During which phase of mitosis do sister chromatids separate

Anaphase and telophase I

Anaphase I of meiosis begins with the release of cohesion between the arms of sister chromatids, much as it does during mitosis. As positioning of bivalent pairs is random, assortment of maternal and paternal chromosomes in each telophase nucleus is also random. Critically, sister centromeres, and thus chromatids, do not separate during anaphase I.

During meiosis I, cytoplasmic division occurs by specialized mechanisms. In females, the division is highly asymmetric, producing one egg and one tiny cell known as a polar body. In males, the process results in production of spermatocytes that remain joined by small cytoplasmic bridges.

D Anaphase A

Anaphase A is the dynamic mitotic stage during which the sister chromatids separate further and migrate along the spindle to opposite spindle poles (Inoué and Ritter, 1975). In filamentous fungi, this occurs within a more or less intact nuclear envelope (Aist, 1969; Aist and Berns, 1981; Aist and Williams, 1972, Bayles et al., 1993). The KCs in F. oxysporum are found at the spindle poles at the end of anaphase A (Aist and Williams, 1972), which verifies that this stage is functionally equivalent to that in higher eukaryotes. In Fusarium spp. it is sometimes possible to observe individual sister chromatids separating to opposite spindle poles in living preparations (Fig. 5; Aist, 1969; Aist and Bayles, 1988). From their different starting points along the middle one-half to two-thirds of the spindle (Fig. 4), the sister chromatids begin their poleward migration asynchronously, creating a momentary mitotic figure in which the chromatids are strung out along most or all of the spindle length, sometimes in two rows, as individual chromatids pass each other on their way to their respective poles (Aist, 1969; Aist and Morris, 1999). This phase has been referred to as the “two-track” or “double-track” stage, and its true identity as anaphase A was not recognized until an accurate description of it, as seen in living cells of F. oxysporum, was published (Aist, 1969). Individual chromatids of wild-type Fusarium spp. are so clearly imaged using differential interference-contrast optics (Fig. 5) that it is possible to measure accurately their rates of migration to the spindle poles at anaphase A with computer-assisted video microscopy techniques (Aist and Bayles, 1988). Different chromatids within a given mitotic nucleus migrate to the spindle pole at different rates. Their migration is typically punctuated by brief moments when the chromatid pauses before completing its journey to the pole. The fastest average rate of anaphase chromatid migration ever recorded for any organism was 7.5 μm/min reported for F. solani f. sp. pisi (Aist and Bayles, 1988). Anaphase A requires about 13 s in F. oxysporum (Aist and Williams, 1972) and 30–45 s in F. solani f. sp. pisi (Aist and Bayles, 1988). The time for anaphase A in a basidio-mycete was about the same as in F. solani f. sp. pisi (Bayles et al., 1993), and in budding yeast cells the initial poleward movement of chromatids—comprising most of anaphase A—lasts about 25 s (Straight et al., 1997).

During anaphase A, the MTs of the mitotic apparatus undergo significant changes as well. Mitotic asters are developed during this stage (Aist and Bayles, 1988; Aist and Williams, 1972) as MTs are polymerized at the cytoplasmic face of the SPB. The development of asters is correlated with a marked increase in the rate of spindle elongation, from 0.6 μm/min during metaphase to 3.6 μm/min during anaphase A in F. solani f. sp. pisi (Aist and Bayles, 1988), suggesting that the asters may play a role in the deployment of forces driving spindle elongation (This point will be further discussed later).

In addition to the increase in the rate of spindle elongation in F. solani f. sp. pisi noted previously, other changes in the spindle occur during anaphase A. Typically, the spindle is composed mainly of two or three bundles of MTs at mid-anaphase A (Fig. 5), but by the end of this phase usually the bundles have been drawn together into one central bundle of MTs (Fig. 6; Aist and Bayles, 1991b; Aist and Berns, 1981). MT cross-bridging occurs in the anaphase A spindle and would be expected to play a role in MT bundling (Jensen et al., 1991). Both the number and the total length of spindle MTs drop precipitously during anaphase A—changes that are too great to be accounted for solely by the depolymerization of KC MTs, which in F. solani f. sp. pisi would number only about 15 per genome (Aist and Bayles, 1991b). Thus, anaphase A clearly represents a transition phase with respect to mitotic MT dynamics, as intranuclear spindle MTs are depolymerizing while cytoplasmic, astral MTs are polymerizing.

