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

A cell cycle is one round of growth and division mitosis cytokinesis Growth and division must be carefully regulated Unregulated cell growth = cancer Cells only come from pre-existing cells

Division occurs in “M-phase:” “mitosis” and “cytokinesis” (<1 hr) Most cell growth occurs during “G 1 ” (6-20+ hrs; duplicate organelles, double in size) DNA replication occurs during “S-phase” (4-10+ hrs)… “G 2 ” prepares cells for division (1-6+ hrs)… G 1 +S+G 2 =“Interphase” Division = “M-phase” A “typical” cell cycle for animal cells is hrs long, but varies… The eukaryotic cell cycle is partitioned into four “phases” ECB C (unreplicated DNA, diploid chr #) 4C (DNA replicated, diploid chr #) 4C 2C 2C 4C

Cell cycle times vary

Adapted from MBoC figures 17-5 and 17-6 DNA content (arbitrary units) 12 Number of cells Cells in G 1 Cells in G 2 /M Cells in S Can determine phase of cell cycle from DNA content Where are cells in G1, S, G2 and M on plot? Which phase has most cells in it? Lasts longest? 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 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 See HWK

1. Fission yeast “cell division cycle (cdc)” mutants define a master regulator (tigger) of the G 2 /M transition cdc2 - (loss of function) WEE2 = cdc2 D (gain of function) “cdc” “wee” cdc13 - (loss of function) cdc cdc25 - (loss of function) cdc wee1 - (loss of function) wee “Wild-type” fission yeastWT Mutant Phenotype cdc2 cdc25 cdc13 wee1 G2G2 M Genetic pathway

Nucleus Egg in “M-phase” Oocyte in “interphase” Transfer M-phase cytoplasm to interphase oocyte… Oocyte “matures” (enters M-phase)… ECB figure Frogs: unfertilized eggs contain an M-phase Promoting Factor Transfer of cytoplasm from egg to oocyte induces M-phase: “M-phase promoting factor (MPF)” Not restricted to egg cytoplasm - Any M-phase cytoplasm will induce M-phase Control expt; Transfer interphase cytoplasm to interphase cell - no effect ECB 18-9

MPF activity cycles during the cell division cycle Time MPF activity MPF peaks in M-phase Interphase M-phase Interphase Peak MPF induces M-phase ECB 18-10

Time MPF activity MPF peaks in M-phase 3. Surf clams and sea urchins: the abundance of “cyclin” proteins varies with the cell cycle “Cyclin” abundance varies with cell cycle: continuously synthesized… degraded at end of M- phase Cyclin B mRNA induces M-phase when injected into Xenopus oocytes Continuously label fertilized eggs with 35 S-methionine Analyze incorporation into proteins by SDS-PAGE ECB 18-6 Ribonucleotide reductase (control) Cyclin A Cyclin B Interphase M-phase Interphase Peak MPF induces M-phase Cyclin synthesisCyclin degraded

Cdc2 gene product is a master regulator of the G 2 -M transition cdc2 cdc25 cdc13 wee1 G2G2 M Three models of the eukaryotic cell cycle MPF regulates entry into M-phase Abundance of “cyclins” in clam eggs varies with the cell cycle Bringing it all together Cyclin B mRNA (clam) induces M- phase in frog oocytes cdc13 encodes a yeast cyclin MPF consists of frog cdc2 homolog and cyclin B

Cell cycle control: from models to molecules Inactive (weakly active) Active MPF (CDK1) “MPF” contains two components: cdc2 gene product = catalytic subunit of protein kinase cyclin B (CLB = cdc13): regulatory subunit activates kinase MPF = “Cyclin-dependent kinase (CDK1)” Remove inhibitory phosphate ECB and Phosphorylate M-phase substrates Histones Lamins MAPs etc cdc2 CLB (cdc13) cdc2 CLB (cdc13) P P cdc2 CLB (cdc13) P cdc2 CLB (cdc13) P cdc25 (inactive) wee1 P Positive feedback CDK1 Inactive Inhibitory kinase Activating kinase MPF (CDK1) activity is also regulated by phosphorylation wee 1 is inhibitory kinase cdc25 is activating phosphatase “Switching on” CDK1 (MPF) drives cell into M-phase

