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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

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Molecular Biology of the Cell. 4th edition.

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The segregation of the replicated chromosomes is brought about by a complex cytoskeletal machine with many moving parts—the mitotic spindle. It is constructed from microtubules and their associated proteins, which both pull the daughter chromosomes toward the poles of the spindle and move the poles apart.

As we have seen, the spindle starts to form outside the nucleus while the chromosomes are condensing during prophase. When the nuclear envelope breaks down at prometaphase, the microtubules of the spindle are able to capture the chromosomes, which eventually become aligned at the spindle equator, forming the metaphase plate (see Panel 18-1). At anaphase, the sister chromatids abruptly separate and are drawn to opposite poles of the spindle; at about the same time, the spindle elongates, increasing the separation between the poles. The spindle continues to elongate during telophase, as the chromosomes arriving at the poles are released from the spindle microtubules and the nuclear envelope re-forms around them.

Both the assembly and the function of the mitotic spindle depend on microtubule-dependent motor proteins. As discussed in Chapter 16, these proteins belong to two families—the kinesin-related proteins, which usually move toward the plus end of microtubules, and the dyneins, which move toward the minus end. In the mitotic spindle, the motor proteins operate at or near the ends of the microtubules. These ends are not only sites of microtubule assembly and disassembly; they are also sites of force production. The assembly and dynamics of the mitotic spindle rely on the shifting balance between opposing plus-end-directed and minus-end-directed motor proteins.

Three classes of spindle microtubules can be distinguished in mitotic animal cells (Figure 18-10). Astral microtubules radiate in all directions from the centrosomes and are thought to contribute to the forces that separate the poles. They also act as “handles” for orienting and positioning the spindle in the cell. Kinetochore microtubules attach end-on to the kinetochore, which forms at the centromere of each duplicated chromosome. They serve to attach the chromosomes to the spindle. Overlap microtubules interdigitate at the equator of the spindle and are responsible for the symmetrical, bipolar shape of the spindle. All three classes of microtubules have their plus ends projecting away from their centrosome. The behavior of each class is thought to be different because of the different protein complexes that are associated with their plus and minus ends.

Figure 18-10. The three classes of microtubules of the fully formed mitotic spindle in an animal cell.

Figure 18-10

The three classes of microtubules of the fully formed mitotic spindle in an animal cell. (A) In reality, the chromosomes are proportionally much larger than shown in this drawing, and multiple microtubules are attached to each kinetochore. Note that the (more...)

Microtubule Instability Increases Greatly at M Phase

The mitotic spindle begins to self-assemble in the cytoplasm during prophase. In animal cells, each of the replicated centrosomes nucleates its own array of microtubules, and the two sets of microtubules interact to form the mitotic spindle. We see later that the self-assembly process depends on a balance between opposing forces that originate within the spindle itself and are generated by motor proteins associated with the spindle microtubules.

Many animal cells in interphase contain a cytoplasmic array of microtubules radiating out from the single centrosome. As discussed in Chapter 16, the microtubules of this interphase array are in a state of dynamic instability, in which individual microtubules are either growing or shrinking and stochastically switch between the two states. The switch from growth to shrinkage is called a catastrophe, and the switch from shrinkage to growth is called a rescue (see Figure 16-11). New microtubules are continually being created to balance the loss of those that disappear completely by depolymerization.

Prophase signals an abrupt change in the cell's microtubules. The relatively few, long microtubules of the interphase array rapidly convert to a larger number of shorter and more dynamic microtubules surrounding each centrosome, which will begin to form the mitotic spindle. During prophase, the half-life of microtubules decreases dramatically. This can be seen by labeling the microtubules in living cells with fluorescent tubulin subunits (Figure 18-11). As the instability of microtubules increases, the number of microtubules radiating from the centrosomes greatly increases as well, apparently because of an alteration in the centrosomes themselves that increases the rate at which they nucleate new microtubules. How does the cell-cycle control system trigger these dramatic changes in the cell's microtubules at the onset of mitosis?

