Junctions between cells creates pathways for communication, so that cells can exchange signals that coordinate their behavior and can regulate their

pattern of gene expression

Two main ways in which animal cells are bound together;

•  connective tissue (depends on the extracellular matrix)

•  epithelial tissue (depends on cytoskeleto of the cells, linked from cell to cell by anchoring junctions)

Cell junctions – diverse in structure and with multiple assignments

1.  Anchoring junctions

Including both cell-cell adhesion and cell-matrix adhesion. Transmits stresses and are fasten to cytoskeletal filaments inside the cell

2.  Occluding junctions

Seal the gap between cells in the epithelia, and makes the cell sheet into a selective semiperimable barrier.

Cell junctions – diverse in structure and with multiple assignments

3.  Channel-forming junction

Create passages between cells, and thereby linking the cytoplasm

4.  Signal-relaying junctions

Allow signals to be relayed from cell to cell across their plasma membrane, at sites of cell-cell contact.

For example; chemical synapses in the nervous system and immunological synapses

CADHERINS AND CELL-CELL ADHESION

Cell-cell adhesion are most clearly seen in mature epithelia, where the cells are held together by strong anchorage of cell to cell

Cell-cell adhesion

•  thight/occluding junctions: close to the apex. Prevents leaking across cells

•  adherens junctions: anchorage sites for actin

•  desmosome junctions: anchorage sites for intermediate filaments

•  gap /channel-forming junctions: passageway for small water-soluble molecules

For all types of anchoring junctions the central role is played by

Transmembrane adhesion proteins

•  spans the membrane

•  one end linked to the cytoskeleton inside the cell, and the other linked to structures outside

•  two superfamilies of proteins

•  CADHERIN SUPERFAMILY (cell-cell)

•  INTEGRIN SUPERFAMILY (cell-matrix)

•  specialization within each superfamily

Cadherin and cell-cell adhesion

•  Cadherins are present in all multicellular animals, and absent in fungi and plants as well as bacterias.

•  Cadherins are dependent of Ca2+ ions (thereby the name);

Removing the calcium ions from the extracellular medium causes cadherin mediated adhesion.

•  all cells in vertebrates express one or more proteins of the cadherin family

The protein E-cadherin is importen in embryo development, making the embryo cells stick together. Mutations in E-cadherin is embryo lethal.

Cadherin superfamily

•  all members have a similar extracellular cadherin motif (green ovals)

•  the intracellular varies, depending on the type of intraction

•  nonclassical cadherins are also found, some primarily involved in dignaling (T-cadherin)

•  the cadherin superfamily consist of 180 members

Cadherin

Binding between cadherins are homophilic,

Cadherin in one cell bind to the same type of cadherin in the adjecent cell

Binding occurs at the N-terminal end, the end that lies away from the membrane.

Cadherin

1.  The cadherin domain in presence of Ca2+ is a rigid rod

2. When Ca2+ is removed the domain can flex, and the conformation is changed so a enhancing bindig to the opposite cell.

Cadherins bind with low affinity, but the formation of many weak bonds in parallel give a strong attachment afterall. Making and breaking of anchoring junctions is important for the constant turnover of tissues!

Cadherin – selective cell-cell adhesion

Cadherins are not like any glue, they mediate highly selective recognition.

Cells from amphibian embryos dissociated, mixed, and then allowed to reassociate. Mesoderm cells (green), neural plate cells (blue) and epidermal cells (red) sort out in a normal arrangement.

Cells depend on guidance from tissue along the path of ”movement”, involving;

•  chemotaxis

•  chemorepulsion movement under the influence of chemicals that attract or repel migrating cells

•  contact guidance migrating cells touch other cells or extracellular matrix components, making transient adhesion that govern the selected track.

In the processes of sorting out, contact guidance and tissue assembly cadherins play a crucial part!

Cadherin – involved in epithelial-mesenchymal transitions

Assembly of cells into an epithelium is a reversible process.

With active adhesion molecules, mesenchymal cells (dispersed unattached cells) can come together and form an epithelium.

An epithelium can also disperse and migrate away as separate cells.

Epithelial-mesenchymal transitions play an important role in normal development

A control system seems to be critical for EMT, consisting of the following gene regelatory components:

•  Slug

•  Snail

•  Twist

These three have E-cadherin as a downstream component.

