Cell cycle checkpoints in yeast
- 1. Cell division cycle in Yeast • Submitted by- Seema Mehra Msc. Microbiology 2nd semester
- 2. Introduction • The cell cycle is the succession of events whereby a cell grows and divides into two daughter cells that each contain the information and machinery necessary to repeat the process. • Between one cell division and the next, all essential components of cell must be duplicated, the most important component is genetic material which must be accurately replicated and the two copies carefully segregated to two daughter cells. • Division cycle of most cells consist of four coordinated processes- Cell growth DNA replication Distribution of the duplicated chromosomes to daughter cells Cell division.
- 3. Contd… • In eukaryotes cell cycle is more complex. • Although cell growth is usually a continuous process, DNA is synthesized during only one phase of the cell cycle and the replicated chromosomes are then distributed to daughter nuclei by a complex series of events preceding cell division. • Progression between these stages of the cell cycle is controlled by a conserved regulatory apparatus.
- 5. Phases of cell cycle Cell cycle is divided into two basic parts- Interphase Mitosis INTERPHASE Chromosomes are decondensed and distributed throughout the nucleus, so nucleus appears morphologically uniform. At molecular level interphase is the time during which both cell growth and DNA replication occur in an orderly manner in preparation for cell division. Cell grows at steady rate throughout interphase
- 6. Contd… It consist of 3 phases- 1. G1 phase(gap 1) - Corresponds to interval (gap) b/w mitosis and initiation of DNA replication. - Cell is metabolically active and continuously grows but does not replicate its DNA. 2. S phase (synthesis) - DNA replication takes place. 3. G2 phase (gap 2) - cell growth continues and proteins are synthesized in preparation for mitosis. - The duration of these cycle phases varies considerably in different kinds of cells. - In budding yeast, all 4 stages of cell cycle gets completed in approx. 90 mins.
- 8. Mitosis • The cell cycle is traditionally divided into several distinct phases, of which the most dramatic is mitosis, the process of nuclear division, leading up to the moment of cell division itself. • In mitosis the nuclear envelope breaks down, the contents of the nucleus condense into visible chromosomes, and the cell's microtubules reorganize to form the mitotic spindle that will eventually separate the chromosomes. • As mitosis proceeds, the cell seems to pause briefly in a state called metaphase, in which the chromosomes, already duplicated, are aligned on the mitotic spindle, poised for segregation. • The separation of the duplicated chromosomes marks the beginning of anaphase, during which the chromosomes move to the poles of the spindle, where they decondense and re-form intact nuclei. • The cell is then pinched in two by a process called cytokinesis, which is traditionally viewed as the end of the mitotic phase, or M phase, of the cell cycle
- 9. Fig..
- 10. • Some cells in adult animals cease division altogether (eg. Nerve cells) and many other cell division only occasionally, as needed to replace cells that have been lost because of injury or cell death( eg. Skin fibroblast, liver). • These cells exit G1 to enter a quiescent stage of cycle called G0, where they remain metabolically active but no longer proliferate unless called on to do so by extracellular signals.
- 11. • Identification of cells at mitotic stage can be done microscopically while cells in other phases of the cycle (G1, S and G2) must be identified by biochemical criteria. • Cells in S phase can be readily identified because they incorporate radioactive thymidine, which is used exclusively for DNA synthesis.
- 12. Regulation of cell cycle by cell growth and extracellular signals Progression of the cells through the division cycle is regulated by extracellular signals from the environment as well as by internal signals that monitor and coordinate the various processes that take place during different cell cycle phases. Cell cycle progression is accomplished by a series of control points that regulate progression through various phases of the cell cycle. A major cell cycle regulatory point in many types of cells occurs late in G1 and controls progression from G1 to S.
- 13. Contd… This regulatory point was first defined by studies of budding yeast (Saccharomyces cerevisiae) where it is known as START. Once cells have passed START, they are committed to enter S phase and undergo one cell division cycle. Passage through START is highly regulated event in yeast cell cycle where it is controlled by external signals, such as availability of nutrients, mating factors, as well as by cell size.
- 14. Eg. If yeasts are faced with a shortage of nutrients, they arrest their cell cycle at START and enter a resting state rather than proceeding to S phase. Polypeptide factors that signal yeast mating also arrest the cell cycle at START, allowing haploid yeast cells to fuse with one another instead of progressing to S phase.
- 16. Contd… • Also, START is a point at which cell growth is coordinate with DNA replication and cell division. • It is evident in budding yeast in which cell division produce progeny cells of very different sizes- a large mother cell and a small daughter cell. • In order for yeast cell to maintain a constant size, the small daughter cell must grow more than the large mother cell does before they divide again. • The cell size must be monitored in order to coordinate cell growth with other cell cycle events.
- 17. • Some cell cycles are controlled principally in G2. • Eg. In Schizosaccharomyces pombe , cell cycle is regulated primarily by control of the transition from G2 to M, which is a principal point at which cell size and nutrients availability are monitored.
