Nuclear Changes in Prophase

Chromosome condensation, the landmark event at the onset of prophase, often begins in isolated patches of chromatin at the nuclear periphery. Later, chromosomes condense into two threads, termed sister chromatids, which are closely paired along their entire lengths. Although chromosome condensation was first observed more than a century ago, the biochemical mechanism remains a mystery. Protein kinases are thought to drive mitotic chromosome condensation by phosphorylating a number of the hundreds of proteins associated with mitotic chromosomes. The onset of condensation correlates with phosphorylation of histones H1 by Cdk1 kinase and H3 by Aurora-B protein kinase, and both are widely used as physiological markers for mitotic cells. However, chromosome condensation still occurs when both of these phosphorylation events are blocked. Possibly, some combination of histone modifications might provide a “code” that promotes chromatin condensation (see Fig. 13-3).

Two pentameric protein complexes, condensin I and II, are major constituents of mitotic chromosomes with an essential role in chromosome architecture (see Fig. 13-19). The two complexes share a pair of ABC ATPases, SMC2, and SMC4 (structural maintenance of chromosomes) but have two different sets of three auxiliary proteins. Condensin II enters the cell nucleus during prophase, where it is required for prophase chromosome condensation. However, chromosomes of vertebrate cells that lack condensin condense rapidly once the nuclear envelope breaks down at the onset of prometaphase, so condensin is not directly responsible for mitotic chromosome condensation. Chromosomes that lack condensin separate normally at the beginning of anaphase but appear to fall apart while moving toward the spindle poles. This appears to reflect defects in their underlying structure, and condensin is required for proteins of the chromosome scaffold to assemble properly (see Fig. 13-19). The molecular explanation for these effects is not known. In vitro, condensin complexes can promote coiling and compaction of DNA, but the significance of this is also unknown.

Cytoplasmic Changes in Prophase

Most of the cytoskeleton reorganizes during prophase. Most notably, the microtubule array changes from an extensive network permeating the cytoplasm into two dense, radial arrays of short, dynamic microtubules around the duplicated centrosomes (see Chapter 34). Each of these asters eventually becomes one pole of the mitotic spindle. During prophase, the two asters usually migrate apart across the surface of the nucle-ar envelope, signaling the start of spindle assembly (Fig. 44-3).

Mitotic microtubules behave like interphase microtubules in many ways (see Chapter 34). They are nucleated at their minus ends, they grow by addition of tubulin subunits at their free plus ends, and they undergo random catastrophes during which they rapidly shorten. To a large extent, the prophase changes in microtubule organization can be explained by two simple biochemical changes: (1) increased microtubule-nucleating activity of centrosomes and (2) altered dynamic instability properties of the microtubules (Table 44-1; also see Chapter 34). Interphase microtubules have a high probability of recovering from catastrophes, so they grow quite long. Mitotic microtubules grow more rapidly but exist only transiently. This is because when they undergo a catastrophe, they usually shorten all the way back to the centrosome, with little chance of rescue. These differences in dynamic instability can be reproduced in vitro in mitotic and interphase cellular extracts. They appear to arise, at least in part, from counterbalancing interactions between microtubule-associated proteins, which promote microtubule stability, and kinesin-13 (see Fig. 36-13), which promotes microtubule disas-sembly.

Table 44-1 COMPARISON OF MICROTUBULE DYNAMICS IN INTERPHASE AND MITOTIC NEWT LUNG CELLS

* Most cellular microtubules grow constantly by addition of subunits to their free ends but occasionally stop growing and begin shrinking rapidly (a “catastrophe”). Unless shrinking is reversed (a “rescue”), the microtubule completely disappears.

(Data from Gliksman NR, Skib-bens RV, Salmon ED: How the transition frequencies of microtubule dynamic instability regulate microtubule dynamics in interphase and mitosis. Mol Biol Cell 4:1035–1050, 1993.)

Other cytoskeletal elements that disassemble during prophase include most, but not all, classes of intermediate filaments (including the nuclear lamins) and specialized actin filament structures, such as stress fibers. However, the junctional complexes between adjoining cells are maintained in epithelial cells. As a result of the cytoskeletal reorganization, most cells round up during prophase. This is particularly evident for animal cells that are cultured on a flat substrate, but cells in tissues also change their shape dramatically during mitosis.

RNA transcription of the chromosomes stops during mitosis. Although a number of mechanisms contribute to this change, phosphorylation of components of the transcriptional machinery by Cdk1 kinase appears to be the predominant mechanism. Cdk1 kinase phosphorylation of ribosomal elongation factor EF2a also stops ongoing protein synthesis and assembly of new ribosomes. Phosphorylation of several nucleolar proteins leads to disassembly of the nucleolus.

The Golgi apparatus and endoplasmic reticulum fragment or vesiculate during prophase (Fig. 44-4). In ad-dition, many membrane-mediated events, including fluid-phase pinocytosis, endocytosis, exocytosis, and intracellular sorting of membrane components (see Chapters 21 and 22), greatly decrease. Golgi disassembly is driven by several kinases, including Cdk1. The first step of the process is fragmentation of the Golgi into smaller mini-stacks. The second step is still being investigated. Some evidence argues that Cdk1 phosphorylation of key components prevents the fusion of transport vesicles back into Golgi stacks (see Chapter 21), the net result being that the Golgi buds apart into small vesicles. Other evidence suggests that an imbalance of vesicle flow between the Golgi and the endoplasmic reticulum results in the Golgi being absorbed into the ER during mitosis. Whatever the mechanism of its disassembly, Golgi reassembly begins again during anaphase, following inactivation of Cdk1 kinase. Some Golgi derived vesicles contribute to the plasma membrane during cleavage furrow ingression at the end of mitosis (see later).

Figure 44-4 golgi apparatus dynamics in interphase and mitosis. Disassembly in mitosis is driven by phosphorylation of components blocking fusion of Golgi membranes.

Nuclear Envelope Disassembly in Prometaphase

Nuclear envelope disassembly involves the removal of two membrane bilayers coupled with disassembly of the nuclear pores and the fibrous nuclear lamina meshwork that underlies the inner bilayer (Fig. 44-6). Protein phosphorylation triggers breakdown of the nuclear envelope but the critical targets and mechanisms are not entirely known. Phosphorylation of the nuclear lamins at two sites flanking the central coiled-coil causes the lamina network to disassemble into subunits and might contribute to disassembly of the envelope. Cdk1 kinase can phosphorylate these lamin residues in vitro, but other kinases might participate in vivo. Additionally, phosphorylation of nucleoporins leads to nuclear pore disassembly. This fenestrates the nuclear envelope, dissolving the barrier between nucleus and cytoplasm. Interaction between microtubules and dynein associated with the nuclear envelope may also rip holes in the envelope.

Figure 44-6 disassembly of the nuclear envelope during mitosis. a– B, Two contrasting models explaining the fate of the nuclear envelope during the transition from interphase to mitosis in a higher eukaryote. C, Micrographs showing solubilization of lamin A fused to GFP during mitosis. Scale bar is 10μm. D–E, Reversible disassembly of lamins A, C, and B is driven by posttranslational modifications of the lamin polypeptides.

