Cellular Reproduction PDF

Cellular Reproduction Cellular Reproduction The cell cycle An overview of the eukaryotic cell cycle. This diagram of the cell cycle indicates the stages through which a cell passes from one division to the next. The cell cycle is divided into two major phases: M pha...

Cellular Reproduction Cellular Reproduction The cell cycle An overview of the eukaryotic cell cycle. This diagram of the cell cycle indicates the stages through which a cell passes from one division to the next. The cell cycle is divided into two major phases: M phase and interphase. M phase includes the successive events of mitosis and cytokinesis. Interphase is divided into G1, S, and G2 phases, with S phase being equivalent to the period of DNA synthesis. The division of interphase into three separate phases based on the timing of DNA synthesis was first proposed in 1953 by Alma Howard and Stephen Pelc of Hammersmith Hospital, London, based on their experiments on plant meristem cells. One might guess that a cell engages in replication throughout interphase. However, studies in the early 1950s on asynchronous cultures (i.e., cultures whose cells are randomly distributed throughout the cell cycle) showed that this is not the case.,DNA replication can be monitored by the incorporation of [3H]thymidine into newly synthesized DNA. Experimental results demonstrating If [3H]thymidine is given to a culture of cells for a short period that replication occurs during a (e.g., 30 minutes) and a sample of the cell population is fixed, dried defined period of the cell cycle. HeLa cells were cultured for 30 minutes in onto a slide, and examined by autoradiography, only a fraction of medium containing [3H]thymidine and then incubated (chased) for various times in the cells are found to have radioactive nuclei. Among cells that unlabeled medium before being fixed and were engaged in mitosis at the time of fixation (as evidenced by prepared for autoradiography. Each culture dish was scanned for cells that were in their compacted chromosomes) none is found to have a mitosis at the time they were fixed, and radioactively labeled nucleus. If labeling is allowed to continue for the percentage of those mitotic cells whose chromosomes one or two hours before the cells are sampled, there are still no were labeled was plotted as shown. (FROM A cells with labelled mitotic chromosomes. We can conclude from STUDY BY R. BASERGA AND F.WIEBEL.) Compiled by Manoj Kumar Singh, PhD 1 Cellular Reproduction these results that there is a definite period of time between the end of DNA synthesis and the beginning of M phase. This period is termed G2 (for second gap). The duration of G2 is revealed as one continues to take samples of cells from the culture until labeled mitotic chromosomes are observed. We can recognize three broad categories of cells: 1. Cells, such as nerve cells, muscle cells, or red blood cells, that are highly specialized and lack the ability to divide. 2. Cells that normally do not divide but can be induced to begin DNA synthesis and divide when given an propriate Stimulus (e.g. lymphocytes, which can be induced to proliferate by interaction with an appropriate antigen). 3. Cells that normally possess a relatively high level of mitotic activity. Included in this category are stem cells of various adult tissues, such as hematopoietic stem cells. Potu Rao’s and Robert Johnson’s Experiment The results of this experiment suggested that the cytoplasm of a mitotic cell contained diffusible factors that could induce mitosis in a nonmitotic (i.e., interphase) cell. This finding suggested that the transition from G2 to M was under positive control. FIGURE. Experimental demonstration that cells contain factors that stimulate entry into mitosis. The photographs show the results of the fusion of an M-phase HeLa cell with a rat kangaroo PtK2 cell that had been in (a) G1 phase, (b) S phase, or (c) G2 phase at the time of cell fusion. As described in the text, the chromatin of the G1-phase and G2-phase PtK2 cells undergoes premature compaction, whereas that of the S-phase cell becomes Compiled by Manoj Kumar Singh, PhD 2 Cellular Reproduction pulverized. The elongated chromatids of the G2-phase cell in c are doubled in comparison with those of the G1 cell in a. (From Karl Sperling And Potu N. Rao, Humangenetik 23:437, 1974.) Role of Protein Kinases Entry of a cell into M phase is initiated by a protein called maturation promoting factor (MPF). MPF consists of two subunits: (1) a subunit with kinase activity that transfers phosphate groups from ATP to specific serine and threonine residues of specific protein substrates & (2) a regulatory subunit called cyclin. The term cyclin was coined because the concentration of this regulatory protein rises and falls in a predictable pattern with each cell cycle. Fluctuation of cyclin and MPF levels during the cell cycle --► This drawing depicts the cyclical changes that occur during early frog development when mitotic divisions occur rapidly and synchronously in all cells of the embryo. The top tracing shows the alternation between periods of mitosis and interphase, the middle tracing shows the cyclical changes in MPF kinase activity, and the lower tracing shows the cyclical changes in the concentrations of cyclins that control the relative activity of the MPF kinase. (Reprinted With Permission From A. W. Murray And M. W. Kirschner, Science 246:616, 1989; _ Copyright 1989; American Association For The Advancement Of Science.) It has been seen that : (1) Progression of cells into mitosis depends on an enzyme whose sole activity is to phosphorylate other Proteins & (2) The activity of this enzyme is controlled by a subunit whose concentration varies from one stage of the cell cycle to another. It has been found that cyclin-dependent Kinases (Cdks) are not only involved in M phase but are the key agents that orchestrate activities throughout the cell cycle. A simplified model for cell cycle regulation in fission yeast The cell cycle is controlled primarily at two points, START and the G2–M transition. In fission yeast, cyclins can be divided into two groups, G1 cyclins and mitotic cyclins. Passage of a cell through these two critical junctures requires the activation of the same cdc2 kinase by a different type of cyclin. A third major transition occurs at the end of mitosis and is triggered by a rapid drop in Compiled by Manoj Kumar Singh, PhD 3 Cellular Reproduction concentration of mitotic cyclins. (Note: cdc 2 is also known as Cdk1.) Cyclin-dependent kinases are often described as the “engines” that drive the cell cycle through its various stages. The activities of these enzymes are regulated by a variety of “brakes” and “accelerators” that operate in combination with one another. These include: Cyclin Binding When a cyclin is present in the cell, it binds to the catalytic subunit of the Cdk, causing a major change in the conformation of the catalytic subunit. & Cdk Phosphorylation/dephosphorylation : Phosphorylation is carried out by Kinases (eg. CAK, i.e Cdk activating kinase and Weei) and dephosphorylation is carried out by Phosphatase (eg. Cdc25) Fig. Progression through the fission yeast cell cycle requires the phosphorylation and dephosphorylation of critical cdc2 residues. (a) During G2, the cdc2 kinase interacts with a mitotic cyclin but remains inactive as the result of phosphorylation of a key tyrosine residue (Tyr 15 in fission yeast) by Wee1 (step 1). A separate kinase, called CAK, transfers a phosphate to another residue (Thr 161), which is required for cdc2 kinase activity later in the cell cycle.When the cell reaches a critical size, an enzyme called Cdc25 phosphatase is activated, which removes the inhibitory phosphate on the Tyr 15 residue. The resulting activation of the cdc2 kinase drives the cell into mitosis (step 2). By the end of mitosis (step 3), the stimulatory phosphate group is removed from Thr 161 by another phosphatase. The free cyclin is subsequently degraded, and the cell begins another cycle. (The mitotic Cdk in mammalian cells is phosphorylated and dephosphorylated in a similar manner.) (b) Identification of Wee1 kinase and Cdc25 phosphatase was made by studying mutants that behaved as shown in this figure. Line 1 shows the G2 and M stages of a wild-type cell. Line 2 shows the effect of a mutant wee1 gene; the cell divides prematurely, forming small (wee) cells. Line 3 shows the effect of a mutant cdc25 gene; the cell does not divide but continues to grow. The red arrow marks the time when the temperature is raised to inactivate the mutant protein. (A:Aftert. R.Coleman Andw.G.Dunphy, Curr. Opin. Cell Biol. 6:877, 1994.) Compiled by Manoj Kumar Singh, PhD 4 Cellular Reproduction Combinations between various cyclins and Cdks at different stages in the mammalian cell cycle Fig. Cdk activity during early G1 is very low, which promotes the formation of prereplication complexes at the origins of replication. By mid-G1, Cdk activity is evident due to the association of Cdk4 and Cdk6 with the D-type cyclins (D1, D2, and D3). Among the substrates for these Cdks is an important regulatory protein called pRb). The phosphorylation of pRb leads to the transcription of a number of genes, including those that code for cyclins E and A, Cdk1, and