Figure 18-11. The half-life of microtubules in mitosis.

Figure 18-11

The half-life of microtubules in mitosis. Microtubules in an M-phase cell are much more dynamic, on average, than the microtubules at interphase. Mammalian cells in culture were injected with tubulin that had been covalently linked to a fluorescent dye. (more...)

M-Cdk initiates the changes by causing the phosphorylation of two classes of proteins that control microtubule dynamics (discussed in Chapter 16). These include microtubule motor proteins and microtubule-associated proteins (MAPs). The roles of these regulators in controlling microtubule dynamics have been revealed by experiments using Xenopus egg extracts, which reproduce many of the changes that occur in intact cells during M phase. If centrosomes and fluorescent tubulin are mixed with extracts made from either M-phase or interphase cells, fluorescent microtubules nucleate from the centrosomes, permitting the behavior of individual microtubules to be analyzed by time-lapse fluorescence video microscopy. The microtubules in mitotic extracts differ from those in interphase extracts primarily by the increased rate of catastrophes, where they switch abruptly from slow growth to rapid shortening.

Proteins called catastrophins destabilize microtubule arrays by increasing the frequency of catastrophes (see Figure 16-36A). Among the catastrophins is a kinesin-related protein that does not function as a motor. In general, MAPs have the opposite effect of catastrophins, stabilizing microtubules in various ways: they can increase the frequency of rescues, in which microtubules switch from shrinkage to growth, or they can either increase the growth rate or decrease the shrinkage rate of microtubules. Thus, in principle, changes in catastrophins and MAPs can make microtubules much more dynamic in M phase by increasing total microtubule depolymerization rates, decreasing total microtubule polymerization rates, or both.

In Xenopus egg extracts, the balance between a single type of catastrophin and a single type of MAP can be shown to determine the catastrophe rate and the steady-state length of microtubules. This balance, in turn, governs the assembly of the mitotic spindle, as microtubules that are either too long or too short are incapable of assembling into a spindle (Figure 18-12). One way in which M-Cdk may control microtubule length is by phosphorylating this MAP and reducing its ability to stabilize microtubules. Even if the activity of the catastrophin remained constant throughout the cell cycle, the balance between the two opposing activities of the MAP and catastrophin would shift, increasing the dynamic instability of the microtubules.

Figure 18-12. Experimental evidence that the balance between catastrophins and MAPs influences the frequency of microtubule catastrophes and microtubule length.

Figure 18-12

Experimental evidence that the balance between catastrophins and MAPs influences the frequency of microtubule catastrophes and microtubule length. (A) Interphase or mitotic Xenopus egg extracts were incubated with centrosomes, and the behavior of individual (more...)

The cell contains a variety of MAPs, catastrophins, and motor proteins, each with subtly different activities. It is the balance between the opposing activities of these proteins that is responsible for the dynamic behavior of the mitotic spindle. We see later how changes in this balance help the spindle to segregate the chromosomes at anaphase.

Interactions Between Opposing Motor Proteins and Microtubules of Opposite Polarity Drive Spindle Assembly

While a shift in the balance between MAPs and catastrophins early in M phase creates more dynamic microtubules, a different sort of balance, between minus-end-directed and plus-end-directed motor proteins, helps assemble the mitotic spindle. Because some of these motor proteins form oligomers that can cross-link adjacent microtubules, they can move one microtubule relative to the other, with the direction of movement dependent on the polarity of both the motor protein and the microtubules. In this way, these motor proteins can form foci by bringing together a group of microtubule ends (Figure 18-13A). Alternatively, such motor proteins can slide antiparallel microtubules past each other (Figure 18-13B). These two different motor protein functions play a crucial part in the assembly and function of the spindle: they create the foci of microtubule minus ends that form the two spindle poles, and they slide antiparallel microtubules past each other in the overlap zone of the spindle (see Figure 18-10).

