16 The Cell Cycle. Chapter Outline The Eukaryotic Cell Cycle Regulators of Cell Cycle Progression The Events of M Phase Meiosis and Fertilization

Transcription

1 The Cell Cycle

2 16 The Cell Cycle Chapter Outline The Eukaryotic Cell Cycle Regulators of Cell Cycle Progression The Events of M Phase Meiosis and Fertilization

3 Introduction Self-reproduction is perhaps the most fundamental characteristic of cells. All cells reproduce by dividing in two, each parental cell gives rise to two daughter cells on completion of a cycle of cell division. Cell division must be carefully regulated and coordinated. In eukaryotic cells, progression through the cell cycle is controlled by protein kinases that have been conserved from yeasts to mammals. Defects in cell cycle regulation are a common cause of the abnormal proliferation of cancer cells.

4 In bacteria, cell growth and DNA replication take place throughout most of the cell cycle; duplicated chromosomes are distributed to daughter cells in association with the plasma membrane.

5 In eukaryotes, the cell cycle has four phases: M phase: Mitosis (nuclear division), usually ending with cell division (cytokinesis). Interphase the period between mitoses, divided into 3 phases: G 1, S, and G 2. G 1 phase (gap 1), the interval (gap) between mitosis and DNA replication. The cell is metabolically active and growing. S phase (synthesis) DNA replication takes place. G 2 phase (gap 2) cell growth continues and proteins are synthesized in preparation for mitosis.

6 Figure 16.1 Phases of the cell cycle

7 The duration of the phases varies considerably in different kinds of cells. Budding yeasts can progress through all 4 phases in 90 minutes. Early embryos may have cell cycles of 30 minutes, but there is no growth (G 1 or G 2 ) phase.

8 In contrast, some cells in adult animals cease division altogether (e.g., nerve cells). Others may divide only occasionally, to replace cells that have been lost. Cell cycle analysis requires identification of the phases. Phases of interphase must be identified biochemically, usually by DNA content. Animal cells in G 1 are diploid (two copies of each chromosome), their DNA content is 2n.

9 During S phase, replication increases the DNA content of the cell to 4n. Cellular DNA content can be determined by incubation of cells with a fluorescent dye that binds to DNA, then analysis of fluorescence intensity of individual cells in a flow cytometer or fluorescence-activated cell sorter.

10 Progression of cells through the division cycle is regulated by both extracellular and internal signals. Cellular processes, such as growth, DNA replication, and mitosis, are regulated by a series of control points. A major control point called START controls progression from G 1 to S in yeast cells. Once cells pass START, they are committed to entering S phase and undergoing one division cycle.

11 Passage through START is highly regulated by external signals, such as nutrient availability and cell size. If there is a shortage of nutrients, cells can arrest the cycle at START and enter a resting phase.

12 In order to maintain constant size, yeast cells must reach a minimum size to pass START. The small daughter cells of budding yeasts spend a longer time in G 1 and grow more than the large mother cell.

13 In most animal cells, the restriction point in late G 1 functions analogously to START. Passage through the restriction point is regulated by extracellular growth factors. Once it has passed the restriction point, the cell is committed to proceed through S phase and the rest of the cell cycle.

14 If appropriate growth factors are not present in G 1, progression stops at the restriction point and cells enter a resting stage called G 0. Skin fibroblasts are arrested in G 0 until stimulated by plateletderived growth factor to proliferate and repair wound damage.

15 Some cell cycles are controlled principally in G 2. The fission yeast Schizosaccharomyces pombe cell cycle is controlled by the transition from G 2 to M, the point at which cell size and nutrient availability are monitored.

16 Vertebrate oocytes can remain arrested in G 2 for long periods (decades in humans). Progression to M phase is triggered by hormonal stimulation. Events in different stages of the cell cycle must be coordinated so that they occur in the appropriate order. E.g., it is critically important that the cell not begin mitosis until replication of the genome has been completed.

17 Coordination between different phases of the cell cycle is dependent on a series of cell cycle checkpoints. DNA damage checkpoints ensure that damaged DNA is not replicated and passed on to daughter cells. The cell cycle is arrested in response to damaged or unreplicated DNA.

18 The spindle assembly checkpoint arrests mitosis at metaphase if the chromosomes are not properly aligned on the mitotic spindle. It is also important to ensure that the genome is replicated only once per cell cycle. Once DNA has been replicated in S phase, control mechanisms prevent re-initiation of DNA replication until the cell cycle has been completed.

19 The MCM helicase proteins bind to origins of replication together with ORC (origin recognition complex) proteins and are required for the initiation of DNA replication. Once initiation has occurred, the MCM proteins are displaced from the origin so that replication cannot initiate again until after mitosis.

20 Regulators of Cell Cycle Progression Recent studies have revealed that the cell cycle of all eukaryotes is controlled by a conserved set of protein kinases, which trigger the major cell cycle transitions. Three experimental approaches contributed to identification of the molecules responsible for cell cycle regulation. 1. Studies of frog oocytes, which are arrested in G 2 until hormonal stimulation triggers entry into the M phase.

21 Regulators of Cell Cycle Progression In 1971, researchers found that oocytes could be induced to enter M phase by microinjection of cytoplasm from oocytes that had been hormonally stimulated. The cytoplasmic factor responsible for this was called maturation promoting factor (MPF). Later work showed that MPF is also present in somatic cells, where it induces entry into M phase. MPF thus appeared to act as a general regulator of the transition from G 2 to M.

22 Figure 16.9 Identification of MPF

23 Regulators of Cell Cycle Progression 2. Genetic analyses of yeasts: investigators found temperature-sensitive mutants that were defective in cell cycle progression (called cdc for cell division cycle mutants). The cdc genes are required for passage through START and for entry into mitosis, and they encode protein kinases. The protein kinase has since been shown to be a cell cycle regulator conserved in all eukaryotes, known as Cdk1.

24 Regulators of Cell Cycle Progression 3. Protein synthesis in early sea urchin embryos: In 1983, Tim Hunt and colleagues identified two proteins (cyclins) that accumulate throughout interphase and are then rapidly degraded toward the end of each mitosis, suggesting a role in inducing mitosis.

25 Regulators of Cell Cycle Progression The three experimental approaches converged in 1988 when MPF was purified and shown to composed of two subunits: Cdk1 and cyclin B. Cyclin B is a regulatory subunit required for catalytic activity of the Cdk1 protein kinase.

26 Regulators of Cell Cycle Progression Further studies demonstrated the regulation of MPF by phosphorylation and dephosphorylation of Cdk1. Cyclin B is synthesized and forms complexes with Cdk1 during G 2. Cdk1 is phosphorylated and inhibited, leading to accumulation of inactive Cdk1/cyclin B complexes throughout G 2. Dephosphorylation activates Cdk1, which phosphorylates several proteins that initiate the events of M phase. Cyclin B is degraded by ubiquitin-mediated proteolysis.

27 Figure MPF regulation

28 Regulators of Cell Cycle Progression Further research has established that Cdk1 and cyclin B are members of protein families. Different members of these families control progression through the phases of the cell cycle. Cdk1 controls passage through START and entry into mitosis in yeasts, in association with G 1 cyclins or Cln s. In higher eukaryotes, there are multiple cyclins and multiple Cdk1-related protein kinases, known as Cdk s for cyclin-dependent kinases.

29 Figure Complexes of cyclins and cyclin-dependent kinases