Chapter 17 – The cell cycle; Molecular Biology of THE CELL
Mitosis is the process of nuclear division. Cytokinesis is the process of cell division. Both happen in the M phase. The interphase is much longer, this includes S phase and interphases. At the transition from metaphase to anaphase an abrupt change in the biochemical state of the cell occurs. After it passes this point the cell carries on to the end of cytokinesis into the interphase. Chromosomes are duplicated in the S phase while most other cell components are duplicated continuously throughout the cycle. If extracellular conditions are favourable and signals to grow and divide are present, cells in early G1 or G0 progress through a commitment point near the end of G1 known as Start. The cell-cycle control system triggers the major events of the cell cycle. It depends on cyclically activated cyclin-dependent protein kinases, Cdks. When cyclin forms a complex with Cdk, the protein kinase is activated to trigger specific cell-cycle events. Without cyclin, Cdk is inactive. The concentrations of the three major cyclin types oscillate during the cell cycle, while the concentrations of Cdks do not change and exceed cyclin amounts. A separate regulatory protein complex, the APC/C, initiates the metaphase-to-anaphase transition. There are three major classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. - G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels drop in S phase. - S-cyclins bind Cdks soon after progression through S tart and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events. - M-cylins activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels drop in mid-mitosis.
In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop. The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk. Phosphorylation of Cdk by Cdk-activating kinase, CAK, at a threonine residue in the T-loop further activates the enzyme by changing the shape o f the T-loop, improving the ability of the enzyme to bind its protein substrates. The Cdks activity is regulated by phosphorylation. The active cyclin-Cdk complex is turned off when the kinase Wee1 phosphorylates two closely spaced sites above the active site. Removal of these phosphates by the phosphatase Cdc25 activates the cyclin-Cdks complex. Cdk activity can be suppressed by inhibitory phosphorylation and Cdk inhibitor proteins, CKIs.
The p27 binds to both the cyclin and Cdk in the complex distorting the active site of the Cdk. It also inserts into the ATP-binding site, further inhibiting the enzyme activity.
Regulated proteolysis triggers the metaphaseto-anaphase transition. APC/C, anaphase promoting complex, or cyclosome is the key regulator of the metaphase-to-anaphase transition. It is a ubiquitin ligase family of enzymes. The APC/C is activated in mitosis by association with Cdc20, which recognizes specific amino acid sequences on M-cyclin and other target proteins. With the help of two additional proteins called E1 and E2, the APC/C assembles polyubiquitin chains on the target protein. The polyubiquitylated target is then recognized and degraded in a proteasome. The activity of the ubiquitin ligase SCF depends on substrate-binding subunit called F-box proteins, of which there are many different types. The phosphorylation of a target protein, such as the CKI shown, allows the target to be recognized by specific F-box subunit.
Preparations for DNA replication begin in late mitosis and G1, when DNA helicases are loaded by multiple proteins at the replication origin, forming the prereplicative complex (preRC). S-Cdk activation leads to activation of the DNA helicases to initiate DNA replication. After duplication of the chromosomes, they are segregated in M phase. S-Cdk activation in S phase also prevents assembly of new prereplicative complexes at any origin until the following G1 – thereby ensuring that each origin is activated only once in each cell cycle. A key player in the initiation of DNA replication is a large multiprotein complex called origin of recognition complex, ORC. The replication origin is bound by the ORC throughout the entire cell cycle. In early G1, Cdc6 associates with the ORC, and these proteins bind the DNA helicase, which contains six closely related subunits called Mcm proteins. The helicase also associates with a protein called Cdt1. Using energy provided by ATP hydrolysis, the ORC and Cdc6 proteins load two copies of the DNA helicase, in an inactive form, around the DNA next to the origin, thereby forming the preRC. At the onset of S-phase, S-Cdks stimulates the assembly of several initiator proteins on each DNA helicase. While another protein kinase, DDK, phosphorylates subunits of the DNA helicase. As a result, the DNA helicases are activated and DNA replication beings. S-Cdks and other mechanisms also inactivate the preRC components ORC, Cdc6 and Cdt1, thereby preventing formation of new preRCs at origins until the end of mitosis.
M-Cdk drives the entry into mitosis. Dephosphorylation activates M-Cdk at the onset of mitosis. Cdk associates with M-cyclin as the levels of M-cyclin gradually rise. The kinases CAK and Wee1 phosphorylate several sites of the inactive M-Cdk. At the end of G2, the M-Cdk complex is activated by the phosphatase Cdc25. Cdc25 is further stimulated by positive feedback. And Wee1 is inhibited by positive feedback.
Principle stages of M phase (mitosis and cytokinesis)
Cohesins hold sister chromatids together.
Condensin helps configure duplicated chromosomes for separation.
The mitotic spindle is a microtubule-based machine. The plus ends of the microtubules project away from the spindle poles, which in this example are organized by centrosomes. One pair of centrioles is located in each centrosome. Kinetochore microtubules connect the spindle poles with the kinetochores of sister chromatids, while interpolar microtubules from the two poles interact with each other. Astral microtubules radiate out from the poles into the cytoplasm. Microtubule-dependent motor proteins govern spindle assembly and function. Kinesine related proteins usually move towards the plus end of microtubules. Dyneins usually move toward the minus end.
Centrosome duplication occurs early in the cell cycle. The centrosome consists of a centriole pair and associated pericentriolar matrix. At a certain point in G1, the two centrioles of the pair separate. During S phase, a daughter centriole begins to grow near the base of each mother centriole and at a right angle to it. The elongation of the daughter centriole is completed in G2. The two centriole pairs remain close together in a single centrosomal complex until the beginning of M phase, then the complex splits and the two daughter centrosomes begin to separate.
