During which phase do the chromosomes begin moving to the poles of the cell?

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.

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 the rate of chromosome motion is limited by kinetochore microtubule disassembly. 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.

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.

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.