Anaphase

During anaphase, the sister chromatids separate and begin to migrate to opposite poles of the cell, and a cleavage furrow begins to develop.

Anaphase begins when the cohesion proteins located between the sister chromatids disappear; the sister chromatids, located at the equator of the metaphase plate, separate and begin their migration toward the opposite poles of the mitotic spindle. The spindle/kinetochore attachment site leads the way, with the arms of the chromatids simply trailing, contributing nothing to the migration or its pathway.

It has been postulated that the observed movement of the chromatids toward the pole in anaphase may be the result of shortening of the microtubules via depolymerization at the kinetochore end. This, coupled with the discovery of dynein associated with the kinetochore, may be analogous to vesicle transport along microtubules. Inlate anaphase, a cleavage furrow begins to form at the plasmalemma, indicating the region where the cell will be divided during cytokinesis.

Anaphase

During anaphase, sister chromatids separate and move to the spindle poles (Figures 2 and 3). Anaphase consists of two phases, anaphase A and B. During anaphase A, the chromosomes move to the poles and kinetochore fiber microtubules shorten; during anaphase B, the spindle poles move apart as interpolar microtubules elongate and slide past one another. Many cells undergo both anaphase A and B motions, but, in some cases, one or the other motion dominates.

Separation of the paired sister chromatids is required for poleward motion in anaphase. Chromatid separation results from the proteolytic degradation of components that link the chromatids at the centromere. Degradation is triggered by the activity of the anaphase-promoting complex, which regulates cell-cycle progression. Chromatid separation is not the result of tugging by microtubules and motor proteins, and can be observed even in the absence of microtubules.

Although the motion of the chromosomes to the spindle poles in anaphase has fascinated biologists for many years, the molecular basis for this motion remains controversial and incompletely understood. During anaphase A, kinetochore microtubules must shorten as the chromosomes move poleward. Measurements of spindle flux show that subunit loss from microtubules occurs at the spindle poles during anaphase. In many cells, however, the rate that chromosomes move exceeds the rate of subunit loss at the pole, and, thus, subunit loss must also occur at the kinetochore.

Pioneering studies of mitosis in living embryonic cells demonstrated that assembly and disassembly of microtubule polymers result in chromosome motion. This work led to the hypothesis that microtubule disassembly drives chromosome motion. Later work identified molecular motors at the kinetochore, leading to the alternative hypothesis that forces generated by molecular motors drive chromosome motion. One possibility is that molecular motors power chromosome motion, but kinetochore microtubule disassembly limits the rate of chromosome motion. Alternatively, disassembly may be responsible for chromosome motion, and motors may tether the chromosomes to the shortening fiber. The presence of potentially redundant mechanisms for chromosome motion may reflect the fact that mitotic fidelity is of utmost importance.

98.1

Methods of Chromosome Analysis

Cytogenetic studies are usually performed on peripheral blood lymphocytes, although cultured fibroblasts obtained from a skin biopsy may also be used. Prenatal (fetal) chromosome studies are performed with cells obtained from the amniotic fluid (amniocytes), chorionic villus tissue, and fetal blood or, in the case of preimplantation diagnosis, by analysis of ablastomere (cleavage stage) biopsy, polar body biopsy, or blastocyst biopsy. Cytogenetic studies of bone marrow have an important role in tumor surveillance, particularly among patients with leukemia. These are useful to determine induction of remission and success of therapy or in some cases the occurrence of relapses.

Chromosome anomalies include abnormalities of number and structure and are the result of errors during cell division. There are 2 types of cell division: mitosis, which occurs in most somatic cells, and meiosis, which is limited to the germ cells. Inmitosis, 2 genetically identical daughter cells are produced from a single parent cell. DNA duplication has already occurred duringinterphase in the S phase of the cell cycle (DNA synthesis). Therefore, at the beginning of mitosis the chromosomes consist of 2 double DNA strands joined together at the centromere, known assister chromatids. Mitosis can be divided into 4 stages: prophase, metaphase, anaphase, and telophase.Prophase is characterized by condensation of the DNA. Also during prophase, the nuclear membrane and the nucleolus disappear and the mitotic spindle forms. Inmetaphase the chromosomes are maximally compacted and are clearly visible as distinct structures. The chromosomes align at the center of the cell, and spindle fibers connect to the centromere of each chromosome and extend to centrioles at the 2 poles of the mitotic figure. Inanaphase the chromosomes divide along their longitudinal axes to form 2 daughter chromatids, which then migrate to opposite poles of the cell.Telophase is characterized by formation of 2 new nuclear membranes and nucleoli, duplication of the centrioles, and cytoplasmic cleavage to form the 2 daughter cells.

