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Cell_Biology_Alberts. M-Cdk activation begins with the accumulation of M-cyclin (cyclin B in vertebrate cells; see Table 17–1). In embryonic cell cycles, the synthesis of M-cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from the high stability of the protein in interphase. In most cell types, however, M-cyclin synthesis increases during G2 and M, owing primarily to an increase in M-cyclin gene transcription. The increase in M-cyclin protein leads to a corresponding accumulation of M-Cdk (the complex of Cdk1 and M-cyclin) as the cell approaches mitosis. Although the Cdk in these complexes is phosphorylated at an activating site by the Cdk-activating kinase (CAK), as discussed earlier, the protein kinase Wee1 holds it in an inactive state by inhibitory phosphorylation at two neighboring sites (see Figure 17–13). Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that


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Cell_Biology_Alberts. neighboring sites (see Figure 17–13). Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that block the active site of the kinase.


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Cell_Biology_Alberts. What, then, triggers the activation of the M-Cdk stockpile? The crucial event is the activation of the protein phosphatase Cdc25, which removes the inhibitory phosphates that restrain M-Cdk (Figure 17–20). At the same time, the inhibitory activity of the kinase Wee1 is suppressed, further ensuring that M-Cdk activity increases. The mechanisms that unleash Cdc25 activity in early mitosis are not well understood. One possibility is that the S-Cdks that are active in G2 and early prophase stimulate Cdc25.


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Cell_Biology_Alberts. Interestingly, Cdc25 can also be activated, at least in part, by its target, M-Cdk. M-Cdk may also inhibit the inhibitory kinase Wee1. The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves positive feedback loops (see Figure 17–20). According to this attractive model, the partial activation of Cdc25 (perhaps by S-Cdk) leads to the partial activation of a subpopulation of M-Cdk complexes, which then phosphorylate Cdc25 and Wee1 molecules. This leads to more M-Cdk activation, and so on. Such a mechanism would quickly promote the activation of all M-Cdk complexes in the cell. As mentioned earlier, similar molecular switches operate at various points in the cell cycle to promote the abrupt and complete transition from one cell-cycle state to the next.


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Cell_Biology_Alberts. At the end of S phase, the immensely long DNA molecules of the sister chromatids are tangled in a mass of partially catenated DNA and proteins. Any attempt to pull the sisters apart in this state would undoubtedly lead to breaks in the chromosomes. To avoid this disaster, the cell devotes a great deal of energy in early mitosis to gradually reorganizing the sister chromatids into relatively short, distinct structures that can be pulled apart more easily in anaphase. These chromosomal changes involve two processes: chromosome condensation, in which the chromatids are dramatically compacted; and sister-chromatid resolution, whereby the two sisters are resolved into distinct, separable units (Figure 17–21). Resolution results from the decatenation of the sister DNAs, accompanied by the partial removal of cohesin molecules along the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids appear in the microscope as compact, rodlike structures that are joined


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Cell_Biology_Alberts. removal of cohesin molecules along the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids appear in the microscope as compact, rodlike structures that are joined tightly at their centromeric regions and only loosely along their arms.


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Cell_Biology_Alberts. Figure 17–20 The activation of M-Cdk. Cdk1 associates with m-cyclin as the levels of m-cyclin gradually rise. The resulting m-Cdk complex is phosphorylated on an activating site by the Cdk-activating kinase (Cak) and on a pair of inhibitory sites by the Wee1 kinase. The resulting inactive m-Cdk complex is then activated at the end of g2 by the phosphatase Cdc25. Cdc25 is further stimulated by active m-Cdk, resulting in positive feedback. This feedback is enhanced by the ability of m-Cdk to inhibit Wee1. Figure 17–21 The mitotic chromosome. scanning electron micrograph of a human mitotic chromosome, consisting of two sister chromatids joined along their length. The constricted regions are the centromeres. (Courtesy of Terry d. allen.)


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Cell_Biology_Alberts. PROPHASE1centrosome forming mitotic spindle condensing replicated chromosome, consisting of two sister chromatids held together along their length intact nuclear envelope kinetochore At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense. Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have replicated and moved apart. For simplicity, only three chromosomes are shown. In diploid cells, there would be two copies of each chromo-some present. In the fuorescence micrograph, chromosomes are stained orange and microtubules are green.PROMETAPHASE 2 Prometaphase starts abruptly with the breakdown of the nuclear envelope. Chromosomes can now attach to spindle microtubules via their kinetochores and undergo active movement. centrosome at spindle pole kinetochore microtubule chromosome in active motion fragments of nuclear envelope METAPHASE 3 At metaphase, the chromosomes are aligned at the equator of


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Cell_Biology_Alberts. active movement. centrosome at spindle pole kinetochore microtubule chromosome in active motion fragments of nuclear envelope METAPHASE 3 At metaphase, the chromosomes are aligned at the equator of the spindle, midway between the spindle poles. The kinetochore microtubules attach sister chromatids to opposite poles of the spindle. centrosome at spindle pole kinetochore microtubule PANeL 17–1: The Principle Stages of M Phase (Mitosis and Cytokinesis) in an Animal Cell 980


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Cell_Biology_Alberts. ANAPHASE4 At anaphase, the sister chromatids synchronously separate to form two daughter chromosomes, and each is pulled slowly toward the spindle pole it faces. The kinetochore microtubules get shorter, and the spindle poles also move apart; both processes contribute to chromosome segregation. TELOPHASE5 During telophase, the two sets of daughter chromo-somes arrive at the poles of the spindle and decondense. A new nuclear envelope reassembles around each set, completing the formation of two nuclei and marking the end of mitosis. The division of the cytoplasm begins with contraction of the contractile ring. CYTOKINESIS6 During cytokinesis, the cytoplasm is divided in two by a contractile ring of actin and myosin flaments, which pinches the cell in two to create two daughters, each with one nucleus. shortening kinetochore microtubule spindle pole moving outward set of daughter chromosomes at spindle pole contractile ring starting to contract centrosomeinterpolar microtubules nuclear


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Cell_Biology_Alberts. one nucleus. shortening kinetochore microtubule spindle pole moving outward set of daughter chromosomes at spindle pole contractile ring starting to contract centrosomeinterpolar microtubules nuclear envelope reassembling around individual chromosomes daughter chromosomes completed nuclear envelope surrounds decondensing chromosomes contractile ring creating cleavage furrow re-formation of interphase array of microtubules nucleated by the centrosome (Micrographs courtesy of Julie Canman and Ted Salmon.) 981


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Cell_Biology_Alberts. The condensation and resolution of sister chromatids depend, at least in part, on a five-subunit protein complex called condensin. Condensin structure is related to that of the cohesin complex that holds sister chromatids together (see Figure 17–19). It contains two SMC subunits like those of cohesin, plus three non-SMC subunits (Figure 17–22). Condensin may form a ringlike structure that somehow uses the energy provided by ATP hydrolysis to promote the compaction and resolution of sister chromatids. Condensin is able to change the coiling of DNA molecules in a test tube, and this coiling activity is thought to be important for chromosome condensation during mitosis. Interestingly, phosphorylation of condensin subunits by M-Cdk stimulates this coiling activity, providing one mechanism by which M-Cdk may promote chromosome restructuring in early mitosis. The mitotic spindle Is a microtubule-Based machine


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Cell_Biology_Alberts. The mitotic spindle Is a microtubule-Based machine The central event of mitosis—chromosome segregation—depends in all eukaryotes on a complex and beautiful machine called the mitotic spindle (see Panel 17–1). The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase, thereby segregating the two sets of chromosomes to opposite ends of the cell, where they are packaged into daughter nuclei (Movie 17.6). M-Cdk triggers the assembly of the spindle early in mitosis, in parallel with the chromosome restructuring just described. Before we consider how the spindle assembles and how its microtubules attach to sister chromatids, we briefly review the basic features of spindle structure.