Figure 18-13. Two functions of multimeric motor proteins that are important for mitotic spindle assembly and function.

Figure 18-13

Two functions of multimeric motor proteins that are important for mitotic spindle assembly and function. Microtubule-dependent motor proteins hydrolyze ATP and move along a microtubule toward either its plus or its minus end. If the motor protein is multimeric, (more...)

During prophase in animal cells, microtubules growing from one centrosome engage with the microtubules of the adjacent centrosome. Because the plus ends of the microtubules are oriented away from the centrosomes, these two sets of microtubules have opposite polarities. Plus-end-directed motor proteins cross-link the two sets of microtubules and help push the centrosomes apart to begin to form the two poles of the mitotic spindle (Figure 18-14). A balance between plus-end-directed motor proteins and minus-end-directed motor proteins is crucial for spindle assembly: whereas plus-end-directed motor proteins operating on overlap microtubules tend to push the two halves of the spindle apart, some minus-end-directed motor proteins tend to pull them together.

Figure 18-14. Separation of the two spindle poles in prophase in an animal cell.

Figure 18-14

Separation of the two spindle poles in prophase in an animal cell. In this model, plus-end-directed motor proteins operating on interacting antiparallel microtubules help separate the two poles of a forming mitotic spindle. New microtubules grow out in (more...)

At least seven families of kinesin-related motor proteins have been localized to the mitotic spindle in vertebrate cells. In the budding yeast S. cerevisiae, five such motor proteins have been shown to work together in the spindle. Increasing the level of one of the plus-end-directed motor proteins produces abnormally long spindles, whereas increasing the level of one of the minus-end-directed motor proteins produces abnormally short spindles (Figure 18-15). Thus, the balance between plus-end-directed and minus-end-directed motor proteins seems to determine spindle length. A similar balance between motor proteins of opposite polarities occurs in human mitotic cells. At least, one of the motor proteins in human cells has to be phosphorylated by M-Cdk to bind to the spindle, suggesting one way in which M-Cdk might control the balance between opposing motor proteins.

Figure 18-15. The influence of opposing motor proteins on spindle length in budding yeast.

Figure 18-15

The influence of opposing motor proteins on spindle length in budding yeast. (A) A differential-interference-contrast micrograph of a mitotic yeast cell. The spindle is highlighted in green, and the position of the spindle poles is indicated by red arrows. (more...)

Kinetochores Attach Chromosomes to the Mitotic Spindle

Prometaphase in animal cells begins abruptly with the breakdown of the nuclear envelope. The breakdown is triggered when M-Cdk directly phosphorylates the nuclear lamina that underlies the nuclear envelope (see Figure 12-20). The disassembly of the nuclear envelope allows the microtubules access to the condensed chromosomes for the first time. Now, the assembly of a mature mitotic spindle can begin (see Panel 18-1, pp. 1034–1035).

The attachment of the chromosomes to the spindle is a dynamic process. When viewed by video microscopy, it seems to involve a “search and capture” mechanism, in which microtubules nucleated from each of the rapidly separating centrosomes grow outward toward the chromosomes. Microtubules that attach to a chromosome become stabilized, so that they no longer undergo catastrophes. They eventually end up attached end-on at the kinetochore, a complex protein machine that assembles onto the highly condensed DNA at the centromere (discussed in Chapter 4) during late prophase. The end-on attachment to the kinetochore is through the plus end of the microtubule, which is now called a kinetochore microtubule (Figure 18-16).

Figure 18-16. Kinetochore microtubules.

Figure 18-16

Kinetochore microtubules. (A) A fluorescence micrograph of a metaphase chromosome stained with a DNA-binding fluorescent dye and with human autoantibodies that react with specific kinetochore proteins. The two kinetochores, one associated with each chromatid, (more...)

