What is the phase where sister chromatids are pulled towards the opposite pole?

Abstract

Sister chromatid cohesion depends on cohesin, a tripartite complex that forms ring structures to hold sister chromatids together in mitosis and meiosis. Meiocytes feature a multiplicity of distinct cohesin proteins and complexes, some meiosis specific, which serve additional functions such as supporting synapsis of two pairs of sister chromatids and determining the loop–axis architecture of prophase I chromosomes. Despite considerable new insights gained in the past few years into the localization and function of some cohesin proteins, and the recent identification of yet another meiosis-specific cohesin subunit, a plethora of open questions remains, which concern not only fundamental germ cell biology but also the consequences of cohesin impairment for human reproductive health.

Biochemical Mechanism of Sister Chromatid Separation

Separation of sister chromatids is regulated by the chromosomes themselves, not by the mitotic spindle. Under certain circumstances, sister chromatids can separate in the absence of microtubules, ruling out a requirement for forces from the spindle in the process.

Three factors regulate sister chromatid separation: a protein complex known as cohesin, a protease known as separase, and an inhibitor of separase known as securin (Fig. 44.16). This system is conserved from yeast to human. Chapter 8 discusses the functions of cohesin in interphase.

Cohesin is a complex of four proteins that resembles the condensin complex (see Fig. 8.18). Like condensin, cohesin has two large subunits from the SMC ATPase family. These proteins, SMC1 and SMC3, are complexed with proteins called Scc1 (which has other names omitted here for simplicity) and Scc3. Additional proteins are required to stabilize the loading of this complex onto DNA. Cells with mutations in cohesin components separate sister chromatids prematurely in mitosis, resulting in chaotic chromosome missegregation. This system is very ancient, as bacteria depend on an SMC-related protein for orderly chromosome segregation.

A variety of evidence suggests that cohesin forms a ring with a diameter of 35 nm, large enough to encircle two sister chromatids like a lasso. In yeast, the complex functions only if it binds chromosomes during DNA replication. Cohesin accumulates at preferred sites on the chromosomes, often near centromeres in budding yeast or in regions of heterochromatin in fission yeast. In vertebrates, most cohesin dissociates from the chromosome arms by late metaphase, owing to the action of the protein kinases Plk1 and Aurora B. Importantly, a critical fraction remains associated with heterochromatin flanking centromeres where it is protected from cleavage by shugoshin until the onset of anaphase (see following paragraphs and Chapter 45).

Sequential cleavage of two key proteins triggers sister chromatid separation at anaphase. This proteolysis makes anaphase onset an irreversible transition. The first target, securin, inhibits the separase protease. After the last chromosome forms an amphitelic attachment to the spindle, the spindle checkpoint is silenced. This allows APC/CCdc20 to tag securin with ubiquitin, leading to its destruction by proteasomes throughout metaphase. When securin levels fall below a critical threshold, separase is unleashed to cleave the Scc1 subunit of cohesin. Cleavage of Scc1 breaks the cohesin ring, allowing the sister chromatids to separate triggering the onset of anaphase (Fig. 44.16B).

Efficient Scc1 cleavage requires that the protein be phosphorylated near its cleavage site. This allows a mode of regulation where shugoshin (Japanese for “guardian spirit”) recruits PP2A to centromeres. PP2A keeps Scc1 dephosphorylated. This inhibits its cleavage and protects cohesin until shugoshin is released following amphitelic attachment of the chromosome. This mechanism is absolutely essential during meiosis, as without it, it would not be able to segregate homologous chromosomes from each other (see Fig. 45.12).

Securin can act as an oncogene in cultured cells and is overexpressed in some human pituitary tumors. Overexpression of securin may dis­rupt the timing of chromosome segregation, leading to chromosome loss and, ultimately, contributing to cancer progression.

G2/M Checkpoint

Separation of sister chromatids during mitosis is a potential danger point for a cell. After DNA is replicated each chromosome consists of paired sister chromatids held together by cohesin. Therefore, if the DNA is damaged, the cell can use information present in the undamaged chromatid to guide the repair process. However, once sisters separate, this corrective mechanism can no longer operate. In addition, if a cell enters mitosis before completing replication of its chromosomes, attempts to separate sister chromatids damage the chromosomes. To minimize these hazards, a checkpoint operates in the G2 phase to block mitotic entry if DNA is damaged or DNA replication is incomplete.