EMT also occur during cancer. Mutations that disrupt the production of E-cadherin are found in cancer cells, and are thought to help make them malignant.

Signalling events during EMT

Catenins links classical Cadherins

•  The cadherin linkage to the cytoskeleton is indirect and depends on a cluster of intracellular anchor proteins.

•  The components vary according to type of anchorage, but in general you will find beta-catenin and/or gamma-catenin to play a central part

•  In adherens junctions, you will also find a remote catenin relative – p120-catenin

Adhesion junctions

Adherens junctions occur in various forms

1.  In nonepithelial tissue they are small punctate attachments, indirectly connecting cortical actin filaments of to interacting cells

2. In epithelial cell they form an adhesion belt close beneath the apical face of the epithelium

•  The contractile bundle of actin filaments lies adjecent to the adhesions belt, parallel to the plasma membrane.

•  The bundle is ancored with the help of cadherin and other associated anchoring proteins.

•  This network, together with the myosin motor proteins, can contract and help folding the epithelial cell sheet into tubes, vesicles and so on.

Desmosome junctions

Desmosome junctions are similar to adherens junctions, but they link intermediate filaments instead of actin.

The general structure of a desmosome Proteins in the desmosome

The bundles of intermediate filaments that are anchored to the desmosome have a great tensile strenght. What kind of intermediate filament anchored to the desmosome depend on the cell type (keratine in epithelial cells, desmin in heart muscle cells..)

Selectins

One of three other superfamilies (integrins, selectins and Ig), in addition to the cadherins, that is central in cell-cell adhesion

•  Selectins are cell-surface carbohydrate-binding proteins (lectins)

•  mediates cell-cell adhesion interactions in the bloodstream

•  important in inflammatory responses and in trafficking of the white blood cells

•  selectins are transmembrane proteins, with a conserved lectin domain

Three types;

1) L- selectin (on white blood cells)

2) P- selectin (blood platelets/endothelial cells)

3) E- selectin (on activated endothelial cells)

Selectin act together with integrins.

The selectins control the binding of white blood cells to the endothelial cells lining blood vessels.

There they can ”help” the blood cells migrating out of the bloodstream and into the tissue ( in collaboration with integrin)

Selectins

•  Selectin mediates a weak binding, and the white blood cells are rolling along the surface of the vessel…

•  …until the blood cell activates its integrin!

The white blood cells integrin will then adhere to proteins on the surface of the endothelial cells

•  and the blood cell can crawl into the tissue

Immunoglobulin (Ig) superfamily

The endothelial cell proteins that are recognized by the white blood cell integrins (ICAM and VCAM) are members of the Ig superfamily of surface proteins

•  contain one or more extracellular Ig-like domains (characteristic to antibodies)

•  unrelated to immune defenses

•  not at strong as cadherin

•  contributes to ”fine-tuning” adhesion interactions

•  important in specialized adhesive phenomena (e.g. white blood cells)

Cell adhesion molecules involved in synapse formation

Especially the nervous system rely on complex systems of adhesion molecules ( in addition to chemotaxis/signal factors) guide axon outgrowth, and thereby specific nerve connections

Synapse formation include:

1.  pre- and postsynaptic cell recognition/adherens

2.  signal receptores

3.  ion channels

4.  synaptic vesicles

5.  docking proteins

SO HOW ARE THEY RECRUITED AND HELD IN PLACE?

Scaffold proteins are thought to be involved in both processes

Scaffold proteins contain several PDZ domains, that recognize and bind the C-terminal intracellular tails of specific transmembrane molecules

Cell adhesion molecules involved in synapse formation

The scaffold molecules can also bind to eachother, and make up a mat of proteins needed in the synapse

The scaffold proteins are also essential in; !- formation of occluding junctions !- control of cell polarity !- control of cell proliferation!