- 18. Cell cycle checkpoints • The events that take place during different stages of cell cycle must be coordinated with one another, so that they occur in appropriate order. • Cell cycle checkpoints prevent entry into the next phase of the cell cycle until the events of the preceding phase have been completed. • Several cell cycle checkpoints function to ensure that incomplete or damaged chromosomes are not replicated and passed onto daughter cells . • These checkpoints sense unreplicated or damaged DNA and coordinate further cell cycle progression with the completion of DNA replication or repair.
- 19. Various checkpoints are- 1. G2 phase checkpoint – Checkpoint in G2 prevents initiation of mitosis until DNA replication is completed. – It senses unreplicated DNA, which generate a signal that leads to cell cycle arrest. – Thus operation of G2 checkpoint prevents initiation of M phase. It also senses DNA damage, such as that resulting from irradiation. 2.G1 phase checkpoint – Arrest at the G1 checkpoint, allows repair of damage to take place before cell enters S phase, where the damaged DNA would be replicated.
- 20. Contd… 3. S-phase checkpoint • Provides continual monitoring of the integrity of DNA to ensure that damaged DNA is repaired before it is replicated. • It also provides a quality control monitor to promote repair of any errors that occur during DNA replication. 4.Spindle assembly checkpoint • Maintains integrity of genome, occurs towards the end of mitosis. • Monitors the alignment of chromosomes onto the mitotic spindle, ensuring that a complete set of chromosomes is distributed accurately to daughter cells.
- 22. Contd.. • Cell cycle arrest at G1, S and G2 checkpoints is mediated by two related protein kinases, ATM and ATR that recognize damaged or unreplicated DNA and are activated in response to DNA damage. • ATM and ATR activate a signalling pathway that leads not only to cell cycle arrest, but also to activation of DNA repair and in some programmed cell death.
- 23. Restriction of DNA replication to once per cycle It is important to ensure that the genome is replicated only once per cell cycle. Thus once a segment of DNA has been replicated in S phase, control mechanisms must exist to prevent re-initiation of DNA replication until cell cycle has been completed. Thus this mechanism involves action of MCM helicase proteins that bind to replication origins together with the origin recognition complex(ORC) proteins. MCM proteins act as ‘licensing factors’ that allow replication to initiate. MCM proteins bind to replication origins during G1, allowing DNA replication to initiate when cell enters S phase. Once initiation has occurred, MCM proteins are displaced from the origin, so replication cannot initiate again.
- 25. Regulation of cell cycle progression • Cell cycle of all eukaryotes is controlled by a conserved set of protein kinases, which are responsible for triggering major cell cycle transitions. Protein kinases and cell cycle regulation I. Maturation promoting factor(MPF)
- 27. Contd.. 2. Second approach was genetic analysis of yeasts. • Pioneered by Hartwell and colleagues in early 1970s. • Investigators identified temperature sensitive mutants of Saccharomyces cerevisiae that were defective in cell cycle progression. • These mutants called cdc mutants underwent growth arrest at specific ponts in the cell cycle. • Eg. Cdc28 mutants in S.cerevisiae caused the cell cycle to arrest at START, indicating cdc28 protein is required for passage through this critical regulatory point in G1.
- 28. Contd… • cdc2 mutant of Schizosaccharomyces pombe arrest cell cycle both in G1 and at G2 to M transition. • Further studies demonstrated that cdc2 and cdc28 encoded a protein kinase-first indication of prominent role of protein phosphorylation in regulating cell cycle. • The protein kinase encoded by the yeast cdc2 and cdc28 genes has since been shown to be a conserved cell cycle regulator in all eukaryotes, which is known as cdk1.
- 30. Contd.. 3. Third line of investigation • It converged with identification of MPF and yeast genetics. • Studies with protein synthesis inhibitors had revealed that entry into M phase of these embryonic cell cycle requires new protein synthesis. • In 1983, Tim Hunt identified 2 proteins. These proteins accumulate throughout interphase and rapidly degraded toward end of each mitosis. • Hunt called these proteins cyclins ( cyclin A and B) and suggested that they might function to induce mitosis, with their periodic accumulation and destruction controlling entry and exit from M phase.
- 31. Contd… • When MPF was purified from frog eggs in 1988, its molecular characterization showd that this conserved regulator of cell cycle is composed of 2 key subunits: cdk1 and cyclinB. • Cyclin B is a regulatory subunit required for catalytic activity of cdk1 protein kinase. • MPF activity is controlled by periodic accumulation and degradation of cyclin B during cell cycle progression. • Once activated, the cdk1 protein kinase phosphorylates a variety of target proteinsthat initiate the events of M phase. • cdk1 activity triggers the degradation of cyclin B (ubiquitin mediated proteolysis). This proteolytic destruction of cyclin B then inactivates cdk1, leading the cell to exit mitosis, undergo cytokinesis and return to interphase.
- 33. Families of cyclins and cyclin- dependent kinases • cdk1 controls passage through START as well as entry into mitosis in yeast. It does so, however in association with distinct cyclins. • G2 to M transition is driven by cdk1 +mitotic B type cyclins (clb1, clb2, clb3 and clb4). • Passage through START is controlled by cdk1 +G1 cyclinsor cln’s. • cdk1+clb5 and clb6- required for progression through S phase. • These associations of cdk1 with distinct B typeand G1 cyclins direct cdk1 to phosphorylate different substrate proteins. • Required for progression through specific phases of cell cycle.
