01/29/16

MCB 104

Cell cycle conceptsDiscovery of the cell cycle machineryYeast genetics cdc genesSea urchin molecular biology cyclin proteinsXenopus biochemistry m-phase promoting factorCdk activity cyclingCheckpoints

The cell doctrine Where a cell arises, there must be a previous cell, just as animals can only arise from animals and plants from plants All cells from cellsThe cell cycle in its most basic representation and definition The continuity of life New cells are produced by duplication, not de novo Cells have to duplicate their genetic information (replication) and segregate it to the daughter cells (mitosis) Three phases Cell growth and chromosome replication Chromosome segregation Cell division Cell cycle events must occur in the appropriate order Cells have to ensure that these processes occur without mistakes (checkpoints)

The cell cycle in animals a few points to keep in mind Some cells can continuously reenter the cell cycle (e.g. stem cells) Other cells enter a quiescent but reversible state (e.g. liver cells) No longer activity dividing Trigger to enter the cell cycle Many undergo terminal terminal and irreversible differentiation (e.g. neuronal cells) Cell division is often but not always coupled with cell growth (biosynthesis) Cell can modify the cycle to undergo DNA replication without cell division (endoreplication rare in human cells)Phases of the cell cycle Timing mechanisms ensure that transitions are unidirectional S before M S = DNA synthesis: genome replication M = mitosis: chromosome segregation dn cell division (cytokinesis) G2 = gap2: growth and preparation for mitosis G1 = gap1: growth G0 = quiescient state Exit before G1, don't exit in G2 Normally when you enter S phase, the rest occurs G1 + S + G2 = interphase or IDiscovery: M phase is induced by a cytoplasmic factor Fuse cells in different cell cycle states Discover that mitotic cells contain M-phase promoting factor = MPF Took a cell in mitosis and fused cell in G1 (what happens when cell share same cytoplasm) G1 chromosomes condensed prematurely Something in M-phase cytoplasm that causes condensationThe cell cycle must be essentially error free Premature chromosome condensation is a disaster If DNA condenses during replication deathCell cycle control: cyclin-dependent kinases CDK/ cyclin = protein kinase complex activity rises and falls during cell cycle Has ceyclin-dependent kinase (Cdk) (active) and cyclin Cyclical changes in phosphorylation of substrates by cdk/cyclins leads to specific events DNA replication then MitosisNobel prize Lee Hartwell Identified cdc genes that regulate the cell cycle and showed that humans also have them Tim Hunt Identified cyclin proteinsThe cell cycle in the budding yeast Saccharomyces cerevisiae Stages of cell cycle can be idenfied by morphology alone Cell cycle is coupled to cell growth Yeast cell good at growingIdentification of cell cycle regulatory genes Make a lot of plates each with one gene mutant Screen mutant and look for ones where cell division is defectiveCdc mutants: arrest at a particular cell cycle Permissive (23 C) All stages of the cell cycle can be observed Restrictive temperature (36 C) Cells arrest at a particular stage of the cell cycle (Eg with large buds, no cytokinesis = G2/M) Or Everyone arrests at G1 cells grow but dont duplicateGenetic control of the cell division cycle in yeast Cdc28 defines start: entry into S phase Cdc28 from yeast is homologous to human CDK1 (cyclin-dependent kinase) First of component that drive cell cycle Introducing cdk1 into cdc28 mutant yeast can rescue the cell cycle phenotype, restoring normal cell division Most cdc genes are conserved across eukaryotes: conservation of cell division processThe discovery of cyclins Proteins that accumulate during interphase and are destroyed abruptly at end of mitosis Fertilize clam or sea urchin eggs megabolic labeling of newly synthesized with S-Met take sample: SDS Page, autodiography when cells synthesize protein, that radioactivity would be incorporated into synthesized protein new proteins cyclin A cyclin B level of proteins accumulate (levels are cycling)The cell cycle control machinery: biochemistry in frogs Matsui Combination of cyclins and hartwells cdks that drive cell cycle Can collect large numbers of oocytes Unfert oocytes arrested in g2 Very rapid cell cycle Can inject oocytes easily Large cytoplasm = easy exracts for protein purificationThe cell cycle control machinery MPF sufficient to drive frog oocytes into M phase (maturation/mitosis/M-phase promoting factor) Injecting cytoplasm from m-phase into non-cyclic cell and cell would be driven into m-phase Also works from human cells in m-phase Mpf purified from eggs: two components Cyclin b and cyclin dependent kinase (CDK) Cdc28 = cdk1 ser/thr kinase, highly conserved Cyclin from sea urchin/clam: levels regulate kinase activityCyclins and cyclin dependent kinases (CDK) control cell cycle progression Binding to a cyclin unlocks the kinase activity of a cdk Phosphorylation of target proteins by cdks drive cell cycle events Cyclins are synthesized and destroyed in a cyclic fashion How? Cdk dont change much in concentration Rise and fall of cyclin that regulates cyclic activityCyclin levels are regulated by proteolysis Modification by polyubiquitylation signals destruction Cyclin is modified by covalent coupling to ubiquitin, a small protein that is an important general post-translational modification Polyubiquitylation (multiple ubiquitins added) Signal for destruction by proteasome Ubiquitin ligase forvcycb is apc = anaphase promoting complex02/01/16Checkpoints Points in cell cycle where progress can be halted until ready to proceed Helps keep process error-free Assessment of environment and if everything is going correctly or if environment is favorableCell cycle control system: timing and checkpoints Checkpoint Microtubules attached to kineticores tension from spindles pulling from each side Is DNA replicatedOne key example: DNA damage checkpoint Make sure DNA is repaired accurately Cell has enzymes that can detect certain aspect of DNA damage Large double stranded space Improper pairing DNA damage activation of ATM Kinase (detection of double stranded break) phosphorylation and stabilization of p53 (acts as transcription factor) transcription of p21 = Cdk inhibitor inactivation of Cdk/Cyclin stop cell cycleP53 is a tumor suppressor gene: Guardian of genome Wen its working great, able to stop cycle when DNA is damaged Accumulate DNA damage DNA damage