In late prophase the mitotic spindle poles have moved to opposite sides of the nuclear envelope. After the nuclear envelope breakdown, the sister-chromatids pairs are exposed to the large number of dynamic plus ends of microtubules. In most cases, the kinetochores are first attached to the sides of these microtubules, while at the same time the arms of the chromosomes are pushed outward from the spindle interior, preventing the arms from blocking microtubule access to the kinetochores. Eventually the laterally-attached sister chromatids are arranged in a ring around the outside of the spindle. Most microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. When the kinetochores are captured in an end-on orientation with the microtubules, additional microtubules are attached to the kinetochores. Alternative forms of kinetochores attachment to the spindle poles can lead to unstable configurations. When microtubule from the same pole attach to both kinetochores the result is unstable. When microtubule from different poles attach to the same kinetochores the result is unstable. When the microtubules are oriented in an unstable way, one of the microtubules tends to dissociate. Only when microtubule from different poles attach to different kinetochores it results in stable binding which will increase the affinity. The APC/C triggers sister-chromatid separation and the completion of mitosis. The activation of APC/C by Cdc20 leads to the ubiquitylation and destruction of securing, which normally holds seperase in an inactive state. The destruction of securing allows seperase to cleave Scc1, a subunit of the cohesion complex holding the sister chromatids together. The pulling forces if the mitotic spindle then pull the sister chromatids apart. Phosphorylation by Cdks also inhibits seperase. Thus, Cdk inactivation in anaphase, resulting from cyclin destruction, also promotes separase activation by allowing its dephosphorylation.
The spindle assembly checkpoint is when unattached chromosomes block sister-chromatid separation. This system depends on a sensor mechanism that mo nitors the strength of icrotubule attachment. Any kinetochores that is not properly attached to the spindle send out a diffusible negative signal that blocks Cdc20 and APC/C activation throughout the cell and thus blocks the metaphase-to-anaphase transition. Mad2 and other proteins are recruited to unattached kinetochores. The unattached kinetochores act likes an enzyme that catalyzes a conformational change in Mad2 which can bind and inhibit Cdc20 and APC/C. In anaphase A, the separated sister chromatids move toward the poles. In anaphase B, the two spindle poles move apart.
M-Cdk triggers the events of early mitosis, inc luding chromosome condensation, assembly of the mitotic spindle, and bipolar attachment of the sister-chromatid pairs to microtubules of the spindle. Spindle formation in animal cells depends largely on the ability of mitotic chromosomes to stimulate local microtubule nucleation and stability, as well as on the ability of motor proteins to organize microtubules into a bipolar array. Many cells also use centrosomes to facilitate spindle assembly. Anaphase is triggered by the APC/C, which stimulates the destruction of the proteins that hold the sister chromatids together. APC/C also promotes cyclin destruction and thus the inactivation of MCdk. The resulting dephosphorylation of Cdk targets is required for the events that complete mitosis, including the disassembly of the spindle and the re-formation of the nuclear envelope.
Cytokinesis is the division of the cytoplasm in two. The actin-myosin bundles of the contractile ring are oriented as shown, so that their contraction pulls the membrane inward.
The contractile ring is regulated by the GTPase RhoA. RhoA is activated by a RhoGEF protein and inactivated by a Rho GTPase-activating protein, RhoGAP. The active GTP-bound form of RoA is focused at the future cleavage site. By binding formins, activated RhoA promotes the assembly of actin filaments in the contractile ring. By activating Rho-activated protein kinases, such as Rock, it stimulates myosin II filament formation and activity, thereby promoting contraction of the ring.
Meiosis is the sexual reproduction of cells. It produces haploid cells carrying only a single copy of each chromosome. There are two rounds of chromosome segregation in meiosis. The structure formed by two closely aligned duplicated homologs is called a bivalent . The centromere is where the two sister chromatids are fixed together. Chiasma is where two chromosomes cross-over.
Mitogens bind to cell-surface receptors to initiate intracellular signaling pathways. This results in the expression of the transcription regulatory protein Myc. Myc increases the expression of many delayed response genes, including some that lead to increased G1-Cdk activity. This triggers the phosphorylation of members of the Rb family of proteins, which inactivates these proteins. This results in the freeing of the gene regulatory protein E2F to activate the transcription of G1/S genes, including the genes for G1/S-cyclin and S-cyclin. The resulting complexes with Cdks form a positive feedback loop by further phosphoylating Rb protein. E2F itself stimulates the transcription of their own genes, another positive feedback loop.
DNA damage blocks cell division and arrests the cell cycle in G1. When DNA is damages, various protein kinases are recruited to the site of damage and initiate a signaling pathway. The first kinase is either ATM or ATR. Additional protein kinases, Chk1 and Chk2, are then recruited and activated. This results in the phosphorylation of the transcription regulatory protein p53. Mdm2 normally binds to p53 and promotes its ubiquitylation and destruction. Phosphorylation of p53 blocks its binding to Mdm2. As a result, p53 accumulates to high levels and stimulates transcription on several genes, including the gene that encodes the CKI protein p21.This protein binds and inactivates G1/S-Cdk and S-Cdk complexes, arresting the cell in G1.
Cell-cycle arrest or apoptosis is induced by excessive stimulation of mitogenic pathways. Abnormally high levels of Myc cause the activation of Arf, which binds and inhibits Mdm2 and thereby increasing p53 levels. This either causes apoptosis or cell-cycle arrest.
The occupation of cell-surface receptors by growth factors leads to the activation of PI 3-kinase, which promotes protein synthesis through a complex signaling pathway that leads to the activation of the protein kinase TOR. Extracellular nutrients also help activate TOR. TOR phosphorylates multiple proteins to stimulate protein synthesis, it also inhibits protein degradation
