1. Lecture 10: Cell Biology interactive  media  ”video” or ”animation ” The eukaryotic kingdom 1 Cell biology 2014 (revised 12/2-13)

2. The four major tissues in the human body - Epithelial - Muscle - Connective - Nerve Cells Cells + ECM Metazoan cells form organs with specialized tissues: 2

3. Different types of cell adhesion Homophilic bindingHeterophilic binding 3

4. Cell-cell contacts in columnar epithelia Gap junction Tight junction Adherens junction Desmosome Connection allowing local communication Cell-cell adhesion 4 hemidesmosome Cell-ECM adhesion Basal lamina Restricting movement of extra-cellular fluids

5. Tight junctions seal epithelial sheets to block passage of fluids in between cells I. Tight junctions: function Intestine Blood vessels Glucose Active and selective transport through the cytosol of cells by e.g., the Na + driven glucose symport 5

6. II. Tight junctions: Architecture The appearance of tight junctions resemble stitches across the plasma membranes of the two cells Tight junctions are made up by occludin and claudin. These are transmembrane proteins, which form tight connections across the extracellular space Linking protein attaches occludin and claudin to the cortical actin cytoskeleton 6

7. P.M.  -catenin Cadherin (calcium-dependent adhesion ) Cell #1 Cell #2 P.M.  -catenin Actin filament Many cadherins are known: E-cadherin in Epithelia N-cadherin in Neural cells Linkers of cadherins to the actin cytoskeleton Adherence junctions from stable cell-cell adhesion points between adjacent cells video 19.1- adhesion_junctions 7 Ca 2+ I. Cadherins: adherence junctions

8. II. Cadherins: growth arrest at cell-cell contact P.M. G1 Wnt G1 cyclin gene..but is stabilized by Wnt signaling  -catenin  -catenin TCF  -catenin Cytosolic  -catenin is by default unstable..... (Ubiq. dep. degradation) 1. 2. 3.  -catenin enters the nucleus:  G1 cyclin transcription  cell proliferation 4. Sequestering of cytosolic  -catenin at the adherence junctions formed after cell proliferation (i.e., at ”density arrest”) 8 Ca 2+

9. III. Cadherins: organization of cells into organs Cells expressing different cadherins Cells expressing different amounts of the same cadherin Cadherins are important for organ formation during development 9 +Ca 2+

10. Desmosomes hold cells together like rivets. Through linkage to IFs, they distribute shear forces evenly within the cell P.M. Cell #1 Cell #2 P.M. Linkers Intermediate filament (IF) Cadherin family protein Structure and function of the desmosome animation 16.4- intermediate_filament 10 Ca 2+

11. Structure and regulation of gap junctions Connexon = ~1.5 nm Free passage of: Amino acids Nucleotides Sugars Ions ”2nd messengers” Connexin PP PP PP Different connexins – different pore size Cell #1 Cell #2 Regulation of pore size 11

12. I. Integrins: Structure and ligand specificity i)Hetero-dimeric proteins consisting of  and  chains ii) At least 21 cell-type specific isoforms of  chain pairs iii) Integrin ligands include ECM components (collagen, fibronectin, laminin) and structures on neighboring cells 12 ECM: Basal laminaECM: connective tissue  x  x (ligand: laminin)  y  y (ligand: fibronectin) Integrins linked to IF (hemidesmosomes: epithelia) Integrins linked to actin (focal adhesions: fibroblasts)

13. II. Integrins: Anchorage to ECM ECM: connective tissue (contains residual migratory cells) 13 ECM: Basal lamina Inactive integrin Integrins linked to IF (hemidesmosomes)  Static cell-ECM interactions, e.g. epithelial sheets Integrins linked to actin (focal adhesions)  Dynamic cell-ECM interactions, e.g., during migration of fibroblasts or leukocytes Basal lamina: barrier towards connective tissue

14. III. Integrins: Architecture of the focal adhesion ECM P. M. Active integrin Focal 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 signaling 14  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) Talin Linker FAK Tyr- P Talin FAK Tyr- P P

15. IV. Integrins: Regulation of ligand-affinity 15 Outside-in activation of ECM-binding Inside-out activation of ECM-binding 1. Default state: The  and  chains are tightly associated Activated state:  and  chains are pushed apart and clustered by talin  High affinity/avidity ECM- association 1. 2. The  and  chains of integrins have affinity for both “each other” and ECM ligands  the concept of competing affinities

16. V. Integrins: Inside-out activation FAK P 2. 1. 3. 2. 3. Activated talin: i)Pushes  and  chains apart ii)Clusters the cytosolic parts of integrin  chains iii)Links  -chains with actin filaments iv)Recruit 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  and  chains High affinity ECM-binding Integrin clustering  increased avidity Albert et al. Fig 19-49 4. 16 Inactive talin

17. VI. Integrins: Outside-in activation Binding 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. 3. inactive talin ECM 4. 17

18. VII. Integrins: survival and cell proliferation signals Plasma membrane MAPK P P P PKB/Akt P PI-3 K P myc Cell cycle entry FAK Bad P 14-3-3 18 Ras GTP Motile cell types requires ECM for both growth and survival Survival P P 3 -Tyr- P P P

19. - 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 molecules I. The extra-cellular matrix (ECM) 19

20. II. The extracellular matrix (ECM) Proteoglycan molecules form highly hydrated gel-like “ground substance” in which the fibrous proteins are embedded Structural proteins, such as collagen and elastin, strengthen and organize the matrix Composed of polymeric networks of 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 ECM The aqueous phase of the ECM permits diffusion of nutrients 20

21. III. ECM: general structure of proteoglycan H2OH2O Na + - - - - - Ca 2+ Protein core Polysaccharide sidechain Negatively charged saccharides attract counter ions and water, giving the ECM the property to resist compression and bounce back to its original shape Osmosis Linking saccharides Glucosamino- glycans (GAGs) linear polymers of repeating disaccharides O-linked sugar 21

22. IV. ECM: Proteoglycan aggregates Hyaluronan, up to 50 000 repeating disaccharides Linker protein Proteoglycans can form huge aggregates onto hyaluronan. These aggregates can be up to 4  m in length These aggregates have a very high shock absorbing capacity and are highly enriched in cartilage 22

23. V. ECM: Collagen architecture Collagen  -chain (single helix) Collagen molecule (triple helix) Collagen fibril Collagen fiber Assembled outside the cell Assembled in ER Collagen is the most common protein in body, it forms strong and flexible fibers. Many types (at least 15) 23

24. VI. ECM: Elastic elastin networks Single elastin molecule In 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! StretchingRelaxation Crosslinking 24

25. VII. ECM: different types of connective tissue ”Normal” connective tissue Cartilage Fibroblast Chondrocyte Ca 10 (PO 4 ) 10 (OH) 2 Bone Osteoblast Physical properties of the tissue depend on the content of the ECM, which is determined by the residual cell type 25

26. Summary: ECM – a sticky business! 26 Laminin - Present in basal lamina of epithelia and the ligand for hemidesmosomes Fibronectin - Present in all ECM and primary high-affinity ligand for focal adhesions

27. Fig. 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. 27 Differential 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 stress

28. “Recommended reading” Alberts et al 5th edition Chapter 19 1131-1145 1150-1162 1164-1194 Focus on the general principles and topics highlighted in the lecture synopsis 28