In newt lung cells, where the initial capture event can be readily visualized, the kinetochore is seen first to bind to the side of the microtubule and then to slide rapidly along it toward one of the centrosomes. The lateral attachment to the chromosome is rapidly converted to an end-on attachment. At the same time, microtubules growing from the opposite spindle pole attach to the kinetochore on the opposite side of the chromosome, forming a bipolar attachment (Figure 18-17). Then begins a truly mesmerizing stage of mitosis. First, the chromosomes are tugged back and forth, eventually assuming a position equidistant between the two spindle poles, a position called the metaphase plate (Figure 18-18). In vertebrate cells, the chromosomes then oscillate gently at the metaphase plate, awaiting the signal to separate. The signal is produced after a predictable lag time after the bipolar attachment of the last of the chromosomes (discussed in Chapter 17).

Figure 18-17. The capture of microtubules by kinetochores.

Figure 18-17

The capture of microtubules by kinetochores. The red arrow in (A) indicates the direction of microtubule growth, while the gray arrow in (C) indicates the direction of chromosome sliding.

Figure 18-18. Chromosomes at the metaphase plate of a mitotic spindle.

Figure 18-18

Chromosomes at the metaphase plate of a mitotic spindle. In this fluorescence micrograph, kinetochores are labeled in red, microtubules in green, and chromosomes in blue. (From A. Desai, Curr. Biol. 10:R508, 2000. © Elsevier.)

As we discuss later, kinetochores play a crucial part in moving chromosomes on the spindle. They have a platelike organization when viewed in the electron microscope (Figure 18-19), and they are associated with both plus-end-directed and minus-end-directed microtubule motor proteins. But it remains a mystery how the plus ends of microtubules are attached to the kinetochore, especially because these ends are continuously polymerizing or depolymerizing, depending on the stage of mitosis.

Figure 18-19. The kinetochore.

Figure 18-19

The kinetochore. Electron micrograph of an anaphase chromatid with microtubules attached to its kinetochore. While most kinetochores have a trilaminar structure, the one shown here (from a green alga) has an unusually complex structure with additional (more...)

Microtubules Are Highly Dynamic in the Metaphase Spindle

The metaphase spindle is a complex and beautiful assembly, suspended in a state of dynamic equilibrium and tensed for action that will begin in anaphase. All of the spindle microtubules, except the kinetochore microtubules, are in a state of dynamic instability, with their free plus ends shifting stochastically between slow growth and rapid shrinkage. In addition, the kinetochore and overlap microtubules exhibit a behavior called poleward flux, with a net addition of tubulin subunits at their plus end, balancing a net loss at their minus ends, near the spindle poles.

The poleward flux in kinetochore and overlap microtubules in metaphase spindles has been studied directly by allowing the microtubules to incorporate tubulin that has been covalently coupled to photoactivatable, “caged” fluorescein. When such spindles are marked with a beam of UV light from a laser, the marks move continuously toward the spindle poles (Figure 18-20). The fluorescent marks get dimmer with time, indicating that many of the overlap and kinetochore microtubules depolymerize completely and are replaced. The dynamics of individual spindle microtubules of all classes (astral, kinetochore, and overlap) can be studied by an ingenious method in which very low amounts of fluorescent tubulin are injected into living cells (Figure 18-21). In these studies, a poleward flux is seen in both kinetochore and overlap microtubules, but not in astral microtubules. The function of the poleward flux, which does not occur in simple spindles such as those in yeasts, is unknown, although it might aid chromosome movement in anaphase.

Figure 18-20. The dynamic behavior of microtubules in the metaphase spindle studied by photoactivation of fluorescence.

Figure 18-20

The dynamic behavior of microtubules in the metaphase spindle studied by photoactivation of fluorescence. A metaphase spindle formed in vitro by adding Xenopus sperm to an extract of Xenopus eggs (see Figure 17-9) has incorporated three fluorescent markers: (more...)

Figure 18-21. Visualizing the dynamics of individual microtubules by fluorescence speckle microscopy.