proteins involved in replication. The G1–S transition, which includes the initiation of replication, is driven by the activity of the cyclin E–Cdk2 and cyclin A–Cdk2 complexes. The transition from G2 to M is driven by the sequential activity of cyclin A–Cdk1 and cyclin B1–Cdk1 complexes, which phosphorylate such diverse substrates as cytoskeletal proteins, histones, and proteins of the nuclear envelope. (The mammalian Cdk1 kinase is equivalent to the fission yeast cdc2 kinase, and its inhibition and activation are similar to that indicated in Figure 14.6.) (After C. J. Sherr, Cell 73:1060, 1993 Nature Revs. Mol. Cell Biol. 8:667, 2007.) Compiled by Manoj Kumar Singh, PhD 5 Cellular Reproduction M PHASE: MITOSIS AND CYTOKINESIS Fig. The stages of mitosis in an animal cell (left drawings) and a plant cell (right photos). (Micrographs From Andrew Bajer.) Compiled by Manoj Kumar Singh, PhD 6 Cellular Reproduction Prophase Formation of the Mitotic Chromosome The extended state of interphase chromatin is ideally suited for the processes of transcription and replication but not for segregation into two daughter cells. Before segregating its chromosomes, a cell converts them into much shorter, thicker structures by a remarkable process of chromosome compaction (or chromosome condensation), which occurs during early prophase Fig. Model for the roles of condensin and cohesin in the formation of mitotic chromosomes. Just after replication, the DNA helices of a pair of sister chromatids would be held in association by cohesin molecules that encircled the sister DNA helices, as shown at the top of the drawing. As the cell entered mitosis, the compaction process would begin, aided by condensin molecules, as shown in the lower part of the drawing. In this model, condensin brings about chromosome compaction by forming a ring around supercoiled loops of DNA within chromatin. Cohesin molecules would continue to hold the DNA of sister chromatids together. It is proposed (but not shown in this drawing), that cooperative interactions between condensin molecules would then organize the supercoiled loops into larger coils, which are then folded into a mitotic chromosome fiber. The top and bottom insets show the subunit structure of an individual cohesin and condensin complex, respectively. Both complexes are built around a pair of SMC subunits. Each of the SMC polypeptides folds back on itself to form a highly elongated antiparallel, coiled coil with an ATP-binding globular domain where the N- and C-termini come together. Cohesin and condensin also have two or three non-SMC subunits that complete the ring-like structure of these proteins. Compiled by Manoj Kumar Singh, PhD 7 Cellular Reproduction In vertebrates, cohesin is released from the chromosomes in two distinct stages 1. Most of the cohesion ( except centromere) dissociates from the arms of the chromosomes as they become compacted during prophase. 2. Release of cohesin from the centromeres is normally delayed until anaphase Centromeres and Kinetochores Centromere: Constricted region of a mitotic chromosome that holds sister chromatids together. This is also the site on the DNA where the kinetochore forms so as to capture microtubules from the mitotic spindle. Kinetochore: Large protein complex that connects the centromere of a chromosome to microtubules of the mitotic spindle. METAPHASE Once all of the chromosomes have become aligned at the spindle equator—with one chromatid of each chromosome connected by its kinetochore to microtubules from one pole and its sister chromatid connected by its kinetochore to microtubules from the opposite pole—the cell has reached the stage of metaphase. Microtubules: 3 types Astral microtubules →Radiate outward from the centrosome into the region outside the body of the spindle. →Determine the plane of cytokinesis Chromosomal (or kinetochore) microtubules →Extend between the centrosome and the kinetochores of the chromosomes →Help maintaining chromosomes in the equatorial plane and its separation during Anaphase Polar (or interpolar) microtubules →Extend from the centrosome past the chromosomes →Maintains the mechanical integrity of the spindle Compiled by Manoj Kumar Singh, PhD 8 Cellular Reproduction Anaphase Anaphase begins when the sister chromatids of each chromosome split apart and start their movement toward opposite poles. Microtubule dynamics during anaphase. Tubulin► subunits are lost from both ends of the chromosomal microtubules, resulting in shortening of chromosomal fibers and movement of the chromosomes toward the poles during anaphase A. Meanwhile, tubulin subunits are added to the plus ends of polar