Cellular and Molecular Biology Topics
Cell Cycle
The cell cycle consists of four phases:
The cell cycle is regulated by “breaks” that stop the cycle at specific checkpoints. There are three major checkpoints: G1 just before entry into S phase, G2 at entry to mitosis, and M phase during the metaphase to anaphase transition. At these checkpoints, the cell surveys environmental signals and monitors whether required processes were completed prior to entering into the next phase of the cycle.
A cell below threshold size, or DNA damage will be stoped at the G1 checkpoint until addditional synthesis or repair is completed. Incompleately replicated DNA, DNA damage or incomplete protein synthesis will stop the cycle at the G2 checkpoint. Irregularities in the chromosome arrangement will stop the cycle at the M phase checkpoint.
G0 is a quiescent state in which cells remain metabolically active but do not replicate. This happens to many cell types after embryonic proliferation. Some cells re-enter G1 as needed for cell proliferation in the mature animal. Cell proliferation is regulated at a particular point in G1 called the restriction point. In the presence of the appropriate growth factors, cells pass the restriction point and enter S phase. Once it has passed the restriction point, the cell is committed to proceed to S phase. On the other hand, if appropriate growth factors are not present, progression through the cell cycle stops at the restriction point and the cell enters G0.
The cyclin-dependent protein kinases (Cdk) are the master controllers of the cell cycle. The kinase catalytic subunit of Cdk associates with a family of activating proteins called cyclins. The name cyclin reflects the fact that they undergo a cycle of synthesis and degradation during the phases of cell division. There are two main types of cyclins: mitotic and G1. Mitotic cyclins bind to Cdk during G2 and are required for entry into M phase. G1 cyclins bind Cdk during G1 and are required for entry into S phase.
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Maturation Growth Factor
The Cdk/cyclin complex is also known as the maturation promoting factor (MPF). Researchers realized that a cytoplasmic factor present in hormone-treated frog oocytes was sufficient to trigger transition from G2 to M. The injection of cytoplasm from an hormone-treated oocyte into G2-arrested oocytes was enough to make them enter M phase. This cytoplasmic factor was called MPF. It was also observed that MPF activity oscillates as the cell cycle progresses.
Studies in yeast identified these cell cycle proteins as encoded by cell-division cycle genes (cdc). A cell with a defective cdc gene cannot complete a division cycle and is arrested at specific checkpoints. Since cells that cannot complete a cell division cycle cannot be propagated, cdc mutants can be selected for and maintained only if their mutant phenotype is conditional. In other words, the gene products fail to function only under certain conditions (temperature, nutritional deficiency, etc). Most conditional cell cycle mutants are temperature-sensitive, where the mutant protein fails to function at high temperatures but works well enough at low temperatures.
MPF regulates the activities of multiple proteins by phosphorylation, modulating their activity as required for cell division. Cyclins bind with Cdk and assist in the recognition and phosphorylation of specific substrates.
During prophase, lamin phosphorylation by Cdk/cyclin disassembles the nuclear envelope, resulting in a mixture of phosphorylated lamin A and C dimmers, nuclear envelope vesicles with phosphorylated lamins attached, and free nuclear pore complex. During anaphase/early telophase, lamins are dephosphorylated by a phosphatase, allowing the nuclear envelope to reform. The endoplasmic reticulum and Golgi are also fragmented into small vesicles by Cdk/cyclin (unknown mechanism), so they can be distributed to daughter cells at cytokinesis.
Another regulatory action of Cdk/cyclin is the phosphorylation of retinoblastoma protein (Rb), which regulates the G1 to S phase transition in mammalian cells. E2F is a transcription factor required for expression of various enzymes involved in the synthesis of dNTPs and DNA. Dephosphorylated Rb binds E2F and prevents its action. Phosphorylated Rb cannot bind E2F. Rb is encoded by a tumor suppressor gene.
MPF also plays a role during cytokinesis. Early in mitosis, MPF phosphorylates myosin light chain, inhibiting the ATPase activity and association with F-actin. As MPF is deactivated during late anaphase due to mitotic cyclin degradation, phosphatases can dephosphorylates myosin light chain. This allows the activation of the contractile machinery, formation of the cleavage furrow and onset of cytokinesis. The regulatory mechanism assures that cytokinesis does not occur until anaphase is completed.