Figure 18-21

Visualizing the dynamics of individual microtubules by fluorescence speckle microscopy. (A) The principle of the method. A very low amount of fluorescent tubulin is injected into living cells so that individual microtubules form with a very small proportion (more...)

One of the most striking aspects of metaphase in vertebrate cells is the continuous oscillatory movement of the chromosomes at the metaphase plate. These movements have been studied by video microscopy in newt lung cells and are seen to switch between two states—a poleward (P) state, which is a minus-end-directed pulling movement, and an away-from-the-pole (AP) state, which is a plus-end-directed movement. Kinetochores are thought to pull the chromosomes toward the poles, while an astral ejection force is thought to push the chromosomes away from the poles, toward the spindle equator (Figure 18-22A). Plus-end-directed motor proteins located on the chromosome arms are believed to interact with the astral microtubules to produce the ejection force (Figure 18-22B). Interestingly, spindles without centrosomes, including those in higher plants and some meiotic spindles, do not display these oscillations, which might reflect the absence of astral microtubules and, consequently, the absence of the astral ejection force.

Figure 18-22. How opposing forces may drive chromosomes to the metaphase plate.

Figure 18-22

How opposing forces may drive chromosomes to the metaphase plate. (A) Evidence for an astral ejection force that pushes chromosomes away from the spindle poles toward the spindle equator. In this experiment, a prometaphase chromosome that is temporarily (more...)

Functional Bipolar Spindles Can Assemble Around Chromosomes in Cells Without Centrosomes

Chromosomes are not just passive passengers in the process of spindle assembly. By creating a local environment that favors both microtubule nucleation and microtubule stabilization, they play an active part in spindle formation. The influence of the chromosomes can be demonstrated by using a fine glass needle to reposition them after the spindle has formed. For some cells in metaphase, if a single chromosome is tugged out of alignment, a mass of new spindle microtubules rapidly appears around the newly positioned chromosome, while the spindle microtubules at the chromosome's former position depolymerize. This property of the chromosomes seems to depend on a guanine-nucleotide exchange factor (GEF) that is bound to chromatin; it stimulates a small GTPase in the cytosol called Ran, inducing Ran to bind GTP in place of GDP. The activated Ran–GTP, which is also involved in nuclear transport (discussed in Chapter 12), releases microtubule-stabilizing proteins from protein complexes in the cytosol, thereby stimulating the local nucleation of microtubules around chromosomes.

In cells without centrosomes, the chromosomes direct the assembly of a functional bipolar spindle. This is how spindles form in cells of higher plants, as well as in many meiotic cells. It is also how they assemble in certain insect embryos that have been induced to develop from eggs without fertilization (i.e., parthenogenetically); as the sperm normally provides the centrosome when it fertilizes an egg (discussed in Chapter 20), the mitotic spindles in these parthenogenic embryos develop without centrosomes (Figure 18-23). Remarkably, in artificial systems, spindles can self-assemble without either centrosomes or centromeres. When beads coated with DNA that lack centromere sequences (and therefore lack kinetochore complexes) are added to Xenopus egg extracts in the absence of centrosomes, bipolar spindles assemble around the beads (Figure 18-24A).

Figure 18-23. Bipolar spindle assembly without centrosomes in parthenogenetic embryos of the insect Sciara..

Figure 18-23

Bipolar spindle assembly without centrosomes in parthenogenetic embryos of the insect Sciara.. The microtubules are stained green, the chromosomes red. The top fluorescence micrograph shows a normal spindle formed with centrosomes in a normally fertilized (more...)

Figure 18-24. Bipolar spindle assembly without centrosomes or kinetochores.

Figure 18-24

Bipolar spindle assembly without centrosomes or kinetochores. (A) A fluorescence micrograph of a spindle (green) that self-assembled around beads (red) coated with bacterial DNA in Xenopus egg extracts. (B) The steps in spindle assembly directed by DNA-coated (more...)

