Chapter 18:  The Mechanics of Cell Division

Cohesins and Condensins Help Configure Replicated Chromosomes for Segregation

When the chromosomes are duplicated in S phase, the two copies of each replicated chromosome remain tightly bound together as identical sister chromatids. The sister chromatids are glued together by multisubunit protein complexes called cohesins, which are deposited along the length of each sister chromatid as the DNA is replicated. This cohesion between sister chromatids is crucial to the chromosome segregation process and is broken only late in mitosis (at the start of anaphase) to allow the sisters to be pulled apart.

The first readily visible sign that a cell is about to enter M phase is the progressive compaction of the replicated chromosomes, which become visible as threadlike structures—a process called chromosome condensation. In humans, for example, each interphase chromosome becomes compacted after replication into a mitotic chromosome that is about 50 times shorter (Figure 18-2). As discussed in Chapter 4, proteins called condensins do the work of chromosome condensation. Activated M-Cdk phosphorylates some of the condensin subunits, triggering the assembly of condensin complexes on DNA and, thereby, the progressive condensation of the chromosomes. The condensins can use the energy of ATP hydrolysis to promote DNA coiling in a test tube, and they are thought to do the same in cells during chromosome condensation. By a mechanism that is still poorly understood, they eventually produce fully condensed mitotic chromosomes, with each of the two sister chromatids organized around a linear central axis, where the condensin complexes are concentrated (see Figure 18-2).

The cohesins and condensins are structurally related, and they work together to configure the replicated chromosomes in preparation for mitosis. Genetic studies show that, if chromatid cohesion is not established properly in S phase, full condensation cannot occur in M phase and chromosomes are abnormally segregated at anaphase. A model for how cohesins can glue two DNA molecules together, while the closely related condensins can bind to a single DNA molecule and induce its supercoiling and condensation, is illustrated in Figure 18-3.

In budding yeast, the sudden degradation and release of the cohesin complexes allows sister chromatids to separate at anaphase. In vertebrate cells, by contrast, most of the cohesin is released from the chromosomes at the start of mitosis, when condensins bind to drive condensation. The small amount of cohesin that remains, however, is enough to hold sister chromatids together until anaphase, when the residual cohesins are degraded, allowing the chromatids to separate, as we discuss later.

Cytoskeletal Machines Perform Both Mitosis and Cytokinesis

After the chromosomes have condensed, two distinct cytoskeletal machines are assembled in sequence to perform the mechanical processes of mitosis and cytokinesis. Both machines disassemble rapidly after they have completed their tasks.

To produce two genetically identical daughter cells, the cell has to separate its replicated chromosomes and allocate one copy to each daughter cell. In all eucaryotic cells, this task is performed during mitosis by a bipolar mitotic spindle, which is composed of microtubules and various proteins that interact with them, including microtubule-dependent motor proteins (discussed in Chapter 16).

Different cytoskeletal structures are responsible for cytokinesis. In animal cells and many unicellular eucaryotes, it is the contractile ring; in most plant cells, it is the phragmoplast. The contractile ring contains both actin and myosin filaments and forms around the equator of the cell, just under the plasma membrane; as the ring contracts, it pulls the membrane inward, thereby dividing the cell in two (Figure 18-4). Plant cells, which have a cell wall to contend with, divide their cytoplasm by a very different mechanism. As we discuss later, instead of using a contractile process that acts on the plasma membrane, the phragmoplast constructs a new cell wall from within the cell, between the two sets of replicated chromosomes.

Two Mechanisms Help Ensure That Mitosis Always Precedes Cytokinesis

In most animal cells, M phase takes only about an hour—a small fraction of the total cell-cycle time, which often lasts 12–24 hours. The rest of the cycle is occupied by interphase. Under the microscope, interphase appears as a deceptively uneventful interlude, in which the cell simply continues to grow in size. Other techniques, however, reveal that interphase is actually a busy time for a proliferating cell, during which elaborate preparations for cell division are occurring in a tightly ordered sequence. Two critical preparatory events that are completed during interphase are DNA replication and duplication of the centrosome.

