During which stage of mitosis sister chromatids are now in opposite poles and start to Decondense back to chromatin fibers?

19.1.5 Telophase

Telophase begins when the decondensing daughter chromatids arrive at the poles, the kinetochore microtubules disappear, and the nuclear envelope reforms around the decondensing chromosomes to form the two daughter nuclei (Benavente, 1991). Nuclear lamins may specifically interact with chromatin to promote nuclear envelope reassembly (Glass and Gerace, 1990). The chromatin begins to decondense and the nucleoli reappear. During telophase, all the nuclear proteins, including RanGTP (Pay et al., 2002), must be rounded up and brought back to the nucleus (see Chapter 16).

2 Connecting Cell Cortex to Nuclei

At telophase of divisions when two daughter nuclei are formed, WRM-1 localized preferentially to the posterior than anterior nuclei (Fig. 3.1C) (Takeshita & Sawa, 2005; Nakamura et al., 2005). This is in good contrast to its anterior cortical localization that is still observed during telophase. Photobleaching experiments revealed that WRM-1 in the anterior cytoplasm and nucleus as well as that in the posterior side accumulates in the posterior nucleus and that the nuclear export rates of WRM-1 are higher in the anterior nucleus. This nuclear asymmetry of WRM-1 is regulated by WRM-1 itself on the anterior cortex, as expression of WRM-1::CAAX that uniformly localized to the cortex inhibits WRM-1 localization in both nuclei (Mizumoto & Sawa, 2007a). Cortical WRM-1 recruits APR-1 to the anterior cortex. In apr-1 mutants, WRM-1 nuclear export is inhibited, resulting in its localization in both nuclei. Thus, APR-1 on the cortex mediates the effects of WRM-1 in the inhibition of WRM-1 nuclear localization.

In other organisms, it is well known that APC functions in the degradation of β-catenin (Cadigan & Peifer, 2009; MacDonald, Tamai, & He, 2009). However, in asymmetric cell division in C. elegans, levels of WRM-1/β-catenin are not affected in apr-1 mutants. APC is also known to stabilize microtubules by binding to their plus ends in mammalian cells (Dikovskaya, Zumbrunn, Penman, & Näthke, 2001). Although this function of APC has not been shown to regulate β-catenin, we have recently showed that APC regulates β-catenin nuclear localization through microtubules in the EMS blastomere (Sugioka et al., 2011). APR-1 on the anterior cortex stabilizes astral microtubules, creating asymmetry of spindle (more astral microtubules from the anterior spindle pole than from the posterior one) (Fig. 3.1C). Disruption of this spindle asymmetry by laser irradiation of the anterior spindle pole disrupted nuclear asymmetry of WRM-1, while the enhancement of the spindle asymmetry by the posterior irradiation caused concomitant increase of WRM-1 nuclear asymmetry. Further, the posterior irradiation in mom-2/Wnt mutants in which asymmetry of spindle and nuclear WRM-1 is disrupted rescued asymmetric POP-1/TCF localization (see below) regulated by nuclear WRM-1. These results showed that spindle microtubules stabilized by APR-1 enhance export of WRM-1 from the anterior nucleus, creating its nuclear asymmetry. How spindle regulates WRM-1 nuclear export is not known. The requirements of kinesin for WRM-1 localization raised the model that microtubule-dependent transport of WRM-1 toward the cell cortex removes it from the perinuclear region, enhancing its nuclear export (Sugioka et al., 2011).

F Telophase

The main events of telophase include a reappearance and enlargement of the nucleolus, enlargement of the daughter nuclei to their interphase size, decondensation of the chromatin resulting in a brighter appearance of the nuclei with phase-contrast optics, and a period of rapid, postmitotic nuclear migration during which the daughter nuclei become positioned prior to septum formation (Aist, 1969, 1995). Although the natural breaking of the spindle is used to define the onset of telophase (Bayles et al., 1993), telophase events involving the nucleolus, the chromatin, and nuclear size frequently begin moments before the spindle breaks. Thus, there is sometimes overlap between the anaphase B and telophase stages regarding the behavior of the various nuclear components. This is one reason why it is helpful to use only one of several available criteria, (i.e., spindle breakdown) to define the starting point for telophase. The other reason is that the daughter nuclei are not truly independent of each other until spindle breakdown; therefore, technically, the nucleus is still dividing.

