4BBY1030 Cell cycle 2023.pdf

The purpose of the cell cycle To copy the genome and partition the copies equally between the daughter cells (unicellular and multicellular organisms). To enable a multicellular organism to grow to adult size. To maintain the total cell number of an adult organism. To replace lost or damaged cells....

The purpose of the cell cycle To copy the genome and partition the copies equally between the daughter cells (unicellular and multicellular organisms). To enable a multicellular organism to grow to adult size. To maintain the total cell number of an adult organism. To replace lost or damaged cells. 3 Prokaryotes divide by binary fission nucleoid DNA attached to cytoplasmic membrane cell enlarges & DNA duplicates septum forms cell divides in two, DNA partitioned into each cell cells separate 4 Two pathways must be co-ordinated. ①Replication of DNA (and the partition of the two copies) ②Cytokinesis (cell separation) ①Replication of DNA ori Circular chromosome of prokaryotes has one origin (ori) of replication Two replication forks (RF) form at the origin; replication is bidirectional theta structure Two identical copies of the circular chromosome ② Cytokinesis early step in bacterial cytokinesis is formation of a ring of a protein, FtsZ, on the inner surface of the cytoplasmic membrane at the future division site FtsZ is distributed randomly throughout the cytoplasm of the cell FtsZ ring contracts Two pathways must be co-ordinated. ①Replication of DNA (and the partition of the two copies) ②Cytokinesis (cell separation) However: Cell cycle of rapidly growing bacteria is shorter than the time needed to copy DNA Cell division takes 20 minutes DNA replication takes 40 minutes Implies that some cells will not contain DNA, because DNA replication can’t keep up with cell division. The paradox implied by this mis-match in timing is resolved by DNA replication being initiated before completion of the previous round (multifork replication) 1 round of replication 2 rounds of replication (multifork) Multifork replication ensures that at least one round of replication is finished before cytokinesis. Eukaryotic cell cycle The additional complications: ①Genome is composed of multiple linear chromosomes (necessitating co-ordinated replication of all of them as well as their faithful segregation). ②Multicellularity: Cells in the context of organs and tissues. ③Numerous organelles (must partition into daughter cells). Details of the cell cycle vary from organism to organism and at different times in an organism’s life. Certain characteristics are universal o DNA must be faithfully replicated. o Replicated chromosomes must be accurately segregated. G1 (Gap 1): growth phase, doubling the mass of organelles and protein, including synthesis of enzymes that will drive DNA replication. S: DNA Synthesis phase. Chromosome duplication. At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids. The sister chromatids must not be allowed to separate from each other, otherwise bipolar attachment to the mitotic spindle would be difficult to achieve. Cohesin ensures that sister chromatids do not drift apart. SMC3 kleisin SMC1 Figure 17-24c Molecular Biology of the Cell (© Garland Science 2008) Structural Maintenance of Chromosomes G2: preparation for mitosis. The beginning of mitosis is marked by two events: 1st event: Chromosome condensation a b a. Interphase: Chromosomes not visible b. M phase begins: duplicated chromosomes condense & become visible 1st event: Chromosome condensation Condensin encircles loops of DNA and compresses the sister chromatids….to give a compact structure. A model for condensin action: a coil of DNA loops 2nd event: Formation of mitotic spindle. Kinetochore – complex of proteins attached to the centromere Spindle pole body Mitotic spindle a bipolar array of microtubules The spindle pole bodies lie outside the nucleus The nuclear membrane must breakdown early in mitosis so that the spindle has access to the chromosomes. http://depts.washington.edu/biowww/pages/faculty-Davis.shtml 2nd event: Formation of mitotic spindle. The tension in the spindle is pulling the chromosomes away from each other…but the sister chromatids are still held together by cohesin. ……concentrated along the chromosome axis (red fluorescent antibody against cohesin). Figure 4-74 Molecular Biology of the Cell (© Garland Science 2008) Chromatids segregated when the kleisin subunit of cohesin is cleaved by a protease: Intact cohesin Cleaved cohesin Cytokinesis - the end of the cell cycle Once the sister chromatids have reached opposite poles of the cell: Nuclear membrane begins to re-form. Cytoplasm is divided in two by a contractile ring of filaments composed of actin and myosin II. Pinches the cell in two, giving two daughters, each with one nucleus. Figure 17-49a Molecular Biology of the Cell (© Garland Science 2008) Cytokinesis - the end of the cell cycle In animal cells the contractile ring divides the cytoplasm from the outside in. In plants, a contractile ring does not form. Instead a new cell wall is constructed between the daughter nucleii, so cytoplasm is partitioned from the inside out. New wall synthesis guided by the phragmoplast – contains microtubules derived from the mitotic spindle. Golgi derived vesicles are transported along these microtubules Figure 17-57 Molecular Biology of the Cell (© Garland Science 2008) Details of the cell cycle vary from organism to organism and at varying times, but the basic processes common to all cycles are faithful replication and segregation of chromosomes. Variations include: I. Timing 2 II. Early embryonic cycles – division without growth. Somatic cells maintain a constant size, because cells grow after each division. The early embryo divides without growing, producing smaller cells with each successive cycle. Early embryonic cycles are 20x faster than those of somatic cells 3 III. Nuclear envelope dynamics. Multicellular organisms operate an open mitosis – nuclear envelope breaks down and then reforms. …because SPB is outside the nucleus. So the membrane must break down so that spindle has access to chromosomes. Unicellular organisms operate a closed mitosis – nuclear envelope remains intact throughout. In baker’s yeast (Saccharomyces cerevisiae), the spindle pole body (SPB) is the sole site of microtubule organization. The SPB is embedded in the nuclear envelope. 