![• c.) EdU= Ethyl Deoxy Uridine
• It is a synthetic Thymidine Analog and can be detected by Fluorescent Azide.
• d.) Flocytometry/FACS (Fluorescence Activated Cell Sorter).
• Based on the DNA content, the staining occurs and the phase can be identified.
• The quantitative stain includes:
• Propidium Iodide
• DAPI (Diamidino Phenyl Indole)
• 7-AAD[Amino Actinomycin-D]
• Hoechst.
17](https://image.slidesharecdn.com/cellcycle-200123115340/85/Cell-cycle-studies-17-320.jpg)
Cell cycle studies.
- 1. CELL CYCLE 1
- 2. Cell Cycle studies. • Cell cycle regulation can be studied at three levels. 1. Genetic level 2. Biochemical level & 3. Molecular level 2
- 3. 1.) Genetical level/ Genetical mechanisms • The Genetical mechanisms of cell cycle regulation are studied in 2 types of Yeasts. • Saccharomyces Cerevesiae - Budding Yeast. • Schizosaccharomyces Pombe- Fission Yeast • Mutants of these Yeasts called cdc-mutants are employed. 3
- 4. • The cdc-mutants are conditional mutants, specifically for ‘temperature sensitive mutants’. • At normal temperatures, the proteins are functional and there is no mutation= Permissive temperature/condition. • At higher temperatures, proteins become bob-functional and mutation is manifested = Restrictive temperature/condition. 4
- 5. • Due to the defective protein, the cell-cycle is arrested at a particular event. • The arrested event indicates the function of wild-type protein. 5
- 6. • Thus by manipulating these two temperatures, the functions of different cell proteins/genes can be determined. • Drosophila is employed for analysis of genetical mechanisms that control and co-ordinate growth and division. (This is cell cycle analysis by Genetic level.) 6
- 7. 2.) Biochemical Level • Animal Oocytes/egg and Embryo are employed for this Biochemical analysis of Cell-cycle. • Ex: Xenopus/Frog Oocytes and Sea Urchin embryos. 7
- 8. • Embryos are employed because of their higher rates of cell division and synchronous division. • Oocytes are employed as they contain stock pile of cell-cycle mRNAs that in future in Embryonic cells will translate into cell cycle proteins, which drive the divisions in Embryonic cells. 8
- 9. 9 Oocytes Ovum Zygote Embryonic cell Cell-cycle(Protein regulators) Embryonic cell division Fig: Depicting the development of Embryonic Stem Cells (This is the Biochemical level of Cell-cycle studies.)
- 10. 3.) Molecular Level • Cultured immortal Mammalian cell-lines are employed in studying the Molecular mechanism of Cell-cycle. 10
- 11. Detection of Phases of Cell-cycle • The following ways can be employed to detect particular phase of cell cycle. 1.)Light Microarray • Less condensed indicates Interphase. • Highly condensed indicates M-Phase. • Mitotic Index= (No.of Cells taken/Total no.of cells)*100 11
- 12. • 2.) Cultured Mammalian cells • Flat-shaped cells indicates ‘Interphase’. • Spherical shaped cells indicates ‘M-Phase’ 12
- 13. • 3.) Cell-cycle markers assays:- • The phases of cell-cycle can be determined by detecting Cell-cycle markers. • Cell-cycle markers are proteins that are specific to a particular shape. • Cell-cycle markers are detected by “Immunofluorescence technique”. Cell-cycle markers + MAbs + Fluorescent dye (or) Cell-cycle markers + 1֯Ab + 2֯Ab + fluorescent dye 13
- 14. • The best Cell-cycle markers are “Cyclins”. • Cyclin-D = G1-Phase • Cyclin-A = S-Phase • Cyclin-B = G2+M Phase • “Ki-67” is a Nuclear protein and can be used as a Cell-cycle marker of proliferative cells= S, G2 & M. 14
- 15. • Phospho Rb protein is a cell-cycle marker used to detect the G1-phase. • Phospho H3 protein is used to detect the S-phase. 15
- 16. • 4.) DNA Synthesis-Cell proliferative assays: • S-phase can be recognized by detecting/observing DNA Replication. • This observation can be done by employing: • a) Tritium labelled Thymidine. • b.) BrdU=Bromo Deoxy Uridine. • i.) It is a synthetic Thymidine Analog. • ii.) Anti-BrdU antibodies + Fluorescent dye. 16
- 17. • c.) EdU= Ethyl Deoxy Uridine • It is a synthetic Thymidine Analog and can be detected by Fluorescent Azide. • d.) Flocytometry/FACS (Fluorescence Activated Cell Sorter). • Based on the DNA content, the staining occurs and the phase can be identified. • The quantitative stain includes: • Propidium Iodide • DAPI (Diamidino Phenyl Indole) • 7-AAD[Amino Actinomycin-D] • Hoechst. 17