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Cell Cycle Regulation
Mammals have multiple Cdks as well as cyclins that associate in different combinations at specific points in the cell cycle. Cdk1-cyclinB and Cdk1-cyclinA are present around S phase. Cdk4-cyclinD and Cdk6-cyclinD are present during the second half of G1. Cdk2-cyclinE is present during late G1 and early S phase.
Cdk becomes associated with cyclin as the level of cyclin gradually increases. The activity of Cdk-cyclin can be further activated by phosphorylation by an activating kinase. Alternatively, the activity of Cdk can be inhibited by phosphorylation at a different inhibitory site. This inhibition may be removed by a specific phosphatase.
Cdk association with different cyclins can alter selection of substrate for phosphorylation. In yeast, cdc2 encodes a Cdk catalytic subunit that combines with either mitotic or G1 cyclins. Which cyclin is associated with the Cdk is thought to determine the selection of proteins it will phosphorylate.
Early in G1, mitogens activate synthesis of cyclin D that assembles with Cdk4. Cyclin E is expressed later in G1 and complexes with Cdk2. Both complexes require phosphorylation on the catalytic subunit for activation. Cdk4/cyclinD and Cdk2/cyclinE regulate transcription factors and proteins required for entry to S phase.
Cdk activity also triggers the ubiquination and proteolitic degradation of cyclins. At the onset of anaphase, mitotic cyclins are polyubiquinated on lysine residues flanking a destruction sequence. The anaphase-promoting complex (APC) is activated only when MPF activity is high. Binding of APC and E2 covalently linked to ubiquitin leads to polyadenylation of cyclin.
Some Cdk protein inhibitors physically bind to Cdk-cyclin, inhibiting the kinase activity. Others inhibitors bind to Cdk and prevents its association with cyclin. It is suspected that mutation in such inhibitory proteins can contribute to uncontrolled cell proliferation and tumor formation.
Cell cycle checkpoints are triggered by intracellular processes that are not complete or damaged . The proteins p21 and p27 inhibit Cdk/cyclin in its phosphorylated or dephosphorylated states, inhibiting progression through the G1 checkpoint. For example, DNA damage induces elevated levels of the transcription factor p53, which in turn induces the Cdk inhibitor p21, causing cell cycle arrest at the G1 checkpoint. P21 inhibits PCNA, a subunit of DNA polymerase (?). This inhibition prevents DNA synthesis but does not prevent PCNA's role in DNA repair. Some cell types execute an alternative program leading to apoptosis, which appears to depend on other p53-regulated genes.
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Mitosis
Mitosis is the cellular process by which chromosomes are evenly partitioned between two daughter cells. Mitosis is a phase of the cell cycle (M phase), subdivides into 4 stages: prophase, metaphase, anaphase and telophase.
Interphase
refers to the stages of the cell cycle that occur between mitosis events. The
centrosome, the microtubule organizing center in animal cell, duplicate during
interphase in anticipation of the next mitosis cycle. Cell growth and DNA synthesis
(G1, S phase and G2), also occur during interphase.
During prophase, centrosomes separate to form spindle poles and the mitotic spindle starts to develop. Chromosomes condense, each into two sister chromatids held together at the centromere, to which proteins attach forming a kinetochore. Myosin regulatory light chain (RLC) is phosphorylated by MPF to prevent cytokinesis.
During
prometaphase, breakdown of the nuclear envelope, ER and Golgi occurs. Microtubules
of the mitotic spindle attach to kinetochores and chromosomes start to align.
At metaphase, the
chromosomes are aligned on the metaphase plate in the center of the spindle.
During anaphase, the linkage between sister chromatids is broken, and they are separated towards opposite poles of the spindle. The nuclei reform and chromosomes decondense during telophase.
Cytokinesis,
the separation of the daughter cells, actually begins during late anaphase but
is not completed until mitosis is over. A
contractile actin and myosin ring known as the cleavage furrow is formed. Cytokinesis
occurs as the result of the contractile force generated by myosin walking along
acting filaments.
The timing of myosin activation is carefully regulated by to protein kinases that phosphorylate myosin regulatory light chain (RLC). MPF phosphorylates an inhibitory site that prevents phosphorylation at the activation site. As MPF levels decrease after mitosis, the MPF site is dephosphorylated by a phosphatase. Then MLCK can phosphorylate the activating site on RLC.