Meiosis begins in the female oocyte during fetal life and is completed years to decades later. In males it begins in a particular spermatogonial cell sometime between adolescence and adult life and is completed in a few days. Meiosis is preceded by DNA replication so that at the outset, each of the 46 chromosomes consists of 2 chromatids. In meiosis, adiploid cell (2n = 46 chromosomes) divides to form4 haploid cells (n = 23 chromosomes). Meiosis consists of 2 major rounds of cell division. Inmeiosis I, each of the homologous chromosomes pair precisely so thatgenetic recombination, involving exchange between 2 DNA strands (crossing over), can occur. This results in reshuffling of the genetic information for the recombined chromosomes and allows further genetic diversity. Each daughter cell then receives 1 of each of the 23 homologous chromosomes. In oogenesis, one of the daughter cells receives most of the cytoplasm and becomes the egg, whereas the other smaller cell becomes the first polar body.Meiosis II is similar to a mitotic division but without a preceding round of DNA duplication (replication). Each of the 23 chromosomes divides longitudinally, and the homologous chromatids migrate to opposite poles of the cell. This produces 4 spermatogonia in males, or an egg cell and a 2nd polar body in females, each with a haploid (n = 23) set of chromosomes. Consequently, meiosis fulfills 2 crucial roles: It reduces the chromosome number from diploid (46) to haploid (23) so that on fertilization a diploid number is restored, and it allows genetic recombination.

3.4 Troubleshooting

While performing live-cell imaging of mitotic cells, it is possible to encounter the following difficulties:

Anaphase timing is prolonged or few cells enter mitosis: Fluorescent light is toxic as it can induce protein, lipid, or in particular DNA damage. This can prevent cells from entering mitosis or prolong anaphase timing. If, compared to previous experiments, control-treated cells rarely enter mitosis or display a significant delay in anaphase timing, it is likely that the cells are experiencing excessive phototoxicity. To avoid this issue, the best recourse is to lower the light exposure (darker neutral density filters and/or shorter exposure times) or to acquire shorter movies at a lower temporal resolution. Another cause for aberrant anaphase timing can be fluctuations in the temperature, in particular for temperatures above 37°C: cells recorded at 35°C will show a delay of a few minutes in anaphase timing; cells recorded at 39°C fail to exit mitosis (A-M.O., unpublished observation). In case of doubt, we recommend independently measuring the temperature on the microscope stage with a high-precision thermometer or directly on the sample with a wet probe thermometer.

Loss of focus: The long duration of such movies can lead to a drift in the z-axis of the microscope stage. To alleviate these types of problems, use software- or hardware-based autofocus systems that will correct for such drifts during the experiment.

Insufficient staining of cells with live-cell dyes: Some tissue culture cell lines might give a weak staining when treated with live-cell dyes. This is often due to the expression of multidrug resistance pumps that expel these dyes. This can be avoided by adding to the growth medium multidrug resistance pump inhibitors, such as Verapamil or Valspodar, that are often provided with the dyes.

Mitotic Spindle Dynamics and Chromosome Movement During Anaphase

Anaphase is dominated by the orderly movement of sister chromatids to opposite spindle poles brought about by the combined action of motor proteins and changes in microtubule length. There are two components to anaphase chromosome movements (Fig. 44.15). Anaphase A, the movement of the sister chromatids to the spindle poles, requires a shortening of the kinetochore fibers. During anaphase B, the spindle elongates, pushing the spindle poles apart. The poles separate partially because of interactions between the antiparallel interpolar microtubules of the central spindle and partially because of intrinsic motility of the asters. Most cells use both components of anaphase, but one component may predominate in relation to the other.

