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