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Cell_Biology_Alberts. The core of the mitotic spindle is a bipolar array of microtubules, the minus ends of which are focused at the two spindle poles, and the plus ends of which radiate outward from the poles (Figure 17–23). The plus ends of some microtubules—called the interpolar microtubules—overlap with the plus ends of micro-tubules from the other pole, resulting in an antiparallel array in the spindle mid-zone. The plus ends of other microtubules—the kinetochore microtubules—are attached to sister-chromatid pairs at large protein structures called kinetochores, which are located at the centromere of each sister chromatid. Finally, many spindles also contain astral microtubules that radiate outward from the poles and contact the cell cortex, helping to position the spindle in the cell.


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Cell_Biology_Alberts. In most somatic animal cells, each spindle pole is focused at a protein organelle called the centrosome (see Figures 16–47 and 16–48). Each centrosome consists of a cloud of amorphous material (called the pericentriolar matrix) that surrounds a pair of centrioles (Figure 17–24). The pericentriolar matrix nucleates a radial array of microtubules, with their fast-growing plus ends projecting outward and their minus ends associated with the centrosome. The matrix contains a variety of proteins, including microtubule-dependent motor proteins, coiled-coil proteins that link the motors to the centrosome, structural proteins, and components of the cell-cycle control system. Most important, it contains γ-tubulin ring complexes, which are the components mainly responsible for nucleating microtubules (see Figure 16–46).


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Cell_Biology_Alberts. Some cells—notably the cells of higher plants and the oocytes of many vertebrates—do not have centrosomes, and microtubule-dependent motor proteins and other proteins associate with microtubule minus ends to organize and focus the spindle poles. Figure 17–23 The metaphase mitotic spindle in an animal cell. The plus ends of the microtubules project away from the spindle pole, while the minus ends are anchored at the spindle poles, which in this example are organized by centrosomes. kinetochore microtubules connect the spindle poles with the kinetochores of sister chromatids, while interpolar microtubules from the two poles interdigitate at the spindle equator. astral microtubules radiate out from the poles into the cytoplasm.


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Cell_Biology_Alberts. The function of the mitotic spindle depends on numerous microtubule-dependent motor proteins. As discussed in Chapter 16, these proteins belong to two families—the kinesin-related proteins, which usually move toward the plus end of microtubules, and dyneins, which move toward the minus end. In the mitotic spindle, these motor proteins generally operate at or near the ends of the micro-tubules. Four major types of motor proteins—kinesin-5, kinesin-14, kinesins-4/10, and dynein—are particularly important in spindle assembly and function (Figure 17–25).


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Cell_Biology_Alberts. Kinesin-5 proteins contain two motor domains that interact with the plus ends of antiparallel microtubules in the spindle midzone. Because the two motor domains move toward the plus ends of the microtubules, they slide the two anti-parallel microtubules past each other toward the spindle poles, pushing the poles apart. Kinesin-14 proteins, by contrast, are minus-end directed motors with a single motor domain and other domains that can interact with a neighboring microtubule. They can cross-link antiparallel interpolar microtubules at the spindle midzone and tend to pull the poles together. Kinesin-4 and kinesin-10


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Cell_Biology_Alberts. Figure 17–24 The centrosome. (a) Electron micrograph of an s-phase mammalian cell in culture, showing a duplicated centrosome. Each centrosome contains a pair of centrioles; although the centrioles have duplicated, they remain together in a single complex, as shown in the drawing of the micrograph in (B). One centriole of each centriole pair has been cut in cross section, while the other is cut in longitudinal section, indicating that the two members of each pair are aligned at right angles to each other. The two halves of the replicated centrosome, each consisting of a centriole pair surrounded by pericentriolar matrix, will split and migrate apart to initiate the formation of the two poles of the mitotic spindle when the cell enters m phase. (a, from m. mcgill, d.p. Highfield, T.m. monahan, and B.R. Brinkley, J. Ultrastruct. Res. 57:43–53, 1976. With permission from academic press.) spindle microtubule dynein kinesin-4,10 kinesin-14 kinesin-5 dynein sister chromatids centrosome+ + +


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Cell_Biology_Alberts. and B.R. Brinkley, J. Ultrastruct. Res. 57:43–53, 1976. With permission from academic press.) spindle microtubule dynein kinesin-4,10 kinesin-14 kinesin-5 dynein sister chromatids centrosome+ + + + + + + – – – – – – – plasma membrane Figure 17–25 Major motor proteins of the spindle. Four major classes of microtubule-dependent motor proteins (yellow boxes) contribute to spindle assembly and function (see text). The colored arrows indicate the direction of motor protein movement along a microtubule—blue toward the minus end and red toward the plus end.


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Cell_Biology_Alberts. proteins, also called chromokinesins, are plus-end directed motors that associate with chromosome arms and push the attached chromosome away from the pole (or the pole away from the chromosome). Finally, dyneins are minus-end directed motors that, together with associated proteins, organize microtubules at various locations in the cell. They link the plus ends of astral microtubules to components of the actin cytoskeleton at the cell cortex, for example; by moving toward the minus end of the microtubules, the dynein motors pull the spindle poles toward the cell cortex and away from each other. multiple mechanisms Collaborate in the assembly of a Bipolar mitotic spindle


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Cell_Biology_Alberts. multiple mechanisms Collaborate in the assembly of a Bipolar mitotic spindle The mitotic spindle must have two poles if it is to pull the two sets of sister chromatids to opposite ends of the cell in anaphase. In most animal cells, several mechanisms ensure the bipolarity of the spindle. One depends on centrosomes. A typical animal cell enters mitosis with a pair of centrosomes, each of which nucleates a radial array of microtubules. The two centrosomes provide prefabricated spindle poles that greatly facilitate bipolar spindle assembly. The other mechanisms depend on the ability of mitotic chromosomes to nucleate and stabilize microtubules and on the ability of motor proteins to organize microtubules into a bipolar array. These “self-organization” mechanisms can produce a bipolar spindle even in cells lacking centrosomes.


