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Lecture 7

Lecture 7 + 8 - Mitosis and Cell Cycle Control

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Western University
Biology 2382B
Sashko Damjanovski

LECTURE 7/8: MITOSIS AND CELL CYCLE CONTROL Function of the Cell Cycle • Essential mechanism by which all living things reproduce and pass on genetic information to next generation of cells • Ensures that DNAin each chromosome is faithfully replicated to produce 2 copies • Replicated chromosomes must be accurately distributed (segregated) to 2 genetically identical daughter cells • Coordination of growth (increase in cell mass) with division The Cell Cycle and its Phases • Cell cycle is the ordered sequence of events in which a cell duplicates its chromosomes and divides into two genetically identical cells. • Four phases • G1-S-G2 = interphase, the cell increases in size • Interphase is the period of time between each round of division • Most dramatic events observed microscopically occur during the M phase Phases of the Cell Cycle • G1(Gap 1) Phase: o Generalized growth and metabolism of the cell o Where most cells arrest when not dividiog (G ) o Variable length (~11 hours in mammalian cells) • S (Synthesis) Phase: o DNAreplication (~6-8 hours) • G2 (Gap 2) Phase: o Preparation for chromosome segregation and cell division (~4 hours) • M (Mitotic) Phase: o Chromatin condensation o Nuclear envelope breakdown o Sister chromatids attach to mitotic spindle o Segregation of chromatids o Decondense and reformation of intact nuclei o Cytokinesis (~1 hour) • Recall: o Hoechst stain allows us to quantify the number of cells in different phases of the cell cycle o Hoechst dye binds to DNA; the more DNApresent, the more the dye binds, the more it fluoresces o The amount if fluoresces is proportional to the amount of DNA present o Cells in2G m have double the fluorescence as cel1s in G o Cells that start to synthesize DNAhave more intensity o Cells that have doubled the DNA, but have not divided, 2re in G m o Most cells are i1 G because that is the longest phase of the cell cycle; cells spend 1ore time in G phase Some Eukaryotic Cell-Cycle Times Cell Type Cell Cycle Time Early frog embryo cells 30 minutes • Liver cells can fall out of the cell cycle a0d go to G for a Yeast cells 1.5 – 3 hours break, then return Intestinal epithelial cells About 12 hours • Brains cells, once differentiated, 0nter G permanently Mammalian fibroblasts in cultureAbout 20 hours Human liver cells About 1 year and never divide again • Post-mitotic: divided during embryogenesis Human nerve cells Terminally differentiated (post-mitotic) M Phase (Mitosis) • Prophase: nuclear envelope breaks down, spindle apparatus forms, chromosomes condense • Metaphase: chromosomes align in plane in center of cell • Anaphase: sister chromatids separate, pulled towards spindle poles • Telophase: chromosomes decondense, reassembly of nuclear membranes • High accuracy and fidelity are required to assure that the chromosomes will be segregated proper • Spindle apparatus consists of a microtubule structure made of tubulin – these form and latch on to sister chromatids, at which point, chromosomes start to align at metaphase • The spindle apparatus pills apart chromosomes in anaphase – they pull towards spindle poles • An equal complement of chromosomes to each daughter cell is desired; accuracy of chromosomes segregation is also really important Cytological features of cycling cultured Human HeLa cells • Time-lapse microscopy shows that we start to see nuclei reforming at around 160 minutes The Budding Yeast S. cerevisiae • Cell cycle stage can be inferred by the size of the bud • Budding yeast have long G1 phase • We can determine what state/phase the cell is in, based on bud size The Fission Yeast S. pombe • Fission yeast grow by elongation of ends, grow big rods • Cytokinesis occurs by formation of septum • Have longer G2 and M phases • In both S. cerevisiae and S. pombe temperature sensitive mutants exist which cause defects in specific proteins required to progress through the cell cycle. • cdc –cell division cycle mutants