After studying this chapter, the reader should be able to:

• Discuss the G₁-S-G₂-M features of the cell cycle.
• Explain the role of cyclins and cyclin-dependent kinases in cell cycle regulation.
• Describe the steps involved in mitosis and cytokinesis.

1. General information
1. The human body is composed of 10¹³ cells, with 200 different cell types
2. A typical mammalian cell has a cell cycle time of 24 hours
3. The cell cycle has four phases: mitosis (M), synthesis of DNA (S), gap 1 (G₁), and gap 2 (G₂); together, the latter three phases are called interphase
4. For some non-dividing or resting cells, cells leave the G₁ phase and enter G₀, a quiescent phase
5. The cell cycle can be analyzed by pulse-chase autoradiography, flow cytometry, cell fusion and synchronization, and classical and molecular genetic techniques
2. Discovery of the cell cycle
1. The cell cycle was discovered by the use of radioisotopes and light microscopic autoradiography
2. In 1953, Alma Howard and S. R. Pelc proposed the following terms for the cell cycle: division (D), gap 1 (G₁), synthesis (S), and gap 2 (G₂); "G" stands for time intervals or gaps
1. This was based on their work on the incorporation of radioactivity (³²P) into the DNA of plant cells
2. The researchers were studying the meristem of the main root of seedling of the bean plant Vicia faba
3. They found a 30 hr cell cycle for these plant cells, with 4 hr for mitosis (M, or D), 6 hr for DNA synthesis, plus a time interval from post-division to DNA synthesis (12 hr for G₁), and another time interval from after DNA synthesis to mitosis (8 hr for G₂)
3. Variations of this technique have been commonly used to estimate cell cycle time periods for other species
1. The standard assay is ³H thymidine autoradiography
2. Mammalian cells in tissue culture proliferate asynchronously
3. A short pulse of ³H thymidine, often for 30 minutes, is introduced into the cells
4. The cells are then grown in an excess of nonradioactive thymidine
5. At various times of chase, cells are sampled for autoradiography; the percentage of labeled mitotic figures is scored
6. Total cell cycle time is the time interval of successive waves of labeled mitotic figures; this is the length of time needed for a cell to complete one cell cycle
7. In a sample, mitotic index is the fraction of cells that is in mitosis; it is a measure of the relative length of mitotic phase of the cell cycle
8. Labeling index is the fraction of cells that is labeled; from this number, the duration of S phase as a fraction of the whole cell cycle can be estimated
4. Cells can also be labeled non-radioactively using bromodeoxyuridine (BrdU), a thymidine analog; labeled cells can be detected using an antibody against BrdU
3. Methods to study the cell cycle
1. The cell cycle can be measured by ³H thymidine or bromodeoxyuridine incorporation (as described above)
2. The cell cycle can be analyzed by flow cytometry, using a fluorescence-activated cell sorter
1. Cells are treated with a DNA-binding fluorescent dye, such as propidium iodide
2. The amount of cellular DNA is proportional to the amount of fluorescence detected by dye binding
3. For a proliferating cell population, a plot of cell number versus DNA content yields the percentage of cells in G₁ (with 1 arbitrary unit of DNA or 2n), in G₂+M (with 2 arbitrary units of DNA or 4n), and in S (cells with more than 1 but less than 2 units of DNA)
4. The percentage of cells in G₁, S, and G₂ corresponds to the time spent in each period of the cell cycle
3. Cells in a proliferating population are in different phases of the cell cycle; methods are available to synchronize the cell cycle
1. Probably the best method of cell synchronization is mitotic detachment
1. During mitosis, the attached cells become rounder
2. Mitotic cells can thus be collected by gentle shaking
3. The method is useful only for attached or monolayer-cultured cells
4. The yield is low, because only 5-8% cells are in mitosis at any one time
5. The yield can be increased by drugs, such as colchicine or colcemid, that inhibit mitosis
2. Another method of selection synchrony is by centrifugation
1. Cells increase in size as they grow, in preparation for division
2. The smaller G₁ cells can be separated from other cells of different sizes by centrifugation
3. Both centrifugal elutriation and density gradient centrifugation are possible
4. These methods are useful for cells in suspension
3. Cells can achieve induced synchrony when the appropriate nutrients and drugs are added
1. Treatment with a high dose of thymidine (2 mM) can stop DNA synthesis because of deoxyribonucleotide feedback inhibition
2. Methods such as thymidine addition and Ile deprivation are available for cell synchronization
