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What are the 4 types of extracellular matrix protein 1. Basement membrane 2. Collagens 3. Elastin 4. Proteoglycans/Glycosaminoglycans What are the 2 sublayers of the basement membrane? 1. Basal laminae 2. Reticular laminae What are the 4 things the basement membrane laminins are the binding domains for? 1. Collagen IV 2. Heparin 3. Heparin sulphate 4. Cell binding What 3 structures does collagen provide tensile strength and elasticity in? 1. tendons 2. cartilage 3. bone collagen is an insoluble glycoprotein + carbohydrate what amino acids and carbohydrates is it made up of? aa: glycine, proline, hydroxylysine, hydroxyproline carbohydrates: glucose + galactose what do type 1, 2, 3 collagen form? main fibres, flexible what does type 1 collagen form? bone what does type 2 collagen form? cartilage what does type 4 collagen form? basement membrane what are the two types of fibronectin soluble insoluble where is fibronectin found heart, lung, liver what is fibronectin made by? fibroblasts, epithelial cells, and hepatocytes (plasma) what are the functions of the cell cytoskeleton cell shape, division, migration, muscle contraction, intracellular organelle transport, segregatation of chromosomes in mitosis IF size, where they are found, role, if they are polar or not tough, durable 8-10nm in nearly all cells but mostly cells subjected to stress eg. epithelia, nerve, muscle-principal role is structural NOT polar IF structure amino head, central rod domain, carboxy terminal tail rod domains similar but variable length N and C terminals- involved in binding to other proteins rod ~310 amino acids rod has distinctive heptad repeat motif of repeating series of 7 amino acids coiled into an alpha helix which the hydrophobic amino acids forming a stripe down the rod ~40 heptad repeats IF monomers 2 parallel helices (each a monomer) wind around one another to make a coiled coil dimer which strong interactions between the hydrophobic amino acids dimers associate into tetramers tetramers are soluble- found free in cytosol types of IF keratins vimentin neurofilaments nuclear lamins mechanical properties of protein filaments MT- stretch easily but break when there's 50% increase in lenght AF- much more rigid- need to apply a large force to deform but only get a small increase in length then rupture IF- easily deformed but withstand large stress without rupture function of IFs maintain cell integrity keratins structure each filament is a mixture of acidic and basic keratins (are hetrodimers) keratins are directly connected to neighbouring cells via desmosomes keratin mutations cause several genetic diseases eg. epidermolysis bullosa simplex- EBS (skin blisters on even slight mechanical stress, rupturing basal cells) blistering diseases happen in other areas due to keratins expressed in these tissues cells rupture b/c keratin filaments are disorganised or clumped neurofilaments types 3: NF-L, NF-M, NF-H co assemble into heteropolymers ie. NF-L + one other NF-M and NF-H have long C terminal tails that bind to neighbouring filaments to generate aligned arrays with a uniform spacing NF cross linking provides regular packaging which gives great tensile strength that axons need- they control the speed of electrical signals Neurodegenerative disease amyotrophic lateral sclerosis- accumulation and abnormal assembly of NF in motor neurons- interferes with axonal transport --> muscle weakness, atrophy Vimentin like filaments includes desmin expressed in cardiac, skeletal and smooth muscle. essential role in muscle integrity includes glial fibrillary acidic protein- marker of glial cells, forms filaments in astrocytes in CNS nuclear lamina lining the surface of the inner nuclear membrane made of lamins unusual features of lamins - have a signal sequence directing the cells protein transport machinery to take them to the nucleus - most widespread type of IF - progenitor of all IF proteins - form 2D sheet like lattice (10-20nm thick) lying between the nuclear envelope and chromatin, with interruptions at nuclear pores -post translationally isoprenylated- helps associate with the nuclear envelope - lamins bind to other proteins on inner nuclear membrane- organise chromatin and nuclear pore spacing nuclear lamina 2D lattice -made of lamin A,B, and C tetramers -disassembles at the onset of mitosis, triggered by phosphorylation of specific serine residues near the ends of rod domains - this is mediated by MPF (maturation promoting factor)- causes nuclear envelope to break, on de phosphorylation the lamins reassemble lamin A mutation responsible for progeria- early aging condition other lamin A mutations cause striated muscle diseases, abnormal fat cell functions and peripheral nerve damage IF accessory proteins link IFs to one another into bundles or networks and link them to the plasma membrane eg. with desmosome proteins integrate IFs with rest of cytoskeleton eg. plectin- bundles vimentin filaments and links IFs to MTs and cell membrane eg. Filaggrin- bundles keratin filaments in skin. Mutations compromise the barrier of the skin Actin filament structure tight helix of globular (G) actin monomers (375 amino acids) dynamic equilibrium: 50/50 for F(polymerised)/G (unpolymerised) POLAR have a slow growing (-) end and fast growing (+) end thinner, shorter, and less flexible that MTs where are actin filaments found stable structures- microvilli, muscle sarcomere labile structures- cell cortex may be cross linked into aggregates or bundles structure of G actin "clam shell" cleft between two domains ATP binds at bottom, Mg binds as well subunits stacked in tight helix when monomer is