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Cell adhesion and ECMLecture 10:Cell Biology interactive media video or animationThe eukaryotic kingdom

1Cell biology 2014 (revised 11/2-14)The four major tissues in the human body- Epithelial- Muscle- Connective - NerveCellsCells + ECMMetazoan cells form organs with specialized tissues:23Stratum CorneumBasal layerECM /residual cells*(fibroblasts)EpidermisDermisBasal lamina

1.2.3.1.2.3.*Different types of cell adhesionHomophilic bindingHeterophilic binding4Cell-cell contacts in columnar epitheliaGap junctionTight junctionAdherens junctionDesmosome Connection allowing local communicationCell-cell adhesion5hemidesmosomeCell-ECM adhesionBasal laminaRestricting movement of extra-cellular fluidsTight junctions seal epithelial sheets to block passage of fluids in between cellsI. Tight junctions: functionIntestineBlood vessels

GlucoseGlucoseActive and selective transport through the cytosol of cells by e.g., the Na+ driven glucose symport6II. Tight junctions: ArchitectureThe appearance of tight junctionsresemble stitches across the plasma membranes of the two cellsTight junctions are made up by occludin and claudin. These are transmembrane proteins, which form tight connections across the extracellular spaceLinking protein attaches occludin and claudin to the cortical actin cytoskeleton7P.M.a-catenina-cateninCadherin (calcium-dependent adhesion) Cell #1Cell #2P.M.b-cateninb-cateninActinfilamentMany cadherins are known: E-cadherin in EpitheliaN-cadherin in Neural cellsLinkers of cadherinsto the actin cytoskeletonAdherence junctions from stable cell-cell adhesion points between adjacent cellsvideo 19.1- adhesion_junctionsanimation 20.2 -contact_inhibition8Ca2+I. Cadherins: adherence junctionsWntDishevelledLRPFrizzledTarget genesTCFb-cateninGSK-3bb-cateninPUbUbUbTarget genesTCFGroucho

AxinAPCGSK-3bb-cateninAxinAPCDishevelledZZZ9Cell cycle entry+ etcG1Cytosolic b-catenin is stabilized by Wnt signalingTGFbeta/BMP and Wnt key regulators during developmentConserved pathways in fly, nematodes, mammals (premises for current understanding)Frizzle: a short, crisp curl. dishevel: To throw into disorderHedgehoogKen and Barby, Cheap date etc

9II. Cadherins: growth arrest at cell-cell contactP.M.G1WntG1 cyclin gene..but is stabilized by Wnt signaling

a-cateninb-cateninb-cateninTCFb-cateninCytosolic b-catenin is by default unstable.....(Ubiq. dep. degradation)

1.2.3.b-catenin enters the nucleus: G1 cyclin transcription cell proliferation4.Sequestering of cytosolic b-catenin at the adherence junctions formed after cell proliferation (at density arrest)10Ca2+III. Cadherins: organization of cells into organsCells expressing different cadherinsCells expressing different amounts ofthe same cadherinCadherins are important for organ formation during development11+Ca2++Ca2+Desmosomes hold cells togetherlike rivets. Through linkage toIFs, they distribute shear forcesevenly within the cellP.M.Cell #1Cell #2P.M.LinkersLinkersIntermediatefilament (IF)Cadherin family proteinStructure and function of the desmosomeanimation 16.4- intermediate_filament12Ca2+Structure and regulation of gap junctionsConnexon= ~1.5 nmFree passage of: Amino acids Nucleotides Sugars Ions 2nd messengers ConnexinPPPPPPDifferent connexins different pore sizeCell #1Cell #2Regulation of pore size13I. Integrins: Structure and ligand specificityHetero-dimeric proteins consisting of a- and b- chains

ii) At least 21 cell-type specific isoforms of a/b-chain pairs

iii) Integrin ligands include ECM components (collagen, fibronectin, laminin) and structures on neighboring cells 14ECM: Basal laminaECM: connective tissuex x (ligand: laminin)y y (ligand: fibronectin)Integrins linked to IF (hemidesmosomes: epithelia)Integrins linked to actin (focal adhesions: fibroblasts) II. Integrins: Anchorage to ECMECM: connective tissue (contains residual migratory cells)15ECM: Basal laminaInactiveintegrinIntegrins linked to IF (hemidesmosomes) Static cell-ECM interactions , e.g. epithelial sheetsIntegrins linked to actin (focal adhesions) Dynamic cell-ECM interactions, e.g., during migration of fibroblasts or leukocytesBasal lamina: barrier towards connective tissue III. Integrins: Architecture of the focal adhesionECMP. M.Active integrinFocal adhesions exist only in motile cells (i.e., not in epithelia) The dynamic nature of focal adhesion is dependent on both Inside-out and Outside-in signaling

FAK: Focal adhesion kinase integrin dependant signaling16 recruitment of SH2-domain signaling proteins (Clustering of FAK trans-phosphorylation, i.e. the same principle as for tyrosine kinase receptors, which are dimerized by ligand binding)

