During what phase of mitosis do spindle fibers breakdown and a nuclear envelope forms around chromosomes?

Prometaphase–metaphase

As the nuclear envelope disappears, the spindle microtubules extend into the central region of the cell, attaching to the chromosomes, which subsequently move towards the equator of the spindle (prometaphase). The spindle consists of kinetochore microtubules attached to the kinetochore, a multiprotein structure assembled at the centromeric DNA region, and polar microtubules, which are not attached to chromosomes but instead overlap with each other at the centre of the cell. The grouping of chromosomes at the spindle equator is called the metaphase or equatorial plate. The chromosomes, attached at their centromeres, appear to be arranged in a ring when viewed from either pole of the cell, or to lie linearly across this plane when viewed from above. Cytoplasmic movements during late metaphase effect the approximately equal distribution of mitochondria and other cell structures around the cell periphery.

Prometaphase

Prometaphase begins when the nuclear envelope disassembles, exposing nuclear structures and its contents to the cytoplasm. The nuclear envelope is composed of two membrane barriers, the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). The nuclear envelope is stabilized by nuclear lamina (polymerization of lamin proteins) underlying the INM. NEB is thought to involve early spindle microtubules directly piercing the nuclear envelope causing folds and invaginations that create mechanical tension in the nuclear lamina (Beaudouin et al., 2002; Georgatos et al., 1997). Lamins are subsequently phosphorylated by Cdk1/cyclin B1 triggering their depolymerization, causing the nuclear envelope to vesiculate (Gallant and Nigg, 1992; Heald and McKeon, 1990; Peter et al., 1990). Concurrently, nuclear pore complexes (NPCs) are disassembled by phosphorylation of the core components, nucleoporins, resulting in complete dissolve of the membrane permeability barrier (Terasaki et al., 2001).

Following NEB, the mitotic spindle is assembled for alignment of chromosomes along the center of the cell (metaphase plate) and subsequent equal segregation of sister chromatids (anaphase) into daughter cells (Helmke et al., 2013; Rieder, 2005). The mitotic spindle is a bipolar structure composed of microtubules and associated motor proteins. Microtubules initially emanate from the spindle poles/centrosomes toward the cell equator and with their plus-ends attach to a large protein assembly on the centrometric chromatin of a chromosome called the kinetochore (Figure 3). This population of microtubules is termed kinetochore- or K-fibres. This attachment allows rapid movement of the bound chromosome such that the sister kinetochore attaches to a microtubule growing from the opposing centrosome. The result is a correctly bi-orientated chromosome at the metaphase plate with a stable microtubule–kinetochore attachment. Once all chromosomes have achieved this, the cell is considered to be in metaphase and chromosome segregation during anaphase can proceed. The mitotic spindle consists of two other populations of microtubules: (1) microtubules that do not attach to kinetochores, but also emanate toward the cell center and are called central spindle or non-kinetochore microtubules and (2) microtubules that emanate from the centrosomes circumferentially and anchor at the cell cortex are called astral microtubules, which function to maintain correct spindle orientation. Proteomics of purified mitotic spindles has identified approximately 800 spindle-associated proteins (Sauer et al., 2005), indicating the breadth of signaling pathways required to establish and maintain this structure for correct chromosome attachment and alignment. Future investigation of these proteins in functional studies will aid in our understanding.

Prometaphase

Prometaphase begins when the nuclear envelope disappears.

Prometaphase begins as the nuclear lamins are phosphorylated, resulting in the breakdown and disappearance of the nuclear envelope. During this phase, the chromosomes are arranged randomly throughout the cytoplasm; each chromosome is composed of two sister chromatids held to each other by a complex of proteins, known ascohesins andcondensins. Microtubules that become attached to the kinetochores are known askinetochore microtubules, whereas microtubules that do not become incorporated into the spindle apparatus are calledpolar microtubules.

(c) Prometaphase

At prometaphase (late prophase) the chromosomes condense inside the nuclear envelope and asters of fibers appear on the outside of the chromosomes. When the nuclear envelope has disappeared, a spindle forms in prometaphase. The spindle fibers comprise bundles of microtubules radiating from the opposite ends and referred to as poles of the cell. The chromosomes then migrate to the equatorial plane where they attach to one of the spindle fibers. In animal cells, spindle formation occurs by centrosomes, which are composed of twin centrioles at right angles surrounded by amorphous material. The centrosome is the major microtubule-organizing center during interphase. The centrosome replicates in late G1 and S phases and the pair can be observed just outside the nuclear envelope. However, higher plants have no characterized centrosomes although the nucleation and dynamics of their microtubules suggest that plants possess cell cycle-dependent microtubule organizing center (MTOC) activities. Initiation of microtubule polymerization within a cell usually occurs at specific nucleating sites referred to as MTOCs. In most higher plants, initiation of mitosis is characterized by two successive events involving different microtubule populations. These are the production of the preprophase band and the development of the bipolar spindle. Cytoplasmic microtubules of higher plants radiate from the surface of the nucleus towards the cell cortex. This is one of the particular aspects of the plant cytoskeleton in comparison with other cell types. The plant nuclear surface may comprise an MTOC activity and this may represent one of the important factors in the control of the initiation of mitosis. There is experimental evidence to support the role of the plant nuclear surface as a microtubule nucleation site. When mitosis begins and chromosomes undergo condensation, tubulin incorporation has been shown to increase on the nuclear surface of Haemanthus endosperm cells in prophase, potentially a cell-cycle-dependent control of nucleation. The formation of the bipolar spindle (Fig. 1.22) around the nucleus is achieved through a transient convergence of microtubules forming aster-like centers, which subsequently produce spindle poles. This increased microtubule interaction is believed to be mediated by specific microtubule-associated proteins (MAPs). Calmodulin is found at higher concentration at the centriolar polar regions of animal cells. It is also found in these prophase microtubule centers, suggesting that there is a calmodulin-regulated mechanism in higher plants.

