Book Notes: ECM Part 3

• integrins: transmembrane receptors that connect cells to the ECM
• external conditions and internal signals are connected and transduced by the integrin
• integrin binds tightly in presence of tension and loosely without
• general shape is 2 subunits alpha and beta that span the membrane (short cytosolic C' and long extracellular N')
• cytosolic portions attach to actin filmanets through talin (binds to beta) and vinculin (similar to adherens jxns)
• extracellular portions attach to specific sequences of laminin and fibronectin or things hanging off of other cells
• cell-cell jxns connect cytoskeleton and signals between cells, while cell-matrix jxns connect cytoskeleton to matrix skeleton and signals
• focal adhesions: sites of cell-matrix adhesion
• integrins swap between actin and inactive conformation (migrating macrophages have to alternate between binding and releasing in order to crawl form binding site to binding site)
• "outside-in activation"
• integrin is tightly folded in absence of ligand: when ligand partially associates, the integrin unfolds and ligand binding affinity increases
• when integrin is folded, the intracellular domain is also tightly closed (alpha and beta bound together)
• when integrin unfolds, the alpha and beta chain separate, exposing the talin binding site
• sum up: grabbing ligand causes connection to internal cytoskeleton to form so that force can be applied for movement (it's like if your arm was separated at your elbow, but if your hands grabbed a dumbbell, your arm connects again at the elbow, and then you can use your biceps to lift the dumbbell, eh?)
• "inside-out activation"
• other signals activate signal cascade, producing PIP2
• PIP2 activates talin to bind to beta integrin
• extracellular unfolds, ready to find a ligand
• sum up: connection at the internal cytoskeleton readies the cell to grab a ligand for next action (it's like if you spot a dumbbell, you hook up your arm to your elbow in preparation, then grab the dumbbell and start exercising :D)
• integrins tend to cluster to form strong adhesions
• their "high-affinity binding" conformation is relatively low affinity anyways
• clustering many integrins is like combining weak noncovalent interactions: strength in numbers
• ECM controls cell proliferation and survival through the integrins
• anchorage dependence: cells depend on ECM connection to decide to grow, divide, or even survive
• muscle, epithelial, and endothelial cells commit suicide if they fall off the ECM
• some cells will not grow even if food is plentiful, unless they are attached
• this dependence ensures cells only grow or divide when the situation is right (as determined by the ECM)
• falling off the ECM allows cells to decide their own fate (dangerous rebels!  just look at those naughty cancer cells, growing and wandering about on their own like that), which is no good for the overall health of the organism 
• binding to ECM also spreads cells apart, so they have room to grow and divide (see, ECM provides for all)--if cells are lost, it'll be faster to refill that space if all the remaining cells get spread out and grow than if only the neighbors are able to divide (they would reach senescence much faster that way *insert frown* )
• focal adhesion kinase (FAK): a cytoplasmic Tyr kinase
• talin and paxillin (which binds to alpha) recruit FAK
• FAK molecules at the focal adhesion point cross-phosphorylate each other
• the phospho-tyrosine is a docking site for Src family of Tyr kinases, who then phosphorylate other proteins and more Tyrs on the FAKS, creating more sites for Src binding
• FAK also helps with removing focal adhesions (observed overexpression of FAK in motile cancer cells, and overproduction of focal adhesions in cells with no FAK)
• FAK and similar pathways produce global (entire) cell response, including affecting gene expression
• integrins can also produce small, local effects (i.e. only in the cytoplasm/membrane close to focal adhesion)
• activate local population of Rho GTPases to cause effect (for example, changing direction of a growing axon)
• basal lamina is only one type of ECM, the connective tissue is also a form of ECM
• variations in composition give it different properties (from jellyfish jelly to lobster carapace to teeth and bone)
• formation and orientation of this ECM is controlled by the cells living in it (mostly fibroblasts!)