Microtubule disassembly on its own can move chromosomes (see Fig. 37.8). Energy for this movement comes from hydrolysis of GTP bound to assembled tubulin, which is stored in the conformation of the lattice of tubulin subunits. Microtubule protofilaments are straight when growing, but after GTP hydrolysis protofilaments are curved, so they peel back from the ends of shrinking microtubules (see Fig. 34.6). Several kinesin “motors” influence the dynamic instability of the spindle microtubules. Members of the kinesin-13 class, which encircle microtubules near kinetochores and at spindle poles, use adenosine triphosphate (ATP) hydrolysis to remove tubulin dimers and promote microtubule disassembly rather than movement.

Kinetochores are remarkable in their ability to hold onto disassembling microtubules. In straight (growing) microtubules, the Ndc80 complex is mostly responsible for microtubule binding. It binds to the interface between α and β tubulin subunits. This interface bends in curved (shrinking) microtubules, so Ndc80 cannot bind. This could allow it to redistribute onto straight sections of the lattice and thereby move away from the curved protofilaments at the disassembling end. In metazoans the Ska complex in the outer kinetochore binds α and β tubulin subunits away from the interface, so it can bind to curved (disassembling) protofilaments. At yeast kinetochores the Dam1 ring (green in Fig. 8.21) couples the kinetochore to disassembling microtubules.

Anaphase A chromosome movement involves a combination of microtubule shortening and translocation of the microtubule lattice that result from flux of tubulin subunits (Fig. 44.14). The contributions of the two mechanisms vary among different cell types. When living vertebrate cells are injected with fluorescently labeled tubulin subunits, the spindle becomes fluorescent (Fig. 44.17). If a laser is used to bleach a narrow zone in the fluorescent tubulin across the spindle between the chromosomes and the pole early in anaphase, the chromosomes approach the bleached zone much faster than the bleached zone approaches the spindle pole. This shows that the chromosomes “eat” their way along the kinetochore microtubules toward the pole. In these cells, subunit flux accounts for only 20% to 30% of chromosome movement during anaphase A, and this flux is dispensable for chromosome movement. In Drosophila embryos, in which subunit flux accounts for approximately 90% of anaphase A chromosome movement, the chromosomes catch up with a marked region of the kinetochore fiber slowly, if at all.

Anaphase B appears to be triggered at least in part by the inactivation of the minus-end–directed kinesin-14 motors, so that all the net motor force favors spindle elongation. Four factors contribute to overall lengthening of the spindle: release of sister chromatid cohesion, sliding apart of the interdigitated half-spindles, microtubule growth, and intrinsic motility of the poles themselves (Fig. 44.7). During the latter stages of anaphase B, the spindle poles, with their attached kinetochore microtubules, appear to move away from the interpolar microtubules as the spindle lengthens. This movement of the poles involves interaction of the astral microtubules with cytoplasmic dynein molecules anchored at the cell cortex.

Anaphase B spindle elongation is accompanied by reorganization of the interpolar microtubules into a highly organized central spindle between the separating chromatids (Fig. 44.15). Within the central spindle, an amorphous dense material called stem body matrix stabilizes bundles of antiparallel microtubules and holds together the two interdigitated half-spindles. Proteins concentrated in the central spindle help regulate cytokinesis. One key factor, PRC1 (protein regulated in cytokinesis 1), is inactive when phosphorylated by Cdk kinase and functions only during anaphase when Cdk activity declines and phosphatases remove the phosphate groups placed on target proteins by Cdks and other mitotic kinases. PRC1 directs the binding of several kinesins to the central spindle. The kinesin KIF4A targets Aurora B kinase to a particular domain of the central spindle, where phosphorylation of key substrates then regulates spindle elongation and cytokinesis.

How can protein kinases such as Aurora B continue to function during anaphase while protein phosphatases are removing phosphate groups placed there by Cdks and, indeed, Aurora B during early mitosis? One answer is that the phosphatase activity is highly localized, controlled by specific targeting subunits. Cdk phosphorylation can inhibit targeting subunits such as the exotically named Repo-Man (recruits PP1 onto mitotic chromatin at anaphase) from binding protein phosphatase 1 or localizing to targets, such as chromatin in early mitosis. When Cdk activity drops, Repo-Man (and other similar targeting subunits) is dephosphorylated, and now targets PP1 to chromatin, where it removes phosphates placed there by Aurora B in the CPC. As long as phosphatases are not specifically targeted to the cleavage furrow, Aurora B can continue to control events there during mitotic exit by phosphorylating key target proteins required for cytokinesis.

