BISC305 F19laverty E 1 PDF

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Chapter 11, 12, 17 Chapter 11 Lipid membrane - 5nm thick Plasma membrane is involved in cell communication, import and export of molecules, and cell growth and motility - Receptor proteins for cell communication Phospholipid - hydrophilic head and 2 hydrophobic tails - Most common in animal membranes: phosphatidylcholine (choline attached to phosphate group) - Glycerol attaches head to tail - Cholesterol and glycolipids are amphipathic Fat droplets appear in water (and membranes form) because of free energy - more stable together - Acetone also polar, dissolves in water Fat molecules (triacylglycerols) are entirely hydrophobic (third tail flips to the other side, so it is surrounded by nonfat Free edges are quickly eliminated Phospholipid bilayers spontaneously close in on themselves to form sealed compartments (like spheres) - Pure phospholipids form liposomes

Fluidity of a Lipid depends on its composition - On tails: - Shorter - more fluid - Unsaturated (kinked) - more fluid (farther away from each other) (oil at room temp) - Temperature affects

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Cholesterol thickens the bilayer (also composes 20% of membrane in animals) - Fills in gaps of kinked chains - Increases fluidity at low temps, decreases fluidity at high temps

Phospholipids - manufactured in ER, created from free fatty acids -deposited into cytosol - how does it get to the outside? - scramblase - enables flip flop Golgi membrane contains flippases - remove specific phospholipids from one side of the bilayer facing the exterior space and flip them into the monolayer that faces the cytosol - Maintains asymmetry that is so prevalent in animal cells - One layer always faces the cytosol - other layer either faces in inside of the organelle or the outside membrane

Glycolipids located mainly in plasma membrane, only in noncytosolic half - Face cell exterior - Acquire sugar groups in golgi apparatus Membrane proteins: 50% of the mass of plasma membrane

Integral membrane proteins - can only be removed with detergents Transmembrane proteins are often a helixes in middle

B barrel can also form a hydrophilic pore Detergents - have one single hydrophobic tail, cone shaped, form miscelles - This solubilizes proteins Bacteriorhodopsin - small protein in plasma membrane of salt thing - Pumps hydrogen out of the cell - When retinal absorbs light, it changes shape, causes conformational change to pump out hydrogen Plasma membrane is stabilized by a meshwork of filamentous proteins called the cell cortex - Example - spectrin network in blood cells - Cell cortex also used to move in more complex cells Membrane proteins can diffuse throughout the layers - Chick and mouse cell hybrid - over time, all were integrated - Except in tight junctions Most proteins have sugars attached (glycoproteins) - Form glycocalyx - sugar coating / carbohydrate layer - Protects from mechanical damage - Also give slimy surface because the carbs attract water Lectins - bind to particular oligosaccharide side chains (also involved in signaling) - Lectin recognizes neutrophil (white blood cell) How to measure membrane flow? - Originally, just tagged antibodies and watched - Now - FRAP attack (fluorescence recovery after photobleaching) - Labeled with fluorescent antibody - Bleached, rate of fluorescence recovery is measured - Reason why bad - tracks hundreds or thousands of proteins, not just one individually - Single particle tracking is new answer - Gold nanoparticles, can track exact movement

Chapter 12 Co2 and 02 can diffuse simply across the membrane Transporters shift molecules across the membranes by changing shape Channels have pores - Ion channels

Virtually any charged particles can’t get through without help Na is most plentiful outside the cell, K is most abundant inside - Positive must be balanced by negatives - Na is balanced by Cl- outside - K is balanced by many things inside - Calcium and magnesium higher outside - Electrical balances create membrane potential Resting membrane potential - not zero Channels - discriminate mainly on the basis of size and electric charge Transporters - specific binding shapes Passive transport - no energy needed (gradients)

Active transport - uses pumps, energy can come from ATP hydrolysis, a transmembrane ion gradient, or sunlight Electrogradient - cells want to pull positive charge in For Na - voltage and concentration gradient work together K - opposing electro and chemical gradients Water can diffuse simply, but it’s slow, so aquaporins are used - osmolarity based on solute concentration