TIGHT JUNCTIONS AND THE ORGANIZATION OF EPITHELIA

60% OF THE CELL TYPES IN THE VERTEBRATE BODY ARE EPITHELIAL

•  almost all epithelial cells are anchores to other tissue on one side – the basal side

•  and they are ”free” on the other side – the apical side

•  they are thereby polarized

Almost all epithelial cells have one function in common, they are selective permeability barriers;

Separating one fluid on the basal side

..from another fluid on the apical side

This require that neighboring cells are sealed together, so that molecules can not leak freely across the cell sheet

THIS IS A JOB FOR OCCLUDING JUNCTIONS

Thight junctions

Occluding junctions are called thight junctions, and play and important role in transcellular transport

•  transcellular transport depend upon two sets of transport proteins,

1.  one set transport nutrients from the lumen/gut side and into the cell

2.  another set transport the same molecules out of the cellon the basolateral side (facilitated diffusion)

•  For this transport to be effective spaces between the cells has to be thightly sealed

•  Important to ensure that the transport molecules cannot leack back into the lumen

Thight junctions

•  Thight junctions are impermeable to macromolecules, but the permeability to small molecules varies. Thight junctions in the small intestine have a different permeability ”pattern” than thight junctions in the epithelium of the urinary bladder.

Extracellular molecules are prevented to enter to epithelium by the thight juctions

Thight junctions

Thight junctions can perform paracellular transport,meaning that cells can transiently alter their juctions.

This is important in absorptions of aa and monosaccharides from the lumen after a meal, when concentration is high enough to allow passive tranport

Thight junctions

Thight junctions consist of a branching network of sealing strands.

The sealing strands encircles the apical end of each cell in the epithelial cell sheet

•  each sealing strand is composed of a long row of transmembrane adhesion proteins embedded in each of the plasma membrane •  sealing strand extracellular domains adhere directly to one another •  the main sealing strand proteins are the claudins

Normal thight junctions contain!•  claudins (sealing strands)!•  occludin (uncertain function)!•  tricellulin (seal membranes together, prevents transepithelial leakage)!

Junctional complex

•  Claudins and occludins are held in the right position in the cell by a network just apical to the adherens and desmosome junctions

•  this network is thought to consist of scaffold proteins

Intracellular scaffold proteins in the

Tjp (thight junction protein) family

are important in the junctional complex

Apico-basal polarity in epithelia

Par3 (scaffold protein, bind to Par6 and aPKC)

Par6 (scaffold protein, bind to Par3 and aPKC)

Atypical protein kinase C, aPKC

Binding sites for Rac and Cdc42 Positive feedback and spatial signalling

More Rac and Cdc42

Par3

Par6

aPKC

Rac

Cdc42

Apex

Crumbs complex

Basal

Scribble complex

The cytoskeleton + Rac directs delivery of basal lamina components to the opposite end of the cell

Planar cell polarity

Proteins involved in planar cell polarity (most identified in Drosophila):

•  Frizzled – Wnt signalling pathway

•  Dishevelled – Wnt signalling pathway

•  Flamingo – cadherin superfamily

•  Dachsous – cadherin superfamily

PASSAGEWAYS FROM CELL TO CELL – GAP JUNCTIONS

Gap junctions

Gap junctions has a radically different function than tight junctions.

Gap junctions bridges gaps between adjecent cells to create direct passageways from the cytoplasm of one cell into that of another

•  gap junctions are present in most animal tissue (both connective tissue and epithelia)

•  the gap is spanned by channel-forming proteins

•  allows both inorganic ions and water-soluble molecules to pass

•  connect cells both eletrically and metabolically

•  coupled cells share small molecules, but not macromolecules

Gap junctions

1. connexins 2. innexins

•  four-pass transmembrane protein

•  six connexins = hemichannel/connexon

•  different tissues – different combinations of connexins

A gap-junction is a dynamic structure, and the turnover of connexin is a few hours. A gap junctions is not similar to a ion channel, because it does not remain continuously open

THE BASAL LAMINA

The basal lamina

Tissues are made up by cells + extracellular space

The extracellular space is made up by a network of macromolecules constituting the extracellular matrix

Extracellular matrix found in;

•  bone

•  tendon

•  dermal skin layer

•  basal lamina : flexible sheet of macromolecules underspinning all epithelia

40 -120 nm thick

The basal lamina

- determine cell polarity

-  influence cell metabolism

-  organize membrane proteins

-  promote cell survival

-  promote cell proliferation

-  promote cell differentiation

-  serve as highway for cell migration

-  important mechanical role (epidermis)

Multi-tasking

The basal lamina

The basal lamina is synthesized by the cells on each side:

1.  the epithelial cells (over) contribute with basal lamina components

2.  connective tissue (under) contribute with basal lamina components

GLYCOPROTEINS

laminin

type IV collagen

nidogen

PROTEOGLYCAN

perlecan

Collagen XVIII

fibronection

•  Laminin is the organizer of the sheet structure. Laminin is composed of three long polypeptide chains!

