Book Notes: ECM Part 1

• cells utilize two strategies to build up tough tissues and organs (and ultimately, the multicellular organism)
• extracellular matrix (ECM): network of proteins and saccharides secreted by cells
• cell-cell adhesion: connecting a population of cells by their internal cytoskeletons
• tissues come in two types, depending on what strategy of support they use
• connective tissue: lots of ECM, cells are very scattered in the ECM and attach to the fibrous polymers like collagen rather than to each other
• examples include bone and tendon
• epithelial tissue: lots of cells bound in sheets of "epithelia," ECM is just a thin fibery layer on one side of the cell sheet "basal lamina"
• epithelial cells attach to each other using cell-cell adhesions
• adhesions come in 4 types
• anchoring jxns: tether cells together and connect to cytoskeleton, passes along mechanical stress throughout entire sheet through the cytoskeletal fibers
• adherens cell to cell jxn
• cell to matrix jxn
• desmosome
• hemidesmosome
• occluding jxns: seal gaps between cells of a sheet to make the sheet also a selectively permeable
• tight jxn
• septate jxn
• channel-forming jxns: make pores where cells connect to share cytoplasm
• gap jxn
• plasmodesmata
• signal-relaying jxns: sites of contact where signals get passed along
• neural synapse
• immuno synapse
• the other types of jxns also can participate in signal transmission
• epithelial cells line up next to each other, with "apex" side facing the lumenal space (for example, the inside of your intestine) and "basal" side facing the basal lamina
• from apex to basal lamina, you would find 1st occluding jxns, 2nd cell-cell anchoring jxns, 3rd channel-forming jxns, and finally cell-matrix anchoring jxns where the cell sheet meets the basal lamina
• transmembrane adhesion proteins: proteins that links the cytoskeleton inside the cell to the ECM or counterpart adhesion protein of another cell to form the anchoring jxn
• cadherin family mediates cell to cell attachment
• integrin family takes care of cell to matrix
• cadherins are calcium ion-dependent adhesion proteins
• experiment showed they are the main glue: antibodies that blocked cadherins caused cells to separate while antibodies that blocked other adhesive proteins had little effect
• classical cadherins: closely related in sequence, have 5 cadherin domains on extracellular side
• includes E-cadherin (epithelium) N-cadherin (nerve, muscle, eye) and P-cadherin (placenta, epidermis)
• nonclassical cadherins: less related (more deviation in shape, varying number of domains, etc.)
• take part in specialized gluing (desmosome proteins, special brain jxns, etc) and signaling (T-cadherin doesn't have transmembrane domain, so it doesn't perform gluing function)
• cadherin binding is homophilic: actin linked cadherin will bind only to another actin-linked cadherin
• the proteins bind at the N'-terminus tip, where it has a knob and a pocke
• Let's say cadherin Alice and cadherin Bob want to bind together.  Alice puts her knob in Bob's pocket and Bob puts his knob in Alice's pocket.  :D
• cadherin proteins have multiple subunits of "cadherin domains" (having a chain of units gives flexibility rather than a stiff rod)
• calcium ions bind between the domains to prevent flexing and stabilize binding
• also induces conformational change to increase affinity
• without calcium, cadherins are easily chewed up by proteases
• cadherin binding overall is low affinity (compare to signal receptors, who bind tightly with high affinity)
• strength comes from multiple bonds: cadherins usually group together in a huddle to form a "junction" of multiple cadherins binding their partners on the neighbor cell
• can be disassembled by peeling them apart like a Velcro strip
• know Table 19-2 (pg. 1135) for the 4 types of anchoring jxns and the proteins that make up the jxns
• while cadherins do glue cells together, they do it specifically (exist hundreds of different cadherins for this purpose)
• birds of a feather stick together (I guess biology likes segregation)
• a heterogeneous mix of cells from different organs reassociate into segregated sections
• this function is especially important for development: cell types migrate long distances toward chemicals or down chemical gradient (chemotaxis/repulsion) or by touching cells who signal where to go (contact guidance)
• at the destination, cells have to recognize it is the destination and associate with destination cells
• example is cells from neural crest spreading away to form neural network (so that you can feel your fingers and toes!)
• changing cadherins on your surface changes who you associate with: strategy used in development for migrating cells to bind to origin, and then change cadherins to bind to destination
• example: neural cells have N-cadherin, but they lose it in order to migrate away from neural crest and gain cadherin-7 to stay loosely together as a group
• when they get to destination, they lose cadherin-7 and re-express N-cadherin to tightly bind to each other and make a ganglion
• cells can switch between being mesenchymal and epithelial cells
• mesenchymal cells are unattached cells that are scattered around tissues: by expressing adhesion proteins, they come together to form sheet of epithelium
• epithelial cells can remove adhesion proteins and float away
• 3 regulatory proteins are in charge of this: Twist, Slug, and Snail.  Expressing these proteins swap cells to mesenchyme, turning off these proteins swap cells to epithelium
• malignant cancer is a form of over-expressing the transition to mesenchymal so that they float away to infect other places (metastasis)
• linking cytoskeleton to cadherins requires adaptor proteins: alpha-catenin, beta-catenin, gamma-catenin (plakoglobin), p120-catenin
• p120 regulates assembly of whole adaptor complex: removal of p120 results in cadherin degradation
• linking to actin is also important to cell-cell adhesion: removal of cytosolic domain of cadherins prevents actin-binding and results in weak cell-cell adhesion (more easily pulled apart)
• adheren jxns coordinate movement of entire cell sheets (to fold tubes like the digestive system or pinch off into organs)
• adhesion belt: adheren jxns close to apex side of all cells in a sheet connect to contractile bundles of actin within each cell
• when all the actin in the belt contracts, the whole sheet curls up like my cat (He likes to lie on his back on the cool tiles in the summer, exposing his fluffy belly; when I reach down to scratch the fluffiness, he curls his whole body and hides the fuzz.  One day, I'll be able to pet him on his belly!)
• myosin handles actin contraction (remember muscles contracting?)
• sheet lies flat without contraction
• desmosome jxns provides strength, no motion (because IFs don't have motor proteins)
• cadherins include desmoglein and desmocollin, whose tails bind the adaptors plakoglobin, plakophilin, both of whom associate with desmoplakin
• the adaptors form a thick plaque where IFs bind (not at tips of filaments but along sides of filaments)
• bind mostly to keratin and desmin intermediate filaments
• desmosomes create network of strong and stretchy IF bundles throughout epithelium

