Saturday, April 16, 2011

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

Book Notes: ECM Part 2

  • cell-cell junctions can send signals into the interior
    • disintegrating the adherens jxns frees the adaptor catenins to wander around cytosol to spread a signal
    • nonclassical cadherins may serve as co-receptors for signal receptors (i.e. VEGF is a survival signal that a receptor tyrosine kinase binds, but the receptor cannot bind the signal without VE-cadherin's help)
  • selectins: cell-surface proteins that bind carbs (they are otherwise known as lectins)
    • they also bind junctions, but specialize in temporary cell-cell adhesion
      • example are white blood cells who patrol around and use this temporary binding to follow tracks so they can migrate between the bloodstream and tissue (i.e. mustering of white blood cells to location of inflammation)
      • "rolling" motion along vessel endothelium
    • transmembrane protein with lectin domain that binds specifically to oligosaccharides on another cell
      • L-selectin: on white blood cells
      • P-selectin: on blood platelets and inflamed endothelial cells
      • E-selectin: on endothelial cells
  • integrins: cell-surface proteins that bind other surface proteins
    • also specialize in temporary cell-cell adhesion but is stronger than selectins
    • work with selectins on white blood cells, but the stronger adhesion is necessary to overcome the selectins' affinity for blood vessel endothelium so that the white blood cell can escape from the vessel into tissue to save the day :D
    • both selectins and integrins are heterophilic, they don't bind to selectins and integrins of the same type
      • selectins bind glycoproteins and glycolipids
      • integrins bind transmembrane immunoglobulins!
  • immunoglobulin (Ig) superfamily: cell surface proteins with extracellular domains that are similar to antibodies
    • intracellular cell adhesion molecule (ICAM): type of Ig bound by white blood cell integrin, heterophilic
    • vascular cell adhesion molecule (VCAM): type of Ig bound by white blood cell integrin, heterophilic
    • neural cell adhesion molecule (NCAM): type of Ig on neurons, homophilic, contribute to cell-type specific organization but not as necessary as cadherins
  • synapse formation depends on axon finding the correct target tissue to form synapse with
    • Ig adhesion molecules help by pairing up homotypically with target cells that express the same type of Ig
      • example is Fasciclin3, who leads neuronal growth cones to connect with motor neurons in the muscle
      • another example is Sidekicks, who matches neurons in different layers of the retina to growing axons
  • synapse formation also requires complex assembly of receptors, ion channels, synaptic vesicles, docking proteins, etc, etc.
    • scaffold protein: chains of proteins with various binding domains
      • PDZ domains: about 70 A.A. long domains that bind cytosolic C-term tails of specific transmembrane proteins
      • the various domains connect to the cadherins, channels, exo/endocytosis regulation protein, cytoskeleton, etc, etc
      • multiple scaffold proteins can also unite to form network connecting all the components of a sypnase
    • Discs large (Dlg): protein that assists in synapse formation, occluding jxn formation, control of cell polarity, and control of cell proliferation
  • all epithelial cells (1) are polarized because one side faces basal lamina while the other faces the lumen and (2) form selectively permeable barrier by utilizing occluding jxns to prevent unfiltered leakage between cells
    • tight jxn: the occluding jxn of vertebrates, prevent molecules from seeping past the cells in any direction and prevents membrane pumps from going to the wrong side (i.e. in gut lining, nutrients must go unidirectionally, from lumen to bloodstream, occluding jxn prevents them from leaking backwards and prevents nutrient pumps from going to the wrong side and pumping them backwards)
    • experimentally show that tight jxns seal cell sheets by putting in a traceable molecule of a certain size
      • visualization of molecule will show it never passes through the cell sheet
    • sealing not COMPLETELY impermeable; small molecules can diffuse through
      • but size limit of small molecules varies between tissues: bladder is almost totally impermeable to tiny sodium ions, while small intestine allows their passage