As discussed in Chapter 17, cyclical oscillations in the activities of the Cdks and of proteolytic complexes drive the cell cycle forward. Cdks trigger various steps of the cycle either by directly phosphorylating structural or regulatory proteins or by activating other protein kinases to do so. The proteolytic complexes activate specific steps in the cycle by degrading key cell-cycle proteins such as cyclins and Cdk inhibitor proteins. Like throwing switches, the activation of Cdks and proteolytic complexes triggers cell-cycle transitions that are normally points of no return. Thus, a green light from M-Cdk to enter M phase results in chromosome condensation, nuclear envelope breakdown, and a dramatic change in microtubule dynamics, all triggered by the phosphorylation of regulatory proteins that control these processes.

It is crucial that the two major events of M phase—nuclear division (mitosis) and cytoplasmic division (cytokinesis)—occur in the correct sequence (see Figure 18-1). It would be catastrophic if cytokinesis occurred before all of the chromosomes had segregated during mitosis. At least two mechanisms seem to prevent this catastrophe. First, the cell-cycle control system that activates proteins required for mitosis is thought to inactivate some of the proteins required for cytokinesis; presumably for this reason, cytokinesis cannot occur until M-Cdk is inactivated at the end of mitosis. Second, after the mitotic spindle has segregated the two sets of chromosomes to opposite poles of the cell, the residual central region of the spindle is required to maintain a functional contractile ring (see Figure 18-4); thus, until the spindle has separated the chromosomes and formed a central spindle, the ring cannot divide the cytoplasm in two.

M Phase in Animal Cells Depends on Centrosome Duplication in the Preceding Interphase

Two critical events must be completed in interphase before M phase begins—replication of the DNA and, in animal cells, duplication of the centrosome. DNA is duplicated so that each new daughter cell inherits an identical copy of the genome, while the centrosome is duplicated to help initiate the formation of the two poles of the mitotic spindle and to supply each daughter cell with its own centrosome. As we discuss later, after the chromosomes have been segregated in late mitosis, the microtubules that emanate from the two centrosomes signal to the cell cortex to help establish the plane of cytoplasmic division. This ensures that the division occurs exactly midway between the two separated groups of chromosomes (see Figure 18-4).

The centrosome is the principal microtubule-organizing center in animal cells (discussed in Chapter 16). It consists of a cloud of amorphous material (called the centrosome matrix or pericentriolar material) that surrounds a pair of centrioles (see Figure 16-24). During interphase, the centrosome matrix nucleates a cytoplasmic array of microtubules, with their fast-growing plus ends projecting outward toward the cell perimeter and their minus ends associated with the centrosome. The matrix contains a great variety of proteins, including microtubule-dependent motor proteins, coiled-coil proteins that are thought to link the motors to the centrosome, structural proteins, and components of the cell-cycle control system. Most important, it contains the γ–tubulin ring complex, which is the component mainly responsible for nucleating microtubules (see Figure 16-22).

The process of centrosome duplication and separation is known as the centrosome cycle. During interphase of each animal cell cycle, the centrioles and other components of the centrosome are duplicated (by an unknown mechanism) but remain together as a single complex on one side of the nucleus (Figure 18-5). As mitosis begins, this complex splits in two, and each centriole pair becomes part of a separate microtubule organizing center that nucleates a radial array of microtubules called an aster (Figure 18-6). The two asters move to opposite sides of the nucleus to initiate the formation of the two poles of the mitotic spindle. When the nuclear envelope breaks down (at prometaphase), the spindle captures the chromosomes; it will separate them toward the end of mitosis (Figure 18-7). As mitosis ends and the nuclear envelope re-forms around the separated chromosomes, each daughter cell receives a centrosome in association with its chromosomes.