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Cell_Biology_Alberts. We now describe the steps of spindle assembly, beginning with centrosomedependent assembly in early mitosis. We then consider the self-organization mechanisms that do not require centrosomes and become particularly important after nuclear-envelope breakdown. Centrosome duplication Occurs Early in the Cell Cycle


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Cell_Biology_Alberts. Centrosome duplication Occurs Early in the Cell Cycle Most animal cells contain a single centrosome that nucleates most of the cell’s cytoplasmic microtubules. The centrosome duplicates when the cell enters the cell cycle, so that by the time the cell reaches mitosis there are two centrosomes. Centrosome duplication begins at about the same time as the cell enters S phase. The G1/S-Cdk (a complex of cyclin E and Cdk2 in animal cells; see Table 17–1) that triggers cell-cycle entry also helps initiate centrosome duplication. The two centrioles in the centrosome separate, and each nucleates the formation of a single new centriole, resulting in two centriole pairs within an enlarged pericentriolar matrix (Figure 17–26). This centrosome pair remains together on one side of the nucleus until the cell enters mitosis.


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Cell_Biology_Alberts. There are interesting parallels between centrosome duplication and chromosome duplication. Both use a semiconservative mechanism of duplication, in which the two halves separate and serve as templates for construction of a new half. Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters mitosis with only two copies: an incorrect number of centrosomes could lead to defects in spindle assembly and thus errors in chromosome segregation.


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Cell_Biology_Alberts. The mechanisms that limit centrosome duplication to once per cell cycle are uncertain. In many cell types, experimental inhibition of DNA synthesis blocks centrosome duplication, providing one mechanism by which centrosome number is kept in check. Other cell types, however, including those in the early embryos of flies, sea urchins, and frogs, do not have such a mechanism and centrosome duplication continues if chromosome duplication is blocked. It is not known how such cells limit centrosome duplication to once per cell cycle.


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Cell_Biology_Alberts. Spindle assembly begins in early mitosis, when the two centrosomes move apart along the nuclear envelope, pulled by dynein motor proteins that link astral micro-tubules to the cell cortex (see Figure 17–25). The plus ends of the microtubules between the centrosomes interdigitate to form the interpolar microtubules, and kinesin-5 motor proteins associate with these microtubules and push the centrosomes apart (see Figure 17–25). Also in early mitosis, the number of γ-tubulin ring complexes in each centrosome increases greatly, increasing the ability of the centrosomes to nucleate new microtubules, a process called centrosome maturation.


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Cell_Biology_Alberts. The balance of opposing forces generated by different types of motor proteins determines the final length of the spindle. Dynein and kinesin-5 motors generally promote centrosome separation and increase spindle length. Kinesin-14 proteins do the opposite: they tend to pull the poles together (see Figure 17–25). It is not clear how the cell regulates the balance of opposing forces to generate the appropriate spindle length. M-Cdk and other mitotic protein kinases are required for centrosome separation and maturation. M-Cdk and Aurora-A phosphorylate kinesin-5 motors and stimulate them to drive centrosome separation. Aurora-A and Plk also phosphorylate components of the centrosome and thereby promote its maturation. The Completion of spindle assembly in animal Cells Requires nuclear-Envelope Breakdown


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Cell_Biology_Alberts. The Completion of spindle assembly in animal Cells Requires nuclear-Envelope Breakdown The centrosomes and microtubules of animal cells are located in the cytoplasm, separated from the chromosomes by the double-membrane barrier of the nuclear envelope (discussed in Chapter 12). Clearly, the attachment of sister-chromatid pairs to the spindle requires the removal of this barrier. In addition, many of the motor proteins and microtubule regulators that promote spindle assembly are associated with the chromosomes inside the nucleus, and they require nuclear-envelope breakdown to carry out their functions. Nuclear-envelope breakdown is a complex, multistep process, which is thought to begin when M-Cdk phosphorylates several subunits of the nuclear pore complexes in the nuclear envelope. This phosphorylation initiates the disassembly of nuclear pore complexes and their dissociation from the envelope. M-Cdk


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Cell_Biology_Alberts. Figure 17–26 Centriole replication. The centrosome consists of a centriole pair and associated pericentriolar matrix (green). at a certain point in g1, the two centrioles of the pair separate by a few micrometers. during s phase, a daughter centriole begins to grow near the base of each mother centriole and at a right angle to it. The elongation of the daughter centriole is usually completed in g2. The two centriole pairs remain close together in a single centrosomal complex until the beginning of m phase, when the complex splits in two and the two daughter centrosomes begin to separate. Each centrosome now nucleates its own radial array of microtubules (called an aster), mainly from the mother centriole.


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Cell_Biology_Alberts. also phosphorylates components of the nuclear lamina, the structural framework beneath the envelope. The phosphorylation of these lamina components and of several inner-nuclear-envelope proteins leads to disassembly of the nuclear lamina and the breakdown of the envelope membranes into small vesicles. Most 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. New microtubules are continually being created to balance the loss of those that disappear completely by depolymerization.


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Cell_Biology_Alberts. Entry into mitosis signals an abrupt change in the cell’s microtubules. The interphase array of few, long microtubules radiating from the single centrosome is converted to a larger number of shorter and more dynamic microtubules emanating from both centrosomes. During prophase, and particularly in prometaphase and metaphase (see Panel 17–1), the half-life of microtubules decreases dramatically. This increase in microtubule instability, coupled with the increased ability of centrosomes to nucleate microtubules as mentioned earlier, results in remarkably dense and dynamic arrays of spindle microtubules that are ideally suited for capturing sister chromatids.


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Cell_Biology_Alberts. Microtubule dynamics are controlled in the cell by a variety of regulatory proteins, including microtubule-associated proteins (MAPs) that promote stability and catastrophe factors that destabilize microtubule plus ends. Changes in the activities of these regulatory proteins are responsible for the changes in micro-tubule dynamics that occur during mitosis. Many of these changes result from phosphorylation of specific proteins by M-Cdk and other mitotic protein kinases.


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Cell_Biology_Alberts. 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 micro-tubules 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, at least in part, on a guanine nucleotide exchange factor (GEF) that is bound to chromatin; the GEF stimulates a small GTPase in the cytosol called 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


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Cell_Biology_Alberts. cytosol called 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 and stabilization of microtubules around chromosomes (Figure 17–27). Local microtubule stabilization is also promoted by the protein kinase Aurora-B, which associates with mitotic chromosomes.