added to a growing filament, clam shell snaps shut, triggering hydrolysis of ATP--> ADP which is trapped inside F actin assembly 3 phases: -nucleation: G actin/ATP monomers slowly form stable complexes -elongation -steady state: no change in F-actin mass (ATP binding clefts all facing the same direction) on incorporation into the filament, ATP is immediately hydrolysed to ADP when monomer is released, trapped ADP is released and replaced by ATP. slow process (t1/2 mins) what are alpha, beta, and gamma actin involved in? alpha actin= contractile structures beta actin= cell cortex and leading edge of motile cells gamma actin= filaments in stress fibres treadmilling-AF as G-actin monomers add onto the (+) end and are lost from the (-) end [even tho F-actin length stays constant] there is a flow of units through the filament this mechanism occurs at the leading edge of migrating cells toxins interacting with actin - cytochalasin D- binds to the (+) of F-actin -lantrunculin- binds G-actin both of these promote dissociation of filaments and paralyze cell locomotion but not cell division -phalloidin- locks adjacent subunits in F-actin together and stabilises them preventing depolymerisation prevents cell migration proteins that regulate actin polymerisation by binding G-actin -thymosin B4: small (~5kDa), binds G actin in a 1:1 complex, preventing polymerisation- sequestering protein -profilin: 15kDa binds G actin 1:1- too low conc to act as a sequestering protein. promotes assembly. localised to cytosolic surface of plasma membrane thymosin B4 and profilin controlling actin polymerisation 1. G actin/thymosin dissociate 2. G adds to (+) end of F-actin 3. ATP--> ADP, subunit dissociates 4. ADP-G actin makes complex with profilin 5. profilin accelerates exchange of ADP for ATP --> G actinATP 6. Profilin delivers G to (+) end or 7. Thymosin sequesters G (takes it to a reserve to use later) Severing proteins- AF -promote turnover of actin filaments needed for cell locomotion -break filament then remain bound at (+) end= capping -uncapped (-) end rapidly shortens - capping and severing proteins are regulated by extracellular signals eg. cofilin and gelsolin bind to PIP2 which inhibits their activity PIP2 hydrolysis by PLC increases severing activity -Ca activates gelsolin (more Ca, more PLC activity due to receptor activation) Capping proteins- AF cap severed ends CapZ binds to the (+) end - independent of Ca - activity inhibited by PIP2 - same pathway as cofilin Tropomodulin caps (-) end. works with capZ to stabilize actin thing filaments in the skeletal muscle sarcomere Arp2/3 AF actin related proteins Arp2/3- complex of 7 proteins which stimulates filament assembly binds to the side of actin filament to nucleate a daughter filament; newly created filaments elongate to create the force to push membrane forward in migrating cells ERM family proteins AF -attachment of AF to plasma membrane -ERM (ezrin, radixin, moesin)- maintain cell polarity -C terminal of ERM binds directly to side of AF -N terminal binds to face of transmembrane glycoproteins eg. CD44, which is a receptor for the ECM component, hyaluronan - attachment is regulated: active ERM conformation is generated when ERM is phosphorylated by tyrosine kinases or by binding to PIP2 triggered by receptors/extracellular signals what does the loss of ERM (merlin) cause? neurofibromatosis- multiple benign tumors in auditory and other parts of the NS AF assemblies (3) 1. Gel-like networks 2. Contractile bundles a. focal contacts b. contractile ring 3. tight parallel bundles a. microvilli b. filopodia c. leading edge of migrating cell Gel like networks- AF meshwork of filaments held at ~90 degrees -filamin -actin gel made by filamin is needed for cells to extend the thin sheet like membrane projections (lamellipodia) that help them crawl along a substrate contractile bundles AF -one type of actin bundle -fibres have opposite polarity -loosely spaced (30-60nm apart) by alpha actinin, allows myosin II to enter to contractile apparatus found in focal contacts and contractile rings Focal contacts- AF AF bundles (stress fibres) terminate at focal contacts attachment sites between actin and ECM allow cells to pull on the substratum Contractile rings- AF a transient belt of AF forms under the plasma membrane - belt contains myosin II, a motor protein, generates force which divides cytoplasm Tight parallel bundles- microvilli AF finger like extensions of the plasma membrane in many epithelial cells several thousand on the apical surface of each cell in human small intestine Tight parallel bundles- filopodia AF spine like projections that allow a cell to explore its environment not contractile as close packing by fimbrin excludes access of myosin II Tight parallel bundles- leading edge of migrating cells AF Arp2/3 complex held by other accessory proteins so that they resemble the plus end of an AF Actin subunits assemble, bypassing the rate limiting nucleation step Arp2/3 also binds to pre existing filament and generate a new filament at a 70 degree angle to produce branching Arp2/3 associated with leading edge (lamelipodium) of migrating cells accessory proteins regulate - filament length, locomotion, organisation and dynamic behaviour - extracellular signals regulate activity of accessory proteins and so change cell behaviour Rho protein family intracellular signals activate these proteins they act as molecular switches to control cell processes Cdc42 acting polymerisation/bundling into filopodia Rac actin polymerisation at cell periphery to make lamellipodia (sheets) Rho actin bundling with myosin II into stress fibres + clustering of integrins to make focal contacts