pg. 1131 - 1144 (from Chapter 14)

jxn = junction

IF = intermediate filaments

Book Notes: Membranes Part 5

• lysosomal storage diseases: caused by defect in an acid hydrolase, which results in accumulation of undigested food/waste in the lysosome
• I-cell disease: recessive disorder in which mutant GlcNAc-phosphotransferase doesn't function, so hydrolases don't get imported to lysosome
• studying this disease revealed some cells with this mutation still managed to import hydrolases to the lysosome by some M6P-independent pathway
• lysosomes also perform exocytosis to get rid of undigestible material
• usually cells use it during stress times
• melanocytes export pigments to keratinocytes to color the skin
• endocytosis: how cells take in macromolecules from outside (too big for channel import)
• membrane pulls inward to form a vesicle
• 2 types of endocytosis:
• phagocytosis: grab large particles, form vesicles called "phagosomes" greater than 250 nm diameter but depends on size of food
• pinocytosis: grab fluid and solutes, form pinocytic vesicles approx. 100 nm diameter, uniform
• professional phagocytes: specialized cells that use phagocytosis for purposes other than simply grabbing food
• macrophages and neutrophils are special white blood cells that eat up invaders as part of defense system
• they also scavenge dead cells (either by suicide "apoptosis" or longevity "senescence")
• fuse phagosomes with lysosomes for digestion
• leftover indigestibles aggregate into residual bodies and get exocytosed
• pinocytosis occurs continuously, but phagocytosis must be triggered by surface-receptor binding
• secreted antibodies find and coat antigens (maybe some key molecule on the surface of a bacterium)
• the coat of antibodies are all sticking out a tail region called Fc region
• phagocytes have Fc receptors that bind the antibodies, which triggers phagocytosis
• local actin polymerization (performed by Rho and Rho-GEFs) extend pseudopods that "grab" the antibody-covered-bacterium
• PI(4,5)P2 accumulates in the membrane near the site of receptor binding
• PI 3-kinase converts it to PI(3,4,5)P3, which depolymerizes actin at the base of the incoming vesicle to allow resealing
• the pseudopods fuse together to form the phagosome
• other receptors bind oligosaccharides on microbes w/o Ab, some recognize apoptotic cell surfaces (remember phosphatidylserine "eat me" signal?)
• the "don't eat me" signals also bind macrophages but trigger an inhibitory cascade to block phagocytosis (phagocytosis is a natural response to anything the phagocyte bumps into, from glass beads to fabric bits)
• endocytosis and exocytosis must be balanced to keep cell surface area and volume constant
• in endocytosis, clathrin continuously forms vesicles by forming clathrin-coated pits at the plasma membrane, taking in whatever fluid happens to be out there at the time
• fuse with endosomes within seconds
• caveolae: non-clathrin vesicles, caveolin proteins on cytosolic side of membrane stabilize lipid rafts when they form
• a lipid raft stabilized by caveolins will invaginate, the pinch off performed by dynamin
• caveolae travel to "caveosomes" (similar to endosome) or to the other side of the cell (transcytosis)
• molecules going through caveolae pathway avoid digestion by lysosomes and their acidic environment
• receptor-mediated endocytosis: process of being picky about your food
• cells have receptor proteins that bind to whatever food/macromolecule it wants--increases efficiency (take up more food for same vesicle volume than without special receptors)
• famous example is cells using this pathway to grab cholesterol
• low-density lipoproteins (LDL): form of cholesterol while being transported in blood
• when cell needs cholesterol, it makes LDL receptors
• LDL receptors wander around mosaic membrane until it hits a clathrin pit in the middle of formation (has site that binds to coated pits)
• LDL is released from receptor when fused with endosome (low pH) and the receptor is returned to the membrane
• lysosomal hydrolases cleave cholesterol from LDL to make it available for membrane synthesis
• too much cholesterol shuts of LDL and cholesterol synthesis (cell does make a little cholesterol by itself)
• some receptors don't enter cell unless bound to ligand, some go in whether they grabbed the macromolecule or not