MPF triggers its own inactivation “anaphase promoting complex (APC)”; targets cyclin B for degradation Polyubiquitin Interphase APC is turned off cdc2 CLB (cdc13) P cdc2 CLB (cdc13) P APC Inactive APC Active Cyclin B degraded by proteosome Anaphase Accumulation of cyclin B CLB (cdc13) Metaphase (mid-M) High cyclin B MPF (CDK1) active Telophase (late-M) Low cyclin B MPF inactive Prophase (early-M) Activation of CDK1 by cyclin and cdc25 Cyclin B accumulation activates MPF MPF activates APC APC inactivates MPF by degrading cyclin B A cytoplasmic oscillator

Review: Time MPF activity MPF peaks in M-phase Interphase M-phase Interphase Cyclin synthesisCyclin degraded Accumulation of cyclin B above threshold activates MPF (CDK1) and promotes entry into M-phase Activation of APC by MPF promotes cyclin destruction, MPF inactivation, and exit from M-phase ECB 18-6

Multiple CDKs regulate progression through the cell cycle M G2G2 S G1G1 ECB S-phase cyclins At least 6 different CDKs and multiple cyclins in mammals Done M-phase S-phase CDKs P Active S-phase CDKs Trigger M-phase S-phase cyclins degraded… P Active M-phase CDK (MPF) M-phase cyclin degraded… Trigger S-phase M-phase CDK (CDK1) M-phase cyclins (B) S-phase cyclins and CDKs regulate DNA replication G1-CDKs; drive cells through G1 (won’t discuss) Degradation of S- phase cyclins promotes exit from S-phase into G 2

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 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 ECB 18-14

Completion of critical cellular processes is monitored at cell cycle “check points” 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? Proceed to anaphase Of these, the G1/S checkpoint for damaged DNA is best understood ECB 18-17

RNA pol The DNA damage checkpoint: p53 induced expression of an S-phase CDK inhibitor DNA damage activates p53 Active p53 acts as a transcription factor to turn on genes, including 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) ECB P P p53 (inactive) P21 binds and inactivates S-phase CDK Active S-phase CDK p53 (active) TranslationTranscription p21 DNA p21 gene

If checkpoint is activated Or undergo apoptosis (in a minute) neurons most plant cells Exit cell cycle (temporary or permanent)

Zones of division and growth in plant roots Meristem - zone of active cell division cells remain in cell cycle Zone of cell elongation - growth but not division; Cells in G 0 Zone of differentiation - cells cease growing and terminally differentiate 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) Arabidopsis thaliana Only a fraction of cells still actively dividing

Apoptosis: A tale of tadpole tails and mouse paws what do they have in common? Both processes involve “programmed cell death (apoptosis)” Tadpole tails are resorbed during metamorphosis ECB figure Paws develop from “paddles” ECB figure ECB - “programmed cell death is a commonplace, normal, and benign event. It is the inappropriate proliferation and survival of cells that presents real dangers”

Necrosis (cell death following injury) often results in lysis, spilling the contents into the surrounding space and causing inflamation During apoptosis (“programmed cell death”), cells remain intact and condense Corpses of apoptotic cells are often engulfed by their neighbors or specialized phagocytic cells Apoptosis is visibly distinct from necrosis ECB 18-20

Apoptosis is mediated by a “caspase cascade” “Caspases” are proteases; inactive precursors activated by proteolysis Presence of suicide signals and/or withdrawal of needed survival factor activates first caspase in cascade Death protein Survival factor Inactive Activated caspases degrade nuclear and cytoplasmic proteins (lamins, cytoskeletal proteins, etc)… Activated endonucleases cut chromosomal DNA… Active Caspase (inactive) ECB Initial caspase proteolytically activates downstream caspases …which activate additional caspases, and so on

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