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Cell_Biology_Alberts. Figure 17–27 Activation of the GTPase Ran around mitotic chromosomes. The Ran protein, like other members of the small gTpase family (discussed in Chapter 15), can exist in two conformations depending on whether it is bound to gdp (inactive state) or gTp (active state). The localization of active Ran in mitosis was determined using a protein that emits fluorescence at a specific wavelength when it is activated by Ran-gTp. In the metaphase human cell shown here, Ran activity (yellow and red) is highest around the chromosomes, between the poles of the mitotic spindle (indicated by asterisks). (From p. kaláb et al., Nature 440:697–701, 2006. With permission from macmillan publishers Ltd.) nucleation antiparallel cross-linking outward push focusing of poles by by kinesin-5 by kinesin-4,10 dynein and kinesin-14 – – –


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Cell_Biology_Alberts. Figure 17–28 Spindle self-organization by motor proteins. mitotic chromosomes stimulate the local activation of proteins that nucleate and promote the formation of microtubules in the vicinity of the chromosomes. kinesin-5 motor proteins (see Figure 17–25) organize these microtubules into antiparallel bundles, while plus-end directed kinesins-4 and 10 link the microtubules to chromosome arms and push minus ends away from the chromosomes. dynein and kinesin-14 motors, together with numerous other proteins, focus these minus ends into a pair of spindle poles. The ability of chromosomes to stabilize and organize microtubules enables cells to form bipolar spindles in the absence of centrosomes. Acentrosomal spindle assembly is thought to begin with the formation of microtubules around the chromosomes. Various motor proteins then organize the microtubules into a bipolar spindle, as illustrated in Figure 17–28.


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Cell_Biology_Alberts. Cells that normally lack centrosomes, such as those of higher plants and many animal oocytes, use this chromosome-based self-organization process to form spindles. It is also the process used to assemble spindles in certain animal embryos that have been induced to develop from eggs without fertilization (that is, parthenogenetically); as the sperm normally provides the centrosome when it fertilizes an egg, the mitotic spindles in these parthenogenetic embryos develop without centrosomes (Figure 17–29). Even in cells that normally contain centrosomes, the chromosomes help organize the spindle microtubules and, with the help of various motor proteins, can promote the assembly of a bipolar mitotic spindle if the centrosomes are removed. 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 in the cell.


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Cell_Biology_Alberts. kinetochores attach sister Chromatids to the spindle


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Cell_Biology_Alberts. Following the assembly of a bipolar microtubule array, the second major step in spindle formation is the attachment of the array to the sister-chromatid pairs. Spindle microtubules become attached to each chromatid at its kinetochore, a giant, multilayered protein structure that is built at the centromeric region of the chromatid (Figure 17–30; also see Chapter 4). In metaphase, the plus ends of kinetochore microtubules are embedded head-on in specialized microtubuleattachment sites within the outer region of the kinetochore, furthest from the DNA. The kinetochore of an animal cell can bind 10–40 microtubules, whereas a budding yeast kinetochore can bind only one. Attachment of each microtubule depends on multiple copies of a rod-shaped protein complex called the Ndc80 complex, which is anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of


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Cell_Biology_Alberts. anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of tubulin subunits at this end (Figure 17–31). Regulation of plus-end polymerization and depolymerization at the kinetochore is critical for the control of chromosome movement on the spindle, as we discuss later.


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Cell_Biology_Alberts. Kinetochore attachment to the spindle occurs by a complex sequence of events. At the end of prophase in animal cells, the centrosomes of the growing spindle generally lie on opposite sides of the nuclear envelope. Thus, when the envelope breaks down, the sister-chromatid pairs are bombarded by microtubule


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Cell_Biology_Alberts. Figure 17–29 bipolar spindle assembly without centrosomes in parthenogenetic embryos of the insect Sciara (or fungus gnat). The microtubules are stained green, the chromosomes red. The top fluorescence micrograph shows a normal spindle formed with centrosomes in a normally fertilized Sciara embryo. The bottom micrograph shows a spindle formed without centrosomes in an embryo that initiated development without fertilization. note that the spindle with centrosomes has an aster at each pole of the spindle, whereas the spindle formed without centrosomes does not. Both types of spindles are able to segregate the replicated chromosomes. (From B. de saint phalle and W. sullivan, J. Cell Biol. 141:1383–1391, 1998. With permission from The Rockefeller University press.) plus ends coming from two directions. However, the kinetochores do not instantly achieve the correct ‘end-on’ microtubule attachment to both spindle poles. Instead, detailed studies with light and electron microscopy show that


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Cell_Biology_Alberts. However, the kinetochores do not instantly achieve the correct ‘end-on’ microtubule attachment to both spindle poles. Instead, detailed studies with light and electron microscopy show that most initial attachments are unstable lateral attachments, in which a kinetochore attaches to the side of a passing microtubule, with assistance from kinesin motor proteins in the outer kinetochore. Soon, however, the dynamic microtubule plus ends capture the kinetochores in the correct end-on orientation (Figure 17–32).


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Cell_Biology_Alberts. Another attachment mechanism also plays a part, particularly in the absence of centrosomes. Careful microscopic analysis suggests that short microtubules in the vicinity of the chromosomes become embedded in the plus-end-binding sites of the kinetochore. Polymerization at these plus ends then results in growth of the microtubules away from the kinetochore. The minus ends of these kinetochore microtubules are eventually cross-linked to other minus ends and focused by motor proteins at the spindle pole (see Figure 17–28). The success of mitosis demands that sister chromatids in a pair attach to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when direction of chromatid movement Figure 17–30 The kinetochore.


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Cell_Biology_Alberts. Figure 17–30 The kinetochore. a fluorescence micrograph of a metaphase chromosome stained with a dna-binding fluorescent dye and with human autoantibodies that react with specific kinetochore proteins. The two kinetochores, one associated with each sister chromatid, are stained red. a drawing of a metaphase chromosome showing its two sister chromatids attached to the plus ends of kinetochore microtubules. Each kinetochore forms a plaque on the surface of the centromere. Electron micrograph of an anaphase chromatid with microtubules attached to its kinetochore. While most kinetochores have a trilaminar structure, the one shown here (from a green alga) has an unusually complex structure with additional layers. (a, courtesy of B.R. Brinkley; C, from J.d. pickett-Heaps and L.C. Fowke, Aust. J. Biol. Sci. 23:71–92, 1970. With permission from CsIRO.)


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Cell_Biology_Alberts. Figure 17–31 Microtubule attachment sites in the kinetochore. (a) In this electron micrograph of a mammalian kinetochore, the chromosome is on the right, and the plus ends of multiple microtubules are embedded in the outer kinetochore on the left. (B) Electron tomography (discussed in Chapter 9) was used to construct a low-resolution three-dimensional image of the outer kinetochore in (a). several microtubules (in multiple colors) are embedded in fibrous material of the kinetochore, which is thought to be composed of the ndc80 complex and other proteins. (C) Each microtubule is attached to the kinetochore by interactions with multiple copies of the ndc80 complex (blue). This complex binds to the sides of the microtubule near its plus end, allowing polymerization and depolymerization to occur while the microtubule remains attached to the kinetochore. (a and B, from Y. dong et al., Nature Cell Biol. 9:516–522, 2007. With permission from macmillan publishers Ltd.) + early prometaphase:


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Cell_Biology_Alberts. while the microtubule remains attached to the kinetochore. (a and B, from Y. dong et al., Nature Cell Biol. 9:516–522, 2007. With permission from macmillan publishers Ltd.) + early prometaphase: lateral kinetochore mid prometaphase: metaphase:late prophase attachments, chromosome arms pushed outwards end-on attachment bi-orientation


