Dr.Souvik Chatterjee

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INDEX

Introduction Definitions Classification Tight junctions Adherens junctions Desmosomes Gap junctions Hemidesmosomes and focal contacts Cellular adhesion Techniques for diagnosis of intercellular junctions Summary2

INTERCELLULAR JUNCTIONS It is a type of structure that exists within the tissue of

some multicellular organisms The mechanical integrity of animal tissues such as

epithelia, nerves and muscles depends on the ability of the cells to interact with each other and the extracellular matrix. Plasma membrane specializations called cellular junctions ,mediate this interaction. Intercellular junctions are specialized regions on the

borders of cells that provide connections between adjacent cells.

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Contd Cell junctions consist of protein complexes and provide contact

between neighbouring cells or between a cell and the extracellular matrix. They are also called membrane junctions They also build up the paracellular barrier of epithelia and control

the paracellular transport. Cell junctions are especially abundant in epithelial tissues. On the molecular level intercellular junctions consists of three

components: Transmembrane adhesive protein Cytoplasmic adapter protein Cytoskeletal filament4

CLASSIFICATION OF INTERCELLULAR JUNCTIONSINTERCELLULAR JUNCTIONS

OCCLUDING JUNCTIONS

COMMUNICATING JUNCTIONS

ANCHORING JUNCTIONS

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ContdThe specialized junctions may be further classified as follows: OCCLUDING JUNCTIONS

Tight junctions (Zona Occludens) COMMUNICATING JUNCTIONS

Gap junctions ANCHORING JUNCTIONS

Adhesive junctions Cell to cell Zonula adherens(adheren junctions) Macula adherens (desmosome) Cell to matrix Focal adhesion Hemidesmosomes6

ContdThe various junctions can be defined as: ADHERENS JUNCTIONS : A type of intercellular junction that links cell membranes and cytoskeletal elements within and between cells, connecting adjacent cells mechanically. Transmembrane proteins called cadherins link the neighbouring cells and connect to actin filaments. TIGHT JUNCTIONS :An intercellular junction at which adjacent plasma membranes are joined tightly together by interlinked rows of integral membrane proteins, limiting or eliminating the intercellular passage of molecules.7

.

Composed of branching network of strands, each

strand act independently from others. Each strand is formed from a row of transmembrane protein. Transmembrane protein called claudin joins plasma membrane of 2 cells.

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ContdGAP JUNCTIONS : narrowed portion of intercellular space containing channels. In electrically excitable tissues, these gap junctions transmit electrical impulses via ionic currents and are known as electrotonic synapses. DESMOSOMES: (macula adherences)It is a cell structure specialized for cell to cell adhesionFound in simple & stratified sq. epithelium.9

DESMOSOMES

Ist observed in the spinous layer of epidermis by an

Italian pathologist Giulio Bizzazero. Helps to resist shearing force. Structure that forms the site of adhesion between

2 cells ,consisting of dense plate in each adjacent cells seprated by a thin layer of extracellular material. Desmosomes link 2 cells together.10

HEMIDESMOSOMES:. Small Rivet like structure on the inner basal surface of keratinocyte in the epidermis of

skin. Forms the site of attachment between the basal surface of the cell and the basement membrane.

.

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TIGHT JUNCTIONS Tight junctions, or zonula occludens: closely associated areas of two cells forming a a tight belt like adhesive seal that

selectively limits the diffusion of water, ions & larger solutes as well as migration of cells. It is a type of junctional complex. Thus, separateing the interior of body from the external world.

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In electron microscopic structure of thin sections of tight junctions the plasma membrane of adjacent cells appear to fuse together in a series of one or more contacts.

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Two structural have been identified in the structure of

tight junctions namely: Occludins Claudin

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A.Preliminary model of tight junction structure with claudin linking the two membranes together & peripheral protein zo-1 linking the cytoplasmic tail of claudin to actin filaments. B-C. transmembrane topology of claudin & occludin.

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Tight junctionsExtracellular domains of claudins form rows of pores along tight

junction.Each claudin has a unique selectivity for cations or anions. At the zona occludens the membranes of adjoining cells converge

and are at a distance of 0.1- 0.3 m

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Contd The cytoplasmic tails of claudins

interact with numerous proteins with roles as scaffolds and in actin binding, signalling and cell polarity. Tight junctions are the barrier that

segregates different pumps, carriers, receptors and lipids in the apical and basolateral domains of plasma membrane. Cell attachment is stronger at zonula occludens.23

Tight junctions They may be tight as in distal convoluted tubule of

kidney or leaky as in blood vessels. Transepithelial barrier established by tight junction,

regulated by hormones like: Vasopressin Aldosterone Cytokines

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Tight junctions Several bacterial toxins also effects tight junction barrier. These toxins disrupts tight junction barrier ,thus breaking

the barrier that protects underlying tissues . Mutation in individual claudin gene results in highly

selective defects.

