BIOL 23100 Final Exam Study Guide
BIOL 23100 Final Exam Study Guide BIOL 23100 - 001
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BIOL 23100 - 001
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This 9 page Study Guide was uploaded by Gayatri on Saturday December 12, 2015. The Study Guide belongs to BIOL 23100 - 001 at Purdue University taught by Peter James Hollenbeck in Fall 2015. Since its upload, it has received 165 views. For similar materials see Biology III: Cell Structure And Function in Biological Sciences at Purdue University.
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Date Created: 12/12/15
BIOL 23100 Final Exam Study Guide The Cytoskeleton: Formation of Specialized Actin Structures • Labile vs. stable actin-based structures in the cell o Labile, transient, highly dynamic structures § Eg. Lamellipodium: the leading edge of a crawling cell such as a WBC § Supported by broad, branched actin filament network o Stable, long lasting structures § Eg. Sarcomere: highly ordered array of actin and myosin filaments § Produces muscle contraction • Four additional kinds of actin-binding proteins: o Crosslinking/bundling proteins § Gather, arrange actin filaments into arrays § Have two or more binding sites for wall of actin filament § Cross-linking proteins link actin filaments loosely into a meshwork; have actin binding sites separated by a long, flexible rod § Binding proteins have a shorter connection and link filaments into tighter, stiff bundles o Polymerizing/depolymerizing proteins § Polymerizing proteins stimulate nucleotide exchange (behave like GEFs), bind monomers that have XDP § Depolymerizing proteins stimulate disassembly, bind filaments, sever or interact with ends o Side-binding proteins § Bind along the sides of actin filaments and stabilize them § Restrict access to filament wall from other proteins o Motor proteins § Bind ATP, hydrolyze it, turn chemical energy into mechanical force to move along actin filaments § Specific to actin/microtubules, positive and negative ends • Microvillus: actin filled projections of cell surface, ~1000/epithelial cell o Tightly linked actin filaments in parallel bundles, linked by 2 actin binding proteins: § Villin § Fimbrin • Lamellipodium: a part of the cytoplasm o Very dynamic structure that surges forward, retracts, and changes its direction of extension very o Supported by actin filaments o Properties: § Rapid assembly § Rapid disassembly § Interlinked filaments § Remodeling in response to signaling o Actin-binding proteins involved: § ARP complex: nucleation protein • Sits on existing filament and nucleates new filament at 70 degrees (branched networks) § Capping protein: (+) end • Keeps filaments short § Depolymerizing protein: “cofilin” • Binds sides of filament that have ADP-actin and severs them § Polymerizing protein: “profilin” ADP/ATP exchange protein • Binds ADP actin, flips it and stimulates exchange of ADP (out) and ATP (in), generates polymerase competent actin Molecular Motors & Actin: Muscle and Non-muscle • Myosin o Myosin motor proteins: bind and hydrolyze ATP, use energy to “walk” along actin filaments § 19 families, can be cell/tissue or organism specific § Share same basic structure with a tail region (cargo binding domain, differs with different myosins), a neck region, and a globular head region (motor domain, binds actin filaments and binds and hydrolyzes ATP) o Mechano-chemical cross-bridge cycle of myosin II § Consists of two types of cycles: • Chemical cycle: ATP binds, hydrolyzes, ADP and Pi release • Mechanical cycle: actin binds, translocation occurs, release occurs, conformational change and back to 1 stept • The sarcomere and muscle contraction o Components of the sarcomere o The sarcomere is a repeating sub-cellular unit: hundreds or more of them are stacked up longitudinally to form a myofibril, and many myofibrils are laterally connected within each skeletal muscle cell § The boundaries of each sarcomere are formed by structures called Z discs, where the actin filaments are anchored at their plus ends. § In the middle of the sarcomere are located the bipolar myosin “thick” filaments which slide toward the actin (+) ends to cause muscle contraction • Tropomyosin: side binding protein that sits on actin, blocks myosin’s access to actin • Troponin complex: regulates position of tropomyosin o Role of calcium ion, and how its cytoplasmic concentration is controlled in the muscle cell. § Calcium ions act as intracellular signaling molecules for muscle contraction § [Ca ] is controlled by: 2+ 1. Voltage gated Ca chan2+ls Ø Release Ca into cytosol in close proximity to the sarcoplasmic reticulum, which is the equivalent of an ER in a muscle cell 2. Ca 2+ gated Ca 2+ channels 2+ 2+ Ø Release