Of all the processes that permit the existence and perpetuation of life, perhaps the most critical is the ability of cells to accurately duplicate and then faithfully segregate their chromosomes at each cell division. During the cell cycle, a highly regulated process discussed in Chapter 19, cells duplicate their chromosomes precisely once during a period known as S phase (for DNA synthesis phase). Once the individual chromosomes have been duplicated, they are held together by proteins called cohesins. The cells then pass through a period called G2 (for gap 2) before entering mitosis, the process by which the duplicated chromosomes are segregated to the daughter cells. This process has to be very precise—loss or gain of a chromosome can either be lethal to the cell (in which case it is often not detected) or cause severe complications for the cell. It is estimated that yeast only mis-segregates one of its 16 chromosomes every 100,000 cell divisions, which makes mitosis one of the most accurate processes in biology. To achieve this accuracy, the process must be highly regulated so that it proceeds in an orderly series of steps and errors are not made. The timing and mechanisms that ensure the fidelity of mitosis are closely regulated by the cell cycle circuitry. Here we limit our discussion of this circuitry as it applies to microtubules and the mechanics of mitosis.
Centrosomes Duplicate Early in the Cell Cycle in Preparation for Mitosis
In order to separate the chromosomes during mitosis, cells duplicate their MTOCs—their centrosomes—in a manner coordinated with the duplication of their chromosomes in S phase (Figure 1). the cell cycle is largely driven by the association of cell-cycle-specific cyclins with cyclin-dependent kinases (CDKs). Two kinases— G1/S phase CDKs and another type called Plk4—promote centrosome duplication. The duplicated centrosomes separate as the cells enter mitosis in a process known as centrosome disjunction, and then nucleate the assembly of microtubules to become the two poles of the mitotic spindle. The number of centrosomes in animal cells has to be very carefully controlled. In fact, many tumor cells have more than two centrosomes, which contributes to genetic instability resulting from mis-segregation of chromosomes and hence aneuploidy (unequal numbers of chromosomes).
Fig1. Relation of centrosome duplication to the cell cycle. Centrosome duplication, which is initiated by the G1/S phase CDKs and Plk4 (step 1 ), results in the pair of centrioles (green) separating and a daughter centriole (blue) budding from each. By the G2 phase, growth of the daughter centrioles is complete, but the two pairs of centrioles remain within a single centrosomal complex. Early in mitosis, driven by the activation of M phase CDKs (step 2 ), the centrosome splits, each half nucleates assembly of microtubules, and the two centriole pairs are moved to opposite sides of the nucleus. The amount of pericentriolar material and the microtubule nucleation activity of the centrosomes in creases greatly in mitosis. In mitosis, each MTOC is called a spindle pole.
As cells enter mitosis, the activity of the two MTOCs— their ability to nucleate microtubules—increases greatly as they accumulate more pericentriolar material. Because the assemblies of microtubules radiating from these two MTOCs now resemble stars, they are often called mitotic asters.
Mitosis Can Be Divided into Six Stages
Mitosis has been divided up into several stages for ease of description (Figure 2a), but in reality it is a continuous process. Here we review the major events of each stage.
Fig2. The stages of mitosis. (a) Upper panels show stages in cultured cells stained blue for DNA and green for tubulin. Lower diagrams show the different stages and the events that occur in each. Mitosis is a continuous process that has been divided into stages for ease of description. (b) Parts of a condensed chromosome in mitosis. The duplicated chromosome consists of two sister chromatids (each is a single DNA duplex), held together by cohesins at a constricted region called the centromere. The centromere is also the site where the kinetochore, which will make attachments to the kinetochore microtubules, forms. [Part (a) micrographs courtesy of Torsten Wittmann.]