microtubules, which also slide across one another, leading to separation of the poles during anaphase ▲In this model for budding yeast, the chromosome is able to remain associated with the plus end of the microtubule as it depolymerises by the presence of the Dam1 ring, which encircles the plus end of the microtubule at the kinetochore. The force required for chromosome movement is provided by the release of strain energy as the microtubule depolymerizes. The released energy is utilized by the curled ends of the depolymerizing protofilaments to slide the Dam1 ring along the microtubule toward the pole. ▲ In animal cell, the Dam1 ring is replaced by a different type of proposed Coupling device, the Ndc80 protein complex of the outer kinetochore plate. Compiled by Manoj Kumar Singh, PhD 9 Cellular Reproduction 🔎 How does the cell determine whether or not all of the chromosomes are properly aligned at the metaphase plate? Unattached kinetochores contain a complex of proteins, the best studied of which is called Mad2, that mediate the spindle assembly checkpoint. The presence of these proteins at an unattached kinetochore sends a “wait” signal to the cell cycle machinery that prevents the cell from continuing on into anaphase. Once the wayward chromosome becomes attached to spindle fibers from both spindle poles and becomes properly aligned at the metaphase plate, the signalling complex leaves the kinetochore, which turns off the “wait” signal and allows the cell to progress into anaphase. As long as the cell contains unaligned chromosomes, Mad2 molecules are able to inhibit cell cycle progress. According to a favored model, inhibition is achieved through direct interaction between Mad2 and the APC activator Cdc20. During the period that Cdc20 is bound to Mad2, APC complexes would be unable to ubiquitinate the anaphase inhibitor securin, thus keeping all of the sister chromatids attached to one another by their cohesin “glue.” Cells are able to correct syntelic attachments* (and other types of abnormal microtubule connections) through the action of an enzyme called Aurora B kinase, which is part of a mobile protein complex that resides at the kinetochores during prometaphase and metaphase. Among the substrates of Aurora B kinase are several of the proteins thought to be involved in kinetochore–microtubule attachment, including members of both the Dam1 complex and the Ndc80 complex and the kinesin depolymerise * When two kinetochores of sister chromatids become attached to microtubules from the same spindle pole Telophase During telophase, daughter cells return to the interphase condition: the mitotic spindle disassembles, the nuclear envelope reforms, and the chromosomes become more and more dispersed until they disappear from view under the microscope. CYTokinesis Our present concept of the mechanism responsible for cytokinesis stems from a proposal made by Douglas Marsland in the 1950s known as the contractile ring theory. Marsland proposed that the force required to cleave a cell is generated in a thin band of contractile cytoplasm located in the cortex, just beneath the plasma membrane of the furrow. Actin filaments become assembled in a ring at the cell ► equator. Contraction of the ring, which requires the action of myosin, causes the formation of a furrow that splits the cell in two. Compiled by Manoj Kumar Singh, PhD 10 Cellular Reproduction Cytokinesis in Plant Cells: Formation of the Cell Plate The microtubules of the phragmoplast, which arise from remnants of the mitotic spindle, serve as tracks for the movement of small Golgi-derived secretory vesicles into the region. The vesicles become aligned along a plane between the daughter nuclei. Step 1, the vesicles send out finger-like tubules that► contact and fuse with neighboring vesicles to form an interwoven tubular network in the center of the cell. Step 2, Additional vesicles are then directed along microtubules to the lateral edges of the network. The newly arrived vesicles continue the process of tubule formation and fusion, which extends the network in an outward direction. Step 3, Eventually, the leading edge of the growing network contacts the parent plasma membrane at the boundary of the cell. Ultimately, the tubular network loses its cytoplasmic gaps and matures into a continuous, flattened partition. The membranes of the tubular network become the plasma membranes of the two adjacent daughter cells, whereas the secretory products that had been carried within the vesicles contribute to the intervening cell plate. Once the cell plate is completed, cellulose and other materials are added to produce the mature cell