Concept of the Cell Cycle Control System • System based on cyclically activated kinases • Progression through cell cycle is regulated • Checkpoints ensure things should proceed: o Is all DNAreplicated? o Is cell big enough? o Is environment favorable? o Is DNAdamaged? • Machinery stops if things go wrong • Involves 3 protein families, 2 of which are enzymes o Kinases – add phosphate groups o Phosphatases – take phosphate groups off o Cyclins – interact with kinases, important as regulator of kinases Functional Complementation • Procedure for screening a DNAlibrary to identify the wild-type gene that restores the function of a defective gene in a particular mutant • Cdc28 is a cyclin-dependent kinase (CDK) • The only CDK found in S.cerevisiae • Plasmid that carries WT allele will complement the recessive mutation • Permissive temperature = 25 C o • Nonpermissive temperature = 37 C • Essentially, we are selecting for cells that form colonies when transfected with the right gene • This “right gene” is cyclin kinase Control of G2 → M Transition in S. pombe • Loss of Cdc2 activity (recessive) prevents S.pombe from entering M phase • Gain of Cdc2 activity (dominant) brings on mitosis earlier • Cdc2 is transcribed & translated throughout cell cycle and is also a cyclin dependent kinase (CDK) • Cdc2 is homologous to Cdc28 - - • Human versions of yeast CDKs exist which can functionally complement cdc2 and cdc28 yeast mutants • Proteins controlling cell cycle are highly conserved between all eukaryotic organisms • Cdc2 is a mutation that leads to nonfunctional protein • The cell elongates but it can’t undergo mitosis and is s2uck in G (grows, but can’t divide) • Selection strategy – tried to clone the gene • Found that Cdc2 is homologous to Cdc28; S. pombe and S. cerevisiae are both same proteins • Recessive: mutation in which there is no functional protein • Dominant: mutation in which proteins are produced, but is turned on all the time (cannot be turned off) – Cdc dominant mutation is a really active form • Recessive cells are long because they don’t have a proper functioning CDK that allows for transition from G2to M o Therefore, don’t have breakdown of nuclear membrane o Don’t have segregation of chromosomes o Don’t get turned on at all • Dominant cells are tiny because they enter mitosis too early o Don’t have time to grow o Consequently, it divides prematurely and is small o Gets turned on at the wrong time • Human versions of CDKs are similar to yeast CDKs o These genes are so important that they haven’t diverged and haven’t become different o High degree of conservation Mammalian cells have more than one CDK that are only active in the stages of the cell cycle they trigger • 3 major CDKs o G1/S CDKs – peak at transition fro1 G to S o S CDKs – peak at S o Mitotic CDKs – peak at transition f2om G to M • Activity peaks at early phases of mitosis • Cdc13 encodes a protein called mitotic cyclin, which interacts with CDKs to form a mitotic dimer • The mitotic dimer makes MPF • MPF is composed of these two proteins (Cdc2 and Cdc13) • These two proteins are important for activating kinase activity of MPF • Cdc levels remain constant throughout, but activity is highes2 at the G to early mitosis phase • Mutations in other gene cdc13 were also identified • cdc13 encodes mitotic cyclin • Heterodimer of Cdc2 (CDK) and Cdc13 (cyclin) makes MPF (mitosis-promoting factor) • Cdc2 in MPF is inactive for most of cell cycle: activity is low in S phase, peaks as M begins, & drops off suddenly only to repeat process. How? • Cdc2 remains constant throughout all stages of the cell cycle • Levels of cyclin increase progressively and peak at2late G phase, then suddenly drop as mitosis progresses • Cyclin is greater at one point when MF is the highest • Conclusions: cyclin bands appear greater at one point in cell cycle, disappear & then reappear at same point next cycle Cyclin levels increase during cycle • Cdc2 levels are equivalent during cell cycle but “its activity” fluctuates • At late G2 Cdc2 kinase binds cyclin to form & activate MPF Cyclin Regulati
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