Cell adhesion junctional structures the epithelial cell Prof

Cell adhesion, junctional structures, the epithelial cell Prof. Dr. Pál Röhlich Dr. Anna L. Kiss Department of Anatomy, Histology and Embryology Semmelweis University Budapest 2017

Adhesion Specific bindings between a. ) individual cells b. ) cells and extracellular matrix (ECM). - Integrates cells in a multicellular organism, - crucial for organisation of tissues and organs. - intracellular anchoring to filaments of the cytoskeleton (actin microfilaments or intermediate filaments). I. Adhesion between cells II. Adhesion between cell and extracelluar matrix

I. Adhesion between cells: Adhesion proteins in the plasma membrane (integral membrane proteins). Significance: • in embryonic development (cell associations, switching between cell associations, specific attachment between nerve and muscle, formation of interneuronal connections (synapses), formation of epithelial layers, etc. …) • in adult orgnisms: • long lasting connections (e. g. epithelia, nervous system) • or • pathology: metastasis of cancer cells, … temporal connections (e. g. in the immune system).

Cell Adhesion Molecules (CAMs) - are intramembrane (transmembrane) proteins located on the cell surface; - involved with the binding with other cells or with the extracellular matrix (ECM) in the process called cell adhesion; - many weak bonds are formed between the cells. These proteins are typically transmembrane receptors and are composed of three domains: a. ) an intracellular domain that interacts with the cytoskeleton, b. ) a transmembrane domain, c. ) an extracellular domain that interacts either with other CAMs of the same kind (homophilic or homotypic binding) or with other CAMs or the extracellular matrix (heterophilic or heterotypic binding).

Cell adhesion molecules homotypic binding heterotypic binding

Cell adhesion molecules Ca 2+-dependent: cadherins selectins integrins Ca 2+-independent: Ig. G-like- CAMs (immunoglobulin superfamily)

Cell-cell adhesion molecules Ca 2+-dependent: 1. Cadherins: Adhesion proteins with a single transmembrane portion. The extracellular part usually consists of 5 repeating domains. Extracellular tip of the molecule (last domain) binds a similar domain of cadherin in the opposite membrane (homotypic binding). Stability of the domains is Ca 2+-dependent (without Ca 2+ binding, the extracellular part of the molecule collapses and the cells become dissociated). Cadherins form a relatively strong binding between cells.

Cadherin-superfamily (about 180 cadherin types in human, „variations on a theme”), Nomination according to cell type and characters, recently numbers. Classical cadherins: E-cadherin (epithelial, in cells of epithelium and morula), P-cadherin (placentar, in heart, lung, gut), N-cadherin (neural, in development of the nervous system, synaptogenesis), M-cadherin (muscular, myoblasts), R-cadherin (retinal, outgrowth of nerves), VE-cadherin (vascular endothelial), K-cadherin (kidney), … In a single cell several cadherin types can occur, expression of cadherins. Appearance of E-cadherin in the morula stage of embryonic development. Mouse. Cells of the morula closely adhere to one another: compaction. 1. 5 days 3. 5 days

2. Selectins Ca 2+- dependent, weak heterotypic bindings. The terminal domain has binding site for certain membrane glycoproteins of other cells. Role: recirculation of lymphocytes in the organism, adhesion of leucocyte to the endothelial surface of blood vessels. („rolling”), … L-Selectin (on leucocytes), P-selectin (on blood platelets), E-selectin (on endothelium) anchoring proteins actin filaments

3. Integrins: Ca 2+-dependent, heterotypic binding, α and β chains, • binding site on the extracellular end. • 24 different integrins. Large majority binds to components of the extracellular matrix, but a few of them to cells. These are: LFA-1 (αLβ 2) Integrin (on leucocytes), binds I-CAM and V-CAM adhesion molecules on endothelial cells. Migration of leucocytes through the wall of the blood vessel. Binding site Mac-1 (αMβ 2) Integrin (on macrophages and lymphocytes) α-chain β-chain

Ca 2+-independent Immunoglobulin-like adhesion molecules: CAMs No Ca-dependence! Immunoglobulin-domains. Weak adhesion, fine regulation. N-CAM (neural cell adhesion molecule). Homotypic bindings. Negative charges on the sialic acid molecules can modulate binding. Role: in development of the nervous system, formation of ganglia, outgrowth of nerve fibers. I-CAM, V-CAM (in the lining cells of blood vessels). Heterotypic bindings with integrins of leucocytes. L 1 -CAM (cell migration, differentiation), PECAM (adhesion between blood platelets and endothelium). Disulfide-bond

II. Adhesion between cells and extracellular matrix (ECM) Strong connection between cells and ECM: produced by the cells: 1. ) fibers; 2. ) proteoglycans, glucosaminoglycans; 3. ) matrix binding glycoproteins 1. ) Connective tissue fibers: Collagen fibers: Large family of collagen proteins. Most frequently occur: collagen type I: composed of fibrous collagen molecules, resistant against pull, thin collagen fibrils form bundles of various thicknesses: thick collagen fibers. collagen type III: can form thin fibers often interconnected with each other (reticular fibers).