wall. Compiled by Manoj Kumar Singh, PhD 11 Cellular Reproduction Some More Explanations... Microtubule-Dependent Motor Proteins Govern Spindle Assembly and Function Major motor proteins of the spindle. Four major classes of microtubule dependent motor proteins (in boxes) contribute to spindle assembly and function. The arrows indicate the direction of motor protein movement along a microtubule. Centrosome Duplication Occurs Early in the Cell Cycle Centriole replication. The centrosome consists of a centriole pair and associated pericentriolar matrix. At a certain point in G1, the two centrioles of the pair separate by a few micrometers. During S phase, a daughter centriole begins to grow near the base of each mother centriole and at a right angle to it (with the help of G1/S-Cdk). The elongation of the daughter centriole is usually completed in G2. The two centriole pairs remain close together in a single centrosomal complex until the beginning of M phase, when the complex splits in two and the two daughter centrosomes begin to separate. Each centrosome now nucleates its own radial array of microtubules (called an aster), mainly from the mother centriole. Compiled by Manoj Kumar Singh, PhD 12 Cellular Reproduction Mitotic Chromosomes Promote Bipolar Spindle Assembly Chromosomes are not just passive passengers in the process of spindle assembly.By creating a local environment that favors both microtubule nucleation and microtubule stabilization, they play an active part in spindle formation. This property of the chromosomes seems to depend, at least in part, on a guanine nucleotide exchange factor (GEF) that is bound to chromatin; the GEF stimulates a small GTPase in the cytosol called Ran to bind GTP in place of GDP. The activated Ran-GTP, which is also involved in nuclear transport, releases microtubule-stabilizing proteins from protein complexes in the cytosol, thereby stimulating the local nucleation and stabilization of microtubules around chromosomes. Local microtubule stabilization is also promoted by the protein kinase Aurora-B, which associates with mitotic chromosomes. The ability of chromosomes to stabilize and organize microtubules enables cells to form bipolar spindles in the absence of centrosomes. Acentrosomal spindle assembly is thought to begin with the formation of microtubules around the chromosomes. Various motor proteins then organize the microtubules into a bipolar spindle. Fig. Spindle self-organization by motor proteins. Mitotic chromosomes stimulate the local activation of proteins that nucleate and promote the formation of microtubules in the vicinity of the chromosomes. Kinesin-5 motor proteins organize these microtubules into antiparallel bundles, while plus-end directed kinesins-4 and 10 link the microtubules to chromosome arms and push minus ends away from the chromosomes. Dynein and kinesin-14 motors, together with numerous other proteins, focus these minus ends into a pair of spindle poles. Bi-orientation Is Achieved by Trial and Error The success of mitosis demands that sister chromatids in a pair attach to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when they separate in anaphase. How is this mode of attachment, called bi-orientation, achieved? What prevents the attachment of both kinetochores to the same spindle pole or the attachment of one kinetochore to both spindle poles? Part of the answer is that sister kinetochores are constructed in a back-to-back orientation that reduces the likelihood that both kinetochores can face the same spindle pole. Nevertheless, incorrect attachments do occur, and elegant regulatory mechanisms have evolved to correct them. Compiled by Manoj Kumar Singh, PhD 13 Cellular Reproduction Fig. Microtubule attachment sites in the kinetochore. (A) In this electron micrograph of a mammalian kinetochore, the chromosome is on the right, and the plus ends of multiple microtubules are embedded in the outer kinetochore on the left. (B) Electron tomography used to construct a low-resolution three- dimensional image of the outer kinetochore in (A). Several microtubules (in multiple colors) are embedded in fibrous material of the kinetochore, which is thought to be composed of the Ndc80 complex and other proteins. (C) Each microtubule is attached to the kinetochore by interactions with multiple copies of the Ndc80 complex (blue). This complex binds to the sides of the microtubule near its plus end, allowing polymerization and depolymerization to occur while the microtubule remains attached to the kinetochore. (A and B, from Y. Dong et al., Nature Cell Biol. 9:516–522, 2007. Fig. Chromosome attachment to the mitotic spindle in animal cells. (A) In late prophase of most animal cells, the mitotic spindle poles have moved to opposite sides of the nuclear envelope, with an array of overlapping microtubules between them. (B) Following nuclear envelope breakdown, the sister- chromatid pairs are exposed to the large number of dynamic plus ends of microtubules radiating from the spindle poles. 