Ca into cytoplasm rapidly when stimulated by Ca release 2+ from T-tubules 2+ § [Ca ] affects troponin complex because it has a Ca binding protein that causes it to go through a conformational change, which then causes tropomyosin to move off of the actin binding site, giving access to myosin. • Organelle transport and other motor-driven movements § Movement of organelles along actin • Organelles move because motor proteins attach to their surface and interact with a cytoskeletal filament at the other end • Motor proteins bind and hydrolyze ATP, and translocate the filaments in a specific direction • Myosin V drives the most organelle movement o Shorter helical alpha tail, forms dimers o Has + ends toward plasma membrane to drive specific direction transport § Movement of actin filaments relative to each other • Myosin II motor proteins work to cause small scale actin contractions in non- muscle cells too • Anti-parallel actin filaments and bipolar myosin filaments work together so that actin filaments can slide toward the center of the myosin filament (eg. contractile ring) § Movement of actin filaments relative to the plasma membrane • Myosin I motors are monomeric • Very short tail that interacts with plasma membrane, head interacts with actin filaments to generate force (seen in microvillus) Molecular motors & MTs: ciliary beat and beyond • Cilia and flagella § MTs: 9 arranged in a ring around a central pair of MTs • Ring MTs = doublets (A complete MT, B incomplete MT) • Serve as support structures, are linked by many accessory proteins that link them, cap them, and make them stable § Major accessory proteins in the axoneme • Nexin links: connect adjacent MT doublets • Radial spokes: link doublet MTs to central sheath • Dynein arms: sit on A-MTs and generate force for ciliary movement o Sliding Disintegration Experiment § Electron microscopy: cross sections suggested bending = MT sliding! • When bent, all parts were not visible, when straight, they were § Sliding Disintegration: axonemes were treated with protease, (which cuts up binding proteins such as nexin links which hold MTs together) ATP was added, Result à Axoneme slides apart! • This gave evidence that dynein generates sliding forces between MTs that gives rise to ciliary beating • In intact flagellum, dynein causes MT bending, because linking proteins are still present o Dynein motor protein § More complex structure than myosin. § Similar globular head domain (motor domain) and a tail that attaches to the motor’s cargo (which is the adjacent MT doublet). § Different from myosin since it is a trimer rather than a dimer of large subunits § Also has a number of additional subunits besides those which have the MT- binding/ATPase activity • Kinesin & Dynein motor proteins o We know that movements generated in organelle transport along MTs within cells cannot be driven by dynein motors alone because dynein only moves organelles toward the (-) ends of MTs. However, in nerve axons, MTs are all arranged with their (+) ends toward the synapse, but they move in both directions! § A (+) end directed motor must exist o Outline the experiments using squid axoplasm that lead to the discovery of the kinesin motor proteins § Cytoplasm was squeezed out of a single axon and its organelle transport was reactivated under a microscope • Observation: motor proteins sometimes stuck to the glass coverslip with motor ends up, facing into solution à gliding of MTs occurred § MTs were also nucleated into an array with (+) and (-) ends, which was done by purifying centrioles (MT organizing centers) to track protein complexes that drove movement of artificial organelles to (+) ends of MTs • A new class of motor proteins, called kinesins, was identified! o Compare and contrast the properties of kinesin with those of dynein and myosin § Kinesins travel toward (+) end on MT, away from center of cell à bring cargos to outside of the cell § Dyneins travel toward (-) end on MT, toward to center of cell à bring cargos to center of cell § Myosins serve to move actin on actin, organelles on actin, or actin on plasma membrane o Explain what organelles must have besides motor proteins in order for the right organelles to be transported in the right direction in cells. § Organized filaments § Correct motors on correct stuff (adaptor proteins) § Regulation of the process Mitosis & Cytokinesis • Events of cell division o Prophase • The ER and Golgi break up into small vesicles and disperse in the cell • Chromosomes get condensed, are now visible two sister chromatids connected by a centromere, and the kinetochore also assembles onto the centromere • Cytoskeleton is disassembled, MTs form the mitotic spindle (transition occurs to next phase after nuclear envelope breakdown) o