(C, Courtesy of William C. Earnshaw.)

Nuclear envelope components are dispersed in the cytoplasm from prometaphase until telophase (Fig. 44-6), but the mechanism may differ in various cell types. In fertilized amphibian eggs, the nuclear membrane breaks up into small vesicles that disperse in the cytoplasm. In vertebrate somatic cells, the nuclear envelope may be absorbed into the endoplasmic reticulum, which remains as an extensive tubular network throughout mitosis. In both cases, lamin B remains associated with the dispersed nuclear envelope, whereas lamins A and C and many proteins of the nuclear pore complexes disperse as soluble subunits.

During prophase, kinetochores transform from nondescript balls of condensed chromatin into organized plaques on the surface of the chromosomes. By early prometaphase, the characteristic trilaminar disk structure (see Fig. 13-20) can be seen. Each sister chromatid has a kinetochore. Thus, sister kinetochores are located on opposite faces of the mitotic chromosome. Only one of the two sister kinetochores faces a given spindle pole at any one time.

Organization of the Mitotic Spindle

The mature metaphase spindle is a bilaterally symmetrical structure with centrally located chromosomes flanked by arrays of microtubules radiating from the poles (Fig. 44-7).

Figure 44-7 role of motors in spindle structure. The mitotic spindle is a dynamic entity whose structure depends on microtubule assembly/disassembly plus the balance of forces that act to slide microtubules relative to one another and to pull the poles together or apart. A, In metaphase, the structure is at steady state. The forces that tend to elongate the spindle, including cytoplasmic dynein (which moves toward microtubule minus ends, pulling the poles out toward the cell cortex) and bipolar kinesin-5 (which moves toward microtubule plus ends, pushing the poles apart), are counterbalanced by kinesin-14, which moves toward microtubule minus ends (and pulls the poles together) and microtubule disassembly at the spindle poles. Dynein and its associated protein, NuMA, also have an important role in organization of the spindle pole. B, In anaphase, the balance of kinesin activity shifts, microtubule disassembly at the poles declines, and the spindle undergoes a dramatic elongation. During anaphase, bipolar kinesin-5 and PRC1 also have important roles in organizing the central spindle, which is essential for subsequent assembly and function of the cleavage furrow.

Three predominant classes of microtubules are present in the metaphase spindle (Fig. 44-12). Kinetochore microtubules have their plus ends embedded in the kinetochore and their minus ends at or near the spindle pole. They characteristically form bundles, called kinetochore fibers, which contain anywhere from 1 microtubule in the budding yeast to more than 200 microtubules in some higher plants. Each human kinetochore binds about 20 microtubules. Up to about 80% of the approximately 2200 spindle microtubules in humans may be present in kinetochore fibers. Interpolar microtubules are distributed throughout the body of the spindle and do not attach to kinetochores. Their minus ends may terminate near the pole but are not physically linked to it so that they appear to be free at both ends. Many interpolar microtubules penetrate between and through the chromosomes and extend for some distance beyond them. Thus, the central spindle contains a large number of interdigitated antiparallel microtubules. Tracking these spindle microtubules by electron microscopy has revealed a tendency for the interdigitated microtubules of opposite polarity to pack next to one another. During late anaphase, these antiparallel microtubules bundle to form a structure, called the central spindle, that appears to have important roles during cytokinesis. Astral microtubules project out from the poles and have a role in orienting the spindle in the cell through interactions with the cell cortex. All of the microtubules within each aster have the same polarity, with their minus ends proximal to the pole. Each unit of a spindle pole, with its associated kinetochore and interpolar and astral microtubules, is referred to as a half-spindle.

Figure 44-12 introduction to metaphase. A, Summary of the major events of metaphase. B, Distribution of DNA (blue), microtubules (red), actin (green), and gamma tubulin (centrosomes [yellow]) in a metaphase PtK1 (rat kangaroo) cell.

(B, Courtesy of Dr. Alexey Khodjakov, Wadsworth Center, Albany, New York.)

Spindle structure is largely determined by a combination of microtubule dynamics plus the action of at least seven different types of kinesins plus cytoplasmic dynein (see Chapter 36). These motors often work in opposition to one another. As a result, the spindle is a highly dynamic structure whose morphology changes as the balance of forces shifts between the various motors. For example, inactivating one or more kinesins with drugs or switching a temperature-sensitive mutant to the nonpermissive temperature can cause the spindle to collapse rapidly on itself. Consequently, chromosomal movements and changes in spindle morphology are complex processes that reflect both the dynamic growth and shrinkage of microtubules plus the net vectorial output of multiple antagonistic and synergistic motors. These various components interact; for example, force exerted by motors can influence the dynamic assembly/disassembly of microtubules.

Spindle Assembly

In metazoans, spindle assembly starts in prophase with the separation of the asters. In most cells, each aster is organized around a centrosome, consisting of a centriole pair and associated pericentriolar material. γ-Tubulin ring complexes in the pericentriolar material efficiently nucleate microtubules (see Fig. 34-16), so each aster acts as a microtubule organizing center. By the end of prophase, the spindle consists of two asters linked by a few interpolar microtubules. Cytoplasmic dynein at the cell cortex exerts an outward force separating the asters, whereas kinesin-14 motors (which move toward microtubule minus ends) on the interpolar microtubules exert a counterbalancing force holding the asters together.

This balance of forces changes when the nuclear envelope breaks down. Bipolar kinesin-5 motors are phosphorylated by Cdk1 kinase and concentrate in the central spindle, where they cross-link adjacent antiparallel interpolar microtubules. Kinesin-5 moves toward the plus ends of microtubules. If such a motor attaches to two adjacent antiparallel microtubules and begins to move, it will cause them to slide apart (Fig. 44-7). Thus, the action of kinesin-5 motors pushes the spindle poles apart. The two half-spindles do not separate because they are physically linked via the chromosomes, with sister kinetochores attached to opposite spindle poles.

Also at this time, the asters mature into focused spindle poles. The pericentriolar material efficiently nucleates the assembly of new microtubules with their minus ends at the pole. In addition, cytoplasmic dynein transports free microtubules that are nucleated throughout the cytoplasm to the centrosome along astral microtubules for incorporation into the spindle. The focused microtubule array at the pole forms partly owing to the tethering of microtubules by centrosomes, and partly due to the concerted action of various motors and microtubule cross-linking proteins such as nuclear mitotic apparatus protein (NuMA). NuMA is released from the nucleus on nuclear envelope breakdown, and it accumulates near the poles at the minus ends of microtubules.