In early embryonic cell cycles, the centrosome cycle can operate even if the nucleus is physically removed or nuclear DNA replication is blocked by a drug that inhibits DNA synthesis. Cycles of centrosome duplication and separation proceed almost normally, first yielding two centrosomes, then four, and then eight, and so on. Egg cell extracts from the frog Xenopus (see Figure 17-9) support multiple rounds of centrosome duplication in a test tube. This system has been used to test the individual protein components of the cell-cycle control system for their ability to stimulate centrosome duplication. Such experiments show that the G₁/S-Cdk (a complex of cyclin E and Cdk2) that initiates DNA replication in S phase (discussed in Chapter 17) also stimulates centrosome duplication, presumably explaining why centrosome duplication begins at the start of S phase.

M Phase Is Traditionally Divided into Six Stages

The first five stages of M phase constitute mitosis, which was originally defined as the period in which the chromosomes are visibly condensed. Cytokinesis occurs in the sixth stage, which overlaps with the end of mitosis. These six stages form a dynamic sequence, in which many independent cycles, involving the chromosomes, cytoskeleton, and centrosomes, have to be coordinated in order to produce two genetically identical daughter cells. The stages that occur during M phase are summarized in Panel 18-1. The complexity and beauty of M phase, however, are hard to appreciate from written descriptions or from a set of static pictures.

The five stages of mitosis—prophase, prometaphase, metaphase, anaphase, and telophase—occur in strict sequential order, while cytokinesis begins in anaphase and continues through telophase. Light micrographs of cell division in a typical animal cell and a typical plant cell are shown in Figures 18-8 and 18-9, respectively. During prophase, the replicated chromosomes condense in step with the reorganization of the cytoskeleton. In metaphase, the chromosomes are aligned at the equator of the mitotic spindle, and in anaphase they are segregated to the two poles of the spindle. Cytoplasmic division is complete by the end of telophase, and the nucleus and cytoplasm of each of the daughter cells then return to interphase, signaling the end of M phase.

Summary

Cell division occurs during M phase, which consists of nuclear division (mitosis) followed by cytoplasmic division (cytokinesis). The DNA is replicated in the preceding S phase; the two copies of each replicated chromosome (called sister chromatids) remain glued together by cohesins. At the start of M phase, cohesin-related proteins called condensins bind to the replicated chromosomes and progressively condense them. A microtubule-based mitotic spindle is responsible for chromosome segregation in all eucaryotic cells. The mitotic spindle in animal cells develops from the microtubule asters that form around each of the two centrosomes produced when the centrosome duplicates, beginning in S phase; at the onset of M phase, the duplicated centrosomes separate and move to opposite sides of the nucleus to initiate the formation of the two poles of the spindle. An actin and myosin-based contractile ring is responsible for cytoplasmic division in animal cells and in many unicellular eucaryotes, but not in plant cells.

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?

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.

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).

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.

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.

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).

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).

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.

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.

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.

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).

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).

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.

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.

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).

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.

Summary

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.

The Microtubules of the Mitotic Spindle Determine the Plane of Animal Cell Division

The mitotic spindle in animal cells not only separates the daughter chromosomes, it also specifies the location of the contractile ring, and thereby the plane of cell division. The contractile ring invariably forms in the plane of the metaphase plate, at right angles to the long axis of the mitotic spindle, thereby ensuring that division occurs between the two sets of separated chromosomes. The part of the spindle that specifies the division plane varies depending on the cell type: in some cells, it is the astral microtubules; in others, it is the overlapping antiparallel microtubules in the central spindle.

The relationship between the spindle microtubules and the placement of the contractile ring has been studied by manipulating fertilized eggs of marine invertebrates. After fertilization, these embryos undergo a series of rapid cleavage divisions, without intervening periods of growth. In this way, the original egg is progressively divided up into smaller and smaller cells. During cytokinesis, the cleavage furrow appears suddenly on the surface of the cell and deepens rapidly (Figure 18-30). Because the cytoplasm is clear, the spindle can be observed in real time through a microscope. If the spindle is tugged into a new position with a fine glass needle in early anaphase, the incipient cleavage furrow disappears, and a new one develops in accord with the new spindle site.