• fibroblast: matrix cells of connective tissue
• secrete matrix macromolecules (chondroblasts make cartilage, osteoblasts make bone, etc)
• matrix similar to lamina in that it has (1) proteoglycans and (2) FIBROUS proteins
• proteoglycans from jelly substance to resist compression and hold the fibroblasts within, while collagen fibers give tensile and resilient strength
• other matrix proteins direct, stabilize, and signal the cells within
• GAGs: glycosaminoglycans, chain of repeats of one disaccharide units, unbranched
• the disaccharide is 1 of either N-acetylglucosamine or N-acetylgalactosamine (simply put: modified glucose or modified galactose) plus a uronic acid
• GAGs are negatively charged due to sulfate/carboxyl groups on sugars
• 4 types:
• hyaluronan
• chondroitin sulfate/dermatan sulfate
• heparan sulfate
• keratan sulfate
• GAGs are hydrophilic and stiff (can't fold like polypeptides), so they take up lots of space and volume for low mass, making porous gels even at low concentrations
• because of this jelly feature, GAGs usually just fill space in the ECM
• their negativity attracts positive ions, who attract water (water likes to follow high ion concentrations, remember hypo and hypertonic?), creating swelling and turgor pressure so that the ECM can resist squishing and compression
• hyaluronan: simplest of GAG, 25000 disaccharide units, no sulfated sugars, no generally linked to any core protein, synthesized on cell surface straight into the ECM (other GAGS are the exact opposite: sulfated, protein-linked, synthesized and exported by exocytosis)
• serves as space filler and shaping (small quantity swells with water to occupy large volume to move aside parts or expand shape)
• synthesized from basal side, it can deform the epithelium to create a cell-free space below for other cells to migrate in
• after cell migration, hyaluronidase degrades the excess hyaluronan
• the other GAGs are attached to protein as proteoglycans
• the core protein goes through the ER processing and receives the sugar chain in the Golgi
• linkage tetrasaccharide: first attachment on protein at a serine, serves as primer for additional sugar attachment
• glycosyl transferases adds sugars one at a time to the sugar primer
• the sugars are also modified in the Golgi: epimerization alters configuration, while sulfations increase negativity
• general proteoglycans can have any number of sugar side chains, but one of them must be a GAG [glycoprotein is less than 60% carb by weight, while proteoglycan is 95% carb by weight]
• variation comes from type of GAG, length of GAG, modifications of GAG, and combos of GAGs on one core protein
• proteoglycans have various functions
• GAG chains form gels of varying charge density and pore size (act as sieves to regulate traffic of molecules through the ECM based on charge and size, just like gel electrophoresis :D )
• control activity of secreted proteins by binding to them:
• binding limits their diffusion, their activity (depending on where they are bound), and their lifetime
• example: heparan sulfate binds FGF (fibroblast growth factor), causing them to oligomerize (aggregate) to enable crosslinking and activation of receptors
• in this way, proteoglycan binding enhances signal transduction
• example: heparan sulfate bind and trap chemokines on inflamed site, chemokines are continuously signaling white blood cells to get here and fix the probelm
• in this way, proteoglycan binding localizes a signal at the needed site
• can act as co-receptors
• some proteoglycans stay attached to the plasma membrane by having their core protein be transmembrane or bound to a GPI anchor (lipid buddy)
• these bound GAGs collaborate with cell-surface receptors or the receptors themselves can be proteoglycans by having a GAG attached
• syndecan: transmembrane proteoglycan, cytosolic domain interacts with actin and signaling molecules in cortex
• bind FGF and pass it on to FGF receptors
• collagen: fibrous proteins
• 3 polypeptide chains (all alpha chains) are wound in superhelix, is long and STIFF
• rich in Pro (which stabilizes the helical conformation), and Gly (at every 3rd residue to help pack tightly since Gly is super small)
• triplet Gly-X-Y sequence
• exist tons of different alpha chains, so different cell types produce different combos if 3 alphas to make different collagen fibers, although the number of possibilities is limited by the individual chains' ability to combine into a superhelix (some A.A. sequences may not fit well with certain other sequences)
• fibrillar collagens: forms fibrils (long rope with no interruptions) 10-300 nm diameter and 100s um long, consists of Type I,II,III,V,XI collagen (bone, cartilage, skin, V similar to I, XI similar to II)
