Zhang notes, both lectures

Recommended reading is from Lodish. He recommends Albert’s molecular biology of the cell. Everything we will discuss is in handout. It should be good enough for the exam.
Cells are assembled into functionally coordinated assemblies called tissue in multicellular organisms. They are connected by cell-cell adhesions. Beside contacting with other cells, cells can also interact with the extracellular matrix.
Interaction categories:
1. cell-cell adhesion
2. cell-matrix adhesion
19-1:
Epithelium is full of cell-cell contact. Also interact with underlying connective tissue. Large molecules are secreted by fibroblasts. They synth and org the ECM.
Today- mechanism to make cells stick together.Epithelium has 3 sections: apical surface, baso-lateral surface, basal. Basal- interact through cell junction and non-cell junction. Interact with basal membrane through ECM adhesion . Junction functions listed in handout. Junction also active in signalling.
Skip a few pages- classification cell adhesion
Junctional mechanism means adhesion based on adhesive structures (cell-cell junction or cell-matrix junction) . Many adhesion mechanisms- nothing visible under EM (Ex: T cell interaction with APC) . When neuron extends neurites, extension based on cell-cell adhesion. Junction mechanisms listed in picture; you can tell functions from name. Tight junction also called occluding junction. Desmosome sticks cells together. Gap junction makes pores between cells for communication.
Cell-matrix adhesion: desmosome and hemidesmosomes are in skin. Desmosome connected to intermediate filaments, thicker than cytoskeleton to resist mechanical stress. Cadherin junction found in many tissues. Non junction adhesion mechanisms- no structural base to visualize under EM.
Tight junction also called occluding junction. Present in most epithelium as well as endothelium. Present at apical end of the basolateral membrane. Network sealing strands account for cell-cell adhesion. EM shows them in the intestinal lumen. The thin strands form the tight junction to seal apical end. Functions- next page.
If you are starving- tight junction is regulated to allow glucose into circulation under extreme conditions. Otherwise it does not allow glucose in.
Tight junction regulated by pore.
Regultion by assembly or disassembly- tight junction comes and goes in previous slide. For individual cells, junction subdivides plasma membrane into different compartments. Basolateral proteins cannot be freely translocated to apical surface or vice versa. Apical membrane has different functions from basolateral. Cancer cells lose tight junction and behave aberrantly due to loss of distinction.
Compsition: Every cell adhesion mechanism can be divided into 3 parts:
1. TM linker proteins called CAMs
2. cytoplasmic adaptor proteins
3. cytoskeleton.
Engage environmental elements and internal elements to create adhesion. All 3 are essential. Adaptor proteins used for regulation and bridging.
Occludin and claudin are 4 transmembrane proteins. Desmosome interacts with intermediate filaments, tight junction with actin filaments.
Extracellular: claudins and occludins. Called type III proteins (N and C terminus intracellular). Matches up with corresponding portions of protein in adjacent cells. Intracellular interact with adaptor proteins. There are many adaptor proteins. Through ZO1 and ZO2 interact with cytoskeleton. Essential components are still controversial.
GAP junction- communicating junctions. Found even in fibroblasts, epithelium, endothelium. Is channel for small molecules between 2 intracellular environments. Is nonspecific. See handout. 10 Amino acids=1 kD. Can label ubiquitin, but it will not pass, which is <1 kD. Can try injecting dye molecules of different sizes into cells and see if they pass.
Difference between gap junction and synapse- chemical in synapse has delay passing from upstream to downstream cell. No delay in gap junction. Fish escape fast not based on synapse, but on diffusion of messages through gap junction. Heart beat transduced through gap junction mechanism.
EM junction is next.
Molecular base: Gap junction formd by connexins with 4 TM domains. 6 of them form oligomer from one cell. One connexon engages another in extracellular domain.