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Adherens junctions and Desmosomes are two types of adhesive junctions using homophilic interactions of cadherins to bind epithelial cells to adjacent cells.

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ADHERENS JUNCTIONS The zonula adherens is a band like specialization of the

membrane and cytoplasm that encircles the apex of adjoining cells and strongly bonds the cells together. In this junction the opposing membranes are 15 20 nm

apart . It is a major site of epithelial cell cohesion.

adherent junction Cytoplasmic actin filaments bind adherens junctions. Homophilic interactions between densely clustered E-cadherens

(the epithelial transmembranic adhesive protein) bind adjacent cells together at adherens junctions. -

catenin(cytoplasmic adapter protein) and Plakoglobin (desmosomal cytoplasmic adapter protein) bind the cytoplasmic domains of E-cadherin.

adherent junction An another cytoplasmic adapter protein, -catenin, binds

cadherins to actin filaments and -catenin to actin filaments. Adherens junctions are first connections that are established

between developing sheets of epithelial cells. The contact begins when cadherins on the tips of filopodia engage

to the cadherins of another cell.

adherent junction

Adherens junctions are a pre requisite for tight junctions that

allow epithelial cells to establish polarity with proteins and lipids in plasma membranes. Zonula adherens is the major site for cell cohesion. It stabilizes the surface of epithelia. The junctions and polarity determine the orientation of mitotic

spindle and the plane of division . This allows for asymmetrical division of stem cells (stratified epithelium).

adherens junction In mature columnar epithelia a belt like adherens junction called

zonula adherens encircles the cell near the apical surface thus maintaining physical integrity of the epithelium.

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DESMOSOMES Desmos means bound , Soma means body.

It is also called Macula Adherens. Provide strong adhesion between the epithelial and muscle

cells. These junctions are small disk shaped spot welds

between adjacent cells.

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desmosomes Cellular

adhesions at transmembrane proteins:

desmosomes

are

mediated

by

Desmogleins

Desmocollins. Plakoglobin also called gamma- catenin. Molecular composition of desmosomes vary in particular tissues

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desmosomes Desmosomes are site for attachment, structural ability of

epithelium linking cytoskeletal structures of two cells. Desmoglein -2 and desmocollin -2

are found in most of the

desmosomes. The devlopment of animal tissues depends on desmosomes &

their constituents proteins.

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GAP JUNCTIONS Gap junctions are plaque that contain large intercellular

channels that connect the cytoplasm of a pair of cells.Half channels in each membrane are called connexons.

Connexons consists of six protein subunits, called connexins.

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gap junctions Connexin are named by their molecular weight. Found exclusively in chordates.

Most connexons pair with identical connexons on the

partner cell to form homotypic gap junctions.

Gap junction communication is conditional It depends on: Number of channels Fraction that are open or closed38

gap junctions Plants lacks gap junctions Cells in plant tissues maintain continuity through

plasmodesmata. Molecules smaller than 1kd diffuse freely through

plasmodesmata.

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gap junctions Oleamide- fatty acid amide produced by the brain , blocks gap

junction and induce sleep in animals. Gap junctions allow osteocytes to maintain cellular supply line to

acquire nutrients from distant blood vessels. White blood cells may also form transient gap junctions with

endothelial cells. Cells in most metazoans communicate by gap junctions.

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gap junctions

Mutations in connexins genes cause human disease. Recessive mutation in the connexin -26 gene are most

common cause of human Deafness. Mutation in connexin-32 gene causes degeneration

of myelin sheets around axons.

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HEMIDESMOSOMES Hemidesmosomes

Basal Cells

TonofilamentsLamina lucida Lamina densa Anchoring filaments Anchoring fibrils

Proteoglycan s

Type IV collagen43

HEMIDESMOSOMES Hemidesmosomes are adhesive

junction that link cytoplasmic filaments to basal lamina. Adhesion to extracellular matrix is different from intercellular adhesion because integrins provide transmembrane link between cytoskeleton and extracellular matrix.44

Contd Two transmembrane proteins mainly found in hemidemosomes

are 64 integrin and type XVII collagen. Outside the cell 64 integrin binds to laminin-5 in basal lamina. The extracellular collagen triple helix forms anchors filaments

between membrane and basal lamina.