    • sometimes cells loosen their junctions to allow nutrients to diffuse through easily, especially after meal, when the concentration gradient is high enough to have nutrients go in the correct direction by themselves: "paracellular transport"
  • sealing strands: chains of adhesion proteins along the length of the cell close to the apex side that bind to partner chains on neighboring cells (not like one pocket or jxn like in cadherins, but chains of jxns all around the cell)
    • claudins: the transmembrane adhesion proteins forming the sealing strands
    • assisted by occulin and tricellulin, who seal the corners where 3 cells meet
    • claudin family has members of varying structure to form different sized pockets to change the permeability
      • paracellular pores: the selective channels formed by various combos of claudins
  • occluding jxns have to occur above adherens and desmosomes, so all three are held together in junctional complexes
    • Tight junction protein (TJP): a.k.a. ZO protein is a cytosolic scaffold protein that organize and anchor the adhesion proteins of the 3 types of jxns
  • septate jxns: occluding jxns of invertebrates, very similar to vertebrate version
  • cell-cell junctions and basal lamina govern the polarity
    • experiment: grow MDCK cells suspended in gel all by itself, shows no polarity
      • let them divide into small colony, the colony will organize itself into a vesicle shape, secrete basal lamina outside the vesicle, and all the cells now show polarity between the outside and inside of the vesicle
      • polarization is spontaneous but depends on contact with neighboring cells
    • experiment: screen for mutations in C. elegans that affect polarization of embryo
      • PAR genes: family of genes that control polarization
      • LKB1: vertebrate homolog of Par4, loss of LKB1 causes loss of polarization, overexpression of LKB1 causes self-polarization (not neighbor dependent), so cells can't orient TOGETHER into a sheet
  • therefore, proteins assisting with polarity come in two categories: producing polarization in general, and orienting groups of cells correctly with landmarks like the basal lamina
    • category 1: produce polarization in individual cell
      • Par3: scaffold protein with PDZ domain, binds to the other two
      • Par6: similar to Par3
      • atypical protein kinase C (aPKC): binds to the two Pars
      • the complex has binding sites for small GTPase Rac and Cdc42 and others
      • Rac and Cdc42 control actin assembly (lack of Rac causes cells to polarize in the wrong direction)
      • assembly of the complex is one specific part of the cell cortex recruits Rac and Cdc42 to polarize the cell towards that one part
      • the complex also controls the Crumbs complex (with scaffolds Discs-lost and Stardust, assembles on apical side) and Scribble complex (with scaffolds Discs-large and Scribble, assembles on basal side)
    • category 2: orient polarized cells to each other and to environment
      • the proteins get coordinated to the different junctions by binding to tails of the transmembrane adhesion proteins
      • from apical to basal:
        • Crumbs complex
        • tight jxn, Par3-Par6-aPKC complex
        • Scribble complex
      • Rac and friends induce cell to secrete basal lamina ECM components to opposite end of cell (since most of the proteins were associated near the apical side with the tight jxns, the "opposite" side is basal lamina)
  • planar cell polarity: cells in a tissue with apico-basal polarity show additional polarity towards another direction perpendicular to the apico-basal axis.
    • example: in trachea, all cells are lined from apical to basal side horizontally, BUT their cilia must sweep in a vertical direction to shift mucus and dust upwards and out
    • studying mutations revealed important proteins whose function is not exactly known yet (they are also oddly named)
      • Frizzled and Dishevelled (these two part of a signal pathway called "Wnt"), Flamingo and Dachsous (these two code for cadherins)
  • gap junction: channel between cells that allow them to share cytoplasm without having to exocytose stuff and the other cell endocytose it (plant counterpart is plasmodesmata)
    • channel is about 2-4 nm long formed by connexins and innexins.