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Mitotic
Spindle
The mitotic spindle, composed of kinetochore microtubules, astral microtubules and polar microtubules, start to polymerize during prophase. Kinetochore microtubules serve to align the chromosomes at the metaphase plate during prometaphase and separate the chromatids. Astral microtubules are pulled towards the centrosomes. Polar microtubules interdigitate at the center of the cell with polar microtubules from the opposite pole, pushing the poles apart.
During prometaphase, kinetochore microtubules attach to chromosomes at kinetochores and align them on the metaphase plate. Kinetochores are specialized structures consisting of proteins attached to a centromere that mediates the attachment and movement of chromosomes along the mitotic spindle. During anaphase A, shortening of the kinetochore microtubules by depolymerization pulls the chromatids to the poles. By telophase, the separated chromosomes arrive at the poles and kinetochore microtubules disappear.
Kinesin in polar microtubules walks to the (+) end of adjacent polar microtubules, pushing them in the (-) direction, during anaphase B. As polar microtubules elongate, the sliding forces push the spindle poles apart.
The astral dynein (-) motors at the poles pull astral microtubules towards themselves during prophase (?). This results in pulling the MTOC's towards the poles. While this happens, the microtubules are de-polymerizing and getting shorter. During anaphase B the astral microtubules are pulled towards the centrosomes.
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Cool Web Site: Animations of Microtubules During Mitosis
Meiosis
In
sexual reproduction, there is a mixing of genomes from two different parents,
producing offspring that differ genetically from one another and from both parents.
In the cycles of fertilization, diploid zygote formation and gametogenesis,
gene combinations are broken up and new arrangements are created.
A homologue or homologous chromosome is one of two copies of a given chromosome in a diploid cell. Diploid cells of the germ line divide by two successive nuclear divisions during meiosis, producing four gametes or haploid daughter cells, containing only one homologue of each chromosome. This process is divided in two stages: meiosis I and meiosis II.
The original diploid germ cell with one parental and one maternal homologue undergoes DNA replication. This yields a pair of duplicated homologues referred to as a bivalent. The bivalent contains four chromatids: two copies of each homologue.
In meiosis I, the two sister homologues align at the metaphase plate. One homologue including its two sister chromatids, is drawn to each cell pole. No division of centromeres occur, so there is no separation of sister chromatids. The cell divides into two, each containing one of the homologues.
Meiosis II occurs without further DNA replication. Chromosomes align on the metaphase plate and the sister chromatids separate, i.e. there is division of centromeres, drawn towards opposite poles before each cell divides into two gametes.
Chromosomal nondysjunction arises from an abnormal meiosis process, whereby chromosomes are not equivalently distributed. Trisomy refers to distribution of two homologue chromosomes to a gamete instead of one. For example, Down’s syndrome is due to an additional chromosome 21. Monosomy occurs when a gamete has no chromosome of a specific homologue, for example in Turner syndrome there is only one X chromosome.
Morphological
changes occur in the chromosome as homologues pair and unpair during prophase
of meiosis I over 4 subphases: leptotene, zygotene, pachytene and diplotene.
Chromosomes condense during leptotene. Synapsis of homologues occurs during
zygotene, i.e. the process of joining, mediated by the synaptonemal complex.
After the two sister chromatids of each homologue condense together, the homologues
are joined by proteins of the synaptonemal complex.
By pachytene the synaptonemal is fully formed and the alignment of homologous chromosomes is thought to facilitate general recombination. Separation of the homologous chromosomes and dissociation of the synaptonemal complex occur during diplotene. As homologues move apart, they remain attached to one another at specific points called chiasmata (singular chiasma), which are crossovers between non-sister chromatids. As the homologous are completely separated, some DNA is exchanged in those regions.
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Formation of Gametes
The
primordial germ cells migrate to the developing gonads, which form ovaries in
females. After a number of mitotic divisions, oogonia begins meiotic division,
becoming primary oocytes. Primary oocytes are formed in the human embryo and
remain arrested in prophase of meiosis I. All primary oocytes are formed by
month 5 of fetal development and will remain dormant unti puberty.
