- aster microtubule
- cell cycle
- cleavage furrow
- contractile ring
- cyclin-dependent kinase (Cdk)
- gap phases (G1 and G2)
- kinetochore microtubule
- M phase
- maturation-promoting factor (MPF)
- minus end
- mitotic spindle
- motor protein
- plus end
- polar microtubule
- S phase
- sister chromatid
- spindle equator
- spindle pole
Introduction and Goals
Cell division requires the replication of DNA and the segregation of this newly synthesized DNA into two daughter cells. In a eukaryotic cell, this process also involves the distribution of chromosomes and the partitioning of the cytoplasm into the two new daughter cells. The duplication and division of chromosomes is a highly regulated, cyclic process that occurs in a precise fashion. This tutorial will describe all of these processes and their regulation.
By the end of this tutorial you should know:
- The phases of the cell cycle
- The important events at each stage of mitosis
- The role of microtubules and motor proteins in chromosome movement and cell division
- The mechanism of the cell cycle, including the roles of cyclin and cyclin-dependent kinases
- The checkpoint regulation of the cell cycle.
The Cell Cycle
Figure 1. The cell cycle.The cell cycle includes the S phase (during which DNA synthesis occurs), the M phase (during which mitosis occurs) and two gap phases (G1 and G2). Mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase.
The duplication of DNA and the subsequent distribution of the newly copied DNA must occur in the correct sequence. The cell undergoes a cycle of DNA replication and division referred to as the cell cycle. The cell cycle has four distinct phases (illustrated in Figure 1): the S phase, during which DNA synthesis occurs; the M phase, during which mitosis and cytokinesis occur; and two gap phases (G1 and G2), which separate the S and M phases. During the gap phases, the cell continues to grow and to monitor intracellular and environmental conditions before proceeding to the next phase. A system of checkpoints in each gap phase ensures that the cell is ready to enter the next phase of the cell cycle. The length of the cell cycle varies for each type of cell. For example, yeast cells have very rapid cell cycle times (1-3 hours to complete one cycle); some human cells have cell cycles that last a few months. Cells spend the majority of their time in the G1 phase.
Figure 2. The mitotic spindle at metaphase. At metaphase, the chromosomes align at the spindle equator. Each sister chromatid of the chromosome is attached to kinetochore microtubules (shown in green). These microtubules emanate from the centrosomes at spindle poles and attach to the chromosomes at the kinetochores (one for each sister chromatid). In addition to the kinetochore microtubules, there are two other distinct types of microtubules in the spindle: the polar microtubules (shown in red), which grow out from the centrosomes and have opposing microtubules overlapping at the spindle equator; and the aster microtubules (shown in blue), which grow out from the centrosomes toward the cortex of the cell. For all three types of microtubules, the minus ends are at the centrosomes and the plus ends (indicated as +) grow away from the centrosomes.
DNA replication, which occurs during the S phase of the cell cycle, was described in some detail in a previous tutorial. Mitosis (M phase), the separation and segregation of chromosomes, occurs in five distinguishable stages (prophase, prometaphase, metaphase, anaphase and telophase), but represents a continuous process in the cell. Mitosis is followed by cytokinesis, the partitioning of the cytoplasm and other organelles into two daughter cells. The rest of the cell cycle (G1, S and G2) is termed interphase.
During interphase, DNA is replicated and centrosomes are duplicated. A centrosome is the microtubule-organizing structure, or the origin of the mitotic spindle that physically separates the chromosomes. During prophase, the chromosomes begin to condense and eventually assume the highly compact and visible form of mitotic chromosomes. The two identical copies of the replicated chromosome, sister chromatids, are held together at the centromere. The mitotic spindle begins to appear between the pair of centrosomes, and the centrosomes begin to move apart to opposite poles (referred to as spindle poles) of the cell. In prometaphase, the nuclear envelope breaks down and the replicated chromosomes attach to the mitotic spindle via the kinetechore, a protein complex associated with the centromeres from each pair of sister chromatids. As metaphase proceeds, the chromosomes align at the midpoint of the mitotic spindle, often referred to as the spindle equator (Figure 2). During anaphase, the sister chromatids of a chromosome are pulled to opposite poles and the spindle poles move even further apart. This serves to separate and segregate the sister chromatids to opposite poles. Each sister chromatid arrives at its spindle pole during telophase and, as it does so, a new nuclear envelope appears around each set of chromosomes, thereby creating two new nuclei. Cell division is complete by cytokinesis, which is the segregation of the cell into two halves.