How does the mitotic spindle control the plane of division? Ingenious experiments in large embryonic cells demonstrate that a cleavage furrow forms midway between the asters originating from the two centrosomes, even when the two centrosomes are not connected to each other by a mitotic spindle (Figure 18-31). Thus, in these cells, the microtubule asters—not the chromosomes or other parts of the spindle—signal to the cell cortex to specify where the contractile ring should assemble. In other cells, the central spindle, rather than the astral microtubules, is apparently responsible for this specification. In either case, it has been speculated that the overlapping microtubules may provide tracks for motor proteins to deliver contractile ring regulators, and perhaps new membrane, to the appropriate region of the dividing cell. But, in fact, the molecular mechanism by which the spindle positions the cleavage furrow remains a mystery.

In some cells, the site of ring assembly is chosen before mitosis, according to a landmark placed in the cortex during a previous cell cycle. In budding yeasts, for example, a ring of proteins called septins assembles before mitosis, adjacent to a bud scar left on the cell surface as the mother and daughter cells separated in the previous division. The septins are thought to form a scaffold onto which other components of the contractile ring, including myosin II, assemble. As we discuss later, in plant cells, an organized band of microtubules and actin filaments assembles just before mitosis and marks the site where the cell wall will assemble and divide the cell in two.

Some Cells Reposition Their Spindle to Divide Asymmetrically

Most cells divide symmetrically. In most animal cells, for example, the contractile ring forms around the equator of the parent cell, so that the two daughter cells produced are of equal size and have similar properties. This symmetry results from the placement of the mitotic spindle, which in most cases tends to center itself in the cytoplasm. The centering process depends both on astral microtubules and on motor proteins that either push or pull on the astral microtubules to center the spindle.

There are many instances in development, however, when cells divide asymmetrically to produce two cells that differ in size, in the cytoplasmic contents they inherit, or in both. Usually, the two daughter cells are destined to develop along different pathways. To create daughter cells with different fates, the mother cell must first segregate some components (called fate determinants) to one side of the cell and then position the plane of division so that the appropriate daughter cell inherits these components (Figure 18-32). To position the plane of division asymmetrically, the spindle has to be moved in a controlled manner within the dividing cell. It seems likely that such spindle movements are directed by changes in local regions of the cell cortex and that motor proteins localiazed there pull one of the spindle poles, via its astral microtubules, to the appropriate region (Figure 18-33). Some of the proteins required for such asymmetrical divisions have been identified through genetic analyses in C. elegans and Drosophila (discussed in Chapter 21), and some of these seem to have a similar role in vertebrates.

Asymmetric division is particularly important in plant cells. As these cells cannot move after division, the selection of division planes is crucial for controlling tissue morphology. We discuss later how the plane of division is determined in these cells.

Actin and Myosin II in the Contractile Ring Generate the Force for Cytokinesis

As the astral microtubules in anaphase become longer lived and less dynamic in response to the loss of M-Cdk activity, the contractile ring begins to assemble beneath the plasma membrane. Much of the preparation for cytokinesis, however, happens earlier in mitosis, before the division of the cytoplasm actually begins. In interphase cells, actin and myosin filaments are assembled into a cortical network and, in some cells, also into large cytoplasmic bundles called stress fibers (discussed in Chapter 16). As cells enter mitosis, these arrays disassemble; much of the actin is reorganized, and myosin II filaments are released. As the chromatids separate in anaphase, myosin II begins to accumulate in the rapidly assembling contractile ring (Figure 18-34).

In many cells, cytokinesis requires the activation of one or more members of the polo-like family of protein kinases. These kinases regulate the assembly of both the mitotic spindle and the contractile ring and are therefore thought to help coordinate mitosis and cytokinesis, but it is uncertain how they do so. The fully assembled contractile ring contains many proteins in addition to actin and myosin II. The overlapping arrays of actin filaments and bipolar myosin II filaments, however, generate the force that divides the cytoplasm in two. They are thought to contract by a mechanism that is biochemically similar to that used by smooth muscle cells; in both cases, for example, the contraction begins when Ca²⁺-calmodulin activates myosin light-chain kinase to phosphorylate myosin II. Once contraction has been stimulated, the ring develops a force large enough to bend a fine glass needle that is inserted in the path of the constricting ring.