Prometaphase § MTs from the two spindle poles attach to each chromosome at the kinetochore § Chromosomes oscillate from (+) and (-) ends § Polar MTs interact and stabilize each other from opposite sides of spindle o Metaphase § Chromosomes align at metaphase plate Each chromosome is attached to both poles (two half spindles engage with each other) o Anaphase § Centromeres split, kinetochores (and sister chromatids) separate § Chromosomes move toward opposite poles (Anaphase A) § Poles separate (Anaphase B) o Telophase § Chromosomes decondense § Daughter nuclei form § Organelles reassemble § Cytokinesis: cleavage furrow pinches and two cells are created • Mitotic spindle o 3 stages of motor proteins § Prometaphase: 3 types of movements - 1. Bipolar kinesin: pole separation (in midzone between) 2. (-) end directed motors at kinetochore 3. (+) end directed motors at kinetochore (All of these work together à chromosome oscillation along MTs) *Transition: (+) end motors turn off* § Metaphase: looks static but is actually not 4. Bipolar kinesin (same as before): exert force to push poles apart but no movement 5. (-) end directed motors: exert force to pull poles together but no movement (These work together à state of dynamic tension/compression) *Transition: centromeres separate* § Anaphase: separation of chromosomes 6. (-) end directed motors: move to poles (Anaphase A) 7. (+) end directed motors: poles separate (Anaphase B) o Assembly/disassembly behavior of the spindle MTs in different regions at metaphase and anaphase § Metaphase: kinetochore is not a (+) end cap, so net tubulin addition, and pole is not a (-) end cap, so net tubulin loss à subunits are treadmilling the length of the MT toward the poles § Anaphase A: kinetochore MTs shorten during chromosome to pole movement because there is net disassembly at (+) ends § Anaphase B: polymerization occurs, assembly at (+) ends • Cleavage furrow o Begins to form perpendicular to long axis of mitotic spindle during anaphase o Formed due to some interaction between mitotic spindle and the furrow, furrow follows position of the spindle but only up until a certain point of commitment § Astral stimulation model § Central spindle stimulation model o Contraction: its diameter decreases as it pinches the cell in half, but SA stays same of actin bundle area à this tells us that disassembly of F-actin must happen during the contraction § Occurs as a result of the actin filaments sliding and depolymerizing o Myosin II (motor protein) drives contraction § Cleavage furrow has actin filaments with mixed polarity, and bipolar myosin filaments engage the actin filaments and cause contraction of actin array à cell is pinched in half o Outline the experiments that showed that myosin drives the cleavage furrow, but not the mitotic spindle § Knecht & Loomis: used antisense RNA (synthetic RNA with sequence complimentary to myosin II RNA) that inhibits its translation à cytokinesis did not occur § Meusen & Cande (1980) and Kiehard & Mabuchi (1982): inhibited myosin à cytokinesis did not occur The Cell Cycle • Stages o M: Mitosis o G1: Metabolism, growth, organelle duplication o S: DNA synthesis o G2: Growth, preparation for mitosis • Central control system o Separate from the events of the cell cycle themselves o A timer with regulation at critical points in the cycle o A protein-kinase machine made of two parts: § Cell cycle kinase (Cdk) § Cyclin, a regulatory protein : (+) control, binds to kinase and activates it • Cell cycle checkpoints: places in the cell cycle where signals from cellular events can delay progress of the timer o Environmental information and cellular status are detected and evaluated for approval o A total of 5 or 6 checkpoints exist. Two main ones: § G2/M: enter M? checks for: • Complete and proper DNA replication • Cell doubling in size § G1/S: enter S? checks for: • Availability of enough XTP • Damage to DNA • Experimental evidence for cell cycle control o Rao and Johnson (1970): Cell Fusion Experiments § Grew cultures with all cells in same phase of cell cycle, and selected cells at different phases and fused them to form a hybrid cell 1. G1 with S à G1 nucleus jumps ahead in cycle, activated to replicate DNA 2. G2 with S à G2 nucleus does not jump ahead in cycle § This told them that DNA synthesis can be induced by cytoplasmic factors, but by G2 nucleus it cannot respond 3. M with any other stage à the nucleus in the other stages undergoes chromosome condensation, even when inappropriate * in S à chromatin damage * in G1à single chromatid chromosomes § This told them that transition to M is under positive control o Experiment that identified Maturation Promotion Factor (MPF): Smith and Wasserman § Xenopus oocytes and eggs used § Cytoplasm was injected from an M phase cell into a G2 oocyte à Result: oocyte was driven to M § Cytoplasm was injected from an interphase cell into a G2 oocyte à Result: oocyte not driven to M § This told them that there was some kind of “Maturation Promotion Factor” involved • MPF is the protein (cyclin-Cdk complex) which helps the cell go through checkpoints • Maturation vs donor cell cycle graph o This showed that MPF concentration oscillated as the cycle progressed o But kinase concentration did not change… o Echinoderm egg experiments that lead to the discovery of cyclins: Hunt and Ruderman § When protein synthesis was inhibited, embryos stopped at the G2/M checkpoint à no mitosis § This told them there must be a newly synthesized critical protein § Hunt and Ruderman placed radioactive 35S cysteine-methionine protein in the embryos § Superimposing the graphs of amount of radioactive material in one band of proteins and the maturation graph told them the following • Proteins called “cyclins” existed, and they bound Cdks • Cyclin reaches a threshold that triggers rise in concentration of oocytes going into mitosis o Significance of the oscillation of mitotic cyclin in this system § [Cyclin] oscillates and so does the percent of oocytes going into M; Superimposing the two together à cyclin has a threshold that it reaches which triggers rise in the number of oocytes going into M o Checkpoint hypothesis for cell cycle control § G2/M checkpoint: • [M-cyclin] increases, reaches threshold where most m-Cdk is bound and active • Critical protein substrates are phosphorylated, enter M • Cyclins are destroyed, no Cdk-cyclin complexes exist, exit M • Feedback to the cell cycle checkpoints o Budding yeast is a good experimental system to study the cell cycle because: § Yeast are single celled fungus, easy to grow, and have a well known haploid stage § Unlike embryos, they have M/G1/S/G2 and grow during each cell cycle § They have no maternal protein stores, cell cycle regulation is affected by the environment § Many mutations in cycle cycle processes (Cdc mutants) already exist and are widely available to access and study o Some mutant strains of yeast that had perfectly good cell cycle kinase and cyclin (the mutations were in other genes) nonetheless could not proceed through the cell cycle… why? § This is due to other mutants, in kinases and phosphatases, which were needed (further modification to Cdks and cyclins) to move past checkpoints o Once cells are done using M-Cdk/M-cyclin complex to cross the G2/M checkpoint, specific proteolysis of cyclin occurs § This is done when ubiquitin is present, a “tag” which is added covalently to proteins targeted for degradation § This process is activated by the complex itself… MPF is a self-limiting timer! Epithelial Cells • Epithelia: cells connected to each other in large, continuous sheets o Form boundary between inside and outside of organism o In contact with the outside world o Have same boundary properties for organism as the plasma membrane does for the cell, such as: § Continous § Selectively permeable § Asymmetric § Respond to external stimuli § Mediate interactions with outside world o 2 critical features: § Specialized cell-cell junctions § Specific membrane proteins that are precisely localized • Properties of epithelial cells in different regions of the body o Protective § Eg. skin, esophagus o Absorptive § Eg. stomach, intestine, lung o Secretory § Eg. skin, stomach, intestine, lung • The four major Cell Junctions o Tight junctions § Very close apposition § Form a permeability barriers § Exist as a “belt” around cells o Adherens junctions § Adhesion properties § Interaction with actin and myosin § Possess ability to change shape of sheets § Interact with actin filaments (and myosin), cadherins, catenins o Desmosomes § Adhesion properties § Interact with intermediate filaments, cadherins, intracellular anchor proteins § Mechanical strength; forms a continuum that spans many cells o Hemidesmosome § Cell-membrane interaction § Interact with intermediate filaments, integrins, and basal lamina § Anchor sheet down on basal lamina (ECM) • Secretion and membrane protein recycling must be organized in a more complex way in epithelial cells because epithelial cells put up stronger barriers, and the concentration of molecules is composed differently outside, inside, and within the membranes • Secretion o Apical and basal signal sequences exist, along with vesicle sorting processes o The junctional complex forms a diffusion barrier so that membrane proteins do not get secreted out o Separate recycling endosomes exist for apical and basal molecules • Glucose Transport o Tasks to be completed: § Glucose must be moved from areas of high à low concentration § Glucose must be moved through the cytoplasm, where concentration is very high § Two membranes must be crossed o Proteins involved: § Na/glucose symporter (apical) § Glucose transporter (basal/lateral) § Na/K ATPase (basal/lateral) o Proper placement of proteins § Na/glucose symporter must be in the apical membrane, otherwise it would move glucose from inside of the body (extracellular space) into the cells § Glucose transporter must be in the basolateral domain, otherwise it would allow glucose to leave cells and go to the gut lumen (outside world) instead of extracellular fluid and bloodstream § Na/K ATPase must be in the basolateral domain, otherwise it would pump sodium into the outside world (lumen of gut) and if it encounters a K there, that would make it energetically impossible to run the transporter The Cell Biology of Pathogens • Pathogens: agents that cause infectious diseases • Dedicated pathogens o Have highly evolved specialized mechanisms for breaching our defenses o Ability to change activity of host to their benefit • Opportunistic pathogens o Not normally found in our bodies/cells o Take advantage of skin wounds, immune deficiencies to enter o Change from innocuous à disease-causing • Human Pathogens o Metazoa (Ascaris – roundworm, Taenia solium – tapeworm) o Protists, single celled eukaryotes (toxoplasma gondii) o Fungi (yeast, tinea pedis) o Bacteria (lots!) o Viruses: DNA/RNA genome o Prions: infectious proteins (BSE: Bovine Spongiform Encephelopathy) • Body’s defense against pathogen entry and movement o Epithelial cells secrete mucus that serves as a physical barrier and contains factors such as antibacterial peptides § Lung epithelia have cilia that sweep mucus, along with bacteria trapped in it out of the airway § GI epithelia do the same thing through peristalsis o Tight junctions maintain a barrier to infection o Macrophages patrol all body tissues and destroy pathogens • Intracellular pathogens o Bacteria § DNA genome § Machinery for replication, transcription, translation § Some can be grown in simple abiotic media o Viruses § Simpler than bacteria; possess simpler toolkit § Require a host to replicate § Small DNA/RNA genome wrapped in a protective protein shell, sometimes a membrane which is stolen from the host o Prions § No genome, just one single protein • Bacterial invasion of cell: enter cell by stimulating it to engulf them (phagocytosis) o Zipper mechanism § Adhesins on bacteria bond to cadherins, integrins § This causes the membrane to surround them and create a phagosome o Trigger mechanism § Bacteria injects proteins in the cytoplasm of host cell § These proteins stimulate actin polymerization, which induces cell membrane to engulf the bacteria, phagosome is generated • Viral invasion of cell o HIV (membrane virus): has a membrane, protein coat, and RNA genome § Fuses with plasma membrane § Uncoats proteins, releases genome into cell o Influenza (membrane virus): has a membrane, protein coat, and RNA genome § Enters cell by receptor mediated endocytosis, becomes an endosome § Fuses with endosomal membrane and uncoats proteins, releases genome into cell o Polio (nonmembrane): has a protein coat and RNA genome § Enters cell by receptor mediated endocytosis, becomes an endosome § Protein opens up and genome gets inserted into cytoplasm through the endosome o Adenovirus(nonmembrane): has protein coat and DNA genome § Enters cell by receptor mediated endocytosis, becomes an endosome § Lysis of endosomal membrane occurs § Travels to nucleus, protein coat opens up and inserts genome into nucleus • Mechanisms intracellular pathogens use to escape degradation o Exit phagosome before it becomes a lysosome § All viruses, some bacteria (listeria) do this o Alter compartment and prevent it from becoming a lysosome § Most bacteria, most eukaryotes do this o Survive in phagolysosome after fusion § Rare, coxilla brunettii do this • Prion: infectious protein o Replication mechanism § A normal protein, by rare chance, undergoes misfolding and becomes a misfolded protein § Misfolded proteins are sometimes chaperoned back to normal comformation, or degraded in the cell § But sometimes, these misfolded proteins form aggregates, which are very toxic! • Infectious seeding of misfolded protein aggregates à formation of amyloids • It is difficult to attack pathogens without attacking the infected cells because some pathogens, like viruses, thrive on the cells themselves, so the only way to get rid of them is to destroy the cells • Practical targets for anti-bacterial drugs o Cell wall, protein synthesis, DNA/RNA enzymes • Practical targets for anti-viral drugs o Reverse transcriptase, viral protease (cleaves viral polyprotein)
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