Transporters are super selective Glucose - Glucose transporter - No charge, no electrochemical gradient, only chemical gradient - Concentration changes depending on insulin and glucagon Transmembrane pumps have 3 mechanisms: - Gradient driven pumps - link the uphill transport of one solute across a membrane to the downhill transport of another - Atp driven - Light driven (bacteriarhodopsin) ATP driven Na pump accounts for 30% of ATP consumption - 3 Na out, 2 K in - Phosphate from ATP is used in pump - Ouobain is a toxin that inhibits the pump by preventing the binding of K

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Keeps the steep concentration gradient

Ca2+ like Na is also kept at a low concentration within the cell - High concentration gradient driven by ATP associated Ca2+ pumps - Similar to Na, but no other ion used Gradient driven pumps

Epithelial cells of the gut - transport glucose from the gut lumen across the gut epithelium and into the blood - If they only had passive glucose transports, they would release glucose into the gut lumen after fasting just as freely as they take it up from the gut after a feast - Also possess a glucose-Na symport - Na concentration gradient is so steep, it drags glucose in with it across its concentration gradient - Binding is cooperative - If the gut epithelial cells had only this symport, they would take up glucose and never release it - So they have 2 types of transporters at opposite ends of the cell - In apical domain (faces gut lumen), have the glucose Na symports - Basal and lateral - have passive to release glucose - Tight junctions stop ends from meshing Plant cells, bacteria, and fungi don’t have Na pumps in plasma membrane - Instead they rely on H+ gradient, H+ pumps Channels - one example is gap junctions Ion channels - have ion selectivity - Depends on the diameter and shape and charge distribution by amino acids - Not continuously open, most are gated

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Faster than any transporter (don’t need to change shape)

K+ leak channels are common - Maintains resting potential - Nernst equation calculates resting membrane potential based on ions in vs out Patch clamp recording can determine how individual ions behave - Microelectrode, one single ion channel - Results in jerky movements - channel like a light switch (even when it’s supposed to be open completely) Voltage gated channel - controlled by membrane potential Ligand-gated channel - controlled by binding of molecule Mechanically-gated channel - mechanical force - Auditory hair cells Voltage-gated ion channels have domains called voltage sensors that are sensitive to changes in membrane potential - Changes above a certain threshold exert an electrical force - Induce a change in membrane potential - which affect many other channels downstream Neuromuscular junction - acetylcholine stimulates muscle contraction by binding to the acetylcholine receptor - Channel opens when acetylcholine attaches - Na flows down gradient

Chapter 17

Cytoskeleton- intricate network of protein filaments that extends throughout the cytoplasm - Bones and muscles of cells - Responsible for large scale movements - Built on a framework of the 3 main types of filaments - Intermediate filaments, microtubules, and actin filaments Intermediate filaments: ropelike fibers - One type forms a meshwork called the nuclear lamina - Give cells mechanical strength - Distribute mechanical stress - Very flexible, great tensile strength - Deform under stress but do not rupture

Microtubules: hollow cylinders made of tubulin - Usually have one end attached to a centrosome - More rigid, rupture when stretched Intermediate filaments have great strength - Called intermediate because their diameter is between that of actin and myosin filaments - Toughest and most durable of all cytoskeleton - Often anchored to cell-cell junctions called desmosomes (where plasma membrane is connected to that of another cell) - Nuclear lamina - underlies and strengthens the nuclear envelope - Lacelike structure throughout the cell Like a rope - many long strands are twisted together to provide tensile strength (ability to withstand tension without breaking) - Alpha helical monomer coiled (2 in different directions) and formed a tetramer, then tetramers line up together too

Both ends of the tetramer are the same, as are the two ends of assembled intermediate filaments - This distinguishes these filaments from microtubules and actin because they are polar - Central rod stays same size generally, heads and tail domains alter