• in early endosomes, the new hydrolases are zymogens, proenzymes that hav an inhibitory domain at N-terminus
• fusion with existing lysosome introduces functional hydrolases into the area who cleave the zymogens into functional hydrolases
• also pH in early endosomes is not quite low enough for hydrolase activity
• this delay allows some endocytosed stuff to be sent back out prior to digestion
• specific proteins are sent back to the plasma membrane by not being released from their receptor in the pH of the endosome, they follow the fate of their receptor
• recycling: membrane buds from endosome containing receptors that must go back to plasma membrane, endosome extends tubules that the vesicles bud from
• LDL receptor goes this pathway but deposits its ligand at the endosome
• transferrin receptor also goes this pathway but keeps its ligand
• transferrin is a carrier of iron in the blood, transferrin drops iron in the endosome, but transferrin receptor sends transferrin back to the ECM
• transcytosis: polarized epithelial cells move specific macromolecules from ECM on one side to the ECM of the opposite side
• receptors go to early endosome, then to special "recycling endosome"
• endosomes are unique to special domains: apical vs basolateral side
• signals on receptors either send them back the ECM on the side they came from, to the central degrading lysosome, or to the recycling endosome of the other side, where they finally get dispatched to the ECM of that new side.
• degradation: receptor is also degraded in the lysosome
• example is the EGF receptor: binding of EGF to receptor initiates growth but then the subsequent degradation of EGF receptors lowers the concentration of these receptors on the cell-surface, decreasing cell sensitivity to EGF post-signal (doesn't keep growing and growing)
• multivesicular bodies: endosomes invaginate internal mini-vesicles, receptors destined for digestion are selectively put on the membrane of these minivesicles so hydrolases can reach them
• clathrin-dependent receptors get a ubiquitin tag (different from proteolytic polyubiquitylation)
• other proteins see this tag and help move receptors to clathrin pits for entrance
• also used to guide receptors into multivesicular bodies
• proteins ESCRT-0,I,II,III bind to the ubiquitin in sequential order (pass cargo from one to the next)
• PI is phosphorylated into PI(3)P, who serves as ESCRT docking site
• secondary phosphorylation into PI(3,5)P2 allows ESCRT to form multimeric assemblies for efficient transport of receptors to invagination sites
• exocytosis is the endphase for two types of secretory pathways
• constitutive secretory pathway: operates continuously, from Golgi to ECM
• regulated secretory pathway: stores molecules that are only needed for specific situations in secretory vesicles, exocytosis to ECM is triggered by some stimulus
• default pathway: no special signal for molecules that are going to be secreted, anything w/o a signal will by "default" get secreted
• in non-polarized cell, molecules either (1) return to ER (2) go to lysosomes (3) go to secretory vesicles
• molecules intended for the same secretory vesicle get aggregated and packed together based on their signal patches
• the vesicles have receptors for aggregates, rather than a receptor for each molecule
• immature secretory vesicle: initial budding from Golgi is like a giant balloon with a couple of marbles inside, very loosely wrapped
• maturation comes from budding and returning excess membrane to Golgi and fusing immatures ones together, until the final vesicle is like a small balloon filled to bursting with marbles (high concentration)
• hormones and secreted hydrolytic enzymes are synthesized as inactive precursors
• are proteolytically cleaved and activated during vesicle maturation
• pro-peptide: section of polypeptide that must be cleaved for activation
• some groups of proteins are synthesized as one polypeptide and cleaved off from the giant chain for activation
• why do proteolytic processing during secretion and not anytime before?
• one reason because final proteins are too short to have originated that size: contain the signal for going into ER, contain signal for going into Golgi, contain all the signals for modification, AND contain a cleavage signal (more convenient to have extensive signal chain or group proteins under one length of signals)