TalinLinkerFAKTyr- PTalinFAKTyr- PTyr- PIV. Integrins: Regulation of ligand-affinity17

Outside-in activation of ECM-bindingInside-out activation of ECM-binding 1. Default state: The a- and b-chains are tightly associated

Activated state: a- and b-chains are pushedapart and clustered by talin High affinity/avidity ECM-association 1. 2. 2.The a- and b-chains of integrins have affinity for both each other and ECM ligands the concept of competing affinities

V. Integrins: Inside-out activationFAKP

2. 1. 3. 2. 3.Activated talin:Pushes a- and b-chains apartClusters the cytosolic parts of integrin b-chains Links -chains with actin filamentsRecruit focal adhesions proteins (vinculin, FAK etc) generation of a focal adhesion point 2. 1. 3. 4. Activation of talin by a RTK ligand (e.g. EGF)

Separation of a- and b-chains

High affinity ECM-binding

Integrin clustering increased avidity

Albert et al. Fig 19-49 4.18

Inactive talinVI. Integrins: Outside-in activationBinding by (very) high affinity ECM ligands..

breaks the interaction between the - and -chains

The exposed -chain talin-binding site..

.activates talin Generation of a focal adhesion point

outside-in + inside-out = positive feedback 1. 2. 3. 4.

ECM 1.

ECM 2. 2. 3.inactivetalin

ECM

4.19VII. Integrins: survival and cell proliferation signalsPlasma membraneMAPK PPPPKB/Akt

PPI-3 KPmycCell cycle entryFAKBadP14-3-320RasGTPMotile cell types requires ECM for both growth and survivalSurvivalPP3-Tyr- P-Tyr- P-Tyr- PTalin- Provides mechanical support to tissues

Organizes cells into tissues

Instructs cells as to where they are and what they should do

- Reservoir for extra-cellular signaling moleculesI. The extra-cellular matrix (ECM)21II. The extracellular matrix (ECM) Proteoglycan molecules form highly hydrated gel-like ground substance in which the fibrous proteins are embeddedStructural proteins, such as collagen and elastin, strengthen and organize the matrixComposed of polymeric networksof several types of macromolecules. Secreted by connective tissue cells,such as fibroblasts & chondrocytes.1.2.3.1.2.3.Multi-adhesive proteins, such as fibronectin and laminin, facilitate cell attachment to the ECMThe aqueous phase of the ECM permits diffusion of nutrients22III. ECM: general structure of proteoglycanH2ONa+-----Ca2+Protein corePolysaccharide sidechainNegatively charged saccharides attract counter ionsand water, giving the ECM the property to resist compression and bounce back to its original shapeOsmosisLinking saccharidesGlucosamino-glycans (GAGs)linear polymers of repeating disaccharidesO-linked sugar23IV. ECM: Proteoglycan aggregatesHyaluronan, up to 50 000repeating disaccharidesLinker proteinProteoglycans can form huge aggregates onto hyaluronan. These aggregates can be up to 4 mm in lengthThese aggregates have a very high shock absorbing capacity and are highly enriched in cartilage24V. ECM: Collagen architectureCollagen a-chain (single helix)Collagen molecule(triple helix)Collagen fibrilCollagen fiberAssembled outside the cellAssembled in ERCollagen is the most common protein in body, it forms strong andflexible fibers. Many types (at least 15)

25VI. ECM: Elastic elastin networksSingle elastin moleculeIn cases there ECM is very flexible, e.g., in skin, lungs and blood vessel walls, some of the collagen is replaced by elastin.Cross-linked elastin behaves like a rubber band!StretchingRelaxationCrosslinking26VII. ECM: different types of connective tissueNormal connective tissueCartilageFibroblastChondrocyteCa10(PO4)10(OH)2Ca10(PO4)10(OH)2BoneOsteoblastPhysical properties of the tissue depend on the content of the ECM, which is determined by the residual cell type27Summary: ECM a sticky business!28Laminin - Present in basal lamina of epithelia and the ligand for hemidesmosomesFibronectin - Present in all ECM and primary high-affinity ligand for focal adhesionsFig. 19-1: Epithelial tissue: The intermediate filaments of the cells themselves (linked from cell to cell by desmosomes) provides mechanical strength. Hemidesmosomes (integrin binding to laminin) are only found in the epithelial cells that connect to the basal lamina. These epithelial cells are normally essentially non-motile.

Connective tissue: ECM provides the mechanical strength, the sole role of the residual cells (fibroblasts) is to produce the ECM components. These residual cells move around and may migrate to e.g. a site of tissue damage.

29Differential means to achieve mechanical strength

Epithelial cells

Basal lamina(dense ECM)

Connective tissue(ECM + cells)Cells resistant to mechanical stress

ECM (but not cells) resistent to mechanical stressRecommended readingAlberts et al5th edition

Chapter 19

1131-11451150-11621164-1194Focus on the general principlesand topics highlighted inthe lecture synopsis 30