The first stage of mitosis, called prophase, is signaled by a number of coordinated and dramatic events. First, the interphase array of microtubules is replaced as the du plicated centrosomes become more active in microtubule nucleation. This activity provides two sites of assembly for dynamic microtubules, forming the mitotic asters. Additionally, the dynamics of the growing microtubules them selves increase due to changes in the activities of +TIPs at their (+) ends. The two asters are then moved to oppo site sides of the nucleus by the action of bipolar kinesin-5 motors that push the intermingling microtubules apart. The separated centrosomes will become the two poles of the mitotic spindle, the microtubule-based structure that separates chromosomes. Second, protein syn thesis is switched from being dependent to independent on the mRNA 5′ cap (see Section 5.4), and the internal mem brane systems, normally dependent on the interphase array of microtubules, are disassembled. In addition, endocytosis and exocytosis are halted, and the microfilaments are generally rearranged to give rise to a rounded cell. In the nucleus, the nucleolus breaks down and chromosomes begin to condense. The cohesins holding together each pair of duplicated chromosomes, or sister chromatids as they are called at this stage, are degraded except at the centromeric region, where the two sister chromatids remain linked by intact cohesins (Figure 2b). Also during prophase, specialized structures called kinetochores, which will become sites of microtubule attachment, assemble at the centromeric region of each sister chromatid. all these events are coordinated by a rapid increase in the activity of the mitotic cyclin-CDK complex, which is a kinase that drives cell cycle progression by phosphorylating multiple proteins.
The next stage of mitosis, prometaphase, is initiated by the breakdown of the nuclear envelope and its retraction into the endoplasmic reticulum, the disassembly of nuclear pores, and the disassembly of the lamin-based nuclear lamina. Microtubules assembled from the spindle poles search for and “capture” chromosome pairs by associating with their kinetochores. Each chromatid has a kinetochore, so a sister chromatid pair has two kinetochores, each of which becomes attached through the microtubules to a different spindle pole during prometaphase in a critical process we discuss in detail below. The chromosome pairs then become aligned at a point equidistant between the two spindle poles. Prometaphase continues until all chromosomes have become aligned, at which point the cell enters the next stage, metaphase.
When the cell detects that all chromosomes are attached to the spindle correctly, the next stage, anaphase, is induced by activation of the anaphase-promoting complex or cy closome (APC/C). The activated APC/C (through several intermediary steps) ultimately leads to the destruction of the cohesins that were holding the sister chromatids together, so that each chromatid can be pulled to its respective pole by the microtubules attached to its kinetochore. This movement is known as ana phase A. A separate and distinct movement also occurs: the movement of the spindle poles farther apart in a process known as anaphase B. Now that the chromosomes have separated, the cell enters telophase, when the nuclear envelope re-forms, the chromosomes decondense, and the cell is pinched into two daughter cells by the contractile ring during cytokinesis.
The Mitotic Spindle Contains Three Classes of Microtubules
Before we discuss the mechanisms involved in the remark able process of mitosis, it is important to understand the three distinct classes of microtubules that emanate from the spindle poles, which is where their (−) ends are embedded. The astral microtubules extend from the spindle poles to the cell cortex (Figure 3). By interacting with the cortex, the astral microtubules perform the critical function of orienting the spindle to the axis of cell division. The kinetochore mi crotubules function by a search-and-capture mechanism to link the spindle poles to the kinetochores on sister chromatid pairs. During anaphase A, the kinetochore microtubules transport the newly separated chromosomes to their respective poles. The polar microtubules extend from each spindle pole toward the opposite one and interact in an antiparallel manner. These microtubules are responsible initially for pushing the duplicated centrosomes apart during prophase, then for maintaining the structure of the spindle, and then for pushing the spindle poles apart in anaphase B. 
Fig3. Mitotic spindles have three distinct classes of microtubules. (a) In this high-voltage electron micrograph, micro tubules were stained with biotin-tagged anti-tubulin antibodies to increase their size. The large cylindrical objects are chromosomes. (b) Schematic diagram corresponding to the metaphase cell in (a). Three sets of microtubules (MTs) make up the mitotic apparatus. All the microtubules have their (−) ends at the poles. Astral micro tubules project toward the cortex and are linked to it. Kinetochore microtubules are connected to chromosomes. Polar microtubules project toward the cell center with their distal (+) ends overlapping. The spindle pole and associated microtubules is also known as a mitotic aster. [Part (a) courtesy of J. Richard Mclntosh.]
Note that all the microtubules in each half of the symmetrical spindle have the same orientation except for some polar microtubules, which extend beyond the midpoint and interdigitate with polar microtubules from the opposite pole.