Keratin filaments - most diverse class of IFs - Every kind of epithelium has its own distinctive mixture of keratin proteins - Specialized keratins also occur in hair, feathers, and claws - All form from a different mixture of keratin subunits - Keratin filaments typically span the interiors of epithelial cells from one side of the cell to the other, and connected through cells in desmosomes - Ends of keratin filaments are attached to desmosomes - Stretchy skin - Epidermolysis bullosa simplex - mutation in keratin, skin is highly vulnerable to mechanical injury (small bump can cause huge blisters) - Disease can be reproduced in transgenic mice expressing a mutant keratin Neurofilaments in nerve cells - ALS (amyotrophic lateral sclerosis) is associated with abnormal accumulation of neurofilaments in cell bodies of axons of neurons In nucleus - IFs form 2-dimensional meshwork - Formed by lamins - Nuclear lamina disassembles and reforms at each cell division - Thought to provide attachment sites for the chromosomes - Collapse and reassembly of the nuclear lamina is caused by phosphorylation and dephosphorylation of the lamins - Phosphorylation weakens interactions and causes them to fall apart - Defects in nuclear lamina associated with progeria - aging prematurely - Wrinkled skin, lose teeth and hair, cardio disease by teens Many IFs are further stabilized and reinforced by accessory proteins like plectin - Cross-link the filaments into bundles and connect them to microtubules, actin filaments, and to adhesive structures in desmosomes - Needed to survive mechanical stress

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Mutation in plectin results in mice that die a few days after birth Human disease - mix of muscular dystrophy, ALS, and neurodegeneration

Microtubules - long, relatively stiff hollow tubes can rapidly disassemble in one location and reassemble in another - Usually grow out from an organizing center - Grow out from a centrosome - Responsible for transporting and positioning membrane-enclosed organelles within the cell and for guiding the intracellular transport of various cystolic macromolecules Mitosis - microtubules first disassemble, and then reassemble into the mitotic spindle - Segregates chromosomes into two daughter cells Microtubules can also form stable structures, like cilia and flagella Microtubules are built from subunits of tubulin - Dimer of a tubulin and b tubulin - Stack together to form hollow tube (13) - Each protofilament has a structural polarity, with a tubulin exposed at one end and b tubulin at the other - Overall, whole microtubule has polarity too - B is plus end - A is minus end - Add more rapidly to the plus end - Polarity is crucial for assembly of microtubules and their role once they are formed In animals - centrosomes are the organizing centers - y tubulin ring complexes serve as starting points (nucleation sites) - Minus end embedded into centrosome at nucleation site, growth occurs at plus end - 2 centrioles at center of the centrosome, function is mystery (still made up of microtubules) Why do they need nucleation sites? It’s much harder to produce a microtubule from scratch (by assembling a ring of ab dimers) rather than adding the dimers to existing y tubulin Microtubules change a lot - one minute, many dimers adding to plus end, then all of a sudden they shrink and lose dimers from the plus end - Dynamic instability (switching back and forth between polymerization and depolymerization) allows for rapid remodeling - Only stable by attachment of plus end to something

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The centrosome can thus be compared to a fisherman casting a line: if there is no bite at the end of the line, the line is quickly withdrawn, and a new cast is made; but, if a fish bites, the line remains in place, tethering the fish to the fisherman.