The Types of Microtubules in the Mitotic Spindle
The mitotic spindle is composed of three distinct types of microtubule fibers (kinetochore microtubules, polar microtubules, and aster microtubules; all emanating from the centrosomes), and they serve to pull and push the sister chromatids apart toward opposite spindle poles. The kinetochore microtubules attach to the kinetochore of a chromosome. There are two kinetochores per replicated chromosome, one for each sister chromatid on opposite sides of the chromosome. A kinetochore microtubule from one pole attaches to one kinetochore, while a kinetochore microtubule from the opposite side attaches to the other kinetochore. During prophase, a kinetochore microtubule grows from the centrosome, and by prometaphase it encounters a kinotechore, binds the proteins within the kinetochore and "grabs" the chromosome. The effect of being grabbed by kinetochore microtubules from opposite poles is a balancing of opposing forces, which results in alignment of the chromosomes at the spindle equator during metaphase. In mammalian cells, many kinetochore microtubules are associated with each sister chromatid. The microtubules from opposite poles that do not attach to the kinetochores but do overlap at the midpoint between the two poles are referred to as polar microtubules. The aster microtubulespoint away from the spindle equator and are attached to the cell's cortex. All three of these distinct spindle microtubule fibers grow out from the centrosome, and all are polymers composed of alpha and beta tubulin subunits. A microtubule grows by the addition of tubulin subunits to one end of the polymer. A microtubule has distinct polarity; the growing end is termed the plus end, and the minus end is associated with the centrosome. The plus end can grow by addition of tubulin subunits (polymerization) or shrink by the loss of tubulin subunits (depolymerization) from the plus end. In addition, microtubules are associated with a variety of proteins that can affect the stability of the polymer. Finally, microtubules are associated with motor proteins, which are proteins that can travel along the length of a microtubule in an energy-dependent fashion. Motor proteins move toward either the plus end or the minus end of a microtubule fiber.
How Microtubules Separate Chromosomes During Anaphase
Figure 3. The mitotic spindle at anaphase A. During anaphase A, the pairs of sister chromatids are separated and move toward the spindle poles. This occurs through the action of the kinetochore microtubules (illustrated in the inset). These microtubules shorten at their plus ends, while the motor proteins attached to the kinetochores of the chromatids travel toward the minus ends; thereby, the sister chromatids remain attached to the shortening microtubules.
Figure 4. The mitotic spindle at anaphase B. During anaphase B, the spindle poles move further apart. This occurs through the combined action of the polar microtubules and the aster microtubules. The action of the polar microtubules is shown in the inset on the right. Overlapping polar microtubules grow by polymerization at their plus ends, while the cross-linked motor proteins travel toward the plus ends, thereby pushing the overlapping polar microtubules past each other and the spindle poles further apart. The action of the aster microtubules is shown in the inset to the left. The aster microtubules depolymerize at their plus ends, while the motor proteins linked to the cell's cortex travel toward the minus ends, thereby pulling the attached spindle poles closer to the cortex and further apart from each other.
During anaphase, the microtubules of the mitotic spindle mediate the separation of the chromosomes. This occurs in a two-step fashion, referred to as anaphase A and anaphase B. In anaphase A, the kinetochore microtubules separate the sister chromatids. The kinetochore microtubules shorten by depolymerization at their plus ends, and motor proteins associated with the kinetochores keep the chromatids linked to the shortened plus ends, retaining the attachment of the chromatids to the kinetochore microtubules. During anaphase B, the two spindle poles move further apart. This occurs through the sliding of the interdigitated (interlocked) polar microtubules. The polar microtubules grow by polymerization at their plus ends, while a pair of cross-linked motor proteins travel toward the plus ends of opposing polar microtubules and act to push the spindle poles apart. Finally, motor proteins associated with the plus ends of the aster microtubules pull the spindle poles toward the cell's cortex and even further apart. Through a combination of pushing and pulling, the sister chromatids are separated.