Microtubule Dynamics Increase Dramatically in Mitosis
Although we have drawn static images of the stages of mitosis, microtubules in all stages of mitosis are highly dynamic. As we have seen, as cells enter mitosis, the ability of their centrosomes to nucleate the assembly of microtubules increases significantly (see Figure 1). In addition, microtubules become much more dynamic. How was this determined? In principle, one could label microtubules with a fluorescent tag and watch their individual behaviors, but practically speaking, there are too many microtubules in a mitotic spindle to follow. To get an average value for the dynamic instability of these microtubules, researchers can introduce fluorescently labeled tubulin into cells, which becomes incorporated randomly into all microtubules. They can then bleach the fluorescent label in a small region of the mitotic spindle and measure the rate at which fluorescence comes back using a technique known as fluorescence recovery after photobleaching (FRAP). Since the recovery of fluorescence is due to the assembly of new microtubules from soluble fluorescent tubulin dimers, its rate represents the average rate at which microtubules turn over. In a mitotic spindle, their half-life is about 15 seconds, whereas in an interphase cell, it is about 5 minutes. It should be noted that these are bulk measurements and that individual microtubules can be more stable or dynamic, as we will see.
What makes microtubules more dynamic in mitosis? As we discussed earlier, dynamic instability is a measure of relative contributions of growth rates, shrinkage rates, catastrophes, and rescues. Analysis of microtubule dynamics in vivo shows that the enhanced in stability of individual microtubules in mitosis is generated mainly by an increase in catastrophes and a decrease in rescues, with little change in rates of growth (i.e., lengthening) or shrinkage (i.e., shortening). Studies with extracts from frog oocytes have suggested that the main factor enhancing catastrophes in both interphase and mitotic ex tracts is depolymerization by kinesin-13 proteins. This can be seen in an in vitro assay in which microtubule assembly from pure tubulin is nucleated from purified centrosomes (Figure 4a). If kinesin-13 is added to the assay, many fewer microtubules are formed. However, if the protein XMAP215, which enhances assembly at the (+) end, is added with the kinesin-13, many microtubules are formed due to a dramatic reduction in catastrophe frequency. It turns out that the activity of kinesin-13 does not change significantly during the cell cycle, whereas the activity of XMAP215 is inhibited by its phosphorylation during mitosis (Figure 4b). This results in much more unstable (more dynamic) microtubules as the cell enters mitosis (Figure 4c).
Fig4. Microtubule dynamics increase in mitosis due to loss of a stabilizing MAP. (a) These three panels reveal the ability of centrosomes to assemble microtubules under various conditions: with pure tubulin (left); with tubulin and the destabilizing protein kinesin-13 (middle); and with tubulin, kinesin-13, and the stabilizing protein XMAP215 (Xenopus MAP of 215 kDa) (right). Further analysis shows that the major effect of XMAP215 is to suppress catastrophes induced by kinesin-13. (b) The increased dynamics of microtubules in mitosis is due to the inactivation of XMAP215 by phosphorylation. (c) Diagram comparing the stabilities of microtubules in interphase and in mitosis. Note that in addition to the decrease in stability in mitosis, the ability of MTOCs to nucleate microtubules increases dramatically in mitosis. [Part (a) republished with permission of AAAS, from Kinoshita et al., “Reconstitution of Physiological Microtubule Dynamics Using Purified Components,” Science 294, no 5545,1340-1343 (2001). Part (b) data from Kinoshita et al, 2002, Trends Cell Biol. 12:267–273.]
Mitotic Asters Are Pushed Apart by Kinesin-5 and Oriented by Dynein
As the two mitotic asters form, they generate interdigitated microtubules of opposite polarity between them. During prophase, the bipolar kinesin-5 interacts with the antiparallel microtubules and, by moving toward the (+) end of each microtubule, slides them apart and thereby pushes the two asters apart. The (−) end–directed motor, cytoplasmic dynein, can also contribute to aster separation as well as orienting the spindle appropriately in the cell. Dynein does this by associating with the cell cortex and pulling on microtubules nucleated from the mitotic asters. As we discuss shortly, this same mechanism is used to elongate the spindle during anaphase B.