- 18. • If, DNA content is: • Doubled, then the cell is in G2+M-phase • Intermediate, then the cell is in S-phase • Half, then the cell is in G1 phase. 18
- 19. Synchronization of Cell cultures • Synchronization of cell cycle can be done by arresting the cells in a particular phase. • Cells can be arrested in G1-phase in Yeasts by: • Restricting nutrients • Using anti-mitogens • Mating pheromones 19
- 20. • In Mammalian cells can be arrested in G1-Phase by Serum starvation. • Serum contains Growth factors/Mitogens. • Cell can be arrested in S-phase by: • Hydroxyl Urea/Hydrea/Carbamide. • Fluorodeoxyuridine • Because, Hydrea inhibits Ribonucleotide Reductase. 20 Ribonucleotides DeoxyribonucleotidesRibonucleotide Reductase (DNA Replication)
- 21. • Fluorodeoxyuridine is used to arrest the cell in S-phase because, it inhibits Thymidilate synthetase. 21 Deoxy Uridylic Acid Thymidylic Acid Thymidylate Synthetase
- 22. • Cells can be arrested in M-phase by treatment with: • Colchisine • Benomyl(Benzimidazole group) • Nocodazole 22
- 24. • There are two classes of regulators: • CdKs & • Enzyme complexes. 24
- 25. ‘CdKs’ means “Cyclin Dependent Kinases”.(Hero) • These are major regulators of Cell-cycle. • These are Ser-Thr Kinases. • These phosphorylate several cell cycle proteins and regulate cell cycle. 25
- 26. • The cell cycle proteins in turn control and regulate each event and each phase of Cell-cycle. • CdK levels are constant throughout the Cell- cycle . • CdK are periodically active and inactive. 26
- 27. • CdK are regulated further by following mechanisms: • 1.) Regulation by Cyclins • 2.) Regulation by CAK • 3.) Regulation by Wee1 Kinase. • 4.) Regulation by cdc25 • 5.) Regulation by CdK Inhibitors. 27
- 28. 1.)Regulation by Cyclins(These are major regulators of CdK)(Heroine) • CdK require Cyclins for their activity and hence are called “Cyclin Dependent Kinase”. • Cyclins are so called because, they are cyclically degraded and synthesized. 28
- 29. • Cyclins contains a 9-residue(Amino acid) Cyclin destructive box that makes them liable for destruction. • Cyclins combined with CdKs to form Cyclin- CdK complexes. 29
- 30. 30
- 31. • G1-CdK regulates progression through ‘Commitment point’ and decision to divide. • G1/S-CdK regulates transition from G1 to S-phase and commits cells to DNA replication. • S-CdK does(promotes) Replication and inhibits re-Replication. 31
- 32. • M-CdK regulates M-Phase. • In the absence of Cyclins, CdKs are inactive, because the active site of CdK is blocked by “T-loop” and it is also called “Activation Loop”. • Binding of Cyclins diplaces the T-loop and the active site is exposed. • Inspite of Cyclin binding the CdKs are still inactive. (The reason is given in next slide.) 32
- 33. 2.) Regulation by CAK • An enzyme “CdK Activating Kinase” phosphorylates Thr-160 of CdKs. As a result, CdKs become partially active. 33
- 34. 3.) Regulation of Wee1 Kinase:(Villian) • The CdKs are still partially inactive because, much earlier(even before Cyclin binding) the CdKs were phosphorylated at Thr-14 and Tyr-15. • This dual phosphorylation is inhibitory and hence CdKs are still inactive. • The dual inhibitory phosphorylation is catalyzed by an inhibitory kinase called “Wee-1 Kinase”. 34
- 35. 4.) Regulation by cdc25 Phosphatase(Teacher): • This dual inhibitory Phosphatases are removed by cdc-25 phosphatase. As a result, CdKs become COMPLETELY ACTIVE. • Cyclin-CdK complexes can now regulate cell-cycle. • Synthetic inhibitors or cdc-25 Phosphatase can be employed as “Anti- Neoplastic” and “Anti-Cancer agents”. 35
- 36. 5.) Regulation by CdK-inhibitors • These inhibit CdKs in the following ways: • Prevent Cyclin binding to CdKs. • Prevent ATP binding to CdKs • Distort active site of CdKs. 36
- 37. • These inhibitors inhibit the CdKs: • As a part of normal cell cycle regulation. • In response to anti-proliferative signals, such as • Terminal differentiation • Senescence • DNA damage and • Contact inhibition. 37
- 38. There are two families of CdK inhibitors: • 1.) Kip/Cip family • 2.) Ink4a/ARF Family 38 p21 p27 p57 G1/S - CdK S-CdK M-CdK Note: indicates ‘inhibition’. p15 p16 p18 p19 G1-CdK
- 39. Enzyme complexes • These are Ubiquitin Ligases. • These ubiquitinate proteins and promote their degradation. 39
- 40. 40
- 41. (These are about the Cell-cycle studies and Regulators.) 41
- 42. 42