MULTICELLULAR ORGANISMS • Cell-Cell Adhesion • Cell-Matrix Adhesion • The Extracellular Matrix, ECM Http://www.plab.ku.dk/bock/index.htm Link: Multicellular organisms 2003 MULTICELLULAR ORGANISMS • The appearance of multicellular organisms allows specialization of cells and formation of organs • Vertebrates have more than 100 specialized cell types (plants have more than 15) • A special matrix, the extracellular matrix, ECM, fills out the space between cells • ECM also binds cells together, acts as reservoir for growth factors and hormones, and creates an environment in which molecules and cells can migrate MULTICELLULAR ORGANISMS • By means of cell adhesion molecules, CAMs, cells are capable of recognizing each other • Plasma membrane receptors take care of cell-ECM interactions CELL JUNCTIONS • Adherens junctions • Gap junctions • Tight junctions • Desmosomes/Hemidesmosomes • Focal adhesions CELL-CELL ADHESION MOLECULES • Cadherins • Ig superfamily CAMs • Selectins • Integrins • Connexins • Occludin and claudin proteins NEURONAL CELL ADHESION MOLECULES LEARNING AND MEMORY • Male humans with L1-mutations develop – Mental retardation – Hydrocephalos – Adducted thumbs • NCAM knock-out animals develop – Morphological changes in bulbus olfactorius and hippocampus – Impaired learning – Emotional disturbances • Modulation of NCAM and L1-function interferes with LTP and learning and memory CADHERINS • A family of Ca2+-dependent CAMs • Ca2+ causes dimerization of Cadherins • The binding is homophilic CELL ADHESION MOLECULES AND DISEASES • Auto antibody against the cadherin desmoglein induce the skin disease Pemphigus vulgaris • Mutations of claudin 16 affect paracellular flow of Mg2+ in the kidney • Mutations of claudin 14 lead to deafness due to an altered transport around hair cells in the cochlea • Several bacterial toxins affect tight junctions leading to increased paracellular transport SELECTINS • Selectins are involved in extravasation • Inflammatory signals activate endothelial cells making P-Selectin undergo exocytosis • P-Selectin on the surface of endothelial cells binds a specific carbohydrate ligand (Sialyl Lewis -x) on leukocytes • The leukocytes attach to the endothelial wall and roll slowly on it • PAF and integrins are then activated and the leukocytes start to extravasate GAP JUNCTIONS 1 • A cluster of channels between two plasma membranes • Each membrane contain a “hemichannel” called a connexon made of 6 subunits - connexins • There are 12 different connexin genes • Usually connexons are hetero-oligomeric and the composition determines permeability GAP JUNCTIONS 2 • Allow particles of < 1.2 nm in diameter to pass • Ions, ATP, cAMP can pass; I.e. hormonal stimulation of one cell can spread to connected cells, and thereby organize coordinated functions such as secretion, contraction, movement of cilia • The channels close at increased Ca2+ concentrations allowing regulation of the degree of coupling to surrounding cells CONNEXIN DISEASES Mutations in several connexin genes are accompanied by: – Deafness – Cataract – Heart malformations – Charcot-Marie-Tooth (degeneration of peripheral nerves) CELL-MATRIX ADHESION • Integrins • Collagens • Laminin and Fibronectin • Proteoglycans and Glucosaminoglycans CELL MATRIX ADHESION • Integrins on the cell surface mediate cell-ECM binding • Integrins are composed of an- and a - chain • There are 3 different -chains and more than 10 types of -chains • The chain composition determines the ligand specificity • The affinity is generally low (Kd 10-6 -10-8) INTEGRINS • Integrins can be activated through a signal from the interior of the cell • Activation involves conformational changes of the integrin • Various integrins recognize specific sequences in their ligands. E.g. 41 recognizes EILDV (in VCAM-1 and in fibronectin) and 51 recognizes RGD in many ECM proteins INTEGRIN CONTAINING JUNCTIONS • A junction consists of an exterior ligand, a transmem- brane protein, a linker, and a cytoskeletal component • An adherence junction connects an ECM