CPC in Mitosis

From prophase to telophase, localization of the CPC is dynamic and is an indication of the multiple roles the CPC plays in regulating mitotic progression and cell division. During early mitosis, the CPC is found at centromeres and diffusely localized along chromosome arms. Another key mitotic role of Aurora B is carried out during prophase. Along with Cdk1, Aurora B contributes to sister chromatid resolution by phosphorylating the cohesion-stabilizing protein Sororin (Losada, 2014; Dreier et al., 2011). Phosphorylated Sororin dissociates from the cohesion subunit Pds5, which ultimately results in WapL-mediated release of cohesion from chromosome arms. Polo-like kinase is also essential for prophase removal of cohesin, and works by phosphorylating cohesin subunits (Losada, 2014). Cells in which the prophase removal pathway is inhibited show an increase in chromosome loss upon completion of mitosis, indicating the importance of this pathway in maintaining genomic stability (Haarhuis et al., 2013). As mitosis progresses, the CPC concentrates at inner centromeres where it participates in an essential, evolutionarily conserved surveillance mechanism required for high-fidelity chromosome segregation. The spindle assembly checkpoint (also known as the mitotic checkpoint) blocks entry into anaphase until all chromosomes attain a bipolar attachment to the spindle. The best recognized role of the CPC in this process is in ‘error correction’ where inappropriate spindle–kinetochore attachments are converted to unattached kinetochores that trigger the SAC.

The phragmoplast constructs the cell wall that will partition the cytoplasm and separate the two daughter nuclei of the dividing cell

The phragmoplast forms in late telophase, as the spindle disappears. It consists of two sets of parallel microtubules, both oriented at right angles to the division plane (Fig. 5.31). The two sets of microtubules overlap at their tips and have the same polarity. Their plus ends are in the equatorial plane where they overlap; their minus ends are nearer the poles. Microfilaments also are present in the phragmoplast and connect the phragmoplast to cortical cytoplasm adjacent to the lateral walls at the site of the PPB. The phragmoplast begins to form in late telophase and it represents a new site of microtubule assembly. The microtubules of the mitotic spindle have largely disappeared by the time the phragmoplast is formed, although some polar spindle microtubules may be recruited for the phragmoplast, and many additional microtubules are assembled to form the dense phragmoplast array. The cell plate, which is the new cell wall that will separate the daughter cells, is constructed in the region of the phragmoplast where the ends of the microtubules overlap. It consists largely of noncellulosic polysaccharides, which are synthesized in the Golgi and transported to the cell plate in Golgi-derived vesicles by the phragmoplast microtubules. The vesicles fuse in the equatorial plane and the noncellulosic polysaccharides they contain become the middle lamella of the cell wall that will divide the daughter cells. The vesicle membrane becomes the plasma membrane. Microfilaments radiate out from the phragmoplast to the peripheral cytoplasm, into the cortical cytoplasm (Fig. 5.32). These microfilaments probably orient the growing cell plate, ensuring that it will insert into the site occupied by the preprophase band before the initiation of mitosis.

Telophase

During telophase, the nuclear envelope reforms on the surface of the separated sister chromatids, which typically cluster in a dense mass near the spindle poles (Fig. 44.18). Some further anaphase B movement may still occur, but the most dramatic change in cellular structure at this time is the constriction of the cleavage furrow and subsequent cytokinesis.

2.3.2 In vivo reassembly

During mitosis, nucleoli disassemble during prophase and reassemble in telophase (Sirri et al., 2008). The nucleolus has been described as “an organelle formed by the act of building a ribosome” (Mélèse and Xue, 1995) and when transcription is repressed its components in part stay associated to rDNA in the NOR (Roussel et al., 1996) and in part migrate as chromosomal passengers (Hernandez-Verdun and Gautier, 1994).