In large cells that lack centrosomes, such as eggs, spindle formation depends on an alternative pathway that is also active in cells with centrosomes (Fig. 44-8). Chromosomes stabilize nearby microtubules, which are then organized into a bipolar spindle by motor proteins and NuMA. This spindle assembly pathway involves importin a and b, two proteins that direct traffic through nuclear pores during interphase (see Fig. 16-14). Importin a and b inhibit mitotic spindle formation by sequestering several essential proteins, including NuMA. Chromosomes counteract this by releasing spindle assembly factors such as NuMA from importin a and b. The mechanism depends on the association of the GTP exchange factor RCC1 (Ran-GEF in Fig. 44-17) with chromosomes. RCC1 produces a local gradient of the active GTPase Ran-GTP, which dissociates NuMA from importin a and b just as it does during protein import into the nucleus. In this case, however, the net result is microtubule stabilization and assembly of the mitotic spindle.

Figure 44-8 assembly of a bipolar spindle in the absence of centrosomes. A gradient of Ran-GTP stabilizes microtubules around chromosomes, releasing spindle assembly factors. Microtubules that accumulate around the chromosomes are sorted and organized by motor proteins and are subsequently focused to make poles by motors and (–) end-binding proteins such as NuMA. These spindle poles lack prominent astral microtubules.

Figure 44-17 chromosomes move on shrinking microtubules during anaphase. A, Mitotic cells are injected with a fluorescently labeled tubulin that rapidly becomes incorporated into the spindle. Just after anaphase onset, a laser is used to photobleach a stripe (white) across the spindle near the upper pole. The live cell is monitored over time by fluorescence (B) and phase contrast (C) microscopy. In this mammalian cell, the chromosomes approach the bleached stripe much faster than the stripe approaches the spindle pole. In other organisms with higher rates of microtubule flux in their spindles, the bleached zone would also move appreciably toward the pole. The numbers are time in seconds.

(B–C, Reproduced from Gorbsky GJ, Sammak PJ, Borisy GG: Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol 106:1185–1192, 1988. Copyright 1988 The Rockefeller University Press.)

If centrosomes are removed or destroyed experimentally, somatic cells can also use motor proteins to organize microtubules into bipolar spindles that lack asters but are otherwise remarkably normal. However, only about half of the cells that have lost centrosomes manage to complete mitosis successfully. Thus, centrosomes are not required to form spindles, but they do contribute to successful chromatid separation and cytokinesis by helping to orient the spindle within the dividing cell.

Chromosome Attachment to the Spindle

Dynamic microtubules of prometaphase asters scan the cytoplasm searching for both binding sites that will capture and stabilize their distal plus ends and for other components, including free microtubules. Breakdown of the nuclear envelope makes the condensed chromosomes accessible to the microtubules. Chance encounters with kinetochores during cycles of growth result in the plus ends of microtubules being captured by the kinetochore. Capture probably involves the nine-component KMN complex, which contains two components capable of binding weakly to microtubules. One of these, the helical Ndc80 complex (see Fig. 13-21) binds along the sides of microtubules forming fine hairs visible in the electron microscope. Other members of the complex provide an anchoring site for Ncd80 in the kinetochore. Captured microtubules are about five-fold less likely to depolymerize catastrophically than free microtubules. When catastrophes do occur, the microtubules depolymerize back to the pole, recycling tubu-lin subunits for incorporation into other, growing microtubules.

Initial attachment of a chromosome to a microtubule often involves a lateral interaction between the corona region of the kinetochore (see Fig. 13-20) and the side of a microtubule (Fig. 44-9). Cytoplasmic dynein then slides the chromosome rapidly along the microtubule toward the pole. These steps were first seen in animal cells, and a similar pattern of chromosome attachment and movement also occurs in budding yeast cells.

Figure 44-9 initial chromosomal movements during prometaphase. a–b, Chromosomal movements initiate with the capture of a microtubule by the kinetochore. This results first in movement toward the pole from which that microtubule originated. These images come from a study in which living cells, observed by differential interference microscopy, were subjected to rapid chemical fixation just after a chromosome had attached to the spindle (arrow). C, Attachment of the chromosome to the spindle was confirmed by indirect immunofluorescence staining for tubulin and, ultimately, by thin-section electron microscopy (D). E, The graph shows the movements of the chromosomes before and after attachment.

(Reproduced from Rieder CL, Alexander SP, Rupp G: Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J Cell Biol 110:81–95, 1990. Copyright 1990 The Rockefeller University Press.)

Capture of the first microtubule by a kinetochore causes the chromosome to move initially toward the spindle pole from which that microtubule originated. Historically, it has been thought that subsequent cap-ture of a microtubule emanating from the opposite spindle pole by the sister kinetochore provides a counterforce that tugs the chromosome in the opposite direction. This bipolar attachment produces a balance of opposing forces that, together with the action of kinesin family motor proteins distributed along the chromosome arms, results in the gradual movement of the chromosome toward a point midway between the spindle poles. These movements are accompanied by coordinated shrinkage of the microtubules at the leading kinetochore and growth of microtubules at the trailing kinetochore. More recently, it has been shown that chromosomes attached to only one spindle pole (mono-oriented) can move toward the spindle equator if the unattached kinetochore associates with the kinetochore fiber of a chromosome that has already become aligned at the spindle equator. In this case, the mono-oriented chromosome glides away from the pole to which it is attached toward the spindle midzone, where it is more likely to capture microtubules emanating from the opposite pole. This motion of one chromosome along the kinetochore fiber of another chromosome requires the kinesin-7 motor CENP-E, which is associated with the kinetochore of the moving chromosome.

The attachment of microtubules to kinetochores can be reproduced and studied in vitro by mixing together chromosomes, isolated centrosomes, and tubulin subunits. Under these circumstances, the plus ends of microtubules growing out from centrosomes attach to the chromosomes. Surprisingly, chromosome-bound microtubules can either lengthen or shorten at the at-tached end without detaching from the chromosome. This tethering of dynamic microtubules is an essential aspect of chromosome movements during mitosis.

Correcting Errors in Chromosome Attachment to the Spindle

The goal of mitosis is to partition the replicated chromosomes accurately to two daughter cells. Therefore, all chromosomes must attach correctly to both spindle poles before being segregated. Three sorts of errors are common: (1) chromosomes with one or both kinetochores lacking attached microtubules, (2) chromosomes with both sister kinetochores attached to the same spindle pole, (3) chromosomes with a single kinetochore attached simultaneously to both spindle poles. Correcting these errors takes time, and the spindle checkpoint (see next section) delays mitotic progression to allow the correction process to occur.

Attachment of both sister kinetochores to a single spindle pole is rare, since sister kinetochores are positioned on opposite faces of the chromosome (see Fig. 13-20). When it occurs, one or both kinetochores must detach for the chromosome to achieve a bipolar orientation. Chromosome attachment to opposite spindle poles is more stable than attachment to a single pole, because the tension generated by bipolar attachment (where forces pull a chromosome simultaneously toward opposite spindle poles) preferentially stabilizes microtubule connections to both kinetochores. It follows that the attachment of a single kinetochore to both spindle poles is more insidious, as that kinetochore is under tension, and the attachments are therefore stable. In fact, attachment of a single kinetochore to both poles seems to be the most common cause of chromosome segregation errors in cultured mammalian cells.

