Exam notes. Taxol stabilizes MTs and prevents cell division. Testing the role of MTs using pharmacological agents - inhibitors

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1 Exam notes Means are 76, 70 and? Mean of means is Approximate grades after 3 exams Testing the role of MTs using pharmacological agents - inhibitors Screens for anti-cancer agents have identified a variety of natural and synthetic products that disrupt MT assembly or function Many drugs/treatments disassemble MTs (block assembly) Examples with medical relevance: Vinblastine/vincristine some leukemias from lilly family odophyllotoxin warts Griseofulvin anti-fungal Drugs/agents that stabilize MTs (promote assembly) Taxol ovarian cancer from bark of Western Yew Taxol stabilizes MTs and prevents cell division 1

2 MTs provide a scaffold for organizing the ER and Golgi Green = MTs Red = ER Yel = overlap ER MTs Golgi Centrosome Green = MTs Red = Golgi Yel = overlap ECB microtubule_ER.mov Disassembly of MTs with drugs fragments ER and Golgi MBoC (4) figure Red = MTs Grn = Golgi Control Nocodazole MTs are used for vesicle transport in some cells: Fast axonal transport Cell body ( soma ) - Axon Nerve terminal ( synapse ) + Nucleus Outward ( anterograde ) transport * Inward ( retrograde ) transport Microtubules MTs oriented with plus-ends distal (towards synapse) ECB Kinesin motors power anterograde transport (to synapse) Use AT hydrolysis to walk towards plus-end Numerous kinesin-related proteins 2

3 The kinesin family: Motors for vesicle transport (vesicles not to scale) Transport vesicle 17.5-kinesin.mov Kinesin 2x Light chains bind cargo 2 x Heavy chains N-terminal motor domains Minusend lusend Kinesin uses AT hydrolysis to walk towards the plus-end of MTs ECB Similar to myosin II, may have common evolutionary origin But movement of two heads of kinesin are coordinated, unlike myosin II MTs are used for vesicle transport: Fast axonal transport Cell body ( soma ) - Axon Nerve terminal ( synapse ) + Anterograde transport * Nucleus * * Retrograde transport * * Microtubules MTs oriented with plus-ends distal (towards synapse) Kinesin powers anterograde transport (to synapse) See ECB figure Cytoplasmic dynein powers retrograde transport (to cell body) Uses AT hydrolysis to walk towards minus-end Cytoplasmic dynein: a minus-end motor for vesicle transport Transport vesicle (vesicles not to scale) Transport vesicle Axonemal dynein and cytoplasmic dynein are different, but related, motors Kinesin 2x Light chains - bind cargo Cytoplasmic dynein 2 x heavy chains Multiple light and intermediate chains 2 x Heavy chains N-terminal motor domains Dynactin complex Minusend lusend See ECB figure Cytoplasmic dynein uses AT hydrolyis to walk towards MT minus-ends Cytoplasmic dynein, dynactin complex plus other proteins link MTs to transport vesicles (cargo) 3

4 Tail of motor protein determines cargo specificity ECB organelle_movement.mov Three cytoskeletal arrays are linked to one another Intermediate filaments Microfilaments Microtubules 25 nm 25 nm 25 nm Linkages are via an array of binding proteins and motors END CYTOSKELETON Lectures 21 and 22: The regulation and mechanics of cell division Today - cell cycle (regulation of cell division) Cell proliferation The eukaryotic cell cycle Measuring the cell cycle Models of the cell cycle: from fungi to frogs The cell cycle is regulated by cyclin-dependent kinases Next time - mechanisms of cell division 4

5 A cell cycle is one round of growth and division Cells only come from pre-existing cells cytokinesis CLEAVA~1.AVI CLEAVA~2.AVI mitosis Growth and division must be carefully regulated Unregulated cell growth = cancer The eukaryotic cell cycle is partitioned into four phases C = amount of DNA in haploid before replication 4C (DNA replicated, diploid chr #) 4C 2C Most cell growth occurs during G 1 (6-20+ hrs; duplicate organelles, double in size) DNA replication occurs during Sphase (4-10+ hrs) G 2 prepares cells for division (1-6+ hrs) 2C 4C ECB 18-2 G 1 +S+G 2 = Division = = mitosis 2C and cytokinesis (<1 hr) (unreplicated DNA, diploid chr #) A typical cell cycle for animal cells is hrs long, but varies Cell cycle times vary (ph~1) 5