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Contd STRUCTURE OF HEMIDESMOSOME: Adhesive protein INTEGRIN Cytoplasmic proteins PECTIN, BP 180 Cytoskeletal element - INTERMEDIATE FILAMENTS Target molecule - LAMININ.

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CELLULAR ADHESIONPRINCIPLES OF CELLULAR ADHESION1.

First principle of adhesion

Cells define their capacity for adhesive interactions by selectively

expressing plasma membrane receptors with limited ligand binding activity. For example: Endothelial cells produce E selectin only when stimulated by inflammatory hormones

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Contd2. Second principle of adhesion Many adhesion proteins bind one main ligand and many ligands bind a single type of receptor, for example Most cadherins bind to themselves, such homophilic interactions require Ca+2 ions.

3. Third principle of adhesion It states that cell modulate adhesion by controlling the surface density, state of aggregation and state of activation of their adhesion receptors.

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Contd4. Fourth principle of adhesion The rates of ligand binding and dissociation are important determinants of cellular adhesion. Many cell surface adhesion proteins bind their ligands weakly in comparison with other specific macromolecules. 5. Fifth principle of adhesion Many adhesion proteins interact with the cytoskeleton inside the cell.

6. Sixth principle of adhesion Association of ligands with adhesion receptors can activate intracellular signal transduction pathways, leading to changes in gene expression, cell division, cell activation.50

CADHERIN FAMILY OF ADHESION RECEPTORS Cadherins name is derived from calcium dependent adhesion

protein. Homophilic interactions of cadherins link epithelial and muscle

cells to adjacent cells at specialized junctions called adherens junctions and desmosomes. The cytoplasmic domains of cadherin junctions interact with actin

filaments to maintain physical integrity of tissues.

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Contd Structural Hallmark of Cadherin family is CAD domain

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Cadherins are named according to their location or cells to which they are attached. for eg:Epithelial tissue -E-cadherinNervous tissue - N-cadherin Placenta- P-cadherin

osteoblasts- O-cadherinKidney-K cadherin muscle -M-cadherin

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SIGNIFICANCE OF CADHERIN Cadherins and catenins participate in transduction of

extracellular signals that control migration and differentiation.

cell

proliferation,

How? By contact inhibition.

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cadherin Loss of E- cadherin expression also promotes tumour metastasis

to lymph nodes and higher frequency of blood- borne (bone and lung) distant metastasis. Loss of E -cadherin can contribute to transition from benign to

malignant tumors. Genetic defects in E-cadherin causes stomach cancer. Epithelial structures are not formed if cadherin expression is

impaired.56

INTEGRIN- FAMILY OF ADHESION RECEPTORS Integrins are the main cellular receptors for extra cellular matrix. Integrins tend to be more promiscous than most adhesion

receptors as some bind to several protein ligands & many matrix molecule bind to 1 integrin.

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Integrins are hetrodimers of two transmembrane

polypeptides called & chains which imparts ligand binding specifity.

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Figure

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integrins

The ligand binding domain of & chains form a

globular head connected to the plasma membrane by 16 nm legs. Both the chains participate in binding at least 2 sites

on ligands. With the exception of red blood cells integrins are

present in plasma membrane of most animal cells.60

Ligands on both side

of plasma membrane influence the conformation of integrins. Cytoplasmic tails of

integrins interact directly or indirectly with a remarkable variety of signaling & structural protien.61

Integrin binding to matrix ligands initates signals that

modify cellular adhesion, locomotion & gene expression.

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SELECTIN - FAMILY OF ADHESION RECEPTORS The defining feature of selectins is a calcium dependent lectin

domain. The lectin domain sits at the end of a rod shaped projection that is

anchored to the plasma membrane by a single transmembrane sequence. Natural ligand for selectins are mucin like glycoproteins

expressed on WBCs and endothelial cells.

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selectin Bonds between selectins and their mucin ligands have high tensile

strength but form and dissociate rapidly. Few selectin mucin bonds are required to togehter WBCs to

endothelium, brief lifetime of this bond allows rolling motion over the surface of endothelium. Inflammatry mediators also regulate selectin in several ways. How?

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TECHNIQUES FOR DIAGNOSIS OF INETRCELLULAR JUNCTIONSHISTOCHEMISTRY Histochemistry has gained lot of importance in recent

years. Many histochemical and special stains can be carried out on formalin-fixed, paraffin embedded material . The periodic acid- Schiff (PAS) stain demonstrates the

presence of glycogen, mucoproteins.