2. ) Glycosaminoglycans, proteoglycans. Glycosaminoglycans (GAGs) are long sugar chains, composed of disaccharide units and carrying acidic groups (carboxyl, sulfate), therefore they contain many negative charges. High water-binding capacity! Proteoglycans (PGs): consist of a core polypeptide chain and of GAG chains laterally bound with covalent bonds.

3. ) Matrix-binding glycoproteins: heterogenous proteins many binding sites for ECM components and cells. They form bridges in between them. The most promeinent matrixbinding proteins: laminin és a fibronectin.

II. Adhesion between cells and extracellular matrix (ECM): integrins Functions of integrins: : • Adhesion between cell and ECM (Attachment of the cell onto the underlying structure is important!) • Local integration of the cytoskeleton with the ECM • Signal transduction from inside outward and reversed Indirect significance: cell migration, cell division, development, differenciation

Integrin as signal transduction molecule Active and inactive forms. Binding of a ligand to the integrin molecule on one side of the membrane activates the molecule on the other side of the membrane (transduction of a signal across the membrane). The role of talin. Activation of signal transduction pathways (binding to the β-chain activates extracellular part of the molecule which in turn strongly binds ECM components. ) ligand binding talin binding

Integrins play important role in cell migration Integrin internalistion: via caveolae Recycling/or lysosomal degradation

Integrins in signal transduction

The epithelial cell Surface epithelium: Epithelial cells are attached to each other laterally and form a continuous layer on free surfaces. General characteristics of the epithelial cell are introduced on the example of the columnar epithelium. The epithelial cell is a polarized cell: cell organelles, cytoskeletal constituents, membrane domains and cell junctions are arranged in a characteristic pattern and orientation. Apical (luminal) surface apical surface Golgi gap junction centrosome lateral surface Intestinal epithelium basal lamina basal surface Columnar epithelial cell showing localisation of cell junctions and surfaces.

Epithelial cells Poligonal cells apical lateral basal

Apical surface • • glykocalyx microvilli (brush border) stereocilium cilia, flagellum Function: – – – protection (mechanical, biological) diffusion barrier absorption, movements secretion (exocytosis) transcytosis

Glycocalyx Function cell-cell recognition (MHC, blood groups) enzyme-function (intestine: enterocytes) cell membrane

Microvilli (brush border) Microfilaments Glykocalyx 0. 08 m 1 m Terminal web – bundles of actin

Microvilli (brush border) To increase the surface

Stereocilia length: 8 -10 µm diameter: 1 µm actin filaments stereocilia Ductus epidydymis

Cilia length: 5 -10 µm diameter: 0, 2 µm Function: movements Structure: microtubules (tubulin) nexin (connects microtubules) dynein (outer and inner)

Cilia (9 x 2 +2) and basal body (9 x 3)

Basal surface Basement membrane: inhibits the free diffusion induces polarity regeneration filter (kidney) epithelial cell • lamina basalis lamina lucida (rara) lamina densa • lamina fibro-reticularis

Basal surface Basal or basement membrane (light microscope) – Basal lamina: Type IV-collagen + adhesion molecules Glycoproteins (laminin, fibronectin) + proteoglycans • Lamina rara externa (lamina lucida) • Lamina densa • Lamina rara interna – Lamina fibroreticularis: Type. III-collagen

Basement membrane A thin layer (40 -120 nm) under the basal surface of the epithelium, only seen with the electron microscope lamina rara lamina densa Electron microscopy lamina fibroreticularis

Molecular structure of the basal lamina. Components: Collagen type IV (these fibrous protein molecules form a multilayered network: lamina densa, a resistant and plastic layer) Laminin (matrix-binding molecule) Perlecan (proteoglycan) Nidogen (a small protein with multiple binding sites) Fibronectin (matrix-binding molecule) Binding to the plasma membrane by: integrin (laminin-receptor), dystroglycan and syndecan (membrane proteoglycans) Integrin Perlecan Dystroglycan Laminin Nidogen Fibronectin lamina densa collagen Type IV