Chromosomes Are Captured and Oriented During Prometaphase
Kinetochores, the structures that mediate attachment between chromosomes and microtubules, assemble on each sister chromatid at a region called the centromere. The centromere is a constricted region of the condensed chromosome defined by a centromeric DNA sequence. Centromeric DNA can vary enormously in size; in budding yeast it is about 125 bp, whereas in humans it is on the order of 1 Mb. Kinetochores contain many protein complexes to facilitate the linkage between centromeric DNA and microtubules. In animal cells, the kinetochore consists of a centromeric DNA layer and inner and outer kinetochore layers, with the (+) ends of kinetochore mi crotubules terminating in the outer layer (Figure 5). Yeast kinetochores are attached by a single microtubule to their spindle pole, human kinetochores are attached by about 30 microtubules, and plant chromosomes by hundreds.
Fig5. The structure of a mammalian kinetochore. Diagram and electron micrograph of a mammalian kinetochore. [Republished with permission of Springer, from McEwen et al., “A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution,” Chromosoma, 1998, 107: 6-7 pp. 366-375; permission conveyed through Copyright Clearance Center, Inc.]
How does a kinetochore become attached to microtubules in prometaphase? Microtubules nucleated from the spindle poles are very dynamic, and when they contact a kinetochore, either laterally or at their ends, this contact can lead to chromosomal attachment (Figure 6a, steps 1a and 1b). Microtubules that “capture” kinetochores are selectively stabilized by a reduction in the rate of catastrophes, which increases the chance that the attachment will persist.
Fig6. Chromosome capture and congression in prometa phase. (a) In the first stage of prometaphase, chromosomes become attached either to the end of a microtubule (1a ) or to the side of a micro tubule (1b ). The chromosome is then drawn toward the spindle pole by dynein-dynactin that becomes associated with one of the kinetochores of the chromosome and moves toward the (−) end of the microtubule ( 2 ). Eventually, a microtubule from the opposite pole finds and becomes attached to the free kinetochore, and the chromosome is now said to be bi-oriented ( 3 ). Once some chromosomes are bi-oriented, others, having established one kinetochore-pole interaction, use CENP-E/kinesin-7 on their free kinetochore to aid in orientation ( 4 ). The bi-oriented chromosomes then move to a central point between the spindle poles in a process known as chromosome congression. Note that during these steps, chromosome arms point away from the closest spindle pole: this is due to chromokinesin/kinesin-4 motors on the chromosome arms moving toward the (+) ends of the polar microtubules. In animal cells, many microtubules associate with each kinetochore. For ease of presentation, only single kinetochore microtubules are shown here. (b) Congression involves bidirectional oscillations of chromosomes, with one set of kinetochore microtubules shortening on one side of the chromosomes and the other set lengthening on the other. On the shortening side, a kinesin-13 protein stimulates microtubule disassembly and a dynein dynactin complex moves the chromosome toward the pole. On the side with lengthening microtubules, kinesin-7 protein holds on to the growing microtubule. The kinetochore also contains many additional protein complexes not shown here. See Cleveland et al., 2003, Cell 112:407–421.
Recent studies have uncovered a mechanism involving Ran, a small GTPase, that enhances the chance that micro tubules will encounter kinetochores. Recall from Chapter 13 that during interphase, the Ran GTPase cycle is involved in the transport of proteins into and out of the nucleus through nuclear pores. During mitosis, when the nuclear membrane and pores have disassembled, a guanine nucleotide exchange factor for the Ran GTPase is bound to chromosomes, thereby generating a higher local concentration of Ran⋅GTP in the vicinity of the chromosomes. Because the enzyme that stimulates GTP hydrolysis on Ran—the Ran GAP—is evenly distributed in the cytosol, this generates a gradient of Ran⋅GTP centered on the chromosomes. Ran⋅GTP induces the association of cytosolic microtubule-stabilizing factors with the microtubule, resulting in enhanced microtubule growth, and in this way biases growth of microtubules nucleated from spindle poles toward chromosomes.
Once a kinetochore is attached laterally or terminally to a microtubule, dynein-dynactin associates with the kinetochore to move the duplicated chromosome down the microtubule toward the spindle pole. This movement eventually results in an end-on attachment of the microtubule to one kinetochore (Figure 18-41a, step 2). This movement helps orient the sister chromatid so that the unoccupied kinetochore on the opposite side is pointing toward the distal spindle pole. Eventually a microtubule from the distal pole will capture the free kinetochore; at this point the sister chromatid pair is said to be bi-oriented (Figure 6a, step 3). With the two kinetochores attached to opposite poles, the duplicated chromosome is now under tension, being pulled in both directions by the two sets of kinetochore microtubules. When one or a few chromosomes are bi-oriented, other chromosomes use these existing kinetochore microtubules to contribute to their orientation and movement to the spindle center. This orientation is mediated by kinesin-7 (also known as CENP-E) associated with the free kinetochore, which moves the chromosome to the (+) end of the kinetochore microtubule (Figure 6a, step 4).