    • ions and small molecules flow easily through them (dyes and electrical currents transfer quickly throughout a sheet of cells)
    • channel is about 1.5 nm wide (ions, sugars, A.A, N.T., vitamins, cyclic AMP and other second messengers) but can't share macromolecules (proteins, DNA, etc)
  • connexon: a hemichannel formed by six connexins (4 pass transmembrane protein)
    • the two connexons of two cells align and stick together to form whole channel
    • gap junction consists of a lot of connexons, so it looks more like a sieve of pores rather than one giant channel
    • connexons can be assembled out of combos of different kinds of connexins to produce different permeabilities
    • even two hemichannels can be made of radically different connexins and still connect together
    • connexin have half life of a few hours, connexons are always added around existing gap jxns and removed from center of existing gap jxns
    • connexins are inserted into membranes and delivered to the plasma membrane by exocytosis
      • they diffuse around the plasma membrane until they hit an existing gap jxn and get trapped there
      • unpaired hemichannels are either closed or can be opened to serve as channels for release or entrance of small molecules
  • gap jxns enable instantaneous transfer of action potential between joined cells
    • useful in nerve cells where speed and synchrony is important in the response (i.e. escaping from surprise attack)
    • useful also in maintaining homeostasis of a tissue (small molecule concentration can fluctuate from cell to cell, but having that channel allows them to diffuse out of high concentration cells to boost low concentration cells)
  • even whole channels can flip between open and closed, and permeability is reduced by low pH or high calcium concentration
    • ECM is full of calcium, so if one cell is punctured, calcium rushes in, the gap junction senses this and closes, protecting neighbors from the damaged cell (remaining connected means whatever gets into the puncture cell can also get into the healthy cells)
  • plasmodesmata: the only jxn for plants, is a pore in which the plasma membranes of different cells are CONNECTED, not just connection by a protein channel
    • channel about 20-40 nm diameter
    • desmotubule: narrow cylindrical tubule that connects to the ER in the center of the channel
      • cytosol is shared around the desmotubule
  • basement membrane (basal lamina): thin, tough, but flexible sheet of ECM that is essential to all epithelia
    • 40-120 nm thick, surrounds muscle and fat and epithelial cells
    • function in forming structure, filtering between its two sides, influence metabolism, promote cell survival, proliferation/differentiation, act as pathway for migratory cells
    • skin depends on connection the basal lamina which holds it fast to the underlying dermis
  • basal lamina made of FIBROUS PROTEINS (glycoproteins with short oligosaccharides) and GLYCOSAMINOGLYCANS (GAGs, linked to proteins to make proteoglycans)
    • lamina composition varies, but there are key components
      • glycoproteins: laminin, type IV collagen, nidogen
      • proteoglycan: perlecan
      • associate with other molecules: collagen XVIII, fibronectin
    • laminin-1: classical laminin, has 3 polypeptide chains arraned like bunch of flowers in a vase
      • exist various isoforms of the each chain type, so laminins come in various combos like connexons
      • interact with each other at heads to form a sheet (PRIMARY ORGANIZER)
    • type IV collagen: 3 long protein chains that form superhelix, interrupted in several places to allow bending
      • interact with each other at terminal domains to form flexible but strong network (TENSILE STRENGTH)
    • laminin binds to perlecan and nidogen and cell laminin receptors, while collagen binds to perlecan and nidogen
    • so perlecan and nidogen connect the networks of laminin and collagens (LINKERS)
    • cell receptors like dystroglycan grab laminin by their feet, laminin heads network together to form a sheet, nidogen and perlecan link the laminin to collagen network
  • examples of diverse functions of basal lamina
    • kidney: superthick lamina blocks maromolecules from leaving blood when filtering for urine
    • epithelium movement: blocks fibroblasts of connective tissue from touching epithelial cells, but allows passage by macrophages and lymphocytes (they have proteases to cut holes through the lamina for them)
    • regeneration: studied at neuromuscular jxn where nerve meets muscle, but they are separated by basal lamina
      • lamina has special isoforms of all of its components
      • when the nerve cell happens to be destroyed, new axons locate the old site by recognizing the special markers of the basal lamina that still marks the junction
      • works similarly for muscle cell regeneration
      • nerve cell deposits agrin proteoglycan at the lamina site, who induces muscle cells to form acetylcholine receptors on their surface, while muscle cells deposit special laminins that bind to voltage gated ion channels of nerve cells
      • when either or both nerve and muscle gets destroyed, muscle regenerates first by natural wound response, but gets positioned correctly by the existing agrin, then growing axons come down to find the old site by binding to the existing laminins

pg. 1145 - 1169
A.A. = amino acids
N.T. = nucleotides