At the moment of rDNA transcription restart, nucleoli are again formed via PNB formation (Dundr et al., 2000) via a progressive recruitment of proteins involved in early and late processing. PNBs, with their content of nucleolar processing proteins, pre-rRNAs and small nucleolar RNAs (snoRNA), play a role that has not yet been completely clarified. Moreover, it seems clear that proteins with a different functional role leave the PNBs at different moments. Recently, Muro et al. (2010) have demonstrated that fibrillarin passes from one incipient nucleolus to another without stopping in PNBs, while other proteins like B23 shuttle between PNBs and nucleoli. The difference in this traffic would suggest a way of regulating the assembly first of the DFC and then of the GC, and this mechanism would involve the Cajal bodies.

Several factors are probably involved in the rebirth of a nucleolus. Transcription itself is not sufficient to start the event (Section 2.3.1) but nucleolar assembly can start independently of rDNA transcription (Dousset et al., 2000). Apparently a paradox: transcription arrest means disassembly, reassembly does not mean transcription restart. Other factors, such as CDK, may intervene to regulate both transcription and processing (Sirri et al., 2008). The final assembly is rather rapid and very probably a “prenucleolar” interaction of processing proteins is required.

If one considers the incredible amount of proteins that disassemble and reassemble during mitosis, and that most of them redistribute at different locations and then are recruited to PNBs, it is not clear what could be the driving force behind. Diffusion is the easy answer for the movements, and indeed a part of nonribosomal proteins show a nucleolar localization signal (NLS), but not all of them possess this feature (Jacobson and Pederson, 1998).

Diffusion can account for a series of movements, although mediated by signal recognizing sequences, but necessity of order and time might involve other mechanisms. It is known that some proteins are recruited from PNBs in a specific, sequential order (Louvet et al., 2008). It is difficult in this case to imagine diffusion as the only mechanism. As described for other nucleolar functions such as transcription (Dundr et al., 2002) or ribosome subunit movement (Cisterna et al., 2006, 2009) there could be place for motor proteins to give directionality (impossible in diffusion mechanisms), time schedule (also possible only in active mechanisms), and releasing order, if any. The coordination found in the movements of nucleolar proteins suggests that they can maintain their interaction during mitosis; however, the mechanisms behind the interactions are still not clear. The interaction has been clearly shown by FRET analysis (Angelier et al., 2005).

Sources and Preparation of Chromosomes from Insects

Mitotic chromosomes undergoing the stages of prophase, metaphase, anaphase, and telophase can be prepared from any insect somatic tissues with dividing cells. Embryos are the best sources of mitotic divisions, but they are also seen in the midgut ceca of adults and juveniles and in the follicle cells covering very early ova in females.

Insect cytogeneticists now usually use colchicine or other mitostatic agents to arrest the chromosomes at metaphase of mitosis by inhibiting the formation of the spindle fibers required for the cells to progress to anaphase. Squashing, under a coverslip, spreads the chromosomes, and for squash preparations the cells are usually prestained. Insect cytogeneticists now often use air-drying to spread the chromosomes, since this process has the advantage of making the chromosomes immediately available for modern banding and molecular cytogenetic methods.

Male meiosis is very commonly used to study the chromosomes of insects and to analyze sex-determining mechanisms. The structure of the insect testis is very favorable to chromosomal studies because each lobe has a single apical cell that divides by a number (s) of spermatogonial divisions (Fig. 1A to yield 2S primary spermatocytes, which then undergo synchronous first and second meiotic divisions to yield 2S+1 secondary spermatocytes and 2S+2 sperm.

First meiotic prophase in insects involves the usual stages (Fig. 1). Replication of the DNA is followed by the prophase stages of leptotene (strand forming), zygotene (chromosome pairing to form bivalents), pachytene (crossing over to yield recombinants), diplotene (repulsion of the homologues), diakinesis (completion of repulsion), and premetaphase (bivalents fully condensed).

Metaphase I is followed by first anaphase, which can be a very informative stage and, in contrast to mammals, is readily available in insects. Second meiotic division is also readily observed in insects (Fig. 1) and can be useful for confirming events in earlier stages.