- 35. Activity of cdk’s during cell cycle progression is reglated by four molecular mechanisms 1. Association of cdk’s with their cyclin partners. 2. Activation of cdk/cyclin complexes require phosphorylation of a conserved cdk threonine residue. Phosphorylation of cdk’s is catalyzed by an enzyme called CAK (cdk activating kinase). 3. Inhibitory phosphorylation of tyrosine residues near cdk amino terminus, catalyzed by wee1 protwin kinase. 4. cdk’s activities are also controlled by binding of inhibitory proteins( called cdk inhibitors or CKIs).
- 37. DNA damage checkpoints • DNA damage checkpoints play a critical role in maintaining the integrity of genome by arresting cell cycle progression in response to damaged or incompletely replicated DNA. • They allowtime for the damage to be repaired before DNA replication or cell division proceeds.
- 39. Mitosis phase • During mitosis, the chromosomes codense, the nuclear envelope of most cells breaks down, the cytoskeleton reorganizes to form the mitotic spindle and the chromosomes move to opposite poles, chromosomes segregation is then usually followed by cell division(cytokinesis). Stages of mitosis 1. Prophase - marked by appearance of condensed chromosomes, each of which consists of two sister chromatids. - daughter DNA molecules produced in S phase. - these newly replicated DNA molecules remain intertwined throughout S and G2, becoming untangled during process of chromatin condensation. - end of prophase corresponds to breakdown of nuclear envelope. - but in yeast, closed mitosis occurs in whih the nuclear envelope remains intact.
- 40. Contd… • In closed mitosis, daughter chromosomes migrate to opposite poles of nucleus, which then divides in two. • In these cells, the spindle pole bodies are embedded within the nuclear envelope and nucleus dividesin two following migration of daughter chromosome to opposite poles of the spindle. 2. Prometaphase • Transition between prophase and metaphase. • Microtubules of mitotic spindle attach to kinetochores of condensed chromosomes.
- 41. Contd… 3. Metaphase • Chromosomes shuffle back and forth until they eventually align on metaphase plate in the center of the spindle. 4. Anaphase • Breakage of link between sister chromatids which then separate and move to opposite poles of the spindle. 5. Telophase • Nuclei reform and chromosomes decondense. 6.Cytokinesis • Begins during late anaphase and completed by the end of telophase. • Resulting in formation of two interphase daughter cells.
- 44. Cdk/cyclinB and progression to metaphase • Events of M phase are initiated by activation of cdk1/cyclinB protein kinase(MPF). Cdk1/cyclinB not only acts as a master regulator of M phase transition, phosphorylating and activating other downstream protein kinase but also acts directly by posphorylating some of the structural proteins involved in cellular organization. • Chromatin condensation is driven by protein omplexex called condensins, which are members of a class of structural maintenance of chromatin(SMC) proteins that play key roles in organization of eukaryotic chromosomes. • Another family of SMC proteins, called cohesins, contribute to chromosome segregation during mitosis. • Cohesins bind toDNA in S phase and maintain linkage between sister chromatids following DNA replication. • As cells enter M phase, condensins are activated by cdk1/cyclinB phosphorylation. • Condensins replace cohesins along the length of chromosomes except at centromere.
- 47. Spindle assembly checkpoint and progression to anaphase • Spindle assembly checkpoint monitors the alignment of chromosomes on metaphase spindle. • Progression from metaphase to anaphase results from ubiquitin- mediated proteolysis of key regulatory proteins, triggered by activation of an E3 ubiquitin ligase called anaphase promoting complex.
- 49. Cytokinesis • Cytokinesis of yeast and animal cells is mediated by a contractile ring of actin and myosinII filaments that forms beneath the plasma membrane. • Cleavage proceeds as contraction of the actin-myosin filaments pulls the plasma membrane inward, eventually pinching the cell in half.
- 51. Meiosis • It is a specialized kind of cell cycle that reduces the chromosome number by half, resulting in theproduction of haploid daughter cells. • Unicellular eukaryotes, such as yeasts, can undergo meiosis as well as reproducing by mitosis. • Diploid S.cerevisiae undrgo meiosis and produce spores when faced with unfavourable environmental conditions.
- 52. Process of meiosis • Meiosis results in the division of a diploid parental cell into haploid progeny, each containing only one member of the pair of homologous chromosomes that were present in the parental cell. • This is accomplished by two sequential rounds of nuclear and cell division (called meiosis I and meiosis II), which follows a single round of DNA replication. • Like mitosis, meiosis I initiates after S phase has been completed and the parental chromosomes have replicated to produce idenical sister chromatids. • During meiosis I, homologous chromosomes first pair with one another and then segregate to different daughter cells. • Sister chromatids remain together, so completion of meiosis I results in the formation of daughter cells containing a single member of each chromosome pair (consisting of 2 sister chromatids). • Meiosis I is followed by meiosis II, which resembles mitosis in that the sister chromatids separate and segregate to different daughter cells.
- 54. Thank you