Glossary

Anaphase A

Stage of mitosis when the chromosomes separate and move towards the spindle poles.

Stage of mitosis when the spindle poles separate.

Diffusion of a particle whose net motion is strongly biased in one direction by an energy source.

Functional center of a chromosome where the sister chromatids are held and where the kinetochore is built.

Organelle serving as the main microtubule organizing center in metazoans.

Kinesin motor located on chromosome arms.

Macromolecular assembly built on the centromere that mediates the attachment of chromosomes to spindle microtubules.

Stage of mitosis when chromosomes are positioned at the spindle equator in a brief steady state.

A microtubule-dependent force that pushes chromosomes away from spindle poles.

Continuous spindle microtubule sliding towards spindle poles.

Stroke of a motor (conformational change) which generates mechanical force from chemical potential.

Stage of mitosis when the spindle begins to form and microtubules begin to attach to kinetochores.

Stage of mitosis when the chromosomes start to condense and the nucleus starts to break down.

Under-labeling of periodic cellular components (e.g., filaments) such that, instead of appearing continuous, they appear as discrete speckles that can reveal component dynamics.

Cellular assembly based on a bipolar array of microtubules that segregates chromosomes during eukaryotic cell division.

A controversial microtubule-independent network proposed to provide a structural scaffold to the spindle.

Stage of mitosis when the spindle disappears and the two nuclei form.

Glossary

Anaphase

A stage of mitosis during which the duplicated chromosomes are segregated to different parts of the cell so they can serve as a complete genome for the next cell cycle. Anaphase is commonly thought of in two parts, A and B. During anaphase A, the chromosomes approach the ends of the mitotic spindle; during anaphase B, the spindle elongates, so the distance between the chromosome sets at the completion of anaphase is greater.

A special case of the general physical phenomenon of diffusion in which the boundary conditions influence the outcome of the many random walks which comprise a true diffusive process. A simple example is diffusion in one dimension with an impermeable boundary; this constrains the otherwise random walks, leading to a nonrandom distribution of particle positions relative to the boundary. A more complicated case, that is directly relevant to biology, is the case in which the boundary moves. Now particle motions driven by thermal fluctuations are biased to produce net particle movement in the same direction as motion of the boundary.

A change in the state of a microtubule such that the polymer goes from a condition of continuous growth to one in which the polymer shortens. Catastrophes are thought to result from the loss of guanosine triphosphate-associated tubulin from the polymer's end. The opposite of a catastrophe is a ‘rescue’.

A chromosomal locus which directs the segregation of that chromosome by serving as a platform for the assembly of a kinetochore.

A macromolecular device which attaches a microtubule or other protein polymer to a load which can then be moved by polymer dynamics.

A proposed mechanism by which the bending tubulin protofilaments, that form at the end of a shortening microtubule, pull on an object which is attached to the polymer wall by an appropriate coupler. Thrust from tubulin bending is thought to push the coupler along the microtubule axis in the direction of microtubule shortening, thereby moving its associated cargo. This mechanism is an alternative to the biased-diffusion mechanism of coupler motion with the end of a shortening microtubule, because the forced walk is driven by chemical energy and it can move even nondiffusing couplers.

A protein complex which forms on eukaryotic chromosomes at their centromeres. It couples a piece of double-stranded DNA to one or more microtubules of the mitotic spindle.

The stage of mitosis at which all the chromosomes have become attached to the mitotic spindle and are situated near its mid plane. The onset of metaphase is not sharply defined because chromosomes move continuously on and off this mid plane while the cell is in metaphase. The end of metaphase occurs at the onset of anaphase, when the duplicate chromosomes separate and begin to move away from each other.

A cytoplasmic polymer, ubiquitous among eukaryotic cells, which assembles from dimers of the proteins α- and β-tubulin. Microtubules are unbranched and comparatively rigid hollow tubes, ∼25 nm in diameter and of lengths which can range from a few tens of nanometers to many micrometers. They are used by cells as frameworks on which to organize many cytoplasmic proteins which perform a diversity of motile and morphogenetic functions.