How the contractile ring constricts is still a mystery. It seems not to operate by a simple “purse-string” mechanism, with actin and myosin II filaments sliding past each other as in skeletal muscle (see Figure 16-71). As the ring constricts, the ring maintains the same thickness in cross-section, suggesting that its total volume and the number of filaments it contains decrease steadily. Moreover, unlike in muscle, the actin filaments in the ring are highly dynamic, and their arrangement changes extensively during cytokinesis.

In addition to specifying the site of contractile ring assembly in early anaphase, in many cells, microtubules also work continuously during anaphase and telophase to stabilize the advancing cleavage furrow. Drugs that depolymerize microtubules, for example, cause the actin filaments in the contractile ring to become less organized. Moreover, if a needle is used to tear microtubules away from the cell cortex, the contractile ring disassembles and the cleavage furrow regresses. It is not known how the microtubules stabilize the ring, although it has been shown that growing microtubules can activate some members of the Rho family of small GTPases, which in turn stimulate actin polymerization (discussed in Chapter 16). One member of this family, Rho A, is required for cytokinesis.

The contractile ring is finally dispensed with altogether when cleavage ends, as the plasma membrane of the cleavage furrow narrows to form the midbody. The midbody persists as a tether between the two daughter cells and contains the remains of the central spindle, which now consists of the two sets of antiparallel overlap microtubules packed tightly together within a dense matrix material (Figure 18-35). Remarkably, in some cells, before cytokinesis has been completed, the mother centriole from one or both daughter cells separates from its daughter centriole (see Figure 18-5c) and migrates into the midbody, where it lingers for minutes, before returning to its daughter cell. Only then do the two daughter cells separate to complete cytokinesis. What the centriole might do in the midbody to trigger the final steps of cytokinesis is not known. After the daughter cells separate completely, some of the components of the residual midbody often remain on the inside of the plasma membrane of each cell, where they may serve as a mark on the cortex that helps to orient the spindle in the subsequent cell division.

Membrane-enclosed Organelles Must Be Distributed to Daughter Cells During Cytokinesis

The process of mitosis ensures that each daughter cell receives a full complement of chromosomes. But when a eucaryotic cell divides, each daughter cell must also inherit all of the other essential cell components, including the membrane-enclosed organelles. As discussed in Chapter 12, organelles like mitochondria and chloroplasts cannot assemble spontaneously from their individual components; they can arise only from the growth and division of the preexisting organelles. Similarly, cells cannot make a new endoplasmic reticulum (ER) unless some part of it is already present.

How, then, are the various membrane-enclosed organelles segregated when a cell divides? Organelles such as mitochondria and chloroplasts are usually present in large enough numbers to be safely inherited if, on average, their numbers roughly double once each cycle. The ER in interphase cells is continuous with the nuclear membrane and is organized by the microtubule cytoskeleton. Upon entry into M phase, the reorganization of the microtubules releases the ER, which fragments as the nuclear envelope breaks down. The Golgi apparatus probably fragments as well, although in some cells it seems to redistribute transiently into the ER, only to re-emerge at telophase. Some of the organelle fragments associate with the spindle microtubules via motor proteins, thereby hitching a ride into the daughter cells as the spindle elongates in anaphase.

Mitosis Can Occur Without Cytokinesis

Although nuclear division is usually followed by cytoplasmic division, there are exceptions. Some cells undergo multiple rounds of nuclear division without intervening cytoplasmic division. In the early Drosophila embryo, for example, the first 13 rounds of nuclear division occur without cytoplasmic division, resulting in the formation of a single large cell containing 6000 nuclei, arranged in a monolayer near the surface (Figure 18-36). This arrangement greatly speeds up early development, as the cells do not have to take the time to go through all the steps of cytokinesis for each division. After these rapid nuclear divisions, cells are created around each nucleus in one round of coordinated cytokinesis called cellularization. Contractile rings form at the cell surface, and the plasma membrane extends inward and pinches off to enclose each nucleus.