Figure 5. Cytokinesis. In an animal cell, the contractile ring (composed of actin and myosin microfilaments) forms during telophase. The contractile ring pinches the cell into two halves as it contracts and forms the cleavage furrow. In a plant cell, vesicles accumulate at the midpoint of the mitotic spindle and begin to form a cell plate, which eventually will partition the cell into two daughter cells.
Once the sister chromatids have reached the opposite poles, the cell can divide into two new cells. This division of the cytoplasm occurs differently in animal and plant cells. In an animal cell, a contractile ringforms around the midpoint of the cell (perpendicular to and centered on the midpoint of the mitotic spindle). This contractile ring is composed of microfilaments of actin and myosin. As this ring contracts, it creates a cleavage furrow that drags the plasma membrane inward, eventually pinching the cell into two new cells (Figure 5). Interestingly, the position of the cleavage furrow is determined by the position of the mitotic spindle, midway between the spindle poles. In a plant cell, a cleavage furrow could not easily pinch the cell into two new cells because of the presence of a rigid cell wall. Instead, a cell plate is initiated perpendicular to and at the midpoint of the mitotic spindle (Figure 5). This cell plate is created by the fusion of vesicles derived from the Golgi complex, which contains the necessary polysaccharides to form the cell plate. The cell plate is bound by a new plasma membrane that becomes continuous with the preexisting plasma membrane of the cell. The cell plate eventually fuses with the cell wall of the parent cell, thereby dividing the parent cell into two daughter cells.
The Role of Cyclins and Cyclin-Dependent Protein Kinases in the Cell Cycle
Figure 6. Maturation-promoting factor (MPF) activity in the cell cycle. Entry into mitosis is regulated by active MPF. MPF is composed of mitotic cyclin (M-cyclin) and mitotic cyclin-dependent kinase (M-Cdk). M-cyclin protein levels rise steadily during G1, S and G2. The levels peak during mitosis, where cyclin associates with M-Cdk. In mitosis, the levels of M-cyclin rapidly drop due to the degradation of the cyclin protein. This triggers the cell's exit from mitosis. The entry into S phase is regulated by S-cyclin and S-Cdk. S-cyclin levels regulate the entry into and exit from S phase in a fashion analogous to M-cyclin.
As stated above, the cell proceeds through a cycle of DNA replication (S phase) and mitosis (M phase). Molecular triggers determine when a cell will enter and exit mitosis. Experiments on the development of frog eggs identified a protein kinase that could promote the maturation of frog eggs; it was named maturation-promoting factor (MPF). Injection of MPF into cells can also promote their entry into mitosis. Furthermore, MPF activity oscillates with the cell cycle, is high during mitosis, and then it dramatically drops. An intense investigation of MPF revealed that this protein kinase is composed of two protein components: cyclin and cyclin-dependent kinase (Cdk). Cyclin protein levels oscillate with the cell cycle, hence its name. Entry into mitosis is regulated by the activity of mitotic Cdk (M-Cdk), which is dependent on M-cyclin levels for activity. Mitotic-cyclin (M-cyclin) levels steadily increase in the G2 phase and peak right before mitosis, at which point M-Cdk is activated, driving the cell into the M phase (Figure 6). In addition to the binding of cyclin, M-CDk must be activated by phosphorylation. The M-Cdk/cyclin complex is phosphorylated by both activating and inactivating kinases, resulting in an inactive M-CDk/cyclin complex. A specific activating phosphatase must remove the inhibitory phosphate group to activate the M-CDk/cyclin complex. Active M-Cdk triggers the phosphorylation of a variety of proteins, including the protein condensin. Condensin associates with DNA in the chromosomes and mediates their condensation in prophase, and with proteins in the nuclear lamina to trigger the break down of the nuclear envelope in prometaphase. In addition, active M-Cdk triggers the degradation of cyclin; so, high M-Cdk activity results in its own inactivation due to the degradation of cyclin. This accounts for the rapid drop in cyclin levels at the end of mitosis, and is the trigger to exit mitosis.
There are a set of cyclins and Cdks specific for the S phase; in a similar fashion, their activity regulates the entry into and exit from the S phase. One activity of the S-Cdk is to phosphorylate and activate proteins associated with the origin of replication (ORI) to trigger the initiation of DNA replication at the origin.
The Regulation of Checkpoints in the Cell Cycle
Figure 7. Cell cycle regulation, and the G1/S and G2/M checkpoints.