A selection of the major extracellular macroproteins

Laminin is the organizer of the sheet structure. !Laminin is composed of three long polypeptide chains!

Figure showing the subunits of laminin. The yellow boxes are the binding sites for different molecules !Laminin is essential to basal lamina assembly and the anchoring to cells!

INTEGRINS AND CELL-MATRIX ADHESION

Matrix receptors tie the matrix outside the cell to the cytoskeleton inside the cell.

Several types of molecules can function as matrix receptors, but the most important one is

INTEGRIN

•  integrins can transmit signals in both directions across the cell membrane

•  integrins ”grab” intracellular and extracellular structures, and loss of tension make molecular signalling complexes to fall aparton either side of the membrane

•  integrins can transmit mechanical and molecular signals

•  integrins can convert one type of signal into the other

Integrin ”working schedule”:

•  head of integrin attach directly to an extracellular protein

•  intracellular integrin tail binds to talin

•  talin bind to actin filament

Intracellular anchor proteins (actinin, vinculin, filamin) adds strenght to the linkage

Integrin

•  24 integrins in humans

•  all consist of an alfa and beta subunit, and both subunits span the cell membrane

•  short intracellular C-terminal tail and large N-terminal extracellular domain

•  the extracellular domain binds to extracellular matrix proteins (laminin, fibronectin, ligands..)

•  the intracellular domain binds to the cytoskeleton (most often via talin and other anchor proteins)

•  in epithelial, most cell-matrix attachment sites found in hemidesmosomer

Linking laminin (outside cell) to keratin (inside cell)

The grip of integrin

Integrin Switches between an active and an inactive state by allosteric regulation

•  folded, inactive integrin has adhered alfa and beta chains

•  when integrin unfolds, the binding site for talin is exposed

•  binding of talin leads to assembly of actin filaments

•  when integrin binds to something, the cell reacts by tying its cytoskeleton to the integrin

OUTSIDE-IN ACTIVATION

•  talin competes with integrin alfa chain for its binding site on the beta chain

•  upon talin binding to beta-chain, the alfa-beta linkage is broken and the two integrin legs can spring apart

•  this also drives the integrin into an active conformation

•  this activation is triggered by intracellular regulatory molecules, such as PIP2 (phosphinositide

INSIDE-OUT ACTIVATION

PIP2 (and others) is produced in response to outside signals transmitted through cell-surface receptors (G-coupled receptors, tyrosine kinase receptors)

Integrin

Integrins usually bind their ligand with low affinity, and strong adhesion depends on clustering of integrins. This is the case in i.e. hemidesmosomes.

Extracellular matrix attachments act through integrins to control cell proliferation/survival

•  the dependence on a substratum to ensure growth/survival is know as anchorage dependence

•  anchorage dependence is mediated by integrins

•  this help cells to grow only when they are in appropriate situations

•  physical spreading is also important for growth

•  this is also forced through by adhesion to different sites (by integrins)

Integrin and FAK (focal adhesion kinase)

In cells grown on normal plastic surfaces focal adhesions are often prominent sites of tyrosine phosphorylation and FAK is found at these sites.

In integrin clusters, FAK is recruited by intracellular anchor proteins (talin, paxillin)

Actin (green), phosphotyrosine (red) and overlap (orange)

Clustered FAK molecules

Cross-phosphorylate eachother

Phosphotyrosine docking site

Src family of cytoplasmic tyrosine kinases

Phosphorylatin FAK on additional tyrosines, and thereby creating docking sites for a lot of intracellular signaling proteins

Outside-in signaling form integrins, via FAK and Src-family kinases, is relayed into the cell

Integrin and FAK (focal adhesion kinase)

- FAK knock-out mice form too many focal adhesions

-  both spreading and migration is slowed

THE EXTRACELLULAR MATRIX OF ANIMAL CONNECTIVE TISSUE

Extracellular matrix found in connective tissues

In connective tissues the extracellular matrix is ”larger” than the cells it surrounds.

