between adjacent cells • Gap junctions consist of clusters of cylindrical gap junction channels, which: – project outward from the plasma membrane – span a 2-3 nm gap between adjacent cells • The gap junction channels consist of two halves, called connexons or hemichannels. – Each consists of six protein subunits called connexins.
  112. 112. Gap junctions allow direct transfer of molecules between adjacent cells • Over 20 different connexin genes are found in humans. – These combine to form a variety of connexon types. • Gap junctions: – allow for free diffusion of molecules 1200 daltons in size – exclude passage of molecules 2000 daltons
  113. 113. Gap junctions allow direct transfer of molecules between adjacent cells • Gap junction permeability is regulated by opening and closing of the gap junction channels, a process called “gating.” • Gating is controlled by changes in – intracellular pH – calcium ion flux – direct phosphorylation of connexin subunits
  114. 114. Plasmodesmata : are cytoplasmic channels that pass through the cell walls of Adjacent cells . Plasmodesmata are lined by plasma membrane and usually contain a dense Central structure called desmotubule, derived from the smooth endoplasmic Reticulum of the two cells
  115. 115. Gap junctions allow direct transfer of molecules between adjacent cells • Two additional families of nonconnexin gap junction proteins have been discovered. – This suggests that gap junctions evolved more than once in the animal kingdom.
  116. 116. CADHERINS The Cadherins are a large family of glycoproteins that mediate Ca2+ dependent Cell- cell adhesion and transmit signals from the ECM to cytoplasm. Cadherins typically join cells of similar type to one another and do so predominantly By binding to the same cadherin present on the surface of neighbouring cell. The best studied cadherins are E-Cadherins (epithelial ), N-cadherins (neural) And P-Cadherin (Placental).
  117. 117. Calcium-dependent cadherins mediate adhesion between cells • Cadherins constitute a family of cell surface transmembrane receptor proteins that are organized into eight groups. • The best-known group of cadherins is called the “classical cadherins.” – It plays a role in establishing and maintaining cell-cell adhesion complexes such as the adherens junctions.
  118. 118. Calcium-dependent cadherins mediate adhesion between cells • Classical cadherins function as clusters of dimers. • The strength of adhesion is regulated by varying both: – the number of dimers expressed on the cell surface – the degree of clustering
  119. 119. Calcium-dependent cadherins mediate adhesion between cells • Classical cadherins bind to cytoplasmic adaptor proteins, called catenins. – Catenins link cadherins to the actin cytoskeleton. • Cadherin clusters regulate intracellular signaling by forming a cytoskeletal scaffold. – This organizes signaling proteins and their substrates into a three-dimensional complex.
  120. 120. Calcium-dependent cadherins mediate adhesion between cells • Classical cadherins are essential for tissue morphogenesis, primarily by controlling: – specificity of cell-cell adhesion – changes in cell shape and movement
  121. 121. Calcium-independent NCAMs mediate adhesion between neural cells • Neural cell adhesion molecules (NCAMs) are expressed only in neural cells. • They function primarily as homotypic cell-cell adhesion and signaling receptors.
  122. 122. Calcium-independent NCAMs mediate adhesion between neural cells • Nerve cells express three different types of NCAM proteins. – They arise from alternative splicing of a single NCAM gene.