6 Can determine phase of cell cycle from DNA content Cells in G 1 Adapted from MBoC figures 17-5 and 17-6 Where are cells in G1, S, G2 and M on plot? Number of cells Cells in G 2 /M Which phase has most cells in it? Lasts longest? Cells in S 1 2 DNA content (arbitrary units) ECB 18-2 Transition from one phase to another is triggered We will take a historical perspective to triggers Regulating the eukaryotic cell cycle: studies in four model organisms Marine invertebrates: Surf clam (Spisula) Sea urchins and starfish See HWK Frog eggs and embryos: Rana pipiens (Northern leopard frog) Xenopus laevis (African clawed frog) Cultured cells HeLa (Human cervical carcinoma) Yeast cell division cycle ( cdc ) mutants: Saccharomyces cerevisiae budding yeast Schizosaccharamyces pombe fission yeast 6

7 1. Fission yeast cell division cycle (cdc) mutants define a master regulator (trigger) of the G 2 /M transition Wild-type fission yeast WT cdc2 - (loss of function) WEE2 = cdc2 D (gain of function) Mutant henotype wee1 - (loss of function) cdc13 - (loss of function) wee cdc cdc wee wee1 cdc25 G cdc2 2 M cdc25 - (loss of function) cdc cdc13 Genetic pathway Cdc2 promotes entry into mitosis 2. Frogs: unfertilized eggs contain an romoting Factor Transfer cytoplasm to interphase oocyte ECB 18-9 Nucleus Egg in Oocyte in interphase Oocyte matures (enters ) Transfer of cytoplasm from egg to oocyte induces : promoting factor (MF) Not restricted to egg cytoplasm - Any cytoplasm will trigger ECB figure 18-5 MF activity cycles during the cell division cycle MF activity MF peaks in eak MF induces Time ECB

8 3. Surf clams and sea urchins: the abundance of cyclin proteins varies with the cell cycle Continuously label fertilized eggs with 35 S-methionine Analyze incorporation into proteins by SDS-AGE Cyclin A Cyclin B Ribonucleotide reductase (control) Cyclin abundance varies with cell cycle: continuously synthesized, degraded at end of M- phase Cyclin B mrna induces when injected into Xenopus oocytes MF activity MF peaks in Cyclin synthesis Time ECB 18-6 eak MF induces Cyclin degraded Three models of the eukaryotic cell cycle wee1 cdc25 G cdc2 2 M cdc13 Cdc2 gene product is a master regulator of the G 2 -M transition Abundance of cyclins in clam eggs varies with the cell cycle MF regulates entry into Bringing it all together Cyclin B mrna (clam) induces M- phase in frog oocytes cdc13 encodes a yeast cyclin B MF consists of frog cdc2 homolog and cdc13 (cyclin B) homolog Cell cycle control: from models to molecules ECB and Inhibitory CLB M-cyclin kinase wee1 CLB Inactive (weakly active) CLB Remove inhibitory Active M-CDK phosphate CLB cdc2 cdc2 cdc2 cdc2 M-CDK (MF) Inactive MF contains two components: cdc25 Histones (inactive) Lamins cdc2 gene product = catalytic subunit of protein kinase MAs M-cyclin = cyclin B (CLB = cdc13): regulatory subunit, cyclins have no enzymatic activity M-CDK = MF = CDK1 Activating kinase M-CDK activity is also regulated by phosphorylation cdc25 ositive feedback wee 1 is inhibitory kinase cdc25 is activating phosphatase, triggers activation of CDK1 Switching on M-CDK drives cell into hosphorylate substrates 8