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Cell_Biology_Alberts. Figure 17-32 Chromosome attachment to the mitotic spindle in animal cells. (a) In late prophase of most animal cells, the mitotic spindle poles have moved to opposite sides of the nuclear envelope, with an array of overlapping microtubules between them. (B) Following nuclear envelope breakdown, the sister-chromatid pairs are exposed to the large number of dynamic plus ends of microtubules radiating from the spindle poles. In most cases, the kinetochores are first attached to the sides of these microtubules, while at the same time the arms of the chromosomes are pushed outward from the spindle interior, preventing the arms from blocking microtubule access to the kinetochores. (C) Eventually, the laterally-attached sister chromatids are arranged in a ring around the outside of the spindle. most of the microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. (d) dynamic microtubule plus ends eventually encounter the kinetochores in an end-on


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Cell_Biology_Alberts. spindle. most of the microtubules are concentrated in this ring, so that the spindle is relatively hollow inside. (d) dynamic microtubule plus ends eventually encounter the kinetochores in an end-on orientation and are captured and stabilized. (E) stable end-on attachment to both poles results in bi-orientation. additional microtubules are attached to the kinetochore, resulting in a kinetochore fiber containing 10–40 microtubules.


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Cell_Biology_Alberts. they separate in anaphase. How is this mode of attachment, called bi-orientation, achieved? What prevents the attachment of both kinetochores to the same spindle pole or the attachment of one kinetochore to both spindle poles? Part of the answer is that sister kinetochores are constructed in a back-to-back orientation that reduces the likelihood that both kinetochores can face the same spindle pole. Nevertheless, incorrect attachments do occur, and elegant regulatory mechanisms have evolved to correct them.


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Cell_Biology_Alberts. Incorrect attachments are corrected by a system of trial and error that is based on a simple principle: incorrect attachments are highly unstable and do not last, whereas correct attachments become locked in place. How does the kinetochore sense a correct attachment? The answer appears to be tension (Figure 17–33). When a sister-chromatid pair is properly bi-oriented on the spindle, the two kinetochores are pulled in opposite directions by strong poleward forces. Sister-chromatid cohesion resists these poleward forces, creating high levels of tension within the kinetochores. When chromosomes are incorrectly attached—when both sister chromatids are attached to the same spindle pole, for example—tension is low Figure 17–33 Alternative forms of kinetochore attachment to the spindle poles. (a) Initially, a single microtubule from a spindle pole binds to one kinetochore in a sister-chromatid pair. additional microtubules can then bind to the chromosome in various ways.


Cell_Biology_Alberts

Cell_Biology_Alberts. a microtubule from the same spindle pole can attach to the other sister kinetochore, or (C) microtubules from both spindle poles can attach to the same kinetochore. These incorrect attachments are unstable, however, so that one of the two microtubules tends to dissociate. When a microtubule from the opposite pole binds to the second kinetochore, the sister kinetochores are thought to sense tension across their microtubulebinding sites. This triggers an increase in microtubule binding affinity, thereby locking the correct attachment in place.


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Cell_Biology_Alberts. and the kinetochore sends an inhibitory signal that loosens the grip of its microtubule attachment site, allowing detachment to occur. When bi-orientation occurs, the high tension at the kinetochore shuts off the inhibitory signal, strengthening microtubule attachment. In animal cells, tension not only increases the affinity of the attachment site but also leads to the attachment of additional microtubules to the kinetochore. This results in the formation of a thick kinetochore fiber composed of multiple microtubules.


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Cell_Biology_Alberts. The tension-sensing mechanism depends on the protein kinase Aurora-B, which is associated with the kinetochore and is thought to generate the inhibitory signal that reduces the strength of microtubule attachment in the absence of tension. It phosphorylates several components of the microtubule attachment site, including the Ndc80 complex, decreasing the site’s affinity for a microtubule plus end. When bi-orientation occurs, the resulting tension somehow reduces phosphorylation by Aurora-B, thereby increasing the affinity of the attachment site (Figure 17–34).


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Cell_Biology_Alberts. Following their attachment to the two spindle poles, the chromosomes are tugged back and forth, eventually assuming a position equidistant between the two poles, a position called the metaphase plate. In vertebrate cells, the chromosomes then oscillate gently at the metaphase plate, awaiting the signal for the sister chromatids to separate. The signal is produced, with a predictable lag time, after the bi-oriented attachment of the last of the chromosomes. multiple Forces act on Chromosomes in the spindle Multiple mechanisms generate the forces that move chromosomes back and forth after they are attached to the spindle, and produce the tension that is so important for the stabilization of correct attachments. In anaphase, similar forces pull the separated chromatids to opposite ends of the spindle. Three major spindle forces are particularly critical, although their strength and importance vary at different stages of mitosis.


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Cell_Biology_Alberts. The first major force pulls the kinetochore and its associated chromatid along the kinetochore microtubule toward the spindle pole. It is produced by proteins at the kinetochore itself. By an uncertain mechanism, depolymerization at the plus end of the microtubule generates a force that pulls the kinetochore poleward. This force pulls on chromosomes during prometaphase and metaphase but is particularly important for moving sister chromatids toward the poles after they separate in anaphase. Interestingly, this kinetochore-generated poleward force does not require ATP or motor proteins. This 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


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Cell_Biology_Alberts. 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).


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Cell_Biology_Alberts. Figure 17–34 How tension might increase microtubule attachment to the kinetochore. These diagrams illustrate one speculative mechanism by which bi-orientation might increase microtubule attachment to the kinetochore. a single kinetochore is shown for clarity; the spindle pole is on the right. (a) When a sisterchromatid pair is unattached to the spindle or attached to just one spindle pole, there is little tension between the outer and inner kinetochores. The protein kinase aurora-B is tethered to the inner kinetochore and phosphorylates the microtubule attachment sites, including the ndc80 complex (blue), in the outer kinetochore as shown, thereby reducing the affinity of microtubule binding. microtubules therefore associate and dissociate rapidly, and attachment is unstable. (B) When bi-orientation is achieved, the forces pulling the kinetochore toward the spindle pole are resisted by forces pulling the other sister kinetochore toward the opposite pole, and the resulting tension


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Cell_Biology_Alberts. bi-orientation is achieved, the forces pulling the kinetochore toward the spindle pole are resisted by forces pulling the other sister kinetochore toward the opposite pole, and the resulting tension pulls the outer kinetochore away from the inner kinetochore. as a result, aurora-B is unable to reach the outer kinetochore, and microtubule attachment sites are not phosphorylated. microtubule binding affinity is therefore increased, resulting in the stable attachment of multiple microtubules to both kinetochores. The dephosphorylation of outer kinetochore proteins depends on a phosphatase that is not shown here.


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Cell_Biology_Alberts. How does plus-end depolymerization drive the kinetochore toward the pole? As we discussed earlier (see Figure 17–31C), Ndc80 complexes in the kinetochore make multiple low-affinity attachments along the side of the microtubule. Because the attachments are constantly breaking and re-forming at new sites, the kinetochore remains attached to a microtubule even as the microtubule depolymerizes. In principle, this could move the kinetochore toward the spindle pole.