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Contd The PAS reaction consists of oxidation of adjacent hydroxyl

groups in 1,2 glycols to aldehydes and staining of aldehydes with fuchsin sulfuric acid. The PAS reaction is of value is studying the thickening of

basement membrane

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IMMUNOFLUORESCENCE Immunofluorescence is a method of determining the

location of antigen (or antibody) in a tissue section or smear by the pattern of fluorescence resulting when the specimen is exposed to the specific antibody (or antigen) labelled with a fluoro chrome. Immunofluorescence

is a technique used for light microscopy with a fluorescence microscope .

This technique uses the specificity of antibodies to

their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample.75

Contd Immunofluorescence

is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualise the location of the antibodies

The immunofluorescent dyes are rhodamine and flurescein.

Immunofluorescence is of two types: DIRECT IMMUNOFLUORESCENCE

INDIRECT IMMUNOFLUORESCENCE

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DIRECT IMMUNOFLUORESCENCE Direct

immunofluorescence testing has a valuable diagnostic role in autoimmune diseases and inflammatory mucocutaneous diseases including auto immune mediated blistering diseases.

Primary, or direct, immunofluorescence uses a single

antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to

it, and the fluorophore it carries can be detected via microscope.

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Contd This technique has several advantages over the secondary

(or indirect) protocol below because of the direct conjugation of the antibody to the fluorophore. This reduces the number of steps in the staining procedure,

is therefore faster, and can avoid some issues with antibody cross-reactivity or non-specificity, which can lead to increased background signal.

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INDIRECT IMMUNOFLURESCENCE Secondary, or indirect, immunofluorescence uses two

antibodies; the first (the primary antibody) recognises the target molecule and binds to it, and the second (the secondary antibody), which carries the fluorophore, recognises the primary antibody and binds to it.

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Contd Antibodies can be prepared by the known antigen into an animal

to provoke immune response. This response can be results in antibody production and it can be

isolated from serum of the injected animal. when a solution containing an antibody is directly applied to a

tissue section containing the antigen, the antibody binds specifically to that antigen. The antigen-antibody complex is subsequently attached to a

second antibody, which is conjugated either by a fluorescent dye (rhodamine) or enzyme (peroxidase-antiperoxidase)81

Contd The antigen antibody complexes are bound to a fluorescent dye

where the antigenic sites fluoresce against a dark background and are immediately photographed on high speed film. The enzyme bound antigen- antibody complexes are further

developed histochemically by exposure to enzyme substrate This results in development of black color which allows

examination of antigenic sites.

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ELECTRON MICROGRAPHY It is a technique where beam of electrons is

transmitted through an ultra thin specimen interacting with the specimen as it passes through. An image is formed from the interaction of the

electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as fluorscent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.84

ELECTRON MICROGRAPHY

Transmission electron microscopy is beneficial where

immunohistochemistry is negative. Using electron microscopy the identification of intercellular

junctions can provide an important diagnostic aid. It is also used in subtype determination of epidermolysis

bullosa. For optimum results the fresh tissue must be fixed in

karnovsky medium (paraformaldehyde and glutaraldehyde) and stored in refrigerator until processing.85

IMMUNOHISTOCHEMISTRY This technique is used to diagnose poorly differentiated

malignant tumours and lymphomas. They can also be beneficial in diagnosis of bullous diseases. With refinement of technique

immunohistochemistry methods have achieved the same sensitivity for many antigens as direct immunofluorescence method.

The immunohistochemical techniques are based on the fact

that protein- based antigens bind to their specific antibody.

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SUMMARY Intercellular junctions are fundamental to the interactions between cells. Mucosal barrier integrity is maintained by the physical interactions of

intercellular junctional molecules on opposing epithelial cells.

In the heart, cell junctions form the low-resistance pathways for rapid

impulse conduction and propagation, enabling synchronous stimulation of myocyte contraction. creating a balance between osmotic gradients.

In kidney, cell junctions help to maintain concentrations of fluid

By means of these junctions, the activities of the individual cells that

make up tissues are co-ordinated, enabling each tissue system to function as an integrated whole.

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Contds They are connected to each other via membrane-associated structures usually transmembrane proteins. These membrane-associated structures help in cohesion and

communication and are called intercellular junctions.

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Contd The defects in intercellular junctions results in various

autoimmune diseases, carcinomas, acute and chronic infections. These results also suggest that a tumor's ability to spread and

metastasize is inversely related to its number of intercellular junctions.

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