Both of these chromosome attachment errors are corrected through the action of Aurora B protein kinase, a member of the chromosomal passenger complex, along with inner centromere protein (INCENP), surviving, and borealin (Fig. 44-10). The other subunits target Aurora B to its various points of action during mitosis and regulate the kinase activity. The complex concentrates in the inner centromeres (the heterochromatin beneath and between the two sister kinetochores) during prometaphase and metaphase. As sister chromatids separate at anaphase, the complex moves to the overlapping interpolar microtubules of the central spindle and to the cell cortex, where the cleavage furrow will form, ultimately winding up in the intercellular bridge during cytokinesis. The chromosomal passenger complex is required to complete cytokinesis but also contributes to the correction of chromosome attachment errors and to the operation of the checkpoint that delays the cell cycle in response to those errors (Fig. 44-10).

Figure 44-10 chromosomal passenger proteins regulate mitotic events. These proteins are present at centromeres in prometaphase (A) and metaphase (B), but transfer to the spindle midzone at anaphase (C) and midbody at anaphase (D). E, Components and some targets of the chromosomal passenger complex, showing the Aurora B protein kinase complexed with INCENP, Survivin, and Borealin. F, If the complex function is inhibited (in this case by RNAi depletion of Borealin), chromosome attachment errors are common and many chromosomes fail to segregate properly in anaphase. Distribution of DNA (blue), microtubules (red), and survivin-GFP (green) in human mitotic cells. Inset in A, Distribution of DNA (blue), kinetochores (red), and Borealin (green) in a prometaphase cell. (A–D, Micrographs by Sally Wheatley and William C. Earnshaw. F and Inset in A, Micrographs by Ana Carvalho, Reto Gassmann, and William C. Earnshaw.

Inset in A and F, Reproduced from Gassmann R, Carvalho A, Henzing AJ, et al: Borealin: A novel chromosomal passenger required for stability of the bipolar mitotic spindle. J Cell Biol 166:179–191, 2004, by copyright permission of The Rockefeller University Press. A–C, From Wheatley SP, McNeish IA: Survivin: A protein with dual roles in mitosis and apoptosis. Int Rev Cytol 247:35–88, 2005.)

Aurora B responds to tension on kinetochores to correct chromosome attachment errors. It phosphorylates both Ndc80 and the Dam1 complex, which are involved in microtubule binding to the kinetochore (see Fig. 13-21). Aurora B phosphorylation strongly inhibits Ndc80 binding to microtubules, and this may signal the kinetochore to let go of the attached microtubule. When a chromosome is correctly attached to both spindle poles, tension may stretch the kinetochore away from the chromosomal passenger complex buried in the chromatin beneath. This might stabilize the chromosome-microtubule interaction by preventing the kinase from phosphorylating the kinetochore.

Finding Time to Fix Chromosome Attachment Errors: The Spindle Checkpoint

Segregation of replicated chromosomes into daughter cells is extremely accurate. For example, budding yeasts lose a chromosome only once in 100,000 cell divisions. Perhaps surprisingly, the frequency of chromosome loss may be 20-fold to 400-fold higher for human cells grown in culture. To achieve even this level of accuracy, most cells must delay entry into anaphase until all chromosomes are attached to both poles of the mitotic spindle. This delay is caused by a cellular quality control pathway or checkpoint—the spindle checkpoint—that senses the completion of chromosome alignment at metaphase (Fig. 44-11). This nomenclature is nearly universal, but it is worth noting that this checkpoint actually monitors kinetochore activity rather than spindle structure. The spindle checkpoint differs from the DNA damage checkpoints in that its default setting is “on” as cells enter mitosis. It shuts off only when every chromosome is properly attached to the spindle.

Figure 44-11 the spindle checkpoint. Signaling by unattached kinetochores stops the cell from entering anaphase until all chromosomes have made a proper bipolar spindle attachment. A, As long as there is a chromosome that is not properly attached to the spindle (beige cells), the cell does not enter anaphase. The cell enters anaphase about 20 minutes after chromosome attachment is complete (green cells). B, In a cell with a persistently maloriented chromosome, anaphase entry is delayed (beige cells). If the unattached kinetochore is destroyed with a high-powered laser, the cell enters anaphase about 20 minutes later. This proves that the unattached kinetochore sends an inhibitory signal. C, The unattached kinetochore sends a signal via the Mad2 protein that ultimately inhibits the APC/C, blocking the degradation of cyclin B and securin and thereby blocking the transition into anaphase. D, Mad2 protein shown in its complex with Mad1 protein.

The spindle checkpoint involves the products of the mitotic arrest–defective genes (MAD) and the budding-uninhibited-by-benzimidazole (BUB) genes. These were originally identified in yeast in genetic screens for cells that continued to try to divide when the spindle was disassembled by drugs. The Mad and Bub proteins are conserved from yeast to human. They accumulate at kinetochores early in mitosis, when the checkpoint is “on” (i.e., during prophase or prometaphase), and most are gradually displaced as microtubules bind and the kinetochores come under tension.

The target of the spindle checkpoint is Cdc20. This protein is thought to be a substrate recognition factor for the APC/C, a ubiquitin-protein ligase (E3 enzyme; see Chapter 23 and Fig. 44-16) that marks target proteins for destruction by proteasomes by decorating them with ubiquitin. Key APC/C substrates include cyclin B and a protein called securin, an inhibitor of the enzyme that triggers separation of sister chromatids at anaphase (see Fig. 44-16). Despite intense study, the mechanistic details are still debated.

Figure 44-16 regulation of sister chromatid pairing by the cohesin complex. a–b, The co-hesin complex forms a ring with a diameter of 35 nm that is loaded onto the chromosomes during DNA replication. The mechanism by which cohesins promote sister chromatid pairing is not known; this is one speculative model. At the onset of anaphase, degradation of its securin inhibitor liberates active separase enzyme. C–E, Separase then cleaves co-hesin subunit Scc1, and the two sister chromatids are able to separate from one another and move toward opposite spindle poles.

Mad1 protein binds to kinetochores that are not properly attached to the spindle (Fig. 44-11). The best guess is that Mad1 then binds Mad2. A loop on Mad2 wraps around Mad1 like a safety belt to make a complex that resides stably at kinetochores. This complex can bind additional Mad2 molecules and convert them to a conformation activated for Cdc20 binding. The “primed” Mad2 molecules are then released either on their own or as part of a “mitotic checkpoint complex”; they bind Cdc20, and the APC/C is inhibited. As each chromosome becomes attached to both poles of the spindle, its inhibitory signals are removed. When the last chromosome has achieved a proper attachment, the last source of inhibitory Mad2 complexes is extinguished, and mitosis can proceed.

In metazoans, two of the checkpoint components are protein kinases, and one of these, BubR1, may be involved in sensing the quality of kinetochore attachments to the spindle. When the nuclear envelope breaks down at prometaphase, the COOH-terminus of the CENP-E kinesin binds to BubR1 located in the outer plate of the kinetochore. This interaction stimulates the BubR1 kinase activity, a possible early step in activating the checkpoint. Subsequent microtubule binding by CENP-E then shuts the kinase off again. Thus, CENP-E may function in recognition of microtubule binding by the kinetochore. The yeast homolog of BubR1 is not a kinase; furthermore, yeasts lack CENP-E. Therefore, how they sense microtubule attachment to kinetochores remains mysterious.