Cell_Biology_Alberts

Cell_Biology_Alberts. A second poleward force is provided in some cell types by microtubule flux, whereby the microtubules themselves are pulled toward the spindle poles and dismantled at their minus ends. The mechanism underlying this poleward movement is not clear, although it might depend on forces generated by motor proteins and minus-end depolymerization at the spindle pole. In metaphase, the addition of new tubulin at the plus end of a microtubule compensates for the loss of tubulin at the minus end, so that microtubule length remains constant despite the movement of microtubules toward the spindle pole (Figure 17–35). Any kinetochore that is attached to a microtubule undergoing such flux experiences a poleward force, which contributes to the generation of tension at the kinetochore in meta-phase. Together with the kinetochore-based forces discussed above, flux also contributes to the poleward forces that move sister chromatids after they separate in anaphase.


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Cell_Biology_Alberts. A third force acting on chromosomes is the polar ejection force, or polar wind. Plus-end directed kinesin-4 and 10 motors on chromosome arms interact with interpolar microtubules and transport the chromosomes away from the spindle poles (see Figure 17–25). This force is particularly important in prometaphase and metaphase, when it helps push chromosome arms out from the spindle. This force might also help align the sister-chromatid pairs at the metaphase plate (Figure 17–36). One of the most striking aspects of mitosis in vertebrate cells is the continuous oscillatory movement of the chromosomes in prometaphase and metaphase. When studied by video microscopy in newt lung cells, the movements are seen to switch between two states—a poleward state, when the chromosomes are pulled toward the pole, and an away-from-the-pole, or neutral, state, when the poleward forces are turned off and the polar ejection force pushes the chromosomes away


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Cell_Biology_Alberts. Figure 17–35 Microtubule flux in the metaphase spindle. (a) To observe microtubule flux, a very small amount of fluorescent tubulin is injected into living cells so that individual microtubules form with a very small proportion of fluorescent tubulin. such microtubules have a speckled appearance when viewed by fluorescence microscopy. (B) Fluorescence micrograph of a mitotic spindle in a living newt lung epithelial cell. The chromosomes are colored brown, and the tubulin speckles are red. (C) The movement of individual speckles can be followed by time-lapse video microscopy. Images of the thin vertical boxed region (arrow) in (B), taken at sequential times, show that individual speckles move toward the poles at a rate of about 0.75 μm/min, indicating that the microtubules are moving poleward. (d) microtubule length in the metaphase spindle does not change significantly because new tubulin subunits are added at the microtubule plus end at the same rate as tubulin subunits are removed


Cell_Biology_Alberts

Cell_Biology_Alberts. (d) microtubule length in the metaphase spindle does not change significantly because new tubulin subunits are added at the microtubule plus end at the same rate as tubulin subunits are removed from the minus end. (B and C, from T.J. mitchison and E.d. salmon, Nat. Cell Biol. 3:E17–21, 2001. With permission from macmillan publishers Ltd.) interpolar plus-end-directed or astral kinesin-4,10 microtubule motor proteins from the pole. The switch between the two states may depend on the degree of tension in the kinetochore. It has been proposed, for example, that, as chromosomes move toward a spindle pole, an increasing polar ejection force generates tension in the kinetochore nearest the pole, triggering a switch to the away-fromthe-pole state and gradually resulting in the accumulation of chromosomes at the equator of the spindle.


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Cell_Biology_Alberts. The apC/C Triggers sister-Chromatid separation and the Completion of mitosis After M-Cdk has triggered the complex processes leading up to metaphase, the cell cycle reaches its climax with the separation of the sister chromatids at the metaphase-to-anaphase transition (Figure 17–37). Although M-Cdk activity sets the stage for this event, the anaphase-promoting complex (APC/C) discussed earlier throws the switch that initiates sister-chromatid separation by ubiquitylating several mitotic regulatory proteins and thereby triggering their destruction (see Figure 17–15A). During metaphase, cohesins holding the sister chromatids together resist the poleward forces that pull the sister chromatids apart. Anaphase begins with the sudden loss of sister-chromatid cohesion, which allows the sisters to separate and move to opposite poles of the spindle. The APC/C initiates the process by targeting the inhibitory protein securin for destruction. Before anaphase, securin


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Cell_Biology_Alberts. Figure 17–36 How opposing forces may drive chromosomes to the metaphase plate. (a) Evidence for a polar ejection force that pushes chromosomes away from the spindle poles toward the spindle equator. In this experiment, a laser beam severs a prometaphase chromosome that is attached to a single pole by a kinetochore microtubule. The part of the severed chromosome without a kinetochore is pushed rapidly away from the pole, whereas the part with the kinetochore moves toward the pole, reflecting a decreased repulsion. (B) a model of how two opposing forces may cooperate to move chromosomes to the metaphase plate. plus-end-directed motor proteins (kinesin-4 and kinesin-10) on the chromosome arms are thought to interact with microtubules to generate the polar ejection force, which pushes chromosomes toward the spindle equator (see Figure 17–25). poleward forces generated by depolymerization at the kinetochore, together with microtubule flux, are thought to pull chromosomes toward the pole.


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Cell_Biology_Alberts. Figure 17–37 Sister-chromatid separation at anaphase. In the transition from metaphase (a) to anaphase (B), sister chromatids suddenly and synchronously separate and move toward opposite poles of the mitotic spindle—as shown in these light micrographs of Haemanthus (lily) endosperm cells that were stained with gold-labeled antibodies against tubulin. (Courtesy of andrew Bajer.) binds to and inhibits the activity of a protease called separase. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of cohesin. The cohesins fall away, and the sister chromatids separate (Figure 17–38). In addition to securin, the APC/C also targets the Sand M-cyclins for destruction, leading to the loss of most Cdk activity in anaphase. Cdk inactivation allows phosphatases to dephosphorylate the many Cdk target substrates in the cell, as required for the completion of mitosis and cytokinesis.