• collagen fibrils aggregate into bundled cables called collagen fibers (several um in diameter)--this method of organization is similar to muscle fibers and fibrils
• Type IX and XII are "fibril-associated collagens" because they decorate surface of fibrils ad link them to one another and other ECM components
• Type IV is part of basal lamina (see previous notes)
• Type VII form dimers that form anchoring fibrils that attach basal lamina to connective tissue
• Type XVII are "collagen-like" proteins that have transmembrane domain and exist in hemidesmosomes
• Type XVIII form core protein of a proteoglycan in basal lamina
• collagen gets lots of post-translational modifications
• pro-alpha chains: large precursor that is synthesized by ribosomes and put into ER, flanked on either end by "propeptides" that will be cleaved
• in ER, Pro and Lys are hydroxylated (hydroxy-Pro and hydroxy-Lys)
• certain hydroxy-Lys get glycosylated
• pro-alpha chains combine into triple helix held by H-bonding = procollagen
• hydroxy-Lys and hydroxy-Pro provide the the H for H-bonding
• propeptides are cleaved post-secretion by enzymes outside the cell
• allows cell to delay collagen fibril formation until outside (otherwise it'd be too big and bulky to get outside)
• propeptides also guide the triple helix formation of procollagen but prevent aggregation into fibrils (characteristic of self-assembly)
• collagen is cross-linked to form fibrils, and there are striations every 67 nm of fibril (produced by staggered packing of collagen)
• cross-linking gives tensile strength
• GAGs resist compression while collagen resists stretching
• tendons have parallel collagen bundles to prevent muscle pulling, while skin has woven network of fibers to resist in all directions (example is toughness of leather)
• arrangement of fibrils is determined by the fibril-associated collagens (i.e. type IX and XII)
• their helix chain is interrupted by nonhelical domains to make it more flexible than regular collagen
• they are not cleaved and keep their propeptides to block aggregation
• they bind periodically to fibrils and mediate interactions btwn fibrils and other matrix stuff
• cells also assist fibril organization by using BRUTE FORCE
• since ECM is connected to cell cytoskeleton, tugging on cytoskeleton impacts outside networks
• fibroblasts can draw in collagen and contract gel to smaller volume, or a cluster of fibroblasts and draw collagen around them into a dense capsule where the collagen goes in circles around them
• elastic fibers: fibers that enable stretching and recovery post-stretching
• inelastic collagen fibrils can be woven in to limit stretching ability and protect from tearing
• elastin: hydrophobic protein that is Pro/Gly rich but not glycosylated and has only hydroxy-Pro
• after secretion, elastin crosslinks to each other and forms network of fibers and sheets
• the chain alternates between hydrophobic segments (gives elasticity by adopting loose random coil conformation) and Ala-Lys alpha helical segments (forms crosslinks)
• elastin core is covered by sheath of microfibrils (10 nm diameter), who are synthesized before elastin and provides scaffold for elastin deposits
• microfibrils are also somewhat elastic
• made of glycoproteins, like fibrillin
• fibronectin: matrix glycoprotein that attach cells to ECM
• dimer with two large subunits joined by disulfide bonds, each subunit has multiple domains, each domain has multiple modules, each module is encoded in one exon
• the fibronectin gene is one large sequence that gets alternatively spliced to make different fibronectins
• one type of module is made of type III fibronectin repeat, which binds to cell integrins
• fibronectin fibrils: insoluble form in which the dimers crosslink by more disulfide bonds and combine with ECM
• not self-assembly, forms where cell integrins stretch fibronectins to expose an extra disulfide binding site, enabling the extra cross-linking
• makes sure fibronectin only forms fibrils where cells need them, not randomly when they're floating through the blood vessel
• RGD sequence (Arg-Gly-Asp): sequence on fibronectin that binds to cells
• anything with an RGD sequence will bind to a cell
• RGD sequence is not the only cell-binding motif, and integrins need to recognize other matrix molecules to form a completely tight bind
• cells can degrade matrix too
• necessary for repair and restructuring whole organs/tissues
• necessary for cell movement and division (making space)
• example: white blood cell localization to injury site

pg. 1169 - 1193