Loss of the spindle checkpoint causes a catastrophic, premature entry into anaphase in higher eukaryotes, regardless of the status of chromosome alignment. This leads to an unequal distribution of sister chromatids to daughter cells, causing a genetic imbalance of the daughter cells known as aneuploidy. Yeasts can live without the checkpoint genes, but their loss is lethal for mice, which die early during embryogenesis. Humans with mutations in BubR1 have the disorder mosaic variegated aneuploidy, and mice heterozygous for various checkpoint components also show increased aneuploidy. Mosaic variegated aneuploidy is associated with an increased cancer risk, but as yet, no clear link has been established between mutations in other spindle checkpoint genes and cancer.

Microtubule Flux within the Metaphase Spindle

Although the average length of the kinetochore microtubules is roughly constant during metaphase, the microtubules change continuously in three ways. First, there is constant net addition of new tubulin subunits (about 10 subunits per second) to the plus end of the microtubules, where they are attached to the kinetochore. Second, a comparable number of tubulin subunits is continuously lost from the minus end of the kinetochore tubules at the spindle poles. Therefore, tubulin subunits slowly migrate through kinetochore microtubules from the kinetochore to the pole (Fig. 44-13). This subunit flux or treadmilling is caused by microtubule depolymerization at the poles driven by kinesin-13 family members. Third, all microtubules attached to each kinetochore change coordinately in length during chromosomal oscillations (see next section).

Figure 44-13 microtubule flux in metaphase. A, Cells entering mitosis are injected with tubulin subunits modified chemically by attachment of a caged fluorescent dye. This type of dye becomes fluorescent after being irradiated with ultraviolet light. When cells enter metaphase and labeled tubulin is incorporated into the spindle, the central spindle is illuminated with a narrow stripe of ultraviolet light. This activates a narrow band of fluorescent tubulin subunits. With time, these subunits approach the spindle poles (P). Because the length of kinetochore microtubules is constant during this time, the labeled tubulin molecules must migrate along the microtubule toward the pole (arrows). This can occur if new subunits are added to the microtubule at the kinetochore and old subunits are removed at the pole. B, Microtubule flux at metaphase in a Drosophila embryo visualized by fluorescence speckle microscopy. Embryos were injected with very low levels of fluorescent tubulin, which, instead of decorating the entire spindle, appears as speckles distributed along the microtubules. If a very sensitive camera is used, these speckles can be seen to move toward the poles, reflecting the flux in the underlying microtubules. Scale bar is 5μm. C, Movement of labeled tubulin speckles toward the spindle poles.

(A, Courtesy of Arshad Desai and the MBL Cell Division Group, Marine Biology Laboratory, Woods Hole, Massachusetts; reprinted by permission from Macmillan Publishers Ltd. from Mitchison TJ, Salmon ED: Mitosis: A history of division. Nat Cell Biol 3:E17–E21, 2001, copyright 2001. B, Courtesy of Paul Maddox and Arshad Desai, University of California, San Diego.)

Chromosome Oscillations during Metaphase

In many cells, even though chromosomes remain, on average, balanced at the middle of the spindle, they jostle one another and undergo numerous small excursions toward one pole or the other throughout metaphase (Fig. 44-14). These oscillations are slow, about 1.5μm/minute (gain or loss of about 40 tubulin subunits per microtubule per second), perhaps because they require the simultaneous shortening or lengthening of the approximately 20 microtubules attached to each kinetochore in vertebrate cells. The oscillatory movements reverse every few minutes. Both active movements by motor proteins and fluctuations in the length of kinetochore microtubules contribute to chromosome oscillations during metaphase, but their relative contributions may vary in different cell types. Although the movements of paired sister chromatids are usually coordinated, uncoordinated movements can stretch or compress centromeric chromatin. Thus, although each kinetochore can act independently, some mechanism (perhaps tension sensors in the kinetochore) usually coordinates their actions.

Figure 44-14 Kinetochore oscillations between P (poleward) and AP (away from the pole) movement during late prometaphase and anaphase in PtK1 (rat kangaroo) cells. A–D, Images showing the movements of several pairs of sister kinetochores, labeled with GFP-Cdc20 (green), combined with phase-contrast images of the cell (red). E and G, Higher-magnification views of sister kinetochores (marked with dashed lines) in prometaphase and anaphase, respectively. F, Kymograph (collage of images of a vertical strip showing the same two kinetochores at various time points during the movie) showing the movements of these two kinetochores. P and AP movements are indicated. Note that oscillations can occur even during anaphase. P movement involves microtubule shrinkage at the leading kinetochore and microtubule growth at the trailing kinetochore (which is undergoing AP movement away from its associated kinetochore). Spindle poles are near the top and bottom of panels E to G.

(Micrographs courtesy of E. D. Salmon, University of North Carolina, Chapel Hill.)

In some cells, microtubules interact with kinesin-4 and kinesin-10 chromokinesins associated with the chromosome arms in addition to kinetochores. These secondary interactions contribute to the stable alignment of chromosomes on the central spindle at metaphase.

Biochemical Mechanism of Sister Chromatid Separation

Separation of sister chromatids is regulated by the chromosomes themselves, not by the mitotic spindle. Under certain circumstances, sister chromatids can separate in the absence of microtubules, ruling out forces from the spindle in the process.

Studies of the budding yeast led to a major breakthrough by revealing three factors that regulate sister chromatid separation: a protein complex known as cohesin, a protease known as separase, and an inhibitor of the protease known as securin (Fig. 44-16). This system is conserved from yeast to human.

Cohesin is a complex of four proteins that resembles the condensin complex (see Fig. 13-19). Like condensin, cohesin has two large subunits from the SMC (structural maintenance of chromosomes) family. These proteins, SMC1 and SMC3, are complexed with proteins called Scc1 (which has other names omitted here for simplicity) and Scc3. Additional proteins are required for the stable loading of this complex onto DNA. Cells with mutations in cohesin components separate sister chromatids prematurely in mitosis, resulting in chaotic chromosome missegregation. This system is very ancient; an SMC-related protein is required for orderly chromosome segregation in bacteria.

Exactly how cohesin holds sister chromatids together is unknown, but a variety of evidence suggests that it could form a ring with a diameter of 40 nm, large enough to encircle two sister chromatids like a lasso. In yeast, the complex functions only if it binds chromosomes during DNA replication, and one factor involved is a specialized form of the RFC complex that loads PCNA rings onto DNA (see Fig. 42-11). Cohesin accumulates at preferred sites on the chromosomes, often near centromeres in budding yeast or in regions of heterochromatin in fission yeast. In vertebrates, most cohesin dissociates from the chromosome arms by late metaphase, owing to the action of protein kinases such as Plk1 and Aurora B, but some remains associated with heterochromatin flanking centromeres until the onset of anaphase.