3. DNA synthesis inhibitors can also be used; these include aphidicolin, which inhibits DNA polymerase, and hydroxyurea, which blocks synthesis of deoxyribonucleotides
4. The effects of these treatments are reversible after the removal of the compound, although they add stress to the cells
4. Experiments with cell fusion yield information on cell cycle controls
1. Cells from different phases of the cell cycle are fused to form heterokaryons (a fused cell with 2 nuclei)
2. For example, in cell fusion between a mitotic cell and an interphase cell (in any phase, G₁, S, or G₂), chromosomes of the interphase nucleus immediately undergo condensation in an attempt to enter mitosis
3. This result suggests the presence of a M-phase inducer from the mitotic cell that influences the behavior of the interphase chromosomes of the heterokaryon
5. Genetic approaches allow the selection of cell cycle mutants
1. The eukaryotic microbes, yeasts, are favored genetic models
2. Both the budding yeast (Saccharomyces cerevisiae) and the fission yeast (Scizosaccharomyces pombe) are used
3. Cycle-division-cycle or cdc mutations are characterized on the basis of conditional mutants, such as temperature sensitive mutants that grow at permissive temperatures and die at non-permissive temperatures, halting at specific points at the cell cycle
4. Based on yeast cdc mutants, a series of dependent and alternate pathways can be plotted as a regulatory cascade for cell cycle control
5. Using elegant yeast molecular genetics, cdc mutants are transformed with plasmids containing random fragments of DNA from normal cells
1. The rare revertants that can grow at the restrictive temperature are selected
2. The "Rescuer" genes are cloned and characterized from the plasmids
6. Over 70 genes, many of which have mammalian counterparts, have been found
1. The fission yeast cdc2 codes a protein kinase
2. The fission yeast cdc13 codes a cyclin
7. Another type of mutation is known as wee, taken from the Scottish word for small
1. A fission yeast cell must reach a critical size to enter mitosis
2. The wee mutant cells are small because they bypass a size checkpoint of the cell cycle
3. The amount of wee protein regulates the entry into mitosis
4. The fission yeast wee1 codes a protein kinase
4. Cell cycle parameters
1. A typical mammalian cell has a cell cycle time of 24 hours, with 12 hr G₁, 6-8 hr S, 3-4 hr G₂, and 1 hr M
2. A human body has 10¹³ cells and perhaps 200 cell types; different cell types have different cell cycle times
3. Skeletal muscle cells and neurons do not divide, liver cells divide about once a year, and some intestinal epithelial cells divide twice a day
4. The shortest cell cycle time is seen in early embryonic cells; during the subdivision of a fertilized egg into smaller cells, the cell cycle time is 8-60 minutes, half in S and half in M
5. In vivo, a mouse duodenum epithelial cell has a cell cycle time of 10 hr; a human stomach epithelial cell, 24 hr; a human bone marrow cell, 18 hr; in culture, a human fibroblast, 18 hr; a HeLa cell, 21 hr, and a Chinese hamster ovary cell, 14 hr
6. The most variable time interval of the cell cycle is the G₁ phase; cells can leave G₁ phase to a G₀ (quiescent) state
1. The ability to enter G₀ accounts for the variability of length of cell cycles in different cell types
2. G₀ cells may have a rate of protein synthesis that is 20% that of proliferating cells
3. At a time interval late in G₁, there is a restriction point
1. This term was coined by Arthur Pardee in 1974
2. The point commits the cell to complete S, G₂, and M, or alternately, to enter the quiescent G₀ state
3. This is also called the commitment point or G₁ checkpoint in mammalian cells, and start in yeasts

1. General information
1. Several checkpoints regulate cell cycle progression
2. Although the best understood is regulation for mitosis, the most important is the G₁ checkpoint
3. A family of protein kinases is involved in cell cycle control
4. The kinases are activated by another family of proteins, known as cyclins
5. The levels of cyclins fluctuate during the cell cycle; cyclins are controlled by their own degradation
2. MPF, kinases, and cyclins
1. Studies on the early embryogenesis of the frog Xenopus laevis led to the discovery of maturation-promoting factor (MPF)
1. Immature oocytes are arrested at the G₂ phase of meiosis I; they are stimulated by the hormone progesterone to enter M phase as mature eggs
2. If the mitotic cytoplasm from a mature unfertilized egg is microinjected into the G₂ phase oocyte, the recepient oocyte is driven into the M phase and completes its maturation
3. Maturation-promoting factor (MPF), is the activity in the egg cytoplasm that can induce an immature oocyte to become a mature egg