Throughout the cell cycle and before a cell can leave a gap phase to enter an M or S phase, the cell must meet certain requirements to proceed to the next phase. If not met, the cell is arrested in the gap phases at points in the cell cycle referred to as checkpoints(see Figure 7). Before entering mitosis, at the G2/M checkpoint, a cell must ensure that DNA replication is complete and that the DNA is intact. At the G1/S checkpoint, a cell must ensure the integrity of the DNA prior to replication and monitor the cell's size and environmental conditions. If the proper criteria are not met, then the cell will be arrested at the checkpoint.
Figure 8. Activation of M-Cdk. M-Cdk bound to cyclin is inactive. The protein complex must first be phosphorylated by two different kinases: an activating kinase and an inhibitory kinase. A phosphatase must remove the inhibitory phosphate from the M-Cdk to be active. This is essentially the G2/M checkpoint, and mitosis will not occur if the criteria to enter mitosis have not been met.
A key regulator of the G2/M checkpoint is the phosphatase that
removes the inhibitor phosphate on M-Cdk. Until this phosphate is
removed, M-Cdk remains inactive (Figure 8). The importance of this
phosphatase is highlighted by a strain of yeast carrying a
temperature-sensitive mutation in the cell division cycle gene cdc25,
which encodes the phosphatase. A temperature-sensitive mutation is a
conditional mutation in a gene; that is, the cells carrying this
mutation are normal at one temperature but are mutant at another
(usually higher) temperature (referred to as the nonpermissive
temperature). At the nonpermissive temperature, cdc25 mutant yeast
cells are blocked at the G2/M checkpoint because the phosphatase
encoded by cdc25 is defective at this temperature and M-Cdk cannot be
activated because the inhibitory phosphate cannot be removed. Once
M-Cdk is active, it actually promotes the activation of the
phosphatase; thus, resulting in further dephosphorylation and
activation of M-Cdk and therefore providing a positive feedback that
accounts for the sharp increase in M-Cdk activity at the beginning of
The protein p53 is an important regulator of the G1/S checkpoint. In response to DNA damage, p53 is activated, and, in turn, stimulates the production of the Cdk inhibitory protein p21. p21 binds and inactivates the S-Cdk/cyclin complex, thus arresting the cell at the G1/S checkpoint to allow repair of the damaged DNA before replication. In the absence of normal p53 activity, replication of damaged DNA will proceed, thus resulting in a higher rate of mutation. This increased rate of mutation can lead to cancer, and, in fact, many human cancers are associated with mutations in p53.
The cell undergoes a cycle of DNA replication and cell division referred to as the cell cycle. Replication occurs in the S phase, and segregation of the duplicated chromosomes and the cytoplasm occurs in the M phase. These are separated by two gap phases, G1 and G2. The five stages of mitosis are prophase, prometaphase, metaphase, anaphase and telophase. Cytokinesis is the separation of the cell into two daughter cells. The separation of the sister chromatids of each chromosome is mediated by the microtubules of the mitotic spindle. In prometaphase, kinetochore microtubules from opposing poles extend and grab hold of the kinetochore of a chromosome. This balancing of the force applied by microtubules from opposite poles positions the chromosomes at the spindle equator, signifying metaphase. During anaphase, the sister chromatids are separated and coincidently pushed and pulled to opposite poles. In anaphase A, the kinetochore microtubules shorten, pulling the sister chromatids apart. In anaphase B, the polar microtubules grow and slide past each other, lengthening the mitotic spindle and pushing the poles apart. The aster microtubules also pull the poles apart. Cytokinesis splits an animal cell into two halves, using a cleavage furrow that is formed by a contractile ring. The position of this ring is determined by the position of the mitotic spindle. Entry into M phase and S phase is regulated by cyclins and cyclin-dependent kinases (M-Cdk and S-Cdk, respectively). M-Cdk is activated in a stepwise fashion. First, it associates with cyclin. Second, it is phosphorylated by two different kinases: one that inhibits and one that activates. Third, a phosphatase must remove the inhibitory phosphate. When M-Cdk is active, it will phosphorylate a variety of proteins that regulate chromosome condensation and nuclear envelope break down. This phosphatase is an important G2/M checkpoint that regulates the activity of M-Cdk and the entry of the cell into mitosis.