Cleavage of two key proteins triggers sister chromatid separation at anaphase. The first of these, securin, is an inhibitor of the separase protease. After the last chromosome forms a bipolar attachment to the spindle, the spindle checkpoint is switched off. This allows APC/C^Cdc20 to tag securin with ubiquitin, leading to its destruction by proteasomes throughout metaphase. When securin levels fall below a critical threshold, separase is unleashed to cleave the Scc1 subunit of cohesin. Cleavage of Scc1 either breaks or disassembles the cohesin ring, allowing the sister chromatids to separate, thereby triggering the onset of anaphase. Phosphorylation of the separase cleavage site on Scc1 by a protein kinase can increase the cleavage of Scc1 by separase. Thus, the proteolysis of Scc1 is integrated and coordinated with the activities of the various mitotic kinases (see Chapter 40).

Human securin is overexpressed in some pituitary tumors, and the protein can act as an oncogene in cultured cells (see Fig. 41-10). Overexpression of securin may disrupt the timing of chromosome segregation, leading to chromosome loss and ultimately contributing to cancer progression.

Mitotic Spindle Dynamics and Chromosome Movement during Anaphase

Anaphase is dominated by the orderly movement of sister chromatids to opposite spindle poles brought about by the combined action of motor proteins and changes in the length of microtubules. Anaphase chromosome movements occur in two phases (Fig. 44-15). Anaphase A, the movement of the sister chromatids to the spindle poles, requires a shortening of the kinetochore fibers. During anaphase B, the spindle elongates, pushing the spindle poles apart. The poles separate partially because of interactions between the antiparallel interpolar microtubules of the central spindle and partially because of intrinsic motility of the asters. Most cells use both components of anaphase, but one component may be strongly exaggerated in relation to the other.

Microtubule disassembly on its own can move chromosomes (see Fig. 37-8). Energy for this movement comes from hydrolysis of GTP bound to assembled tubulin, which is stored in the conformation of the tubulin subunits. Chromosomes appear to use motor molecules such as cytoplasmic dynein in the kinetochore corona to hold onto disassembling microtubules. These motors must “run” toward the poles without losing their grip as the microtubules disassemble behind them. In addition, at yeast kinetochores the Dam1 ring (green in Fig. 44-21) can remain associated with disassembling microtubules. Other kinesin “motors” influence the dynamic instability of the spindle microtubules. Members of the kinesin-13 class, which encircle microtubules near kinetochores and at spindle poles, use ATP hydrolysis to promote microtubule disassembly rather than movement.

Figure 44-21 in eggs, the cleavage furrow forms midway between spindle asters. in animal cells, the central spindle is important. A, A classic experiment in which a sand-dollar egg is caused to adopt a toroid shape. At cytokinesis 2, the egg cleaves into four cells, and a furrow forms between the back sides of the two spindles. (For a description of this and other classic experiments in cytokinesis, see the book by Rappaport in the Selected Readings list.) B, left, A wild-type Drosophila spermatocyte undergoing cytokinesis, with the contractile ring stained in yellow. Right, In a profilin mutant, no central spindle forms, and the cell fails to form a contractile ring.

(Micrographs courtesy of Professor Maurizio Gatti, University of Rome, Italy. B, From Giansanti MG, Bonaccorsi S, Williams B, et al: Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev 12:396–410, 1998.)

Anaphase A chromosome movement involves a combination of microtubule shortening and translocation of the microtubule lattice due to flux of tubulin subunits (Fig. 44-13). The contributions of the two mechanisms vary among different cell types. When living vertebrate cells are injected with fluorescently labeled tubulin subunits, the spindle becomes fluorescent (Fig. 44-17). If a laser is used to bleach a narrow zone in the fluorescent tubulin across the spindle between the chromosomes and the pole early in anaphase, the chromosomes ap-proach the bleached zone much faster than the bleached zone approaches the spindle pole. This shows that the chromosomes “eat” their way along the kinetochore microtubules toward the pole. In these cells, subunit flux accounts for only 20% to 30% of chromosome movement during anaphase A, and this flux is dispensable for chromosome movement. In Drosophila embryos, in which subunit flux accounts for about 90% of anaphase A chromosome movement, the chromosomes catch up with a marked region of the kinetochore fiber slowly, if at all.

Anaphase B appears to be triggered by the inactivation of the minus end-directed kinesin-14 motors, so that all of the net motor force favors spindle elongation. Three factors contribute to overall lengthening of the spindle: sliding apart of the interdigitated half-spindles, microtubule growth, and intrinsic motility of the poles themselves (Fig. 44-7). During the latter stages of anaphase B, the spindle poles, with their attached kinetochore microtubules, appear to move away from the interpolar microtubules as the spindle lengthens. This astral movement involves interaction of the astral microtubules with cytoplasmic dynein molecules anchored in the cortical cytoplasm.

Anaphase B spindle elongation is accompanied by reorganization of the interpolar microtubules into a highly organized central spindle between the separating chromatids (Fig. 44-15). Within the central spindle, an amorphous dense material called stem body matrix stabilizes bundles of antiparallel microtubules and holds together the two interdigitated half-spindles. Proteins concentrated in the central spindle help to regulate cytokinesis. One key factor, PRC1 (protein regulated in cytokinesis), is inactive when phosphorylated by Cdk kinase and functions only during anaphase when Cdk activity declines. PRC1 directs the binding of several kinesins to the central spindle. Each kinesin appears to target a specific protein kinase, such as Aurora B, to a particular domain of the central spindle, where phosphorylation of key substrates then regulates spindle elongation and cytokinesis.

Reassembly of the Nuclear Envelope

Nuclear envelope reassembly begins during anaphase and is completed during telophase (Fig. 44-19). As in spindle assembly, Ran-GTP promotes early steps of nuclear envelope assembly by releasing near the surface of the chromosomes key components that were sequestered by importin b. Most of these factors are not yet identified, but they include several nuclear pore components.

Figure 44-19 scanning electron microscopy of the stages of assembly of membrane vesicles on the surface of chromosomes in a Xenopus egg cytosolic extract. A solution containing membrane vesicles was added to isolated chromatin from Xenopus sperm, fixed, and then imaged by field emission scanning electron microscopy. Each panel shows the time of incubation prior to fixation.

(Micrographs courtesy of K. L. Wilson, Johns Hopkins Medical School, Baltimore, Maryland. A and C, From Wiese C, Goldberg MW, Allen TD, et al: Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent “envelope smoothing” event. J Cell Sci 110:1489–1502, 1997.)

The mechanism of nuclear envelope reassembly is debated, in part because the fate of the nuclear membrane during mitosis is unclear. If the nuclear envelope disassembles to discrete vesicles as it does in eggs, then envelope reassembly is a classic membrane-sorting problem (see Fig. 21-12) that requires the fusion of membrane vesicles. In eggs, Ran-GTP and unknown factors direct the fusion of at least two or three discrete populations of membrane vesicles to re-form the nuclear envelope. On the other hand, if the nuclear membrane is absorbed into the endoplasmic reticulum during mitosis, as in vertebrate somatic cells, then reassembly involves lateral movements of membrane components within the membrane network and their stabilization at preferred binding sites at the periphery of the chromosomes.