The centrosome-independent spindle assembly process is different from the assembly that is directed by centrosomes. In the DNA-coated bead model, for example, the microtubules first nucleate near the surface of the DNA, and then microtubule motor proteins sort the microtubules into bundles of uniform polarity, push the minus ends of the microtubules apart, and focus them into spindle poles (Figure 18-24B).

Even vertebrate cells can use such a centrosome-independent pathway to construct a functional bipolar spindle if the centrosomes are destroyed with a laser beam. Although the resulting acentrosomal spindle can segregate chromosomes normally, it lacks astral microtubules, which are responsible for positioning the spindle in animal cells; as a result, the spindle is often mispositioned, resulting in abnormalities in cytokinesis. If present, however, centrosomes normally direct spindle assembly because they are more efficient at nucleating microtubule polymerization than are chromosomes.

Anaphase Is Delayed Until All Chromosomes Are Positioned at the Metaphase Plate

Mitotic cells usually spend about half of M phase in metaphase, with the chromosomes aligned on the metaphase plate, jostling about, awaiting the signal that induces sister chromatids to separate to begin anaphase. Treatment with drugs that destabilize microtubules, such as colchicine or vinblastine (discussed in Chapter 16), arrests mitosis for hours or even days. This observation led to the identification of a spindle-attachment checkpoint, which is activated by the drug treatment and arrests progress in mitosis. The checkpoint mechanism is used by the cell-cycle control system to ensure that cells do not enter anaphase until all chromosomes are attached to both poles of the spindle (discussed in Chapter 17). If one of the protein components of the checkpoint mechanism is inactivated by mutation or by an intracellular injection of antibodies against the component, the cells initiate anaphase prematurely.

The spindle-attachment checkpoint monitors the attachment of the chromosomes to the mitotic spindle. It is thought to detect either unattached kinetochores or kinetochores that are not under the tension that results from bipolar attachment. In either case, unattached kinetochores emit a signal that delays anaphase until they all are properly attached to the spindle (see Figure 17-27). Drugs that destabilize microtubules prevent such attachment and therefore maintain the signal and delay anaphase. The inhibitory signaling role of the kinetochore can be demonstrated in mammalian cells in culture, where a single unattached kinetochore can block anaphase; destruction of this kinetochore with a laser causes the cell to enter anaphase.

Sister Chromatids Separate Suddenly at Anaphase

Anaphase begins abruptly with the release of the cohesin linkage that holds the sister chromatids together at the metaphase plate. As discussed in Chapter 17, this metaphase-to-anaphase transition is triggered by the activation of the anaphase promoting complex (APC). Once this proteolytic complex is activated, it has at least two crucial functions: (1) it cleaves and inactivates the M-phase cyclin (M-cyclin), thereby inactivating M-Cdk; and (2) it cleaves an inhibitory protein (securin), thereby activating a protease called separase. Separase then cleaves a subunit in the cohesin complex to unglue the sister chromatids (see Figure 17-26). The sisters immediately separate—and are now called daughter chromosomes—and move to opposite poles (Figure 18-25).

Figure 18-25. Chromatid separation at anaphase.

Figure 18-25

Chromatid separation at anaphase. In the transition from metaphase (A) to anaphase (B), sister chromatids suddenly separate and move toward opposite poles—as shown in these light micrographs of Haemanthus (lily) endosperm cells that were stained (more...)

The chromosomes move by two independent and overlapping processes. The first, referred to as anaphase A, is the initial poleward movement of the chromosomes. It is accompanied by shortening of the kinetochore microtubules at their attachment to the chromosome and, to a lesser extent, by the depolymerization of spindle microtubules at the two spindle poles. The second process, referred to as anaphase B, is the separation of the poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apart. Anaphase A depends on motor proteins at the kinetochore. Anaphase B depends on motor proteins at the poles that pull the poles apart, as well as on motor proteins at the central spindle (the bundles of antiparallel overlap microtubules between the separating chromosomes) that push the poles apart (Figure 18-26). Originally, anaphase A and anaphase B were distinguished by their different sensitivities to drugs. These differences are now thought to reflect differences in the sensitivities of the microtubule motor proteins that mediate the two processes.