Duplicated Chromosomes Are Aligned by Motors and Microtubule Dynamics
During prometaphase, the chromosomes come to lie at the midpoint between the two spindle poles, called the meta phase plate, in a process known as chromosome congression. During this process, bi-oriented chromosome pairs often oscillate backward and forward before arriving at the metaphase plate. Chromosome congression involves the coordinated activity of several microtubule-based motors together with regulators of microtubule assembly and disassembly (Figure 6b). These regulators are localized at the kinetochores, but how they are maintained there is poorly understood—they are not part of the stable kinetochore complexes described in the next section. The oscillating behavior of chromosomes involves lengthening of microtubules attached to one kinetochore and shortening of microtubules attached to the other kinetochore, all without losing their attachments. In metazoans, several microtubule-based motors associated with the kinetochore contribute to this process. First, dynein-dynactin provides the strongest force pulling the chromosome pair toward the more distant pole. This movement requires simultaneous shortening of the microtubule, which is enhanced by kinetochore-localized kinesin-13. The microtubules associated with the other kinetochore have to grow as the chromosome moves. Anchored at this kinetochore is the kinesin-related motor kinesin-7, which holds onto the growing (+) end of the lengthening microtubule. Also contributing to congression is another kinesin, chromokinesin/kinesin-4, which associates with the chromosome arms. Kinesin-4, a (+) end–directed motor, interacts with the polar microtubules to pull the chromosomes toward the center of the spindle. When the chromosomes have congressed to the metaphase plate, dynein-dynactin is released from the kinetochores and streams down the kinetochore microtubules to the poles. These different activities and opposing forces work together to bring all the chromosomes to the meta phase plate, at which point the cell is ready for anaphase.
The Chromosomal Passenger Complex Regulates Microtubule Attachment at Kinetochores
We have noted that the segregation of chromosomes at mitosis must be very accurate, so it is crucial that all chromosomes are bi-oriented before anaphase begins. During the random kinetochore-to-microtubule attachment process, it is possible for mistakes to be made; for example, both kinetochores of a sister chromatid pair might attach to microtubules from the same spindle pole. If such attachments persisted during metaphase, it would result in one cell missing a chromosome and the other having an extra one, which would either be lethal or very detrimental. Cells have two important mechanisms to ensure that all chromosomes are correctly bi-oriented before anaphase begins.
The first mechanism ensures that the kinetochore microtubule interactions are weak until bi-orientation occurs. When a chromosome is correctly bi-oriented, tension is produced across the chromosome, and this tension leads to the kinetochore-microtubule attachments becoming stabilized. To understand how this works, we need to look a bit more closely at the molecular components that link a kinetochore to a microtubule. kinetochores assemble on regions of chromosomal DNA marked by a centromere-specific H3 histone variant called CENP-A. This variant marks the site for kinetochore assembly, which is a very complicated process. About half a dozen distinct stable protein complexes, consisting of more than 40 different proteins, have been shown to associate with this centromeric region in yeast. Essentially all these protein complexes are conserved in humans, which is not surprising given the fundamental importance of kinetochores. One of these, the so-called Ndc80 complex, is long and flexible, and many copies of it link the inner kinetochore with the (+) end of the microtubule in a sleevelike arrangement (Figure 7a). The function of Ndc80 and many of the associated factors at the kinetochore is regulated by the chromosomal passenger complex (CPC). This complex associates with the inner kinetochore early in mitosis, and among its components is a protein kinase called Aurora B. Once the CPC is associated with the kinetochore, Aurora B can phosphorylate several com ponents in the near vicinity, including the Ndc80 complex, which loosens the attachment of Ndc80 to the microtubule. The phosphorylation of these components is not stable, however: another protein, the phosphatase PP1, which is associated with the outer kinetochore, can dephosphorylate them. Thus when the kinetochores on a pair of sister chromatids are not under tension, Ndc80 is continually phosphorylated by Aurora B and dephosphorylated by PP1. The result is a weak interaction between the kinetochore and microtubule. However, when bi-orientation occurs, the tension generated pulls on both kinetochores and extends the flexible Ndc80 complex to increase the spacing between the inner and outer kinetochores (Figure 7b, c). As a result of these movements, Ndc80 cannot be phosphorylated by Aurora B, and the dephosphorylated state of Ndc80 renders it more firmly attached to the microtubule. In this way, microtubule attachments to bi-oriented chromosomes are selectively stabilized.