component with an integrin linked to an adapter (e.g. vinculin) and F-actin • A hemidesmosome connects an ECM-component to integrin and via an adapter (e.g. plectin) to intermediate filaments (keratins) INTEGRIN DISEASES • Genetic defects in integrin 2 lead to leucocyte-adhesion deficiency. The patient becomes susceptible to bacterial infections DISINTEGRINS • Disintegrins contain the RGD sequence and interfere with integrin-ECM adhesion allowing deadhesion and cell migration • The ADAMs (A Disintegrin And a Metalloprotease) “remodel” surface proteins; f.x. at the fusion of sperm and egg, the fusion of myoblasts during myogenesis, release of TNF from the surface COLLAGENS • The most abundant animal protein • At least 16 types exist • The structural unit is composed of three 300 nm long coiled subunits in a triple helix • The helical structure depends on the abundant presence of glycin, proline (and hydroxyproline) making a motif gly-pro-x, which is necessary for twisting together the three strands COLLAGENS 2 • Collagens are synthesized as precursors called procollagens • They are glycosylated in ER and Golgi adding Gal and Gly to hydroxy-lysine residues and long oligosaccharides to selected asparagine residues • Proline and lysine are hydroxylated • Disulphide bonds are made between the N- and C- terminal parts of the propeptides • After exocytosis the N- and C-terminals are “trimmed”, only then can the fibrils be formed COLLAGENS 3 • Lack of vitamin C prevents hydroxylation impaired fibrils • Mutations or deletions of -chains in Collagen I can lead to the disease Osteogenesis imperfecta LAMININ •Laminin is a key component of the basal lamina DISEASES OF THE BASAL LAMINA • Alport’s syndrome appears as impaired ultrafiltration in the kidney resulting in renal failure and hearing loss. Mutations in collagen IV -chains result in this syndrome. • Antibodies against 3-chains of collagen IV lead to pulmonary hemorrhage and renal failure (Goodpasture’s syndrome) FIBRONECTIN • Fibronectins attach cells to collagens • Fibronectins are dimers • Fibronectins express the RGD sequence recognized by integrins PROTEOGLYCANS 1 • The Polysaccharides in proteoglycans are long repeating polymers of dissacharides called Glucosaminoglycans (GAGs) • One sugar of the dissacharides is a uronic acid and the other is an aminosugar (e.g. N- acetylglucosamine) • One or both sugars contain one or two sulphate residues PROTEOGLYCANS 2 • Heparin sulphate and chondroitin sulphate are added to a 3-sugar “linker” (Xyl-Gal- Gal) added to a Serine in the core protein • Proteoglycans are found both in ECM and attached to the plasma membrane PROTEOGLYCANS IN THE ECM • In cartilage the key proteoglycan is aggrecan • The central component of aggrecan is a carbohydrate, hyaluronan • At 40 nm intervals aggrecan core proteins are attached (assisted by a linker protein) to a decasaccharide sequence in hyaluronan • Attached to the aggrecan core protein are multiple GAGs (via the trisaccharide linker) • The GAGs in aggrecan are chondroitinsulphate and keratin sulphate • MW of an aggrecan 2 x 108 PROTEOGLYCANS ON THE CELL SURFACE • A typical example is syndecan • The core protein spans the membrane with a short cytosolic domain • The GAGs are attached via the trisaccharide linker to serine residues • The GAGs in syndecan are heparan sulphate chains • Syndecan binds extracellularly to collagens and fibronectin and intracellularly to the cytoskeleton HYALURONAN (HA) • HA is a GAG found in ECM • HA is also a key component of complex proteoglycans • HA consists of approx. 50,000 disaccharides in a random coil. It can be bound to the surface receptor CD44 • HA gives strength, flexibility and smoothness to the ECM and forms a viscous hydrated gel in which cells can migrate • HA makes the ECM able to resist compression DISEASES OF GAG Rare genetic defects in enzymes required for the synthesis of Dermatan sulfate lead to defects in bones, joints, muscles, and skin. The individuals do not grow to normal hight and appear prematurely aged.