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Lecture 7

Biology 2290F/G Lecture 7: Mitosis and Cell Cycle Control

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Biology 2290F/G
Ray Zabulionis

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 DNA in 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 - Mother cell gives rise to 2 genetically identical daughter cells - Important that the DNA on the chromosomes are segregated properly to both daughter cells - We have cell growth and cell division (2 different things) o Growth: cell gets larger o If the cell is to divide, and it doesn't have adequate lipid, proteins, organelles, it won't have enough structure and nutrients to ensure survival - Conditions must be optimal for cell 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 - Most dramatic events observed microscopically occur during the M phase - Longest period of cell cycle is interphase - 4 phases of cell cycle: G1, S, G2 known as interphase, then M (mitosis) - We see dramatic changes mostly only in mitosis - During mitosis, chromosomes are segregated appropriately into daughter cells o Chromatin condenses o Breakdown of nuclear envelope o Chromosome segregation - After mitosis, we have reformation of nuclear membrane, chromosome relaxation Phases of the cell cycle G1: generalized growth and metabolism of the cell - Where most cells arrest when not dividing - Variable length (~11 hrs in mammalian cells) S: DNA replication (6-8 hrs) G2: preparation for chromosome segregation and cell division (4 hrs) M: Chromatin condensation - Nuclear envelope breakdown - Sister chromatids attach to mitotic spindle - Segregation of chromatids - Decondense and reformation of intact nuclei - Cytokinesis (1 hr) - G1: higher percentage of cells in G1 phase than any other phase o Most metabolism occurring here o If there's some event that suggests there's non-ideal conditions for growth, or if there's DNA damage, the DNA will exit out of the cell cycle into G0 o Some cells permanently enter into G0 and no longer divide; known as post-mitotic cells - S: DNA replication - G2: prepare for mitosis - M: chromatin will condense - can be compared to a long garden hose in G1. In G2 the cell gathers up the hose and compresses it (condensation), helping to move the chromatin more easily o Cytokinesis: cells formally pinch off and pull apart - In FACS, we can see that DNA intensity (DNA content) is proportional to Hoechst stain intensity o In population of cells that are dividing and growing, we see a strong G1 peak o In late G2, they've replicated DNA but haven't undergone cytokinesis o Medium amount of fluorescence are the cells that are in the process of replicating their DNA - If we interfered with regulatory mechanisms, peak intensity would change 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 properly - Important that you have equal number of chromosomes distributed to the 2 daughter cells - Aneuploidy is when you have uneven number of chromosomes distributed; cancer causing Cytological features of cycling cultured human HeLa cells - At 0 minutes, there are a lot of cells in interphase - Can see the nucleus, some vesicles, other structures - We start to see cells that start to round up, producing a phase halo (due to phase contrast microscopy) o When cells are flat, we see less phase halo, but when they round up they form a halo due to superimposition of wavelengths - When cells go through mitosis, the cells round and we see two distinct cells formed that then pinch off and flatten once again - Phase ring shows that we are using phase contrast microscopy o Also, stark contrast details, gross morphological detail shows phase contrast microscopy o In electron microscopy, we have better resolution and better detail - Hard to say whether the original cells were in G2 or S - So we see changes in mitosis, but we can't really tell between other stages of the cell cycle The budding yeast S. cerevisiae - Cell cycle stage can be inferred by the size of the bud - Budding yeast have long G1 phase - The budding yeast forms a bud during the cell cycle - The larger the bud, it gives you an idea of what stage of cell cycle it's in - In G2 and M, the bud becomes more pronounced o Until the cell divides - Mother cell is slightly larger than daughter cell - Using budding yeast, we can start to understand what's going on in the cell cycle - This image was formed using scanning electron microscopy o Cells coated with electron-dense stain o We see high detail, 3D structure of cells - Note that the budding yeast have a very long G1 phase The fission yeast S. pombe - Fission yeast grow by elongation of ends - 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 - Other type of yeast called fission yeast (S. pombe) o Large rod-shaped cells (pill-like) - As it progresses through cell cycle, the rod gets longer and longer - Eventually you get formation of septum, which is the final stage of cytokinesis - Fission yeast has much longer G2 and M phases of the cell cycle o So we see more longer-shaped cells - This is important because these two types of yeast models are important for figuring out what regulates how cells go through the stages of the cell