• essentially, you start out as big as you need to be with all the signals, and cleave as you go, instead of starting out short but somehow including all the signals you need and the original info for function
• second reason is because hydrolytic enzymes are dangerous to cell, so you wait til last minute to activate it
• secretory vesicles either directly expel its contents or hang around the plasma membrane to wait for a signal for secretion
• cytoskeletal components guide vesicles to the site of secretion, where constitutive vesicles fuse and expel right away
• an external signal activates intracellular signals like calcium ions that triggers regulated vesicles to fuse and expel its contents
• nerve signals is a prime example, where an action potential releases calcium ions that cause release of neurotransmitters in milliseconds
• this extreme speed is aided by the fact that the vesicles are already bound to the membrane by SNAREs but not fully wound into four-helix bundle for membrane fusion-- "priming"
• only a few vesicles are primed at any one time, so as soon as it's used up, there are more vesicles that are waiting for their turn so that a neural synapse can fire neurotransmitters many times in succession
• exocytosis can be a localized phenomenon (only releasing contents in one area of the cell's membrane)
• example is allergic reaction, where allergen causes mast cells to release histamine
• experiment attached an allergen to a tiny bead, and the cell coughed out histamine only where the bead touched, but when the cells were floating in a solution concentrated with allergen, the cell released histamine from all over its membrane
• another example is killer T lymphocyte releasing death proteins only next to victim so neighbors don't get the death signal
• secretory vesicle's leftover membrane is quickly used again in endocytosis and its membrane proteins are sent to lysosomes for degradation or recycled
• some exocytosis is intended for membrane growth (as opposed to quickly recycling whatever extra membrane you put in with exocytosis)
• wound or puncture to the membrane allows extracellular calcium ions to flood in, causing a signal for emergency plasma fusion to seal the gap
• vesicles fusing to form this gap aren't specially produced by Golgi at this time, the cell just uses whatever membranes happen to be nearby (lysosome, endosome, vesicles, blah)
• polarized cells must direct secretory vesicles toward one side or the other (not just a simple default pathway)
• example is epithelial cells, who have apical side and basolateral side
• tight junctions in the membrane block outer leaflet membrane proteins from diffusing between the two faces (so only outside membrane proteins are unique to one side or the other, cell contents are not necessarily distinct from one half to the other)
• epithelial cells secrete digestive enzymes and mucus on apical side, and send basal lamina components to basolateral side
• these components and enzymes travel together until they reach the trans Golgi network
• apical membrane contains lots of glycosphingolipids to protect from digestion
• membrane proteins intended for apical surface associate with glycosphingolipid rafts in the TGN, so natural lipid aggregation has side effect of sorting proteins!
• membrane proteins intended for basolateral surface have sorting signals in cytosolic tail that direct coat proteins to take them to the surface
• if membrane containing these proteins get involved in some endocytosis and end up on some endosomes, the same tail signals direct their return to the membrane
• nerve cells have specialized secretory vesicles along with the normal ones
• synaptic vesicles: tiny 50nm diameter vesicles storing neurotransmitters
• because they are needed to rapid priming, firing, and re-priming, synaptic vesicles are made not from Golgi but from nearby nerve terminal membrane
• Golgi makes necessary membrane components and they fuse with the membrane at the nerve terminal
• membrane components include the neurotransmitter transporters
• when it's time for firing, the membrane is rescued by endocytosis and imports neurotransmitters to form a synaptic vesicle rather than an endosome
• after firing (vesicle has fused the membrane), the same membrane components get recycled into more synaptic vesicles