After puberty, 5-15 primary oocytes begin maturation with each ovarian cycle, only one reaching full maturity in each cycle. During this maturation processes, the primary oocyte resumes meiosis I, with the alignment and separation of chromosomes. At the end of meiosis I, the cytoplasm is distributed unevenly, producing two cells that differ in size. The larger cells is the secondary oocyte, the smaller is known as the polar body.
The secondary oocyte will remain arrested in metaphase of meiosis II until fertilization occurs, when it will complete meiosis II and split into the mature egg or zygote and a second polar body. The zygote contains one maternal and one paternal pronuclei. Both polar bodies are eventually degraded.
Maturation
promoting factor (MPF) is a general regulator of the transition from G2 to M-phase.
It is a complex of Cdk2 and cyclin B (mitotic cyclin). Initial activation of
MPF results in progression to metaphase I. MPF activity then falls at the transition
from metaphase I to anaphase I. Following completion of meiosis I, MPF activity
again rises and remains high during metaphase II arrest.
Spermatogonia develop from primordial germ cells that migrated to the testis early in embryogenesis. When the animal becomes sexually mature, the spermatogonia beguin to proliferate rapidly, generating some progeny that retain the capacity to continue to divide indefinitely (as stem-cell spermatogonia) and other progeny (maturing spermatogonia) that will, after a limited number of further normal cell divisions, undergo meiosis to become primary spermatocytes.
The
primary spematocytes continue through meiosis I to become secondary spermatocytes.
After completing meiotic division II, they produce haploid spermatids that differentiate
into mature spermatozoa.
Spermatogenesis differs from oogenesis in several ways: (1) new cells enter meiosis continually from the time of puberty, (2) each cell that begins meiosis gives rise to four mature gametes rather than one, and (3) mature sperm form by an elaborate process of cell differentiation that beguins after meiosis is complete.
Mature spermatozoa have three general features: head, midpiece and flagellum. The head contains the nucleus and at the tip an acrosomal vesicle contains hydrolytic enzymes that facilitate penetration into the egg’s outer coat. The midpiece is rich in mitochondria to provide ATP for motility. The flagellum is a dynein motor. Spermatogonia does not contain ER, Golgi or ribosomes.
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Fertilization
The zona pellucida is a specialized extracellular matrix consisting largely of glycoproteins that surrounds the oocyte plasma membrane. Beyond the zona pellucida is the corona radiata, a rich nutrient layer. Once the sperm has penetrated the corona radiata, it binds to the zona pellucida. The zona pellusida contains three main glycoproteins. ZP2 and ZP3 assemble into filaments and ZP1 cross links the filaments into a three dimensional network. ZP3 acts as a specific sperm receptor.
Upon binding the egg, the sperm elicits the acrosomal reaction, releasing proteases and hyaluronidase from the sperm’s acrosome. The acrosomal reaction allows penetration of the sperm across the zona pellucida. After the acrosomal reaction, the inner acrosomal membrane forms the outer surface covering most of the sperm. In mammals, the whole sperm (head, midpiece and tail) penetrates the egg and for a short time may be seen intact in the interior of the egg.
The egg’s cortical reaction ensures that only one sperm fertilizes the egg. There is a rapid depolarization of the eggs plasma membrane in response to fusion with the first sperm. This depolarization is temporary and a second mechanism to prevent polyspermy is provided by the cortical reaction. Mammalian eggs contain specialized secretory vesicles, or cortical granules, just under the plasma membrane in the outer region, or cortex, of the egg’s cytoplasm. In the cortical reaction, the cortical granules release enzymes that can change the structure of the zona pellucida so that there is no further binding of sperm. Among the changes is the hydrolysis of carbohydrates on ZP3, so it can no longer bind the spermatozoid’s plasma membrane. Furthermore, there is proteolitic cleavage of ZP2 that hardens the zona pellucida, making it impermeable to other sperm.
Upon entering the egg, the sperm mitochondria and tail degenerate and the sperm nucleus forms the paternal pronucleus. The secondary oocyte completes meiosis II, forming a mature ovum and a second polar body. The nucleus of the mature ovum is the maternal pronucleus. The paternal and maternal pronuclei then contract, their membranes break down, and their chromosomes intermingle. The maternal and paternal chromosomes become associated with the spindle apparatus in preparation for mitotic division. Fertilization is now complete and the fertilized egg is called a zygote.
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