Just as DNA damage can arrest the cell cycle in G1 phase, damaged or unreplicated DNA also halts the cell cycle temporarily in the G2 phase. Interestingly, the G1 checkpoint—which can be activated by a single DNA break in human cells—is more sensitive than the G2/M checkpoint, which requires 10 to 20 breaks to block cell-cycle progression. The G2/M checkpoint may be less sensitive then the G1 checkpoint, because G2 cells are already primed to enter mitosis. Consequently, human cells can enter mitosis with limited amounts of damaged or unreplicated DNA. These problem regions can be detected and repaired in the daughter cells after division (see later).

Studies of radiation-induced G2 delay in budding yeast identified a major cell-cycle checkpoint that is sensitive to the status of the cellular DNA. Cells defective in this checkpoint are more sensitive than wild-type cells to radiation injury because they continue to divide, despite the presence of broken or otherwise damaged chromosomes (Fig. 43.8). The cells die, presumably from chromosomal defects or loss. In metazoans, the G2/M checkpoint delays entry into mitosis until the damage is either fixed, triggers cell suicide by apoptosis, or causes cells to enter a nonproliferating (senescent) state. The checkpoint works by modulating the activities of the components that control the G2/M transition.

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.

Anaphase

The separation of sister chromatids marks the commencement of anaphase followed by the movement of chromosomes toward the spindle poles. The chromosomes are pulled apart by shortening of the kinetochore microtubules at the centromere attachment sites, and this is thought to be triggered by the loss of tension following cleavage of the cohesin complex. Simultaneously, the non-kinetochore spindle microtubules elongate, thereby pushing the centrosomes farther apart. This is regulated by two forces: (1) plus-end-directed motor proteins, such as end-binding protein-1 (EB1), at the central spindle and between the overlapping microtubules from both poles (Tamura and Draviam, 2012). These motor proteins slide the microtubules at the plus-ends past one another, creating the forces to push the spindle poles farther apart. (2) Minus-end directed motor proteins, such as dynein, that interact with the cell cortex and the astral microtubules to create pulling forces on the spindle poles (Tamura and Draviam, 2012). By the end of anaphase, the kinetochore microtubules have disassembled and a complete set of chromosomes has assembled at each pole and begun to decondense. During this time, cyclin B1 is degraded, resulting in the inactivation of Cdk1 and this is necessary for driving mitotic exit (Hershko, 1999).

Abstract

During mitosis, duplicated sister chromatids are properly aligned at the metaphase plate of the mitotic spindle before being segregated into two daughter cells. This requires a complex process to ensure proper interactions between chromosomes and spindle microtubules. The kinetochore, the proteinaceous complex assembled at the centromere region on each chromosome, serves as the microtubule attachment site and powers chromosome movement in mitosis. Numerous proteins/protein complexes have been implicated in the connection between kinetochores and dynamic microtubules. Recent studies have advanced our understanding on the nature of the interface between kinetochores and microtubule plus ends in promoting and maintaining their stable attachment. These efforts have demonstrated the importance of this process to ensure accurate chromosome segregation, an issue which has great significance for understanding and controlling abnormal chromosome segregation (aneuploidy) in human genetic diseases and in cancer progression.

Molecular Biology of Nondisjunction

During normal mitotic cell division, the sister chromatids are distributed to the daughter cells by attaching their kinetochores to the microtubules from the opposite cell poles. Most of the information on molecular players in chromosome disjunction comes mostly from the genetic and biochemical analysis of budding yeast, Saccharomyces cerevisiae. The cohesins, including the Scc1p protein acts as a glue, holding sister chromatids together. The separation of sister chromatids is regulated by ubiquitin-mediated proteolysis, via three protein complexes, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase). E3 is also called the anaphase-promoting complex or cyclosome (APC/C) in S. cerevisiae. E3 specifically degrades the inhibitor Pds1p to allow sister chromatid separation. The role of Pds1p is to inhibit sister chromatid separation by disabling Esp1p to stimulate Scc1p cleavage. The spindle checkpoint mechanism ensures, through a signal transduction cascade, that the mitosis does not proceed to anaphase if one or more chromatids are not properly attached to spindle microtubules. The kinetochores thus can be viewed as ‘sensors’ that recognize the unattached chromosomes and initiate a signal causing arrest of the cell cycle. The mechanism by which nondisjunction overrides the spindle checkpoint mechanism is not yet clear.