The important chromosomal event of prometaphase is the attachment of the chromosomes to the spindle and their movement towards the center of the spindle. Attachment of the chromosome to the spindle occurs at the kinetochore, which contains proteins for chromatid attachment. The breakdown of the nuclear envelope permits the kinetochores to attach to the spindle microtubules.

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.

3.2 Imaging acquisition and data analysis

Images of prometaphase and metaphase spindles were acquired with a cooled charge-coupled device camera (Andor Technology) mounted on a Nikon Eclipse Ti microscope with a Plan Apo VC 60 ×,1.4 NA, oil immersion objective (Nikon). Image series in the z-axis were obtained using 0.3 μm optical sections (23 slices). For both the nocodazole shock and cold stable assays, raw immunofluorescence images of spindles were classified as either “spindle-like,” “high polymer,” or “low polymer” (Gayek & Ohi, 2014). Image deconvolution and contrast enhancement for representative images was performed using AutoQuant X3 (Media Cybernetics) and Fiji (Schindelin et al., 2012).

Chromosome Attachment to the Spindle

Dynamic microtubules of prometaphase asters scan the cytoplasm effectively “searching” for binding sites that will capture and stabilize their distal plus ends. Captured microtubules are approximately fivefold less likely to depolymerize catastrophically than free microtubules. When catastrophes do occur, the microtubules depolymerize back to the pole, recycling tubulin subunits for incorporation into other, growing microtubules.

Breakdown of the nuclear envelope makes the condensed chromosomes accessible to the microtubules. Chance encounters allow kinetochores to capture microtubule plus ends. Capture probably involves the nine-component KMN network, which includes the rod-shaped Ndc80 complex (see Fig. 8.21) that binds along the sides of microtubules near their plus ends. Another member of the complex, the scaffolding protein Knl1 (its name in vertebrates—the “K” of KMN; see later), anchors Ncd80 in the kinetochore.

Historically, it was thought that forces generated by bipolar attachment of the kinetochores of sister chromatids center chromosomes midway between the two spindle poles. This hypothesis was based on the observation that when a kinetochore first attaches to a microtubule, the chromosome moves along the side of that microtubule toward the spindle pole (Fig. 44.9). Subsequent capture of a microtubule emanating from the opposite spindle pole by the sister kinetochore would provide a counterforce pulling the chromosome in the opposite direction. Chromokinesin family motor proteins distributed along the chromosome arms were also thought to contribute to the gradual movement of the chromosome toward the middle of the spindle. These movements are accompanied by coordinated shrinkage of the microtubules at the leading kinetochore and growth of microtubules at the trailing kinetochore.

More recent studies revealed that chromosomes attached to only one spindle pole can move away from that pole if the unattached kinetochore associates with the kinetochore fiber of a chromosome already aligned at the spindle equator. In this case, the kinetochore of the mono-oriented chromosome glides toward the equator, where it is more likely to capture microtubules emanating from the opposite pole. This motion of one chromosome along the kinetochore fiber of another chromosome requires the kinesin-7 motor centromere protein E (CENP-E) (see Fig. 36.13) associated with the kinetochore of the moving chromosome. Recognition of a tubulin posttranslational modification leads CENP-E to move the chromosome toward the spindle equator, rather than out into the aster.

The attachment of microtubules to kinetochores can be reconstituted in vitro from mixtures of chromosomes, isolated centrosomes, and tubulin subunits. The plus ends of microtubules grow out from centrosomes and attach to the chromosomes. Surprisingly, chromosome-bound microtubules can either lengthen or shorten at the attached end without detaching from the chromosome. Similar experiments with kinetochores isolated from budding yeast cells showed that kinetochores can remain attached to a shortening microtubule plus end even against an applied force of 9 pN (piconewtons). Physiological levels of tension actually stabilize the attachments of kinetochores to microtubules in vitro, as in vivo. This tethering of kinetochores to disassembling microtubules is essential for chromosome movements during mitosis.