cycle - Both yeast generate temperature-sensitive mutants o Defects in specific proteins required to progress through cell cycle 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 DNA replicated? Is cell big enough? Is environment favourable? Is DNA damaged? - Machinery stops if things go wrong - By using these mutants, we learn what key proteins regulate the stages of the cell cycle o Result: cyclically activated kinases (things that put phosphate groups on proteins) - Why do we have to regulate cell cycle? o DNA damage occurs, so there must be mechanisms in place to check that everything's okay Key regulators of the cell cycle - Involves 3 protein families, 2 of which are enzymes: o Kinases, phosphatases, cyclins - In many cancers, things that regulate cell cycle don't work, which allows the cells to grow and divide indefinitely - Hartwell, Hunt and Nurse used yeast to identify the kinases, phosphatases, and cyclin proteins that regulate cell cycle o All discovered using yeast Functional complementation - Plasmid that carries WT allele will complement the recessive mutation - Cdc28 is a cyclin-dependent kinase (CDK) – the only CDK found in S. cerevisiae - Take cdc28 cells grown at 25 degrees, they form colonies - Transform with plasmid library of wild-type S. cerevisiae DNA - Transformed cdc28 cells will grow at 37 degrees - They created mutations randomly in yeast cells o These mutations would interfere with the ability of the cells to divide; didn't know which genes were operating these cells o Mutants didn't grow at the normal optimal temperature of 37 degrees, but if we shifted it down to 25, the mutant would function - The question is: what is the mutation? - Functional complementation assay: introduce a gene (WT) into the protein, which will complement the deficiency in the cell o The deficiency: inability to grow at 37 degrees o Artificial version of the gene will now complement the deficiency, allowing the cell to divide and form colonies - Generated a plasmid library, isolating RNA from normal WT yeast cell, forming complementary DNA into a plasmid vector, then inserting them into the cells - Plate the cells and look for colonies; after formation, we can extract the plasmid and sequence the DNA in the plasmid - Found mutant called Cdc28 - When we analyse the sequence of Cdc28, they realized it was a cyclin-dependent kinase (CDK) - We know we need this kinase in the cell cycle - Note: library has lots of genes (Gene X and Y) that have nothing to do with the cell cycle, so when we grow at 37 degrees, they are not complemented, no colonies formed Control of G2 – M transition in S. pombe - Loss of Cdc2 activity (recessive) prevents S. pombe from entering M phase - Cdc2 is transcribed and 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 the cell cycle are highly conserved between all eukaryotic organisms - This had a mutant called cdc2 o Identified cdc28 in the mutant o The mutant had very long rod-shaped cells that were unable to undergo mitosis o Could grow indefinitely, but no division - Cdc2 and Cdc28 are homologous - In humans, we have CDKs as well o Can take the human version of CDK and introduce into the yeast cells, and they will complement the deficiencies o This indicates that cell cycle regulators are highly conserved (functional conservation) o Throughout evolution, that function is being preserved (important) - Mutations in another gene were also identified o Encodes mitotic cyclin - Heterodimer of CDK and mitotic cyclin (Cdc2) makes MPF (mitosis-promoting factor) - Cdc2 in MPF is inactive for most of the cell cycle: activity is low in S phase, peaks as M begins and drops off suddenly only to repeat the process – how? - Question: is it simply the CDKs that control cell cycle? - Across cell cycle, CDK amount was the same, but activity varied o Activity peaked between G2 and M phases - Found another gene that encoded mitotic cyclin o Found that the CDK and the cyclins interact together as a complex (mitosis-promoting factor) o This complex is critical for transition from G2 to M - The kinase in this complex is inactive during most of cell cycle; peaks as mitosis begins Western Blotting - Mitotic cyclin bands appear greater at one point in cell cycle, disappear and then reappear at the same point next cycle o Cyclin is responsible! - We see increase in amount of protein through cell cycle stages - Cyclin amount peaks at G2, then drops off at M - Suggests that amount of cyclin is regulating kinase activity - When cyclin level peaks, we have optimal amount of kinase activity 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 and activate MPF - When 2 proteins interact together, we see highest interaction - Confusing: the dashed line shows the absolute amount of protein, while red line shows protein activity - Why aren't the proteins active during S phase, when there's quite a bit of cyclin? - Activity doesn't exactly parallel amount of protein present Cyclin regulation of cell cycle - Cyclins bind to and activate CDKs - Cyclins are only present during the cell cycle stage that they trigger and are absent in other cell cycle stages - Cyclins are divided into fo
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