It determines the tissue`s physical properties.

The extracellular matrix has a complex role:

•  regulate the cells

•  survival

•  development

•  migration

•  proliferation

•  shape

•  function

Connective tissue underlying an epithelium

Cells and Macromolecules in the connective tissue

•  macromolecules in the extracellular matrix are mainly produced by cells in the matrix

•  cells in the matrix organize the matrix (via the cytoskeleton)

•  in most connective tissue most macromolecules are sectreted by fibroblasts

Fibroblasts

- Chondroblasts (cartilage)

-  osteoblasts (bone)

Main macromolecules in conncetive tissue

1.  glycosaminoglycan / proteoglycans Form a hydrated gel-like substance, resists compressive forces and allow rapid diffusion

2.  fibrous proteins / collagen Stenghtens the matrix and give resilience

unbranched polysaccharide chain, with repeating disaccharide units

•  one of two sugers is always an amino sugar, most often sulfated

•  GAGs are negatively charged, and strongly hydrofilic

•  GAGs fill most of the extracellular space

•  Attracts cations (Na2+) and osmose causes large amounts of water to be sucked into the matrix

•  the swelling from water is known as the turgor pressure

Main GAGs:!1.  hyaluronan!2.  chondroitin sulfate / dermatan sulfate!3.  heparan sulfate!4.  keratan sulfate!

Hyaluronan is a simple GAG, and found in large quatities in early embryos.

No sulfated sugars, identical disaccharides, and long chained.

It is spun out directly from the plasma membrane. Can deform the epithelium and create a cell-free space beneth it – important in heart development (valves and septa).

Also important in wound healing and as a joint fluid

Glycosaminoglycan (GAG)

Proteoglycan = GAG + core protein

GAG Core protein Polypeptide chain of mebrane-bond ribosomes

Assembled in the Golgi Delivery to the cell by exocytosis

•  linkage tetrasaccharide serve as a primer for polysaccharide growth

•  polarized sugars are covalently modified

•  the linkage tetrasaccharide attached to a serine chain on the core protein

•  one sugar at the time added by a glycosyl transferase

Proteoglycans

•  at least one of the sugar side chains must be a GAG

•  contains as much as 95% carbohydrate by weight

•  limited heterogeneity (every core protein can carry a variable number/types of attached GAGs)

•  proteoglycans can be huge

- Proteglycans have an important role in chemical signalling (bind secreted signal molecules)

-  Bind chemokines in inflammatory responses (bind to the GAGs) Bind chemokines to the endothelial surface on a blood vessel on inflammatory site, stimulating white blood cells to come

-  TGF-beta binds to core protein

Fibrous proteins

Collagens – found in all multicellular animals

Primary structure is a long, stiff triple-stranded helical structure consisting of three alfa-chains

•  left-handed helix

•  3 aa per turn, with glycine every third aa

•  molecule 300 nm

•  42 genes encoding different collagen alfa-chains in humans

•  type I collagen most common (skin and bones) – FIBRILLAR COLLAGENS

•  type IV: network-forming collagen

•  type VII: anchoring fibrils

Collagen fibrils produced by fibroblasts

Collagen

Collagen undergo post-translational modifications

Collagen

After secretion, procollagen is converted into collagen molecules. Collagen molecules assemble into collagen fibrils.

•  The fibrils are strenghtened by formation of covalent cross-links between lysine residues

•  these cross-links are only found in collagen and elastine

•  the extent and type of cross-linking varies from tissue to tissue ( highly cross-linked in achilles tendon)

It is the connective tissue itself that determine the size and arrangement of collagen fibrils by gene regulation!

Elastin

Elastic fibers in the extracellular matrix give tissues resilience so they can recil after transient strech

•  elastin is the main component

•  elastin is a hydrophobic protein, rich in prolines and glycines ( but not glycosylated as in collagen)

•  tropoelastin (precursor) is sectreted into the extracellular space and assembled into fibers close to the plasma membrane

•  elastic fibers consist of elastin+microfibrils (fibrillin ; Marfan`s syndrome)

•  the elastine polypeptide chain adapt a ”random coil” form