Meiotic chromosomes in insect females are difficult to prepare and are usually studied only in special cases, such as parthenogenesis.

Mitosis

Mitosis is divided into four phases: prophase, metaphase, anaphase, and telophase. Interphase is the interval from the end of mitosis until the beginning of the next. Each cell division begins with a phase of DNA replication, referred to as S phase. DNA replication results in two sister chromatids for each chromosome. Prophase is marked by gradual condensation of the chromosomes, disappearance of the nucleolus and nuclear membrane, and the beginning of the formation of the mitotic spindle. At metaphase, the chromosomes become arranged on the equatorial plane, but homologous chromosomes do not pair. In this stage, chromosomes also reach maximum condensation. In anaphase, the chromosomes divide at the centromeric regions, and the two chromatids separate and migrate to opposite poles. Telophase begins with the formation of the nuclear membranes and division of the cytoplasm (Fig. 16.10).

2.5 Mitotic exit and cytokinesis

MTs are also necessary for changes in cell shape and size during anaphase and telophase. Upon anaphase onset, depolymerization of spindle MTs has to be compensated by an increase in astral MT polymerization/elongation (Morrison and Askham, 2001; Strickland et al., 2005b). Elongation of astral MTs is necessary for their interaction with the cell cortex and definition of the cytokinetic furrow, but apparently is not essential for anaphase progression itself, as the cytokinetic furrow can still be formed even in the absence of astral MTs (Rankin and Wordeman, 2010; Strickland et al., 2005a,b; Sullivan and Huffaker, 1992).

MT reorganization during mitotic exit is strictly associated with the inactivation of the mitotic kinase CDK1, which triggers the formation of anaphase MTs and the midbody (Wheatley et al., 1997). A similar phenomenon was also observed in Drosophila S2 cells and shown to involve acentriolar MT-organizing centers (aMTOCs). These aMTOCs were able to nucleate MTs de novo upon CDK1 inhibition at anaphase onset (Moutinho-Pereira et al., 2009), and this was dependent on the activity of Msps/XMAP215 and KLP10A/kinesin-13. This reorganization also depends on the precise regulation of MT dynamics and allows daughter cells to adhere simultaneously to the substrate (Ferreira et al., 2013).

Cytokinesis relies on MTs in several ways. First, definition of the cleavage plane is specifically determined by astral MTs (and not spindle MTs) as furrowing still occurs in the presence of asters without any intervening spindle (Rieder et al., 1997). However, successful completion of cleavage does require interaction of midzone MT bundles with the cell cortex (Wheatley and Wang, 1996). Moreover, if anaphase astral MT formation is suppressed by interfering with the + TIP EB1 or with dynactin, cytokinesis is delayed (Strickland et al., 2005b), which supports the necessity of MT interaction with the cortex to define cleavage plane localization (Bement et al., 2005; Strickland et al., 2005a). At this stage, regulation of MT dynamics seems to be dispensable, as contact of MTs with the cortex is sufficient to trigger the process. In contrast with earlier stages of cytokinesis, MTs are essential for completion of the process (Savoian et al., 1999). MTs that establish the midbody are acetylated, highly stable (Margolis et al., 1990), and resistant to nocodazole treatment (Foe and von Dassow, 2008; Piperno et al., 1987). Nevertheless, some midbody MTs are still able to exhibit a highly dynamic behavior as can be seen by live imaging of MT plus ends with fluorescent-tagged EB proteins, which show comets moving in and out of the midbody (Rosa et al., 2006). Thus, it is not surprising that γ-tubulin was found in the midbody during late cytokinesis (Julian et al., 1993), suggesting active MT nucleation. Notably, γ-tubulin interacts with the Augmin complex during anaphase, and this is required for MT nucleation in the central spindle and successful cytokinesis (Uehara et al., 2009). Final disassembly of the midbody requires that MTs are cut, which is accomplished by a mechanism that involves the MT-severing enzyme spastin (Guizetti et al., 2011).