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Cell_Biology_Alberts. If the APC/C triggers anaphase, what activates the APC/C? The answer is only partly known. As mentioned earlier, APC/C activation requires binding to the protein Cdc20 (see Figure 17–15A). At least two processes regulate Cdc20 and its association with the APC/C. First, Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. Second, phosphorylation of the APC/C helps Cdc20 bind to the APC/C, thereby helping to create an active complex. Among the kinases that phosphorylate and thus activate the APC/C is M-Cdk. Thus, M-Cdk not only triggers the early mitotic events leading up to metaphase, but it also sets the stage for progression into anaphase. The ability of M-Cdk to promote Cdc20–APC/C activity creates a negative feedback loop: M-Cdk sets in motion a regulatory process that leads to cyclin destruction and thus its own inactivation. Unattached Chromosomes Block sister-Chromatid separation: The spindle assembly Checkpoint


Cell_Biology_Alberts

Cell_Biology_Alberts. Unattached Chromosomes Block sister-Chromatid separation: The spindle assembly Checkpoint Drugs that destabilize microtubules, such as colchicine or vinblastine (discussed in Chapter 16), arrest cells in mitosis for hours or even days. This observation led to the identification of a spindle assembly checkpoint mechanism that is activated by the drug treatment and blocks progression through the metaphase-toanaphase transition. The checkpoint mechanism ensures that cells do not enter anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle assembly checkpoint depends on a sensor mechanism that monitors the strength of microtubule attachment at the kinetochore, possibly by sensing tension as described earlier (see Figure 17–34). Any kinetochore that is not properly attached to the spindle sends out a diffusible negative signal that blocks Cdc20–APC/C activation throughout the cell and thus blocks the


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Cell_Biology_Alberts. Figure 17–38 The initiation of sisterchromatid separation by the APC/C. The activation of apC/C by Cdc20 leads to the ubiquitylation and destruction of securin, which normally holds separase in an inactive state. The destruction of securin allows separase to cleave scc1, a subunit of the cohesin complex holding the sister chromatids together (see Figure 17–19). The pulling forces of the mitotic spindle then pull the sister chromatids apart. In animal cells, phosphorylation by Cdks also inhibits separase (not shown). Thus, Cdk inactivation in anaphase (resulting from cyclin destruction) also promotes separase activation by allowing its dephosphorylation. metaphase-to-anaphase transition. When the last sister-chromatid pair is properly bi-oriented, this block is removed, allowing sister-chromatid separation to occur.


Cell_Biology_Alberts

Cell_Biology_Alberts. metaphase-to-anaphase transition. When the last sister-chromatid pair is properly bi-oriented, this block is removed, allowing sister-chromatid separation to occur. The negative checkpoint signal depends on several proteins, including Mad2, which are recruited to unattached kinetochores (Figure 17–39). Detailed structural analyses of Mad2 suggest that the unattached kinetochore acts like an enzyme that catalyzes a change in the conformation of Mad2, so that Mad2, together with other proteins, can bind and inhibit Cdc20–APC/C.


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Cell_Biology_Alberts. In mammalian somatic cells, the spindle assembly checkpoint determines the normal timing of anaphase. The destruction of securin in these cells begins moments after the last sister-chromatid pair becomes bi-oriented on the spindle, and anaphase begins about 20 minutes later. Experimental inhibition of the checkpoint mechanism causes premature sister-chromatid separation and anaphase. Surprisingly, the normal timing of anaphase does not depend on the spindle assembly checkpoint in some cells, such as yeasts and the cells of early frog and fly embryos. Other mechanisms, as yet unknown, must determine the timing of anaphase in these cells.


Cell_Biology_Alberts

Cell_Biology_Alberts. The sudden loss of sister-chromatid cohesion at the onset of anaphase leads to sister-chromatid separation, which allows the forces of the mitotic spindle to pull the sisters to opposite poles of the cell—called chromosome segregation. The chromosomes move by two independent and overlapping processes. The first, anaphase A, is the initial poleward movement of the chromosomes, which is accompanied by shortening of the kinetochore microtubules. The second, anaphase B, is the separation of the spindle poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apart (Figure 17–40).


Cell_Biology_Alberts

Cell_Biology_Alberts. Chromosome movement in anaphase A depends on a combination of the two major poleward forces described earlier. The first is the force generated by micro-tubule depolymerization at the kinetochore, which results in the loss of tubulin subunits at the plus end as the kinetochore moves toward the pole. The second is provided by microtubule flux, which is the poleward movement of the micro-tubules toward the spindle pole, where minus-end depolymerization occurs. The relative importance of these two forces during anaphase varies in different cell types: in embryonic cells, chromosome movement depends mainly on microtubule flux, for example, whereas movement in yeast and vertebrate somatic cells results primarily from forces generated at the kinetochore.


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Cell_Biology_Alberts. Spindle-pole separation during anaphase B depends on motor-driven mechanisms similar to those that separate the two centrosomes in early mitosis. Plus-end directed kinesin-5 motor proteins, which cross-link the overlapping plus ends of the interpolar microtubules, push the poles apart. In addition, dynein motors that anchor astral microtubule plus ends to the cell cortex pull the poles apart (see Figure 17–25).


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Cell_Biology_Alberts. Although sister-chromatid separation initiates the chromosome movements of anaphase A, other mechanisms also ensure correct chromosome movements in anaphase A and spindle elongation in anaphase B. Most importantly, the completion of a normal anaphase depends on the dephosphorylation of Cdk substrates, which in most cells results from the APC/C-dependent destruction of cyclins. If M-cyclin destruction is prevented—by the production of a mutant form that is not recognized by the APC/C, for example—sister-chromatid separation generally occurs, but the chromosome movements and microtubule behavior of anaphase are abnormal.


Cell_Biology_Alberts

Cell_Biology_Alberts. 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 their metaphase length.


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Cell_Biology_Alberts. Figure 17–39 Mad2 protein on unattached kinetochores. This fluorescence micrograph shows a mammalian cell in prometaphase, with the mitotic spindle in green and the sister chromatids in blue. One sister-chromatid pair is attached to only one pole of the spindle. staining with anti-mad2 antibodies indicates that mad2 is bound to the kinetochore of the unattached sister chromatid (red dot, indicated by red arrow). a small amount of mad2 is associated with the kinetochore of the sister chromatid that is attached to the spindle pole (pale dot, indicated by white arrow). (From J.C. Waters et al., J. Cell Biol. 141:1181–1191, 1998. With permission from the authors.) segregated Chromosomes are packaged in daughter nuclei at Telophase


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Cell_Biology_Alberts. By the end of anaphase, the daughter chromosomes have segregated into two equal groups at opposite ends of the cell. In telophase, the final stage of mitosis, the two sets of chromosomes are packaged into a pair of daughter nuclei. The first major event of telophase is the disassembly of the mitotic spindle, followed by the re-formation of the nuclear envelope. Initially, nuclear membrane fragments associate with the surface of individual chromosomes. These membrane fragments fuse to partly enclose clusters of chromosomes and then coalesce to reform the complete nuclear envelope. Nuclear pore complexes are incorporated into the envelope, the nuclear lamina re-forms, and the envelope once again becomes continuous with the endoplasmic reticulum. Once the nuclear envelope has re-formed, the pore complexes pump in nuclear proteins, the nucleus expands, and the mitotic chromosomes are reorganized into their interphase state, allowing gene transcription to resume. A new nucleus has been


Cell_Biology_Alberts

Cell_Biology_Alberts. pore complexes pump in nuclear proteins, the nucleus expands, and the mitotic chromosomes are reorganized into their interphase state, 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.