Figure 18-26. The major forces that separate daughter chromosomes at anaphase in mammalian cells.

Figure 18-26

The major forces that separate daughter chromosomes at anaphase in mammalian cells. Anaphase A depends on motor proteins operating at the kinetochores that, together with the depolymerization of the kinetochore microtubules, pull the daughter chromosomes (more...)

Kinetochore Microtubules Disassemble at Both Ends During Anaphase A

As each daughter chromosome moves poleward, its kinetochore microtubules depolymerize, so that they have nearly disappeared at telophase. We can see this process by fluorescence video microscopy, in which labeled tubulin is injected into cells so that the sites of recent tubulin incorporation can be seen. In such experiments, the kinetochore ends of the kinetochore microtubules are observed to be the primary sites of tubulin addition during metaphase. In anaphase A, however, the kinetochore microtubules shorten mainly by the loss of tubulin from their kinetochore ends. It is not known how this switch from polymerization to depolymerization at kinetochores occurs at anaphase, but it may be triggered by the loss of tension that occurs when the cohesion between the sister chromatids is destroyed.

The poleward flux discussed earlier, with the continuous loss of tubulin subunits from both the overlap and kinetochore microtubules at the poles (see Figure 18-21), continues through anaphase. Thus, the kinetochore microtubules disassemble from both ends in anaphase.

Although it is clear that both microtubule motor proteins and microtubule depolymerization at the kinetochores contribute to chromosome movement during anaphase A, the exact molecular mechanism that drives the movement is still unknown. It is also unclear how kinetochores can remain attached to a microtubule that is losing tubulin subunits at its kinetochore (plus) end. There are two main ideas about how chromosomes move in anaphase A. One is that motor proteins at the kinetochores use the energy of ATP hydrolysis to pull the chromosomes along the kinetochore microtubules, which depolymerize as a consequence. Another is that the depolymerization itself drives the movement, without using ATP (Figure 18-27). The second possibility might seem implausible at first, but it has been shown that purified kinetochores in a test tube, with no ATP present, can remain attached to depolymerizing microtubules and thereby move. The energy that drives the movement is stored in the microtubule and is released when the microtubule depolymerizes; it ultimately comes from the hydrolysis of GTP that occurs after a tubulin subunit adds to the end of a microtubule (discussed in Chapter 16). How motor proteins and microtubule depolymerization at the kinetochore combine to drive chromosome movement remains one of the fundamental mysteries of mitosis.

Figure 18-27. Two alternative models of how the kinetochore may generate a poleward force on its chromosome during anaphase A.

Figure 18-27

Two alternative models of how the kinetochore may generate a poleward force on its chromosome during anaphase A. (A) Microtubule motor proteins at the kinetochore use the energy of ATP hydrolysis to pull the chromosome along its bound microtubules. Depolymerization (more...)

Both Pushing and Pulling Forces Contribute to Anaphase B

In anaphase B, the spindle elongates, pulling the two sets of chromosomes farther apart. In contrast to anaphase A, where the depolymerization of kinetochore microtubules is coupled to chromosome movement toward the poles, in anaphase B, the overlap microtubules actually elongate, helping to push the spindle poles apart. Anaphase B is driven by two distinct forces (see Figure 18-26). The first depends on plus-end-directed microtubule motor proteins in the central spindle that form a bridge between the overlapping microtubules of opposite polarities; the translocation of these motors toward the microtubule plus ends slides the microtubules past one another, pushing the poles apart. As a result, the bundle of overlap microtubules in the central spindle thins out (Figure 18-28). This mechanism is similar to that described earlier, in which motor proteins push the poles apart during spindle assembly in prophase (see Figure 18-14). The second force contributing to anaphase B depends on minus-end-directed motor proteins that interact with both the astral microtubules and the cell cortex and pull the two poles of the spindle apart (Figure 18-29).