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. (C) Eventually, the laterally- attached sister chromatids are arranged in a ring around the outside of the spindle. Most of the microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. (D) Dynamic microtubule plus ends eventually encounter the kinetochores in an end-on orientation and are captured and stabilized. (E) Stable end-on attachment to both poles results in bi-orientation. Additional microtubules are attached to the kinetochore, resulting in a kinetochore fiber containing 10–40 microtubules. Compiled by Manoj Kumar Singh, PhD 14 Cellular Reproduction Incorrect attachments are corrected by a system of trial and error that is based on a simple principle: incorrect attachments are highly unstable and do not last, whereas correct attachments become locked in place. How does the kinetochore sense a correct attachment? The answer appears to be tension. When a sister-chromatid pair is properly bi-oriented on the spindle, the two kinetochores are pulled in opposite directions by strong pole ward forces. Sister-chromatid cohesion resists these poleward forces, creating high levels of tension within the kinetochores. *When both sister chromatids are attached to the same spindle pole, tension is low and the kinetochore sends an inhibitory signal that loosens the grip of its microtubule attachment site, allowing detachment to occur. *When bi-orientation occurs, the high tension at the kinetochore shuts off the inhibitory signal, strengthening microtubule attachment. In animal cells, tension not only increases the affinity of the attachment site but also leads to the attachment of additional microtubules to the kinetochore. This results in the formation of a thick kinetochore fiber composed of multiple microtubules. The tension-sensing mechanism depends on the protein kinase Aurora-B, which is associated with the kinetochore and is thought to generate the inhibitory signal that reduces the strength of microtubule attachment in the absence of tension. It phosphorylates several components of the microtubule attachment site, including the Ndc80 complex, decreasing the site’s affinity for a microtubule plus end. When bi- orientation occurs, the resulting tension somehow reduces phosphorylation by Aurora-B, thereby increasing the affinity of the attachment site. Following their attachment to the two spindle poles, the chromosomes are tugged back and forth, eventually assuming a position equidistant between the two poles, a position called the metaphase plate. In vertebrate cells, the chromosomes then oscillate gently at the metaphase plate, awaiting the signal for the sister chromatids to separate. The signal is produced, with a predictable lag time, after the bi- oriented attachment of the last of the chromosomes. How tension might increase microtubule🖝 attachment to the kinetochore. These diagrams illustrate one speculative mechanism by which bi-orientation might increase microtubule attachment to the kinetochore. A single kinetochore is shown for clarity; the spindle pole is on the right. (A) When a sister chromatid pair is unattached to the spindle or attached to just one spindle pole, there is little tension between the outer and inner kinetochores. The protein kinase Aurora-B is tethered to the inner kinetochore and phosphorylates the microtubule attachment sites, including the Ndc80 complex (blue), in the outer kinetochore as shown, thereby reducing the affinity of microtubule binding. Microtubules therefore associate and dissociate rapidly, and attachment is unstable. (B) When bi-orientation is achieved, the forces pulling the kinetochore toward the spindle pole are resisted by forces pulling the other sister kinetochore toward the opposite pole, and the resulting tension pulls the outer kinetochore away from the inner kinetochore. As a result, Aurora-B is unable to reach the outer kinetochore, and microtubule attachment sites are not phosphorylated. Microtubule binding affinity is therefore increased, resulting in the stable attachment of multiple microtubules to both kinetochores. The dephosphorylation of outer kinetochore proteins depends on a phosphatase that is not shown here. Compiled by Manoj Kumar Singh, PhD 15 Cellular Reproduction Compiled by Manoj Kumar Singh, PhD 16