3.4 CDK1 and exit from meiosis I

Upon entry into prometaphase, rising CDK1 activity drives the formation of the spindle. However, once the bivalents become sufficiently attached to the spindle to activate the APC, a role reversal occurs and the spindle then controls CDK1 activity. During GV arrest, it is APCFZR1 that is used to control cyclin B1 levels (Section 2.3), but soon after GVB, FZR1 levels drop and APCFZR1 activity wanes (Reis et al., 2007). From somatic cell studies, it has been established that FZR1 is negatively regulated by CDK1 activity, thus after GVB, we would predict such a decrease in APCFZR1. However, the rising CDK1 activity actually promotes CDC20 as a coactivator of the APC, and as such it is APCCDC20 that is tasked with degrading both cyclin B1 and securin, so permitting meiotic exit (Jin et al., 2010; Reis et al., 2007). However, as described above, this activity is initially held in check by the SAC.

The satisfaction of the SAC and the consequent increase in APCCDC20 activity starts to lower the levels of cyclin B1 in the oocyte causing a drop in CDK1 activity (Hampl & Eppig, 1995). This, and the equivalent drop in securin permit the activation of separase, which by cleaving the cohesin rings allows the disjunction of the sister chromatid pairs making up each bivalent (Herbert et al., 2003). Due to their opposing poleward orientation, the two chromatid pairs are pulled away from each other along the lengthening spindle. In mitosis, this process is dependent on low CDK1 activity as an artificial anaphase in the presence of high CDK1 activity (in metaphase) results in a very ineffective anaphase (Oliveira, Hamilton, Pauli, Davis, & Nasmyth, 2010).

Unlike dividing somatic cells, which follow division with DNA replication, oocytes enter directly into meiosis II. There is only a brief, partial decondensation of the chromatin, the nuclear envelope does not reform, and there is no DNA synthesis. This may be because during this brief period separating the two meiotic divisions, termed interkinesis, CDK1 activity does not decline completely (Hampl & Eppig, 1995), allowing rapid entry into meiosis II. It is interesting to note that in mouse oocytes, this rapid reactivation of CDK1 activity may be helped, at least in part, by not fully degrading all of the oocyte's cyclin B1. This would allow rapid CDK1 activation by virtue of it being independent of de novo cyclin B1 synthesis. However, this creates a problem, in that the residual CDK1 activity, caused by persistent cyclin B1, could block meiotic exit. It seems likely that this is resolved by free separase, which can bind CDK1 and in so doing inhibit the activity of this kinase (Gorr, Boos, & Stemmann, 2005). In mouse oocytes, when the interaction of free separase with CDK1 is blocked using an antibody raised against the region in separase that binds CDK1, polar body extrusion is inhibited although this does not interfere with the ability of separase to cleave cohesins (Gorr et al., 2006).

Prometaphase

The nuclear membrane fragments during prometaphase and the mitotic spindle, consisting of microtubules and associated proteins, forms between the two centrioles. Bundles of microtubules from the centrioles form spindle fibers that extend between the poles of the cell and interact with kinetochores that are located at the centromere region of each chromosome (Fig. B6a). Some spindle fibers attach to the kinetochores and are the kinetochore microtubules of the mitotic spindle; the nonkinetochore microtubules extend between the poles of the cell without attaching to the chromosomes.

In electron micrographs, centrioles appear as cylindrical structures which occur in pairs lying at right angles to each other (Figs. B6b and B7). Each cylinder is closed at one end and consists of nine parallel, overlapping “blades,” each blade a simple “pipe of Pan” composed of three fused microtubules. The centrioles divide before prophase and, at the beginning of prophase, four centrioles may be seen together. The daughter centriole is always arranged at right angles to the parent.

B Metaphase Arrest to Reduce the Effect of Over-duplicated Centrosomes

Centrosomes are clustered and fused during prometaphase and metaphase in S2 cells (Goshima and Vale, 2003). Therefore, most of the cells arrested in metaphase have two centrosomes. This is achieved through RNAi knockdown of the subunits of APC/C (E3 ubiquitin ligase) that is required for the destruction of anaphase inhibitors (Goshima et al., 2007) or inhibition of the proteasome by MG132 treatment (Kwon et al., 2008). APC/C knockdown [e.g., Cdc27 or Cdc16 (Goshima et al., 2007)] also increases the number of metaphase cells 5–10-fold, and therefore, it becomes dramatically easier to detect mitotic cells under the microscope. However, there are two precautions to bear in mind when using this method. First, later mitotic events, such as anaphase or cytokinesis, are very rarely seen due to strong metaphase arrest. Second, chromosome alignment is often impaired through prolonged arrest (Goshima et al., 2007). The metaphase image galleries displayed at http://rnai.ucsf.edu/mitospindlescreen/index.html (Goshima et al., 2007) suggest that only ∼20% of the cells have perfectly aligned chromosomes in the Cdc27-arrested condition. Nevertheless, metaphase arrest method is a powerful approach for assessing chromosome alignment defects; when essential kinetochore components are knocked down, for example, the extent of misalignment becomes very drastic and can easily be identified.