Timing of Centromere Splitting

In mitosis, the cohesion of sister chromatids at the centromere lapses at the end of metaphase, enabling the daughter chromosomes to move apart towards the two poles of the spindle. In meiosis, in contrast, the chromatids remain joined at the centromere at the first anaphase. The bivalent chromosomes, resulting from pairwise synapsis and chiasma formation, each separate into two dyads, each consisting of two chromatids joined at the centromere (Figure 1B), which is not split until the end of metaphase of the second division. Thus the centromere can be defined genetically as that point in a linkage map that always segregates at the first division of meiosis (reductionally) and never at the second division (equationally).

2.1.3 In situ hybridization

In meiosis I, kinetochores of sister chromatids are tightly associated together, and kinetochores of homologous chromosomes must be bioriented and attached by microtubules emanating from opposite poles. Failure in this process can result in chromosome mis-segregation (McKim & Hawley, 1995; Radford, Nguyen, Schindler, & McKim, 2017). Kinetochores assemble at a particular region of the chromosome called the centromere. Centromeres of each homologous chromosome are pulled toward opposite poles (Dernburg, Sedat, & Hawley, 1996). Pericentromeric heterochromatin contains tandemly repeated sequences, with at least one unique repetitive sequence per chromosome (Abad et al., 1992; Carmena, Abad, Villasante, & Gonzalez, 1993; Dernburg, 2000; Dernburg et al., 1996; Loh et al., 2012; Meireles, Fisher, Colombié, Wakefield, & Ohkura, 2009; Radford et al., 2012; Zhaunova, Ohkura, & Breuer, 2016). Therefore, a specific pair of homologous centromeres can be visualized under a fluorescent microscope by fluorescent in situ hybridization using a fluorescently labeled oligonucleotide that can hybridize to these chromosome-specific sequences (Dernburg, 2000; Dernburg et al., 1996).

To generate a probe, terminal deoxynucleotidyl transferase (TdT) is used to add fluorescently labeled nucleotides to the 3′ terminus of an oligonucleotide. 5 pM of an oligonucleotide is mixed with 0.8 mM of dTTP, 0.1 mM of dUTP conjugated with fluorescent molecules (such as Alexa 546-dUTP), and 1.5 U/μL of terminal deoxynucleotidyl transferase (TdT; #M1871 Promega) in transferase buffer. The solution is incubated at 37°C for 1 h before inactivating TdT at 70°C for 10 min. Labeled oligonucleotides are separated from unincorporated nucleotides using a G25 gel filtration column (Miniquick column #11814397001 Sigma) and stored at − 20°C.

Matured flies are dissected in methanol, ovaries dechorionated by sonication, and oocytes rehydrated and collected, in the same way as for immunostaining, described in Section 2.1.1. 20 × SSC buffer (3 M NaCl, 0.3 M trisodium citrate, pH 7) is prepared. Oocytes are postfixed in 200 μL of PBS + 8% formaldehyde (Paraformaldehyde 32% Solution EM grade #15714 Electron Microscopy Sciences) for 2 min at 37°C and rinsed twice in 300–600 μL of 2 × SSCT buffer (10% 20 × SSC, 0.1% Tween-20). Oocytes are washed at least 10 min in 2 × SCCT. They are then washed for 10 min in 400 μL of freshly made 50% formamide buffer (10% 20 × SSC, 50% formamide, 0.1% Tween-20) and incubated in 400 μL of 50% formamide buffer for 1 h at 37°C. 50% formamide buffer is entirely removed and replaced by 40 μL of the probe solution made by mixing 4 μL of the probes with 36 μL of 1.1 × hybridization buffer (16.67% 20 × SSC, 55.56% formamide, 0.111 g/mL dextran sulfate sodium salt, pH 7). Oligonucleotides are denatured for 2 min at 91°C and hybridized from 1 h to overnight at 30°C. Oocytes are washed twice with 500 μL of 30°C preheated 50% formamide buffer for a total of 30 min at 30°C. Oocytes are rinsed twice with 2 × SSCT and washed 10 min in 2 × SSCT. For simultaneous immunostaining, oocytes are washed in PBS and the immunostaining protocol can be resumed from blocking in PBS–T + 10% FCS followed by antibody incubation. With this method, it is possible to visualize the chromosomes (DAPI), the spindle (DM1A), and the centromere of the third chromosome (dodeca satellite), showing biorientation of centromere three within the bipolar meiotic spindle (Zhaunova et al., 2016; Fig. 2).