Lamin subunits disassembled in prophase are recycled to re-form the nuclear envelope at the end of mitosis. Reassembly of the nuclear lamina is triggered by removal of mitosis-specific phosphate groups and methyl-esterification of several COOH side chains on lamin B (Fig. 44-6). B-type lamins are among the earliest components of the nuclear envelope to target to the surface of the chromosomes during mid-anaphase. Either at this time or shortly thereafter, other proteins associated with the inner nuclear membrane, including BAF, LAP2, and lamin B receptor (see Fig. 14-8), join the forming envelope. Lamin A enters the re-forming nucleus later during telophase, after the reassembly of nuclear pore complexes and reestablishment of nuclear import pathways. Its assembly into the peripheral lamina occurs slowly over a period of several hours in the G₁ phase. Transport of lamins through nuclear pores appears to be essential for nuclear reassembly. If lamin transport is prevented, chromosomes remain highly condensed following cytokinesis, and the cells fail to reenter the next S phase.

Signals Regulating the Position of the Cleavage Furrow

Elegant experimental data from classic studies on fertilized echinoderm eggs suggest that a cleavage stimulus, emitted by the mitotic spindle, specifies the position of the cleavage furrow midway between the poles and perpendicular to the long axis of the spindle, thereby ensuring that the cleavage process separates the daughter nuclei (Fig. 44-21). In fertilized eggs, the poles, with their large astral arrays of microtubules, are regarded as the source of the cleavage stimulus, as furrows can be induced to form midway between two poles, even when no chromosomes are present. In addition, a signal emitted by the bundled microtubules of the central spindle appears to modulate the behavior of the furrow signaled by the poles.

The molecular nature of the cleavage stimulus itself remains a mystery in animals, although several of its features are known:

Although signals from the poles of the mitotic spindle suffice to induce cytokinesis in large invertebrate embryos, other mechanisms contribute information to position the cleavage furrow between the daughter nuclei. In fission yeast, with closed mitosis, the nucleus determines the position of cleavage. In animal somatic cells, the organized central spindle plays a critical role, both early and late in cytokinesis. Drosophila mutants that fail to form a central spindle cannot initiate cytokinesis. In contrast, C. elegans embryos that lack a central spindle can initiate but not complete the process.

Assembly and Regulation of the Contractile Ring

Exposure of the cell cortex to the cleavage stimulus culminates in the assembly of a contractile ring consisting of a very thin (0.1 to 0.2μm) array of actin filaments attached to the plasma membrane at many sites around the equator (Fig. 44-23). Polymerization of the actin filaments depends on formins (see Fig. 33-12). Small, bipolar filaments of myosin-II are interdigitated with actin filaments. The plasma membrane adjacent to this actin-myosin ring undergoes alterations in its lipid composition that are important for the function of the contractile ring.

Figure 44-23 organization of the contractile ring. A, Organization of actin at the cell cortex prior to cytokinesis. B, Distribution of actin and myosin at the start of ring contraction. C, INCENP (red) concentrates at the site where the cleavage furrow will form just before myosin (green). D, INCENP and myosin concentrate in the contractile ring during contraction. E, Confocal micrograph shows the distribution of myosin in a contracting contractile ring. F–G, Dividing invertebrate egg with DNA (blue) and actin (red) in the contractile ring. H, Organization of actin and myosin filaments during cytokinesis. I–J, Electron micrographs showing actin filaments in the contractile ring. Note the thick filaments that are thought to be myosin-II filaments (red arrowheads) and the actin filaments (yellow arrows).

(C–D, Courtesy of William C. Earnshaw. E, I, and J, Courtesy of P. Maupin, Johns Hopkins Medical School, Baltimore, Maryland. F–G, Courtesy of Professor Issei Mabuchi, University of Tokyo, Japan. References: Maupin P, Pollard TD: Arrangement of actin filaments and myosin-like filaments in the contractile ring and actin-like filaments in the mitotic spindle of dividing HeLa cells. J Ultrastr Res 94:92–103, 1986; Maupin P, Phillips CL, Adelstein RS, Pollard TD: Differential localization of myosin-II isozymes in human cultured cells and blood cells. J Cell Sci 107:3077–3090, 1994; Eckley DM, Ainsztein AM, MacKay AM, et al: Chromosomal proteins and cytokinesis. J Cell Biol 136:1169–1183, 1997.)

Membrane furrowing requires actin and the motor activity of myosin-II (see Fig. 36-5). In animals, the small GTPase RhoA regulates actin polymerization by formins as well as constriction of the ring. Many other proteins are required for cytokinesis to go to completion. In their absence, furrowing begins, but the cleavage furrows ultimately regress, producing binucleated cells. This large class of proteins includes anillin, the chromosomal passenger proteins (Aurora-B kinase and its associated subunits [Fig. 44-10]) and a complex of Rho-GAP with a kinesin-6, among many others. Anillin helps to keep active myosin-II focused into an organized contractile ring throughout cytokinesis.

The chromosomal passengers and the Rho-GAP/kinesin-6 complex are required both for animal cells to assemble the central spindle and for the completion of cytokinesis. Although neither fits all criteria to be the cleavage stimulus, it is worth noting that both RhoA and the chromosomal passenger proteins require microtubules to localize to the site of cleavage furrow formation.

Fission yeast assemble a contractile ring along a well-defined pathway by recruiting proteins from cytoplasmic pools (Fig. 44-24). Polo kinase releases an anillin-like protein from the nucleus to mark the cortex in the middle of the cell and recruit myosin-II and a formin. Profilin activates the formin to polymerize actin filaments. Myosin-II pulls the actin filaments together into a ring around the equator of the cell (Fig. 44-23).

Figure 44-24 Cytokinesis in fission yeast Schizosaccharomyces pombe. During interphase, microtubules (red) position the nucleus in the middle of the cell. Actin filaments concentrate in small patches (yellow) in the cortex at the two growing ends of the cell (see Fig. 33-1). The mitotic spindle is inside the nucleus, as the nuclear membrane does not break down during mitosis. As the cell enters mitosis, an anillin-like protein moves from the nucleus to the equatorial cortex, where it sets up nodes of proteins, including myosin-II and a formin. The formin grows actin filaments (yellow), and myosin-II pulls the nodes together into a continuous contractile ring. At the end of anaphase a signaling system consisting of a GTPase and three protein kinases (the septation initiation network, SIN) triggers constriction of the contractile ring and associated synthesis of new cell wall to form a septum. The septum is a three-layered structure, with the primary septum flanked by two secondary septae. Digestion of the primary septum separates the daughter cells.

(Reference: Wu J-Q, Kuhn JR, Kovar DR, Pollard TD: Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev Cell 5:723–734, 2004.)