The process by which eukaryotic cells segregate their already duplicated chromosomes in preparation for cell division. The name derives from the Greek word for ‘thread’, because during the early stages of mitosis, chromosomes become visible in a light microscope as slender threads within the nucleus.

The cellular machine which organizes and segregates a cell's duplicated chromosome during mitosis. In overview, the spindle is a twofold symmetric array of microtubules, some of which interact with chromosomes at their kinetochores and some of which interact with one another to form a mechanical connection between the two spindle ends. The name derives from the resemblance between this structure in some cells and an old-fashioned device for spinning wool into yarn.

A property of biological motions along a polymer when they continue for many consecutive steps or achieve motion for a comparatively long distance.

A strand of α- and β-tubulin dimers connected end-to-end. Most microtubules in cytoplasm are made from 13 protofilaments which run parallel to the microtubule axis. These protofilaments are slightly out of register, so their tubulin monomers form a 3-start left-handed helix. Not all protofilaments end at the same position along the microtubule axis, so microtubule ends are often uneven. When a microtubule end is shortening, the protofilaments bend away from the microtubule axis before losing their subunits. In vivo, the protofilaments on even elongating microtubules are somewhat flared.

Change in the state of a microtubule such that the polymer goes from a condition of shortening to one of net growth. Rescues are the opposite of ‘catastrophes’. They are thought to result from the addition of guanosine triphosphate-tubulin to a previously shortening plus end.

A soluble protein which is ubiquitous among eukaryotic cells. There is a family of tubulins, but the most common members are dimers of α- and β-tubulin. γ-tubulin forms a ring-shaped complex with several nontubulin proteins; this ‘γ-tubulin ring complex’ (γ-TuRC) is the principal initiator of microtubule polymerization in cells. Other tubulin isoforms in eukaryotes are found only in association with centrioles. Recently, several isoforms of tubulin have been found in bacteria and archaea; one such tubulin, FtsZ, is a principal player in bacterial cytokinesis. Both α- and β-tubulin bind guanosine triphosphate (GTP), and this association is necessary for tubulin to polymerize. During the polymerization process, the GTP on β-tubulin becomes hydrolyzed, so the bulk of a microtubule is made from tubulin in which α-tubulin still binds GTP, but β-tubulin has guanosine diphosphate, a form of the dimer which can no longer polymerize.

Anaphase

Anaphase is characterized by the segregation of the chromosomes.161 This event is controlled by the mitotic ubiquitin ligase APC/C-Cdc20. APC/C-Cdc20 ubiquitinates, and thereby triggers the degradation of, cyclin B1 and a protein called securin.39 Both securin and cyclin B1/Cdk1 complexes are able to bind and inhibit a protease called separase.162,163 APC/C-Cdc20 activity results in the degradation of cyclin B and securin and the subsequent separase activation. Once released, separase cleaves the Scc1 component of the cohesin complex, which opens the cohesin ring, unlinking the sister chromatids and allowing them to be pulled to opposite poles (see Fig. 4.5). The spindle poles then move farther apart to ensure that the chromosomes are fully segregated. The separase-dependent cleavage of Scc1 also is essential to link segregation of chromatids with the separation of centrioles during mitotic exit.163 Cyclin B degradation results in the parallel inhibition of Cdk1 activity, thereby releasing the inhibitory mechanism that limit PP1 and PP2A activity during the earlier phases of mitosis.84,90,92 The reactivation of these phosphatases results in the massive dephosphorylation of mitotic phosphoproteins and results in the disassembly of the mitotic spindle, chromosome decondensation, and the reformation of the nuclear envelope.161

During anaphase, Cdh2, which is inhibited by Cdk-dependent phosphorylation during mitosis, is dephosphorylated and replaces Cdc20 as the main APC/C activator.39 APC/C-Cdh2 is responsible for the degradation of multiple cell cycle regulators, including Cdc20. APC/C-Cdh2 also activates the ubiquitination and degradation of geminin, allowing accumulation of Cdt1 for origin relicensing in the subsequent G1 phase, and the mitotic cyclins, allowing loss of Cdk kinase activity. Loss of Cdh2 does not result in major abnormalities during mitotic exit but results in earlier entry into the following S phase because of increased Cdk activity and DNA damage.164,165 Cdh2 therefore is required to prevent unscheduled entry into S phase and genomic instability.43