Back to p.5
Families of CAMs here.
Cadherin is important CAM. Engages homophilic interaction. Counterreceptor is itself. Cadherin engages cadherin from another cell. Depends on Ca or Mg- divalent cation. Only cell adhesion molecule not dependent on Ca or Mg is Ig family.
Cmadh- integrins. Integrin can also engage cell-cell adhesion.
Cadherin is a type 1 TM protein. Functions dependent on Ca. Se handout.Classical form adherens junction.
How do cadherins engage homophilic interaction?Cadherin has affinity for Ca. Conformational change in absence- can be chipped off by protease. Trypsin and EDTA (chelates Ca)used to make cells confluent. Cadherin floppy without Ca. Some cells can be detached only with EDTA iif cell-cell adhesion not well developed (as in cancer cells) Prtottype is E cadherin. Extracellullarly has 5 domains. Each domain contains Ca binding seq. Membrane-proximal has higher Ca affinity than distal. Low [Ca]- binds to proximal dom first. Triggers conformational change resulting in stiffness of subdomain. Higher Ca- distal stiff. Cis interaction must occur first. Dimerization on same cell is intermediated by seq between C1 and C2. Once homodimerization accomplished. Conformation is ready for trans interaction with another pair from adjacent cell to accomplish homophilic interaction.
Functions: see handout.Cadherins serve as sorting mechanism for cells in embryogenesis. Only functions ID cells come together. Prevents intrusion from other tissues. How would you demonstrate interaction homophilically? Simple experiment. Have to find system that does not express E cadherin, express it, and observe. Fibroblasts do not express E-cadherin or other cadherins. Transfected fibroblasts and observed. Fibroblasts with E cadherin came together. Were important for cell-cell adhesion and only cells with E cadherin could be sorted to specific place.
Neural tube development from epithelial cells. E-cadherin is major one experessed in epithelial cells. Neural tube cells stopped expressing E and started expressing N cadherin. Formed 2 organs based on sorting.
Neural crest cells migrate and form lot of organs. When they migrate lose N cadherin expression. Aggregate wih expression of N cadherin again at the end.
Cadherins also play role in pathologic conditions.
When cancer loses E cadherin, can undergo epithelial-mesenchymal transition. Transfection in cancer cells to increase cadherin expression can stop cancer migration.
How cadherin connected to intracellular cytoskeleton- Slide shows classical cadherins interaction with actin. Catenins are adaptor proteins. Beta and gamma engage cytoplasmic domains of cadherins. Alpha interacts with cytoskeleton as a bridge. In some pathological conditions, level of one or more catenins is altered.
Well known signalling pathway-wnt pathway. Ligand engages frizzled receptor. Activates disheveled (names from Drosophila mutation) Dsh inactivates GSk3beta (glycogen synthase kinase) Once GSK3B activated, associates with B-catenin, binds APC (adenomatous polyposis coli protein)GSK phosphorylates N terminal B catenin and targets by APC through ubiquitination to be degraded.
If APC is mutated, B catenin accumulates in cytoplasm. Wnt signalling activation results in inactivated GSK. No B-catenin gotten rid of- accumulates in cytoplasm. Consequent accumulation of B-catenin- saturates cadherin junction. Extra will translocate interaction to nucleus. Can bind a variety of TFs to activate transcription. This is necessary during embryogenesis, but once development is finished, wnt pathway should be deactivated.
Desmosome- homophilic, Ca dependent interaction. Difference between desmosome and cadherin junction is cytoskeletal part. Linked to intermediate filaments. Plakoglobins are gamma catenins. Mutation in desmosome results in disease.Mutation in desmoplakins- blister easily.
Hemidesmosome is not cell-cell adhesion molecule. It is a is cell matrix adhesion molecule. Connects to keratins in skin.

Some molecules have no cytoskeletal structural base. Cell-cell adhesion mediated by Ig family of molecules.Ca independent interaction. Structural Ig subdomains is similar to antibody subdomains. VCAM is Ig superfamily proteins.