Nuclear division without cytokinesis also occurs in some types of mammalian cells. Osteoclasts, trophoblasts, and some hepatocytes and heart muscle cells, for example, become multinucleated in this way.

The Phragmoplast Guides Cytokinesis in Higher Plants

Most higher-plant cells are enclosed by a semirigid cell wall, and their mechanism of cytokinesis is different from that just described for animal cells. Rather than a contractile ring dividing the cytoplasm from the outside in, the cytoplasm of the plant cell is partitioned from the inside out by the construction of a new cell wall, called the cell plate, between the two daughter nuclei (Figure 18-37). The orientation of the cell plate determines the positions of the two daughter cells relative to neighboring cells. It follows that altering the planes of cell division, together with enlargement of the cells by expansion or growth, leads to different cell and tissue shapes that help determine the form of the plant.

The mitotic spindle by itself is not sufficient to determine the exact position and orientation of the cell plate. The first visible sign that a higher-plant cell has become committed to divide in a particular plane is seen in G₂, when the cortical array of microtubules disappears in preparation for mitosis. At this time, a circumferential band of microtubules and actin filaments forms a ring around the entire cell just beneath the plasma membrane. Because this cytoskeletal array appears before prophase begins, it is called the preprophase band. The band becomes thinner as the cell progresses to prophase, and it disappears completely before metaphase is reached. Yet, the division plane has somehow been established: when the new cell plate forms later during cytokinesis, it grows outward to fuse with the parental wall precisely at the zone formerly occupied by the preprophase band. Even if the cell contents are displaced by centrifugation after the preprophase band has disappeared, the growing cell plate tends to find its way back to the plane defined by the former preprophase band.

The assembly of the cell plate begins in late anaphase and is guided by a structure called the phragmoplast, which contains the remaining overlap microtubules of the mitotic spindle that interdigitate at their growing plus ends. This region of overlap is similar in structure to the central spindle in animal cells in late anaphase. Small vesicles, largely derived from the Golgi apparatus and filled with polysaccharide and glycoproteins required for the synthesis of the new cell-wall matrix, are transported along the microtubules to the equator of the phragmoplast, apparently by the action of microtubule-dependent motor proteins. Here, the vesicles fuse to form a disclike, membrane-enclosed structure called the early cell plate (see Figure 18-9G). The plate expands outward by further vesicle fusion until it reaches the plasma membrane and the original cell wall and divides the cell in two. Later, cellulose microfibrils are laid down within the matrix of the cell plate to complete the construction of the new cell wall (Figure 18-38).

The Elaborate M Phase of Higher Organisms Evolved Gradually from Procaryotic Fission Mechanisms

Procaryotic cells divide by a process called binary fission. The single, circular DNA molecule replicates and division occurs by the invagination of the plasma membrane and the laying down of new cell wall between the two chromosomes to produce two separate daughter cells. In E. coli, before the chromosome replicates, the single origin of replication (oriC) is located at one pole of the rod-shaped bacterium. As soon as oriC is replicated, one copy of the sequence is immediately translocated to the opposite pole of the cell, after which the rest of the chromosome is replicated. Like the two spindle-pole asters in an animal cell, the bacterial daughter chromosomes at the cell poles somehow determine the location of the plane of cell division, ensuring that fission takes place at the cell equator, so that each daughter cell inherits one chromosome (Figure 18-39). Although a number of genes and proteins involved have been identified, the mechanisms responsible for the active translocation of oriC and the inhibition of fission everywhere but at the equator remain unknown.

Binary fission in procaryotes depends on filaments made of the FtsZ protein. FtsZ is a cytoskeletal GTPase that is structurally related to tubulin and assembles into a ring at the equator of the cell (Figure 18-40A, and see Figure 16-17). The FtsZ filaments are essential for the recruitment of all the other cell division proteins to the division site. Together, these proteins guide the inward growth of the cell wall and membrane, leading to the formation of a septum that divides the cell into two. Bacteria in which the ftsZ gene is inactivated by mutation cannot divide. A FtsZ-based mechanism is also used in the division of chloroplasts in plant cells (Figure 18-40B) and mitochondria in protists. In fungi and animal cells, another self-assembling GTPase called dynamin (discussed in Chapter 13) has apparently taken over the function of FtsZ in mitochondrial division.