The assembly pathway is not yet understood in animal cells. INCENP and anillin move from the interphase nucleus to the cortex around the cell equator in early anaphase (Fig. 44-23). Some preexisting actin filaments are recruited intact into the contractile ring from adjacent areas of the cortex, whereas other filaments form de novo in the developing ring by assembly from monomers. Formins and profilin are involved, so the pathway resembles that in fission yeast. Myosin-II for the contractile ring is derived from various interphase structures. In cultured vertebrate cells, most of the myosin-II comes from stress fibers (see Fig. 33-1) that break down during prophase. Myosin-II is dispersed throughout the cytoplasm until anaphase, when it concentrates in the cortex, especially around the equator where the furrow forms.

Constriction of the Cleavage Furrow

Cleavage furrow ingression is widely believed to be driven by contraction of the contractile ring, though some researchers believe that alterations in the physical characteristics (e.g., tension and stiffness) of the plasma membrane may also have a role to play. Constriction of the ring probably involves a sliding filament mechanism, similar to muscle (see Figs. 39-12 and 39-20). During the early stages of furrowing, the contractile ring maintains a constant volume, but the structure disassembles completely by the end of cleavage. Thus, during the later stages of cytokinesis, contraction is accompanied by disassembly of the ring.

The role of myosin-II as the motor for cytokinesis was established by microinjection of inhibitory antibodies into echinoderm embryos and confirmed by genetic inactivation in the slime mold Dictyostelium. Slime mold amoebas that lack the myosin-II heavy chain still extend pseudopodia, round up during mitosis, and complete nuclear division but cannot form a normal cleavage furrow. Mutant cells accumulate many nuclei, because the mitotic cycle continues. The mutants can proliferate if grown on a substratum to which they are tightly adherent by using pseudopods to pull themselves apart into smaller cells.

Constriction of the contractile ring is regulated so that it does not begin until after the onset of anaphase B, when sister chromatids are well separated. Local release of calcium appears to initiate constriction of the contractile ring in some cells. The calcium may activate the enzyme myosin light chain kinase, which, in turn, activates myosin-II (see Fig. 39-21). Exposure of dividing cells to agents that stimulate the release of calcium accelerates the appearance and rate of propagation of the cleavage furrow, whereas injection of compounds that bind calcium can inhibit cytokinesis. Other kinases and counterbalancing phosphatases are also involved, so regulation of myosin-II in cytokinesis is actually quite complex.

Membrane Addition and Abscission

As the contractile ring pulls the cell membrane inward, the single cell that entered mitosis is gradually transformed into two daughter cells joined by a thin intercellular bridge (Fig. 44-20). This process requires a significant net increase in the surface area of the cell. New plasma membrane is inserted adjacent to the leading edge of the furrow. The source of the new membrane appears to be secretory vesicles derived from the Golgi apparatus, and addition to the cleavage furrow is a specialized form of exocytosis. Fusion of vesicles providing the new membrane depends on specific syntaxins, t-SNAREs (see Chapter 21) that promote vesicle fusion along the secretory pathway. Targeted endocytosis is also important for cytokinesis, although its role is unclear.

The plasma membrane in the cleavage furrow has a discrete composition. In budding yeast, this compartment is delineated by rings made from polymers of septins, a family of GTP-binding proteins. Septins are essential for cytokinesis in Saccharomyces cerevisiae but not fission yeast.

In most animal cells, contraction of the cleavage furrow ultimately reduces the cytoplasm to a thin intercellular bridge between the two daughter cells. The intercellular bridge contains a highly ordered, antiparallel array of microtubules derived from the spindle with a dense knob, the midbody, at its center (Fig. 44-20). Isolated midbodies contain over 160 proteins, about one third involved in various aspects of membrane trafficking.

The midbody is encircled by a dense ring of proteins that includes the kinesin-6 that is essential for central spindle assembly (see the earlier section titled “Assembly and Regulation of the Contractile Ring”) and a protein known as centriolin, which is associated with the centrosome for the rest of the cell cycle. A related yeast protein regulates the exit from mitosis. The conserved domain of centriolin binds the exocyst, a multisubunit protein complex that targets secretory vesicles to the plasma membrane (see Chapter 21, under the section titled “Tethering Factors”). These secretory vesicles accumulate near the midbody and fuse with the plasma membrane to separate the two daughter cells from one another. The details of this fusion event are not yet understood. The exocyst complex also contributes to cytokinesis in budding and fission yeasts.

In some tissues, intercellular bridges remain open as ring canals. After several rounds of nuclear division with incomplete cytokinesis, the network of cells maintains cytoplasmic continuity as each former contractile ring matures into a larger ring canal. During Drosophila oogenesis, four rounds of nuclear division with persistent ring canals creates 15 nurse cells, all in continuity with the oocytes (Fig. 44-25). The cytoplasmic continuity through ring canals allows nurse cells to transfer their cytoplasm into the developing egg, thus greatly increasing its stockpile of proteins and mRNAs available for use in early development. In mammals, incomplete cytokinesis is notable in the testis, where ring canals connect several hundred developing sperm cells.

Figure 44-25 Incomplete cytokinesis in a Drosophila egg chamber leaves cells joined by ring canals. Colocalization of actin (red) and the ring canal protein kelch (green) in the ring canals makes them appear yellow. In the Drosophila egg chamber, ring canals connect nurse cells to each other and to the oocyte. Late in oocyte development, a contraction of the nurse cells forces much of their cytoplasmic contents through the ring canals and into the oocyte. This is one way in which the oocyte gains the stockpile of compo-nents that are needed for early development of the fly embryo.

(Courtesy of Reed Kelso and Lynn Cooley, Yale University, New Haven, Connecticut.)

Exit from Mitosis

To exit from mitosis, cells must inactivate the Cdk1 kinase. This reverses the biochemical and structural changes that are characteristic of mitosis and prepares the cell for proliferation in the next cell cycle. The exit from mitosis is better understood in the yeasts than in animal cells.

In budding yeast, a signaling pathway called the mitotic exit network (MEN) terminates mitosis, promotes contraction of the contractile ring, and initiates septation. The pathway consists of a small GTPase and protein kinases. Cdk kinase activity suppresses the pathway until anaphase, when Cdk activity drops sharply. The MEN GTPase is associated with one spindle pole body (the yeast version of the centrosome), while its key regulator, a GTP exchange factor, is located in the bud. Elongation of the mitotic spindle during anaphase B moves the GTPase into the bud, where it is activated.

The MEN activates a phosphatase, Cdc14p, by releasing it from sequestration in the nucleolus. Cdc14p inhibits Cdk kinase activity in two ways: First, it inhibits the degradation of a Cdk inhibitor protein, which accumulates and inhibits the Cdk; second, it dephosphorylates Cdh1, which binds the APC/C and triggers the degradation of B-type cyclins and other proteins. Cdc14p also triggers other events during anaphase, including the transfer of chromosomal passenger proteins to the central spindle.

In fission yeast, proteins homologous to the MEN drive the cell out of mitosis. It is not known whether animal cells use a similar program to promote the exit from mitosis. Cdc14 phosphatase is required for cytokinesis in C. elegans, but the contributions of other components of the mitotic exit network have not yet been established.