Figure 18-28. The sliding of overlap microtubules at anaphase.

Figure 18-28

The sliding of overlap microtubules at anaphase. These electron micrographs show the reduction in the degree of microtubule overlap in the central spindle during mitosis in a diatom. (A) Metaphase. (B) Late anaphase. (Courtesy of Jeremy D. Pickett-Heaps.) (more...)

Figure 18-29. A model for how motor proteins may act in anaphase B.

Figure 18-29

A model for how motor proteins may act in anaphase B. Plus-end-directed motor proteins cross-link the overlapping, antiparallel, overlap microtubules and slide the microtubules past each other, thereby pushing the spindle poles apart. The red arrows indicate (more...)

The relative contributions of anaphase A and anaphase B to chromosome segregation vary greatly, depending on the cell type. In mammalian cells, anaphase B begins shortly after anaphase A and stops when the spindle is about twice its metaphase length; in contrast, the spindles of yeasts and certain protozoa primarily use anaphase B to separate the chromosomes at anaphase, and their spindles elongate to up to 15 times the metaphase length in the process.

At Telophase, the Nuclear Envelope Re-forms Around Individual Chromosomes

By the end of anaphase, the daughter chromosomes have separated into two equal groups at opposite ends of the cell and have begun to decondense. In telophase, the final stage of mitosis, a nuclear envelope reassembles around each group of chromosomes to form the two daughter interphase nuclei.

The sudden transition from metaphase to anaphase initiates the dephosphorylation of the many proteins that were phosphorylated at prophase. Although the relevant phosphatases are active throughout mitosis, it is not until M-Cdk is switched off that the phosphatases can act unopposed. Shortly thereafter, at telophase, nuclear membrane fragments associate with the surface of individual chromosomes and fuse to re-form the nuclear envelope. Initially, the fused membrane fragments partly enclose clusters of chromosomes; the fragments then coalesce to re-form the complete nuclear envelope (see Figure 12-21). During this process, the nuclear pore complexes are incorporated into the envelope, and the dephosphorylated lamins reassociate to form the nuclear lamina. The nuclear envelope once again becomes continuous with the extensive membrane sheets of the endoplasmic reticulum. Once the nuclear envelope has re-formed, the pore complexes pump in nuclear proteins, the nucleus expands, and the condensed mitotic chromosomes decondense into their interphase state, thereby allowing gene transcription to resume. A new nucleus has been created, and mitosis is complete. All that remains is for the cell to complete its division into two.


Mitosis begins with prophase, which is marked by an increase in microtubule instability, triggered by M-Cdk. In animal cells, an unusually dynamic microtubule array (an aster) forms around each of the duplicated centrosomes, which separate to initiate the formation of the two spindle poles. Interactions between the asters and a balance between minus-end-directed and plus-end-directed microtubule-dependent motor proteins result in the self-assembly of the bipolar spindle. In higher-plant cells and other cells that lack centrosomes, a functional, bipolar spindle self-assembles instead around the replicated chromosomes. Prometaphase begins with the breakdown of the nuclear envelope, which allows the kinetochores on the condensed chromosomes to capture and stabilize microtubules from each spindle pole. The kinetochore microtubules from opposite spindle poles pull in opposite directions on each duplicated chromosome, creating, together with a polar ejection force, a tension that helps bring the chromosomes to the spindle equator to form the metaphase plate. The spindle microtubules at metaphase are highly dynamic and undergo a continuous poleward flux of tubulin subunits. Anaphase begins with the sudden proteolytic cleavage of the cohesin linkage holding sister chromatids together. The breakage of this linkage allows the chromosomes to be pulled to opposite poles (the anaphase A movement). At about the same time, the two spindle poles move apart (the anaphase B movement). In telophase, the nuclear envelope re-forms on the surface of each group of separated chromosomes as the proteins phosphorylated at the onset of M phase are dephosphorylated.

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Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989, 1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James D. Watson .
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