Fig7. CPC regulation of microtubule-kinetochore attachment. The Ndc80 complex forms a critical and regulated attachment between the kinetochore and microtubule (+) end. (a) Diagram showing the sleevelike arrangement of the Ndc80 complex linking the inner kinetochore to the (+) end of a microtubule embedded in the outer kinetochore. See S. Santaguida and A. Musacchio, 2009, EMBO J. 28:2511–2531. (b) Diagram of the relationship between the chromosomal passenger complex (CPC), which is associated with the inner kinetochore and contains the kinase Aurora B, and the outer kinetochore, where the phosphatase PP1 binds. Notice that when both kinetochores are under tension, the outer kinetochores move away from the CPC; as a result, Aurora B cannot phosphorylate components in the outer kinetochore, which include the microtubule-binding site of the Ndc80 complex. (c) Cell in metaphase stained for tubulin (red), DNA (blue), Aurora B kinase (green), and the outer kinetochore (magenta). Notice how the outer kinetochore is pulled away from Aurora B (inset). [Part (c) reprinted by permission from Macmillan Publishers Ltd: from Ruchaud S. et al., “Chromosomal passengers: conducting cell division,” Nature Reviews Molecular Cell Biology, 2007, 8:798–812.]
While the CPC is important for bi-orientation of each individual chromosome, it does not ensure that all chromosomes are bi-oriented before anaphase begins. The second mechanism to ensure correct chromosome segregation is the spindle assembly checkpoint pathway, a signaling pathway that stops the progression of the cell cycle into anaphase until tension is present at all the kinetochores. Even a single unattached, or inappropriately attached, kinetochore can activate the spindle assembly checkpoint pathway and pause the cell cycle until the error is corrected. This mechanism, guarantees that all the chromosomes are correctly bi-oriented before the cell proceeds into anaphase.
Anaphase A Moves Chromosomes to Poles by Microtubule Shortening
The onset of anaphase A is one of the most dramatic movements that can be observed in the light microscope. When the spindle assembly checkpoint has been passed, APC/C activation induces proteolysis of the remaining cohesins holding the sister chromatids together. Suddenly, the two paired sister chromatids separate from each other and are drawn to their respective poles. The movement is sudden because the kinetochore microtubules are under tension, and as soon as the cohesin attachments between the chromatids are removed, the separated chromatids are free to move.
Experiments with isolated metaphase chromosomes have shown that anaphase A movement can be powered by micro tubule shortening, using the stored structural strain released by removal of the GTP-bound tubulin subunits at the microtubule tip. This mechanism can be nicely demonstrated in vitro. When metaphase chromosomes are added to purified microtubules, they bind preferentially to the (+) ends of the microtubules. Dilution of the mixture to reduce the concentration of free tubulin dimers results in the movement of the chromosomes toward the (−) ends by microtubule depolymerization at the chromosome-bound (+) ends. In addition, recent experiments have shown that in Drosophila, two members of the microtubule depolymerizing kinesin-13 protein family also contribute to chromosome movement in anaphase A. One of these kinesin-13 proteins is localized at the kinetochore and enhances disassembly there (Figure 8, A1), and the other is localized at the spindle pole, enhancing depolymerization there (Figure 8, A2). Thus, at least in the fly, anaphase A is powered in part by kinesin-13 proteins specifically localized at the kinetochore and spindle pole to shorten the kinetochore microtubules at both their (+) and (−) ends, drawing the chromosomes to the poles.