4 IV. Polarity. There are examples where cells divide asymetrically (the daughter cells can differ in size or cytoplasmic content or both) Usually the daughter cells will develop along different pathways. The mother cells must segregate cell fate determinants to one side and then position the plane of division so that one daughter inherits the determinants. e.g. P granule segregation in development of the nematode worm Caenorhabditis elegans IV. Polarity. e.g. P granule segregation in development of the nematode worm Caenorhabditis elegans (a useful model organism; transparent, only 959 cells; including germ cells in the gonad at one end) IV. Polarity. The first cell division of a fertilized C.elegans egg is assymetric blue stain: DNA green stain: P-granules complex of RNA and protein which drives formation of germ cells IV. Polarity. e.g. stem cell division Stem cells are attached to niche cells, blocks their differentiation, but cell division is allowed. One daughter is released and is free to differentiate. The other daughter remains attached to the niche cells and remains a stem cell. Other aspects of cell cycle control anchorage dependence: displayed by many animal cells, in which cells must be attached to a substratum in order to divide density-dependent inhibition: in which cells stop dividing once they contact each other (contact inhibition) In tissue culture: Surface needed for division. Cells divide to form a single layer, and then stop. If some cells are removed, division occurs to fill the gap (and then stops). The cell cycle control system 1. Cell cycle engine The protein complex that drives the cycle 2. Co-ordination e.g. replicated DNA must go through mitosis before replication occurs again 3. Checkpoints The cycle will stop if the cell is deprived of nutrients or DNA is damaged, or if chromosomes fail to attach to the spindle. 1. Cell cycle engine Phases of the cell cycle are driven by action of a protein kinase, the cyclin dependent protein kinase (CDK). Levels of the kinase itself remain constant throughout the cell cycle. It is the activity (not the levels) of the kinase that activates the phases of the cycle. 1. Cell cycle engine The kinase is only active when complexed with another protein, cyclin Cyclins are the key to regulating the cell cycle because: They undergo cycles of synthesis and degradation – so their levels rise and fall There are different CDK’s and cyclins, each pair activating a different phase of the cell cycle. e.g. G1/S phase cyclin (cyclin E) G1/S CDK (levels don’t change) Activity of CDK Cyclin E (levels reflect the CDK activity profile) Triggers G1/S transition – commits cell to the cycle (e.g. commitment to DNA replication). G1 S phase G2 Mitosis e.g. M phase cyclin (cyclin B) M CDK (levels don’t change) Activity of CDK Cyclin B (levels reflect the CDK activity profile) G1 S phase G2 Mitosis activates condensin Promotes entry into mitosis e.g. Induces nuclear membrane breakdown Mitotic CDK phosphorylates nuclear lamin Causes depolymerization of lamin filaments Lamina mesh disintegrates, and no longer supports the nuclear membrane. CDK-cyclin complexes trigger different phases of the cell cycle because the cyclin directs the kinase to specific target proteins. Cyclins are degraded by proteolysis, terminating the cell cycle phase they control. M CDK (levels don’t change) Activity of CDK Cyclin B (levels reflect the CDK activity profile) Cyclin B degradation is required for exit from mitosis and ending the cell cycle G1 S phase G2 Mitosis 2. Co-ordination The cycle is tightly regulated…phases must occur in the proper order. S phase and M phase must only happen once during each cycle. e.g. mechanism must exist to prevent re-replication of G2 DNA existence of such mechanisms was first proven using cell fusion experiments. 2. Co-ordination Fusing an S phase with a G1 phase cell: The S phase nucleus continues DNA replication The G1 DNA is instructed to enter S phase Fusing an S phase with a G2 phase cell: The S phase nucleus continues DNA replication The G2 DNA is NOT forced into S phase 3. Checkpoints Surveillance mechanisms (checkpoints) operate continually to ensure next phase is not initiated unless the previous one has been completed 1. In G1: Restriction point (R). A G1 positive signal (growth factor) R from the outside will instruct the cell to divide (no signal, no division). M S (Mitosis) (DNA Synthesis) G2 2. At G2/M: Is DNA synthesis complete? Cell cycle is suspended if not (passing less DNA to progeny is not permitted) 3. Checkpoints Surveillance mechanisms (checkpoints) operate continually to ensure next phase is not initiated unless the previous one has been completed 3. Spindle checkpoint: is each chromosome attached to the spindle? If not, the cell cycle is suspended. G1 R M S (Mitosis) (DNA Synthesis) G2 4. AND…the DNA damage checkpoint (operates throughout the cycle). Arrests cycle while damage is repaired. Consequences of checkpoint failure. Failure of DNA damage checkpoint: Cycle keeps turning despite DNA damage. Mutations accumulate. Leads to cancer. If spindle checkpoint fails: Unequal segregation of chromosomes Causes human aneuploidies (meiotic spindle) e.g. Down’s Syndrome (extra copy of chromosome 21) % incidence of trisomy Risk of having a Down’s syndrome child increases with maternal age Maternal age Hassold T. and Hunt P. (2001) Nature Reviews Genetics. 2: 280-291. Cancer and the cell cycle All cancers feature a de-regulated cell cycle (as a consequence of mutation) signals that start and G1 stop the cell cycle are R ignored Checkpoints that protect M the genome (DNA damage, mitotic spindle) no longer operate G2 Cells do not communicate with each other (e.g. no contact inhibition). S Summary In the prokaryotic cell cycle, multifork replication compensates for the mismatch in timing between DNA replication and cell division. The eukaryotic cell cycle is divided into four phases, enabling faithful replication and segregation of chromosomes. The cyclin dependent kinase/cyclin complex drives the eukaryotic cell cycle. The cycle is subject to regulation at multiple levels, including checkpoints that guard against accumulation of mutations and mis-segregation of chromosomes