Biological significance of the basal lamina: 1. mechanical: a. ) binds the epithelial layer firmly to the ECM. Important e. g. in the strong binding of the epidermis to the underlying connective tissue. The basal lamina is a continuous layer, b. ) inhibits immigration of connective tissue cells into the epithelium or free emigration of epithelial cells into the connective tissue (cancer!). 2. cell biological: the basal lamina sends information signals through integrins into the cell interior and is therefore important in survival and division of the epithelial cell as well as in the maintenance of cell polarity. During epithelial regeneration it „leads” the epithelial cells to cell -free areas. Without basal lamina no continuous epithelial layer can be formed. Genetic defects of basal lamina proteins are lethal for the embryo in an early developmental stage. 3. molecular sieve: filters blood from the capillaries of the renal glomerulus (ultrafiltrate) and retains proteins and cells in the blood. In genetic defects of one of its molecular components blood or blood proteins appear in the urine. The pore size of the filter depends primarily on proteoglycans in the basal lamina.

Basal striation membrane invaginations+mitochondria

Lateral surface: cell junctions • Mechanical connecting structures: – macula adherens (desmosoma) – zonula adherens – hemidesmosoma • Diffusion barriers – zonula occludens (tight junction) • Communicating junctions – nexus (gap junction) Long-lasting coupling between cells and between cell and ECM with various functions. General structure: adhesion molecules, adaptor proteins, cytoskeleton

Desmosomes and zonula adherens adaptor proteins: (cateins, vinculin, αactinin) adaptor proteins integral membrane protein, adhesion mol.

Desmosomes

Zonula adherens, desmosomes

Hemidesmosome (half-desmosome): Adhesion molecules: integrins (e. g. laminin receptor α 6β 4) Adaptor molecules: plectin, dystonin Cytoskeleton: intermediate filaments (e. g. keratin in epithelium) ECM: laminin (lamina basalis)

Hemidesmosomes • It makes stronger the connection of the epithelial cells to the lamina basalis integrins

Hemidesmosomes

Zonula occludens (tight junction) Integral membrane protein: occludin, claudin adapter molecules: ZO 1, ZO 2 cytoskeleton: microfilaments (actin)í

Zonula occludens (tight junction) ZO 1, ZO 2 protein

Functions of tight junction • Diffusion barrier in the plasma membrane (blocking lateral diffusion in the membrane), the belt- like tight junction divides the plasma membrane into an apical and baso-lateral domain (with different sets of molecules) • Diffusion barrier in the intercellular space: free diffusion of substances between neighboring cells is blocked. • Biological significance: controlled and unidirectional transport across the epithelial layer apical membrane baso-lateral membrane

Zonula occludens (tight junction) Cell junction functioning as a diffusion barrier in the intercellular space and in the plasma membrane. Localisation: belt-like, running circumferentially close to the luminal surface.

Nexus (gap junction) Patch-like contacts between two cells: large number of channels (connexons) in the opposite membranes, that are bound to each other in the intercellular space to form continuous channels between the two cells. cell membranes intercellular space connexon (connexin subunits) 2 connexons

Molecular structure: connexon: a complex composed of 6 transmembrane proteins (connexins), that surround a central canal. A similar complex in the opposite membrane is joined to it in the intercellular space. A continuous canal is formed which leads from one cell into the other making communication by free diffusion of low molecular weight substances possible. opposite plasma membranes Connexin: Transmembrane proteins Gap junction: patch-like membrane domain with densely packed connexons Diversity of connexons, in different cell types. Combinations of different connexins in a connexon. Examples of connexin isoforms: Cx 50 in the crystalline lens of th eye, defects lead to glaucoma Cx 26 in sensory cells of the inner ear, defects cause auditory malfunction Cx 32 in nerve fibers, defects lead to problems in nerve conduction.

Nexus Biological significance: Communication between cells: nutrient transport (cells of the lens, osteocytes, follicular cells surrounding the oocyte in the ovary), synchronized reaction for signals during the development etc. Electrical connection between cells: electrical synapsis: ions can migrate between cells, stimulus can be transmitted without delay; In heart muscle: synchronized contraction

Nexus (gap junction, macula communicans) gap junction

Interdigitation

Cell junctions in polarized cells Hemidesmosom es

Textbook: Essential cell biology, 3 rd ed. p. 689 -690, 693 -707, + szövettan + lecture summarized in the present ppt presentation Sources of figures: Röhlich: Szövettan, 3. edition, Semmelweis, Budapest Alberts – Johnson – Lewis – Raff – Roberts – Walter: Molecular biology of the cell. 5. edition, Garland Science Specimens and/or micrographs, or drawings of P. Röhlich Junqueira – Carneiro: Histologie, 6. edition, Springer