Fig8. Chromosome movement and spindle pole separation in anaphase. Anaphase A movement is powered by microtubule-shortening kinesin-13 proteins at the kinetochore (A1 ) and at the spindle pole (A2 ). Note that the chromosome arms still point away from the spindle poles due to associated chromokinesin/ kinesin-4 members, so the depolymerization force has to be able to overcome the force pulling the arms toward the center of the spindle. Anaphase B also has two components: sliding of antiparallel polar microtubules powered by a kinesin-5 (+) end–directed motor (B1 ), and pulling on astral microtubules by dynein-dynactin located at the cell cortex (B2 ). Arrows indicate the direction of movement generated by the respective forces. See Cleveland et al., 2003, Cell 112:407–421.
Anaphase B Separates Poles by the Combined Action of Kinesins and Dynein
The second part of anaphase involves separation of the spindle poles in a process known as anaphase B. A major contributor to this movement is the involvement of the bipolar kinesin-5 proteins (Figure 8, B1). These motors associate with the over lapping polar microtubules, and since they are (+) end–directed motors, they push the poles apart. While this is happening, the polar microtubules have to grow to accommodate the increased distance between the spindle poles. Another motor—the microtubule (−) end–directed cytoplasmic dynein, localized and anchored on the cell cortex—pulls on the astral microtubules and thus helps separate the spindle poles (Figure 8, B2).
Additional Mechanisms Contribute to Spindle Formation
There are a number of cases in vivo in which spindles form in the absence of centrosomes, including plant-cell mitosis and animal-cell meiosis in females. This observation implies that nucleation of microtubules from centrosomes is not the only way in which a spindle can form. Studies exploiting mitotic extracts from frog eggs—extracts that do not contain centrosomes— show that the addition of beads covered with DNA is sufficient to assemble a relatively normal mitotic spindle (Figure 9). In this system, the beads induce the assembly of microtubules, and factors in the extract cooperate to make a spindle. One of the factors necessary for this reaction is cytoplasmic dynein, which is proposed to bind to two microtubules and migrate to their (−) ends, thereby drawing them together.
Fig9. Mitotic spindles can form in the absence of centrosomes. Centrosome-free extracts can be isolated from frog oocytes arrested in mitosis by centrifuging eggs to separate a soluble material from the organelles and yolk. When fluorescently labeled tubulin (green) is added to extracts of the soluble material together with beads covered with DNA (red), mitotic spindles spontaneously form around the beads from randomly nucleated microtubules. See Kinoshita et al., 2002, Trends Cell Biol. 12:267–273, and Antonio et al., 2000, Cell 102:425. [Micrograph republished with permission of Nature, from Heald, R. et al., “Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts,” Nature, 1996, 382:6590, pp. 420-425.]
As mentioned in Section 18.1, a newly discovered γ-TuRC-associated complex, the augmin complex, also contributes microtubules to the mitotic spindle. In late prometaphase and metaphase, the augmin complex binds the sides of existing spindle microtubules to nucleate the assembly of additional microtubules having the same polarity as the mother microtubule. This activity contributes to both polar and kinetochore microtubule abundance in the spindle.
Recent studies have provided a model for how spindles can form in the absence of centrosomes. The ability of DNA covered beads to induce the assembly of microtubules is de pendent on Ran, the small GTPase that, in interphase cells, drives nuclear import and export. Recall that proteins destined for import into the nucleus associate with a nuclear import receptor, called an importin, that results in the complex passing from the cytosol through nuclear pores and into the nucleoplasm. The level of Ran·GTP is high in the nucleus because its activator, the guanine nucleotide exchange factor for Ran (Ran-GEF), is localized there by binding to chromatin. Ran⋅GTP binds to the importin, changing its conformation to release the transported protein, and then the importin-Ran⋅GTP complex leaves the nucleus to return to the cytosol through a nuclear pore. After nuclear envelope breakdown in mitosis, because the Ran-GEF is associated with chromatin, there is a gradient of Ran⋅GTP around chromosomes. This Ran⋅GTP releases a protein called TPX from importin. TPX binds the augmin complex and γ-TuRC to drive the assembly of microtubules in the vicinity of the chro matin, which can be used to make the spindle. In spindles with centrosomes, this pathway also exists to enhance the number of kinetochore and polar microtubules. It involves an association of the TPX–augmin–γ-TuRC complex with the side of an existing microtubule to nucleate assembly of new ones at a shallow angle to the mother microtubule, and thereby enhance the density of microtubules with the same polarity.