pg. 785 - 809 (from Chapter 13)

PI = phosphoinositide

Biochem Interlude

Someone has requested I post last semester's Biochem notes for any students taking Biochem in the upcoming fall.  I'll do so after Cell Bio is over (but prior to summer!)

Book Notes: Membranes Part 4

• TGN (trans Golgi network) sorts proteins going out to lysosome, exterior, etc.
• lysosome: membrane-bound organelle with hydrolytic enzymes inside
• e.g. proteases, lipases, phosphatases, nucleases, etc.
• acid hydrolase: general term for all of those enzymes, are activated by proteolytic cleavage and acid environment (pH 4.5 - 5.0)
• lysosomal membranes are glycosylated to prevent the organelle from digesting itself (sugar addition blocks cleaving)
• the end products of digestion get sent back to cytosol for reuse
• vacuolar H+ ATPase: a pump in the lysosome membrane, pumps in H+ to keep acid and build concentration gradient, as H+ goes out, small subunits also go out (remember symport?)
• lysosomes are diverse in shape and size but are biochemically identical (a certain Ab stains only organelles with hydrolases, and the images showed that these large or small, round or elliptical bodies were in fact all lysosomes)
• diverse in function: lysosomes digest intra/extracellular debris, make nutrients, and destroy phagocytosed microbes
• diverse in formation: lysosomes progress through different stages from endosome (initial formation of membrane vesicle plus new enzymes plus new food) to endolysosome (fuse with existing lysosome for extra materials) to mature lysosome (most of the new food is all digested)
• vacuoles: very large fluid-filled vesicles in plant/fungi
• store nutrients, degrade waste, cheap volume growth, maintain turgor pressure, maintain chemical homeostasis (store excess chemicals in the vacuole so cytosol concentrations are constant)
• endocytosed molecules first enter the cell in a pinched off vesicle
• vesicle meetes early endosome and fuses
• some stuff (either membrane or content) gets sent back to the plasma membrane
• pump is slowly building up acidity of endosome, becomes late endosome
• either meets a lysosome to form an endolysosome or digests all by itself and mature into a lysosome directly
• bud off endosome membrane to give back to Golgi
• autophagy: self-degradation of organelles
• retiring organelle gets wrapped up in another membrane to form an autophagosome and fuses with a lysosome
• in starving conditions, cell digests some cytosol too
• also helps in restricting development
• enclosed tubular membrane must envelope and then reseal around organelle
• phagocytosis is a similar process
• how do lysosomal proteins reach the lysosome?
• both lysosomal hydrolases and lysosomal membrane proteins get transported into rough ER and then to TGN
• transport vesicles bud from TGN, only lysosomal proteins are brought
• Mannose-6-Phosphate (M6P) receptor: transmembrane receptor in TGN, recognizes the M6P groups added onto only the N-linked oligosaccharides of the lysosomal proteins
• this is delivered to early endosomes
• receptor binds at pH 6.5 - 6.7 and releases at pH 6 (late endosomes)
• an acid phosphatase in the endosome removes the phosphate from the mannose, so the signal is gone
• retromer complex: coat protein that recognizes the cytoplasmic tails of the M6P receptor to form a vesicle
• retromer-coated vesicles return M6P receptors to the Golgi
• some lysosomal proteins may escape receptor binding and end up outside the cell, but they do no harm (ECM pH is 7.4)
• lysosomal proteins have signal patch to receive M6P
• GlcNac phosphotransferase in cis Golgi binds at signal patch and adds GlcNac-phosphate to one or two of the mannose res on each oligosaccharide chain
• second enzyme in trans Golgi cleaves off GlcNac to leave behind the phosphate on the mannose
• since each protein has many oligosaccharide chains, they consequently have many M6P, making the binding to the receptor high affinity (more to love :D)

pg. 779 - 785 (from Chapter 13)