4. Mitotic cell extracts from all eukaryotic cells studied, including yeast, starfish, and humans, can function as MPF in the frog oocyte assay; interphase cell extracts cannot function in this way
5. Thus, MPF is a ubiquitous eukaryotic inducer of mitosis and meiosis and is called M-phase promoting factor
2. Studies on the early embryogenesis of sea urchins led to the discovery of cyclins
1. Protein synthesis during embryogenesis was analyzed by ³⁵S-Met incorporation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and gel autoradiography
2. Whereas most proteins accumulate during the cell cycle, one class of proteins showed a periodic pattern of synthesis
3. The proteins accumulate during interphase, reach a maximum during metaphase-anaphase, and then are suddenly destroyed
4. The proteins are named cyclins; it has been hypothesized that cyclins activate MPF
3. MPF activity is due to cyclin and M-phase kinase
1. The purified MPF has protein kinase activity
2. Using anti-cdc2 antibody and western blot, the frog cdc2 counterpart is found to be a major component of the purified MPF
3. In an in vitro cell-free system from Xenopus egg extract and sperm nuclei that has been depleted of mRNA and is arrested at interphase, addition of purified cyclin mRNA can restore MPF activity
4. MPF or M-phase promoting factor is a heterodimer complex composed of the p34 cyclin-dependent kinase (Cdk, M-phase kinase) and mitotic cyclin
5. Whereas the Cdk protein is always present, MPF activity is transient during the cell cycle
6. This phenomenon occurs because the cyclin level fluctuates; cyclins are destroyed by the ubiquitin-dependent cytosolic protein degradation system
7. Reversible phosphorylation and dephosphorylation reactions are the control mechanisms of the eukaryotic cell cycle
8. Various substrate proteins may be the target of the activated M-phase kinase; these include histone H1 for chromatin condensation, tubulin for spindle formation, and nuclear lamin for nuclear envelope breakdown
4. In addition to controling the M phase, cyclins and kinases are also involved in other cell-cycle control checkpoints
1. For fission yeast, a single cdc2 kinase interacts with different cyclins to control different checkpoints; it interacts with mitotic cyclin for G₂/M control and with G₁ cyclin for G₁ control
2. In mammalian cell cycle, there are at least 7 different kinases (Cdk, Cdk1 is also called cdc2) and 8 different cyclins
1. For M phase, the cdc2 counterpart interacts with cyclin B (mitotic cyclins)
2. For G₁ checkpoint, the Cdk2 interacts with cyclin E (G₁ cyclins)
5. Cell cycle is regulated by proteins known as cyclin-dependent kinase inhibitors or CKIs
1. CKIs bind to Cdk or Cdk-cyclin complex and inhibit the kinase activity
2. Mammalian CKIs are grouped into p21 and p16 families
3. CKIs arrest cell cycle progression at G₁ and their synthesis may lead to cell differentiation
4. The protein p21 is produced after cell injury (such as irradiation) and its gene expression is activated by a protein known as p53, one that can sense cellular DNA damage and then either allow cell repair or cell death (apoptosis)
5. A member of the p21 family is p27, knockout mice with p27 are larger than litter mates and their organs such as thymus are also larger than controls, indicating modulation of cell division regulation
6. Knockout mice with p16 show a greater incidence of cancer, again indicating the importance of cell division regulation
6. MPF can be viewed as internal mitotic inducers; external mitotic inducers called growth factors act on the entire organism
1. About 50 growth factors are known
2. Some have broad activity; for example, platelet-derived growth factor (PDGF) acts on connective tissue cells and neuroglial cells
3. Other growth factors have narrow activity; for example, interleukin-2, IL-2, acts on T lymphocytes
7. The yeast cdc2 kinase and related genes from other organisms have been cloned by recombinant DNA techniques
8. Current findings have shown the conservation of cell cycle controls among all eukaryotes

1. General information
1. Cell division (the M phase) includes both mitosis (nuclear division) and cytokinesis (cytoplasmic division)
2. Reversible protein phosphorylation is responsible for some of the morphological changes seen during cell division
3. Unique cytoskeletal structures, the mitotic spindle and the contractile ring, are involved in cell division
2. Mitosis
1. The process of mitosis has been divided into stages, including prophase, prometaphase, metaphase, anaphase, and telophase
1. During prophase, chromosome condensation, nucleolar disintegration, and nuclear envelope breakdown occur
2. During prometaphase, the mitotic spindle forms and the chromatids attach to the spindle
3. During metaphase, the chromatids attach to the spindle at the equatorial metaphase plate via the kinetochores