Cytokinesis Splits the Duplicated Cell in Two
During late anaphase and telophase in animal cells, the cell assembles a microfilament-based contractile ring attached to the plasma membrane that will eventually contract and pinch the cell into two, a process known as cytokinesis (see Figure 2). The contractile ring is a thin band of actin filaments of mixed polarity interspersed with myosin-II bipolar filaments. On receiving a signal, the ring contracts, first to generate a cleavage furrow and then to pinch the cell into two.
Two aspects of the contractile ring are essential to its function. First, it has to be appropriately placed in the cell. It is known that this placement is determined by a signal provided by the spindle, so that the ring forms equidistant between the two spindle poles. The signal is provided, at least in part, by the chromosomal passenger complex (CPC) that regulates the attachment of microtubules to kinetochores during prometaphase (see Figure 7b). Up to anaphase, the CPC is associated with the inner kinetochores of unseparated chromatids. When anaphase begins, it leaves the centromeres and associates with the overlapping polar microtubules at the center of the spindle (Figure 10). There the CPC recruits another protein complex, centralspindlin, that includes a (+) end directed kinesin motor protein, which concentrates at the middle of the spindle due to its motor activity. As anaphase B continues, centralspindlin recruits a guanine nucleotide exchange factor for RhoA. Recall from Chapter 17 that Rho proteins are small GTP-binding proteins that are activated by exchange factors to catalyze the exchange of GDP for GTP. Once activated, the RhoA⋅GTP activates a formin protein to drive the nucleation and assembly of actin filaments that make up the contractile ring. In this way, the position of the spindle directly defines the site of contractile ring formation, and hence cytokinesis.
Fig10. The chromosomal passenger complex (CPC) remains at the spindle midzone during anaphase and telophase. Micrographs of a cell in late anaphase (left) and telophase (right), showing microtubules (red), DNA (blue), Aurora B kinase (green), and kinetochores (magenta). Notice how the Aurora B, which is part of the CPC, concentrates in the region where the polar microtubules overlap and where the contractile ring will form. Scale bar 5 μm. [Reprinted by permission from Macmillan Publishers Ltd: from Ruchaud S. et al., “Chromosomal passengers: conducting cell division,” Nature Reviews Molecular Cell Biology, 2007, 8:798–812.]
The second important aspect of the contractile ring is the timing of its contraction: if it were to contract before all chromosomes had moved to their respective poles, disastrous genetic consequences would ensue. a signaling pathway has been discovered in budding yeast called the spindle position checkpoint, which pauses the cell cycle to ensure that cytokinesis does not occur until the spindle is appropriately oriented. The mechanism of this coordination in animal cells is still being unraveled.
Plant Cells Reorganize Their Microtubules and Build a New Cell Wall in Mitosis
Interphase plant cells lack a central MTOC that organizes microtubules into the radiating array typical of animal cells. Instead, numerous MTOCs containing γ-tubulin line the cortex of plant cells and nucleate the assembly of transverse bands of microtubules below the cell wall (Figure 11, left). These microtubules, which are of mixed polarity, are released from the cortical MTOCs by the action of katanin, a microtubule-severing protein; loss of katanin gives rise to very long microtubules and misshapen cells. The reason for this is that these cortical microtubules, which are cross-linked by plant-specific MAPs, aid in laying down extracellular cellulose microfibrils, the main component of the rigid cell wall.
Fig11. Mitosis in a flowering plant cell. Immunofluorescence micrographs (top) and corresponding diagrams (bottom) showing arrangement of microtubules in interphase and mitotic plant cells. A cortical array of microtubules girdles a cell during interphase. As the cell enters prophase, the microtubules (green), together with actin filaments (red), assemble under the cell cortex into a preprophase band, which marks the future cortical division site. As the cell enters prometaphase and metaphase, a spindle similar to that seen in animal cells forms. However, due to the cell wall, cytokinesis in plant cells is very different from that in in animal cells. Microtubules deliver vesicles whose membranes are used to assemble a membrane network called a phragmoplast, whose organization is defined by actin filaments linked to the cortical division site. Eventually, the phragmoplast becomes part of the plasma membranes of the two daughter cells. Enzymes secreted from the vesicles then build a cell wall between the two daughter cells. See G. Jürgens, 2005, Annu. Rev. Plant Biol. 56:281–299 [Micrographs courtesy of Susan M. Wick.]
