1. Cell adhesion, junctional structures, the epithelial cell
Prof. Dr. Pál Röhlich
Dr. Anna L. Kiss
Adhesion molecules, glycocalyx, microvilli, cilia, basement membrane, cellular junctions
Department of Anatomy, Histology and Embryology
Semmelweis University
Budapest
2017

2. 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).
Adhesion between cells
Adhesion between cell and extracelluar matrix

3. 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 temporal connections (e.g. in the immune system).
pathology: metastasis of cancer cells, …

4. Cell Adhesion Molecules (CAMs)
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).

5. Cell adhesion molecules
homotypic binding
heterotypic binding

6. Cell adhesion molecules
Ca2+-dependent:
cadherins
selectins
integrins
Ca2+-independent:
IgG-like- CAMs (immunoglobulin superfamily)

7. Cell-cell adhesion molecules
Ca2+-dependent:
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 Ca2+-dependent (without Ca2+ binding, the extracellular part of the molecule collapses and the cells become dissociated).
Cadherins form a relatively strong binding between cells.

8. P-cadherin (placentar, in heart, lung, gut),
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

9. 2. Selectins Ca2+- 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

10. 3. Integrins: Ca2+-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 (αLβ2) Integrin (on leucocytes), binds I-CAM and V-CAM adhesion molecules on endothelial cells. Migration of leucocytes through the wall of the blood vessel.
Mac-1 (αMβ2) Integrin (on macrophages and lymphocytes)
Binding site
α-chain
β-chain

11. Ca2+-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. L1-CAM (cell migration, differentiation), PECAM (adhesion between blood platelets and endothelium).
Disulfide-bond

12. II. Adhesion between cells and extracellular matrix (ECM)
Strong connection between cells and ECM
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).

13. 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.

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

15. 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

16. 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

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

18. Integrins in signal transduction

19. 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.

20. Epithelial cells
Poligonal cells
apical
lateral
basal

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

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

23. Microvilli (brush border)
Microfilaments
Glykocalyx
0.08 m
1 m
Terminal web – bundles of actin

24. Microvilli (brush border)
To increase the surface

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

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

27. Cilia (9x2 +2) and basal body (9x3)

28. 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

29. 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:
TypeIII-collagen

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

31. 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)
Perlecan
Integrin
Dystroglycan
Laminin
Nidogen
lamina densa collagen Type IV 
Fibronectin

32. Biological significance of the basal lamina:
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!).
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.
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.

33. Basal striation
membrane invaginations+mitochondria

34. 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

35. Desmosomes and zonula adherens
adaptor proteins:
(cateins, vinculin, α-actinin)
adaptor proteins
integral membrane protein, adhesion mol.

36. Desmosomes

37. Zonula adherens, desmosomes

38. Hemidesmosome (half-desmosome):
Adhesion molecules: integrins (e.g. laminin receptor α6β4)
Adaptor molecules: plectin, dystonin
Cytoskeleton: intermediate filaments (e.g. keratin in epithelium)
ECM: laminin (lamina basalis)

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

40. Hemidesmosomes

41. Zonula occludens (tight junction)
Integral membrane protein: occludin, claudin
adapter molecules: ZO1, ZO2
cytoskeleton: microfilaments (actin)í

42. Zonula occludens (tight junction)
ZO1, ZO2 protein

43. 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
baso-lateral membrane

44. 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.  

45. connexon (connexin subunits)
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

46. 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.
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:
Cx50 in the crystalline lens of th eye, defects lead to glaucoma
Cx26 in sensory cells of the inner ear, defects cause auditory malfunction
Cx32 in nerve fibers, defects lead to problems in nerve conduction.
opposite plasma
membranes 

47. 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

48. Nexus (gap junction, macula communicans) 

49. Interdigitation

50. Cell junctions in polarized cells
Adhesion molecules, polarized epithelial cells, glycocalyx, microvilli (brush border), cilia, cell junctions, basal lamina
Hemidesmosomes

51. Textbook: Essential cell biology, 3rd 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