4. During anaphase, the centromeres separate suddenly and the chromatids migrate
5. During telophase, the nuclear envelope reforms; this phase is essentially the reverse of prophase
2. The central structure that functions in mitosis is the cytoskeleton-derived mitotic spindle
1. Mammalian cells have a centrosome called the microtubule organizing center (MTOC); it is composed of centrosome matrix and a pair of centrioles
1. g tubulin is unique to centrosomes
2. Plant centrosomes lack centrioles
2. Centrosome duplication occurs during late interphase, with each daughter centrosome consisting of 1 old and 1 new centriole; at the start of M phase, the two centrosomes move apart and act like MTOC with a radial array of microtubules that is known as a star-like structure or an aster
3. This reorganization of the cytoskeleton during M phase causes the formation of the mitotic spindle; this process accounts for the roundness of cells in M phase
4. The two asters migrate opposite to each other to form the two poles; additional microtubule recruitment forms the mitotic spindle
5. The mitotic spindle is composed of three types of microtubules
1. Astral microtubules radiate from the two centrosomes at the two poles
2. Polar microtubules also radiate from the two centrosomes and stretch to the midline of the spindle, where polar microtubules from the two centrosomes meet and overlap
3. Kinetochore microtubules radiate from the kinetochores to the two centrosomes
6. Kinetochores are multilayered structures on the metaphase chromosome that are adjacent to the centromere region; they serve as the attachment points for the kinetochore microtubules
1. There is 1 kinetochore per chromosome at G₁; this is duplicated to 2 kinetochores per chromosome at G₂ (one per chromatid)
2. Each metaphase chromosome consists of two chromatids
3. Human patients with a disease known as scleroderma often have autoantibodies against kinetochores; why this happens has not been determined
4. The disorder is characterized by progressive fibrosis of connective tissues
5. Patient autoantibodies lead to the characterization of kinetochore-specific proteins (centromere binding proteins)
6. One of these proteins is the minus-end-directed microtubular motor
7. Kinetochore proteins assemble at specific centromeric regions of chromosomes
1. Mammalian centromeric DNA contains repetitive DNA that is also known as satellite DNA
2. Each mammalian kinetochore binds about 30-40 microtubules
3. Yeast centromeric DNA has a specific sequence of about 110 nucleotides in size
4. Each yeast kinetochore binds 1 microtubule
8. Microtubules are dynamic in the mitotic spindle
1. Chromosomes in mitosis are like the corpse at a funeral; they provide the reason for the proceedings but do not take an active part
2. Motor proteins such as kinesins are involved in chromosome segregation
9. Anaphase, triggered by cyclin degradation and MPF inactivation, leads to chromatid segregation and migration toward the two poles
1. Anaphase A consists of the shortening of kinetochore microtubules (chromatids move to the poles)
2. Anaphase B consists of the lengthening of polar microtubules (the poles move apart)
3. In addition to chromosome segregation, fragmentation of large cytoplasmic organelles occurs during M phase to ensure that the organelles are properly segregated during cell division
1. ER and Golgi break up into vesicles and fragments during mitosis
2. ER fragments associate with spindle microtubules during mitosis
3. Cytokinesis
1. Cytokinesis is the process by which the cytoplasm of a cell is divided after mitosis is complete
2. The process begins with the furrowing of the plasma membrane at the location of what was the metaphase plate; hence, the mitotic spindle determines the site of cytoplasmic cleavage furrow during cytokinesis
3. Microfilaments are involved in cleavage furrows
4. Actin and myosin generate the force for cleavage
1. A contractile ring, made from a bundle of about 20 actin microfilaments, is formed
2. During cytokinesis, the ring constricts as the cleavage furrow invaginates
3. As the two daughter cells form, the ring narrows to form a structure known as the midbody, composed of the remaining polar microtubules and other matrix materials
4. The midbody disappears by the time the cell division is completed
5. The process of cytokinesis results in a distribution of non-nuclear organelles to two daughter cells

1. Describe the different approaches that have been taken to the investigation of the cell cycle.
2. Discuss the interaction of MPF with cyclin and cdc2 kinase.
3. Differentiate the role of microtubules and microfilaments during M phase.
4. Given a mitotic cyclin cDNA from the fruit fly, discuss means to demonstrate gene complementation for the cdc13 cyclin mutant of fission yeast.