Dynamic instability is driven by GTP hydrolysis - Each free tubulin dimer contains one GTP molecule tightly bound to b tubulin, which hydrolyses the GTP to GDP shortly after the dimer is added to a growing microtubule - GDP remains tightly bound - When polymerization is proceeding rapidly, tubulin dimers add to the end of the microtubule faster than the GTP they carry is hydrolyzed - End of a rapidly growing microtubule is composed of entirely GTP-tubulin dimers, which form a GTP cap - These bind more strongly to their neighbors, and they pack together more efficiently - It will occasionally happen that the tubulin dimers at the free end of a microtubule will hydrolyze their GTP before the next dimers are added, so that the free end of the protofilaments are now composed of GDP-tubulin - These associate less tightly, tipping the balance in favor of disassembly - Because the rest of the microtubule is composed of GDP-tubulin, once depolymerization has started, it will tend to continue, and the microtubule starts to shrink rapidly - Tubulin dimers can be free in cytosol, ready to be taken up Colchicine - binds tightly to free tubulin dimers and prevents their polymerization - When they can’t polymerize, tubulin loss continues until the spindle disappears Taxol - opposite - binds tightly to microtubules and prevents them from losing subunits - Can grow but cannot shrink - Same overall effect tho - arresting dividing cells in mitosis - Conclusion - for the mitotic spindle to function microtubules must be able to assemble and disassemble - Antimitotic cells, including colchicine and taxol, are used to treat human cancers Proteins can attach to microtubules and inhibit dynamic instability - Stabilized microtubules then serve to maintain the organization of the differentiated cell Most animal cells are polarized - Reflection of polarized systems of microtubules in its interior - Help position organelles - Guide streams of vesicular and macromolecular trafficking - Movement by microtubules is way faster than free movement - Mostly unidirectionally in microtubules too

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Depend on microtubule-associated proteins - Stabilize against disassembly

Motor proteins drive intracellular transport - Saltatory movement occurs along either microtubules or actin filaments - Movements are driven by motor proteins - Use energy of ATP hydrolysis, unidirectional - Motor proteins can also attach to other cell components, so they can carry cargo - Kinesins: move towards plus ends - Dyneins: move towards minus ends Both kinesins and ciliary dynein have two globular ATP-binding heads and a single tail Cytoplasmic dyneins interact with microtubules in a stereospecific manner - Motor protein will attach to a microtubule in only one direction - Tails can bind to cell component like vesicle or other cargo - Globular heads have ATPase activity - support conformational changes for binding, release, binding (walking) Microtubules and motor proteins play an important part in positioning organelles within eukaryotic cell - In most cells, tubules of the ER reach almost the edge of the cell - Kinesins pull ER outward along microtubules, stretching it like a net - Dyneins attach to golgi apparatus and pull it in toward the nucleus - When cells are treated with colchicine (microtubule disassembly) - ER and golgi chance location dramatically How we know - Usual form of studying proteins (isolating them) doesn’t really work for motor proteins - 2 advancements - Microscopy - Could assemble working transport system from scratch - Teeming cytoplasm - Long squid axon, squeezed out like toothpaste - New video microscopy - Snaking Tubes - Discovery of kinesin - Incubated axoplasm extract with microtubules and organelles in the presence of AMP-PNP, pulled out microtubules with motor proteins still attached - Added ATP to release proteins, found kinesin that could stimulate the gliding of microtubules along a glass cover slip - Fluorescent tagging of kinesin shows steplike movement Cilia and Flagella

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Cilia grow from basal body which is the organizing center - Motile cilia beat in a whiplike fashion - Found in respiratory tract and oviduct Flagella longer, designed to move the entire cell Cilia and flagella - 9+2 arrangement - 9 doublet tubes in bundle, 2 singles in center Accessory proteins help move - Most important is ciliary dynein - Globular heads interact with other microtubules - Sliding force is converted to a bending motion Kartagener’s syndrome - defects in ciliary dynein - Men infertile, sperm immotile - Increased susceptibility to bronchial infections Single cilia can also be antenna for extracellular signals

Extra notes - addition of double bond to fat makes the melting temp decrease Data point one - low temp induces desaturases (more fluid) Data point 2 - even in aerobic conditions, cancer cells favor metabolism via glycolysis, mpc1 and mpc2 Glucose increases the uptake of sodium and thus water TFR - non lipid rafts, PLAP - lipid rafts BCRP co-localizes with lipid rafts, needs cholesterol Cholesterol mostly stiffens Carbs on layer - n linked (asparagine), o linked (serine) Shaker K ion channel - 4 subunits, positively charged AAs that “feel” the voltage 6X106 ions per second...