Zhanglecture 2
This talk about extracellular matrix and adhesion molecules necessary for ECM adhesion. Complex matrix of large molecules is the extracellular matrix. Produced by fibroblasts. Fibroblasts are migratory and use the fibers they produce to migrate around. Basal lamina is produced by epithelial layer above it.
What are the functions of the ECM?
Fill and provide order to extracellular space.
ECM has ligands for cell adhesion proteins and can communicate signals to cell about survival and differentiation and motility through these molecules. Also reservoirs for growth factors, chemokines, cytokines. Prevent growth factor effects from being too transient or abrupt- buffer them and prolong effects through persistent release. ECM is important for educating stem cells to commit for different lineages.
Provides way for cells to move within tissues. Cells migrate on collagen molecules like a railroad track.
Components ECM?
Complicated. Only going through major components. 2 parts: proteins and polysaccharides or proteoglycans.
Large space in ECM filled by proteoglycans- combination of peptides with polysaccharides. Difference between proteoglycan and glycoprotein- glycoprotein mostly from AAs. Proteoglycan- opposite. Cell culture used to be on the flask, but this is not physiological. In vivo, growth factors come from below, not above. More experiments are done these days on 3-D collagen gel to closely mimic in vivo conditions. Lots of proteoglycans included.
Structure is simple for proteoglycan- GAGs are building blocks of them.
Major component connective tissue- collagen.Basic biochem feature is Gly-X-Y triplet repetitive sequence. Y is typically proline or hydroxyproline. Enzyme prolyl hydroxylase is important for maturation of collagen. Hydroxylation required for lateral association. Each collagen- 3 chains. Form a triplex. Multiple helices for collagen fibers. Hydroxyproline negatively charged- can form H bonds between chains to provide strong bonds in helix. Enzyme to hydroxylate proline require vitamin C.

Fibrillar collagen- connective tissues.
Fibril-associated collagens have more flexibility.
Network form networks instead of fibers.
Fibril assoc collagen attach to fibrillar collagens. Triplets are interrupted by non-triplet sequence- increases flexibility.
Type IV is different- network in basement membrane.Laminin, entactin, proteoglycans, other proteins are also there. C terminal interaction is covalent. Lateral interactions are mediated by H bonds.
Goodpasture’s syndrome- affects glomerulus and lung because endothelium and epithelium interact at basement membrane with blood vessel on one side and lumen on other. Patient develops autoantibody to Alpha 3 chain of collagen IV. Antibody precipitates in lung and kidney.

Different forms basement membrane occur. The muscle form is thin and less well developed than that in other locations, but it is still important for health. It surrounds individual muscle cells. Muscular dystrophy can be caused by many factors- one is when the muscle cell cannot attach securely to basement membrane. Mutation in an integrin keeps muscle from attaching to membrane and cannot retract firmly.
Stereotype is that of epithelium and endothelium. The basement membrane in kidney is fusion of basement membrane of epithelium and endothelium.
Matrigel- constructed from basement membrane to use as 3-D culture system- from mouse tumor thans secretes lots of basement membrane components.
Called recombinant basement membrane- used to culture cancer cells, stem cells, epithelium.
Laminin- isolated from lamina. Large protein formed by 3 beta chains.

Fibronectin is a major non-collagen ECM protein. Typically basic unit is single chain. Dimerizes at C terminal through disulfide bond.Type III fibronectin subdomain is one of the most observed ones in the body. Found in lots of proteins. 3AAs- RGD form a loop projecting from Type III domain- is bonding site for integrin 5 beta1, which is fibronectin receptor.Cell binding region is Type III subdomain where it binds to integrins listed. Leukocytes use Cell Binding Site #2 to migrate into tissues.
Integrins- affinity ECM and proteins is lower than intracellular interactions. Could use affinity chromatography to isolate. Ligand to column and pass cell lysate through. Put fibronectin on column and found the cellular receptor called integrin.
Integrins function as cell-cell adhesion molecules as well.Function as dimer of alpha and beta chains. Relatively large EC domain and short cytoplasmic chains..
Collagen receptors are alpha1 and alpha2 (major ones)See sheet.
RGD binding integrins bind to fibronectins and other RGD-containing proteins. Slide is a headache- have rough concept. Do memorize the ones for collagen and laminin.
Integrins can also be used as receptor to invade cells by bacteria and viruses.

Cytoplasmic view of integrins- has transmembrane linker proteins and intracellular skeletal protein and adapter proteins. Most integrins bind to actin cytoskeleton. Actinin and talin are well-established bridging proteins.
Special case in spleen- intermediate filaments interact with alpha 6 beta 4 integrin in spleen.

Integrins are often physiologically inactive. Dangerous to be active all the time. Only want to activate in neutrophils or platelets under special circumstances. Only under wound condition do you want platelets to aggregate.
Most cells do not survive unless attached because of integrin signalling. Cancer cells have aberrant integrin signalling to avoid apoptosis.