9 M-CDK triggers its own inactivation anaphase promoting complex (AC) ; targets cyclin B for degradation rophase (early-m) Activation of CDK1 by cyclin and cdc25 Metaphase (mid-m) High M-cyclin M-CDK active CLB cdc2 M-cyclin accumulation activates M-CDK M-CDK activates AC AC inactivates M-CDK by ubiquitinating cyclin B Accumulation of M-cyclin CLB AC Inactive AC Active Ubiqutin ligase CLB cdc2 A cytoplasmic oscillator olyubiquitin AC is turned off cdc2 Telophase (late-m) Low M-cyclin M-CDK inactive M-cyclin degraded by proteosome Anaphase Review: ECB 18-6 M-CDK peaks in M-CDK activity Cyclin synthesis Cyclin degraded Time Accumulation of M-cyclin above threshold activates M-CDK and promotes entry into ; cyclin has no enzymatic activity Activation of AC by M-CDK promotes cyclin destruction, M-CDK inactivation, and exit from Multiple CDKs regulate progression through the cell cycle cyclins (B) Active M-CDK Trigger cyclin degraded At least 6 different CDKs and multiple cyclins in mammals G 2 M G1-CDKs; drive cells through G1 (won t discuss) CDK S-phase cyclins degraded ECB S Active S-phase CDKs Trigger S-phase G 1 S-phase CDKs S-phase cyclins S-phase cyclins and CDKs trigger DNA replication Degradation of S- phase cyclins promotes exit from S-phase into G 2 9

10 S-Cdk regulates DNA replication Origin recognition complex - protein scaffolding for assembly of other proteins Cdc6 increases in G1; binds ORC and induces binding of other proteins forming pre-replicative complex ECB Origin is ready to fire Active S-Cdk 1- phosphorylates ORC causing origin to fire = replication 2-phosphorylates Cdc6 leading to ubiquitination and degradation Cdc6 not made until next G1 - prevents origin from double firing Completion of critical cellular processes is monitored at cell cycle check points ECB Is the cell big enough? Is the environment favorable? Is DNA undamaged? Yes? Enter S phase Is DNA undamaged? Is DNA replicated? Is cell big enough? Yes? Enter M phase Have all chromosomes attached to spindle? Yes? roceed to anaphase revents cell from triggering next phase until previous one is finished Of these, the G1/S checkpoint for damaged DNA is best understood The DNA damage checkpoint: p53 induced expression of an S-phase CDK inhibitor p53 (inactive) DNA damage activates p53 DNA p53 (active) p21 gene RNA pol Transcription Active p53 acts as a transcription factor to turn on genes, including p21 ECB Translation p21 p21 protein inhibits G1/S phase CDKs, blocking entry into S-phase Cell arrests in G1 until damage repaired, or undergoes apoptosis (programmed cell death) Active S-phase CDK 21 binds and inactivates S-phase CDK Mutations in p53 in half of human cancers! 10

11 If checkpoint is activated Exit cell cycle (temporary or permanent) neurons most plant cells Or undergo apoptosis (in a minute) Zones of division and growth in plant roots Arabidopsis thaliana Only a fraction of cells still actively dividing Zone of differentiation - cells cease growing and terminally differentiate Zone of cell elongation - growth but not division; Cells in G 0 Meristem - zone of active cell division Regulation of each zone is not well understood in plants but involves hormones In animals: mitogens stimulate cell proliferation (block checkpoints) growth factors stimulate cell growth (stimulate biosynthesis, inhibit degradation) Apoptosis: A tale of tadpole tails and mouse paws what do they have in common? ECB figure Tadpole tails are resorbed during metamorphosis ECB figure aws, hands and feet develop from paddles Both processes involve programmed cell death (apoptosis) ECB - programmed cell death is a commonplace, normal, and benign event. It is the inappropriate proliferation and survival of cells that presents real dangers 11

12 Apoptosis is visibly distinct from necrosis ECB Necrosis (cell death following injury) often results in lysis, spilling the contents into the surrounding space and causing inflammation During apoptosis ( programmed cell death ), cells remain intact and condense Corpses of apoptotic cells are often engulfed by their neighbors or specialized phagocytic cells 18.3-apoptosis.mov Death protein Inactive Apoptosis is mediated by a caspase cascade Survival factor Caspase (inactive) Active Caspases are proteases; inactive precursors activated by proteolysis resence of suicide signals and/or withdrawal of needed survival factor activates first caspase in cascade Initial caspase proteolytically activates downstream caspases which activate additional caspases, and so on Activated caspases degrade nuclear and cytoplasmic proteins (lamins, cytoskeletal proteins, etc) Activated endonucleases cut chromosomal DNA ECB Caspase cascade must be carefully regulated Bcl-2 family of proteins are death proteins Form pores in outer mitochondrial membrane releasing cytochrome c (respiratory chain) Cytochrome c binds adaptor and complex activates first procaspase 12