With the evolution of the eucaryotes, the genome increased in complexity, and the chromosomes increased in both number and size. For these organisms, a more elaborate mechanism for dividing the chromosomes between daughter cells was apparently required. Clearly, the mitotic apparatus could not have evolved all at once. In many primitive eucaryotes, such as the dinoflagellate Cryphthecodinium cohnii, mitosis depends on a membrane-attachment mechanism, in which the chromosomes have to bind to the inner nuclear membrane for segregation. The intermediate status of this large, single-celled alga is reflected in the composition of its chromosomes, which, like those of procaryotes, have relatively little associated protein. The nuclear membrane in C. cohnii remains intact throughout mitosis, and the spindle microtubules remain entirely outside the nucleus. Where these spindle microtubules press on the outside of the nuclear envelope, the envelope becomes indented in a series of parallel channels (Figure 18-41). The chromosomes become attached to the inner membrane of the nuclear envelope opposite these channels, and chromosome segregation occurs on the inside of this channeled nuclear membrane. Thus, the extranuclear “spindle” is used to order the nuclear membrane and thereby define the plane of division. Kinetochores in this species seem to be integrated into the nuclear membrane and may therefore have evolved from some membrane component.

Eucaryotic tubulin and procaryotic FtsZ clearly have a common evolutionary history. But, microtubules are important for chromosome segregation in even the most primitive eucaryotes, where they are also present in flagellar axonemes (discussed in Chapter 16). Whether the flagellum or the spindle evolved first is unclear.

A somewhat more advanced, although still extranuclear, spindle is seen in hypermastigotes, in which the nuclear envelope again remains intact throughout mitosis. These large protozoa from the guts of insects provide a particularly clear illustration of the independence of spindle elongation and the chromosome movements that separate the chromatids. The sister kinetochores become separated by the growth of the nuclear membrane (to which they are attached) before becoming attached to the spindle. Only when the kinetochores are near the poles of the spindle do they acquire the kinetochore microtubules needed to attach them to the spindle. Because the spindle microtubules remain separated from the chromosomes by the nuclear envelope, the kinetochore microtubules, which are formed outside the nucleus, must somehow attach to the chromosomes through the nuclear membranes. After this attachment has occurred, the kinetochores are drawn poleward in a conventional manner (see Figure 18-41).

Organisms that form spindles inside an intact nucleus may represent a further stage in the evolution of mitotic mechanisms. In both yeasts and diatoms, the spindle is attached to chromosomes by their kinetochores, and the chromosomes are segregated in a way loosely similar to that described for animal cells—except that the entire process generally occurs within the confines of the nuclear envelope (see Figure 18-41). It is thought that the “open” mitosis of higher organisms and the “closed” mitosis of yeasts and diatoms evolved separately from a common ancestor resembling the modern hypermastigote spindle. At present, there is no convincing explanation for why higher plants and animals have evolved a mitotic apparatus that requires the controlled and reversible dissolution of the nuclear envelope.

Summary

Cell division ends as the cytoplasm divides into two by the process of cytokinesis. Except for plants, cytokinesis in eucaryotic cells is mediated by a contractile ring, which is composed of actin and myosin filaments and a variety of other proteins. By an unknown mechanism, the mitotic spindle determines when and where the contractile ring assembles and, thereby, when and where the cell divides. Most cells divide symmetrically to produce two cells of the same content and size. Some cells, however, specifically position their spindle to divide asymmetrically, producing two daughter cells that differ in size, content, or both. Cytokinesis occurs by a special mechanism in higher-plant cells—in which the cytoplasm is partitioned by the construction of a new cell wall, the cell plate, inside the cell. The position of the cell plate is determined by the position of a preprophase band of microtubules and actin filaments. The organization of mitosis in fungi and some protozoa differs from that in animals and plants, suggesting how the complex process of eucaryotic cell division may have evolved.