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Cell_Biology_Alberts. We saw earlier that phosphorylation of various proteins by M-Cdk promotes spindle assembly, chromosome condensation, and nuclear-envelope breakdown in early mitosis. It is thus not surprising that the dephosphorylation of these same proteins is required for spindle disassembly and the re-formation of daughter nuclei in telophase. In principle, these dephosphorylations and the completion of mitosis could be triggered by the inactivation of Cdks, the activation of phosphatases, or both. Although Cdk inactivation—resulting primarily from cyclin destruction—is mainly responsible in most cells, some cells also rely on activation of phosphatases. In budding yeast, for example, the completion of mitosis depends on the activation of a phosphatase called Cdc14, which dephosphorylates a subset of Cdk substrates involved in anaphase and telophase.


Cell_Biology_Alberts

Cell_Biology_Alberts. M-Cdk triggers the events of early mitosis, including chromosome condensation, assembly of the mitotic spindle, and bipolar attachment of the sister-chromatid pairs to microtubules of the spindle. Spindle formation in animal cells depends largely on the ability of mitotic chromosomes to stimulate local microtubule nucleation and stability, as well as on the ability of motor proteins to organize micro-tubules into a bipolar array. Many cells also use centrosomes to facilitate spindle assembly. Anaphase is triggered by the APC/C, which stimulates the destruction of Figure 17–40 The two processes of anaphase in mammalian cells. separated sister chromatids move toward the poles in anaphase a. In anaphase B, the two spindle poles move apart.


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Cell_Biology_Alberts. Figure 17–40 The two processes of anaphase in mammalian cells. separated sister chromatids move toward the poles in anaphase a. In anaphase B, the two spindle poles move apart. the proteins that hold the sister chromatids together. APC/C also promotes cyclin destruction and thus the inactivation of M-Cdk. The resulting dephosphorylation of Cdk targets is required for the events that complete mitosis, including the disassembly of the spindle and the re-formation of the nuclear envelope. The final step in the cell cycle is cytokinesis, the division of the cytoplasm in two. In most cells, cytokinesis follows every mitosis, although some cells, such as early Drosophila embryos and some mammalian hepatocytes and heart muscle cells, undergo mitosis without cytokinesis and thereby acquire multiple nuclei. In most animal cells, cytokinesis begins in anaphase and ends shortly after the completion of mitosis in telophase.


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Cell_Biology_Alberts. The first visible change of cytokinesis in an animal cell is the sudden appearance of a pucker, or cleavage furrow, on the cell surface. The furrow rapidly deepens and spreads around the cell until it completely divides the cell in two. The structure underlying this process is the contractile ring—a dynamic assembly composed of actin filaments, myosin II filaments, and many structural and regulatory proteins. During anaphase, the ring assembles just beneath the plasma membrane (Figure 17–41; see also Panel 17–1). The ring gradually contracts, and, at the same time, fusion of intracellular vesicles with the plasma membrane inserts new membrane adjacent to the ring. This addition of membrane compensates for the increase in surface area that accompanies cytoplasmic division. When ring contraction is completed, membrane insertion and fusion seal the gap between the daughter cells. actin and myosin II in the Contractile Ring generate the Force for Cytokinesis


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Cell_Biology_Alberts. actin and myosin II in the Contractile Ring generate the Force for Cytokinesis In interphase cells, actin and myosin II filaments form a cortical network underlying the plasma membrane. In some cells, they also form large cytoplasmic bundles called stress fibers (discussed in Chapter 16). As cells enter mitosis, these arrays of actin and myosin disassemble; much of the actin reorganizes, and myosin II filaments are released. As the sister chromatids separate in anaphase, actin and myosin II begin to accumulate in the rapidly assembling contractile ring (Figure 17–42), which also contains numerous other proteins that provide structural support or assist in ring assembly. Assembly of the contractile ring results in part from the local formation of new actin filaments, which depends on formin proteins that actin and myosin flaments of the


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Cell_Biology_Alberts. Figure 17–41 Cytokinesis. (a) The actin–myosin bundles of the contractile ring are oriented as shown, so that their contraction pulls the membrane inward. (B) In this low-magnification scanning electron micrograph of a cleaving frog egg, the cleavage furrow is especially prominent, as the cell is unusually large. The furrowing of the cell membrane is caused by the activity of the contractile ring underneath it. (C) The surface of a furrow at higher magnification. (B and C, from H.W. Beams and R.g. kessel, Am. Sci. 64:279–290, 1976. With permission from sigma Xi.) contractile ring of actin and myosin flaments in cleavage furrow (A) 0.5 µm nucleate the assembly of parallel arrays of linear, unbranched actin filaments (discussed in Chapter 16). After anaphase, the overlapping arrays of actin and myosin II filaments contract to generate the force that divides the cytoplasm in two. Once contraction begins, the ring exerts a force large enough to bend a fine glass needle that is inserted in


Cell_Biology_Alberts

Cell_Biology_Alberts. and myosin II filaments contract to generate the force that divides the cytoplasm in two. Once contraction begins, the ring exerts a force large enough to bend a fine glass needle that is inserted in its path. As the ring constricts, it maintains the same thickness, suggesting that its total volume and the number of filaments it contains decrease steadily. Moreover, unlike actin in muscle, the actin filaments in the ring are highly dynamic, and their arrangement changes continually during cytokinesis.


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Cell_Biology_Alberts. 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, a large protein structure derived from the anti-parallel interpolar microtubules of the spindle midzone, packed tightly together within a dense matrix material (Figure 17–43). 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. Local activation of Rhoa Triggers assembly and Contraction of the Contractile Ring


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Cell_Biology_Alberts. Local activation of Rhoa Triggers assembly and Contraction of the Contractile Ring RhoA, a small GTPase of the Ras superfamily (see Table 15–5), controls the assembly and function of the contractile ring at the site of cleavage. RhoA is activated at the cell cortex at the future division site, where it promotes actin filament formation, myosin II assembly, and ring contraction. It stimulates actin filament formation by activating formins, and it promotes myosin II assembly and contractions by activating multiple protein kinases, including the Rho-activated kinase Rock (Figure 17–44). These kinases phosphorylate the regulatory myosin light chain, a subunit of myosin II, thereby stimulating bipolar myosin II filament formation and motor activity.


Cell_Biology_Alberts

Cell_Biology_Alberts. RhoA is thought to be activated by a guanine nucleotide exchange factor (Rho-GEF), which is found at the cell cortex at the future division site and stimulates the release of GDP and binding of GTP to RhoA (see Figure 17–44). We know little about how the RhoGEF is localized or activated at the division site, although the microtubules of the anaphase spindle seem to be involved, as we discuss next. The microtubules of the mitotic spindle determine the plane of animal Cell division The central problem in cytokinesis is how to ensure that division occurs at the right time and in the right place. Cytokinesis must occur only after the two sets of chromosomes are fully segregated from each other, and the site of division must Figure 17–42 The contractile ring.


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Cell_Biology_Alberts. Figure 17–42 The contractile ring. (a) a drawing of the cleavage furrow in a dividing cell. (B) an electron micrograph of the ingrowing edge of a cleavage furrow of a dividing animal cell. (C) Fluorescence micrographs of a dividing slime mold amoeba stained for actin (red) and myosin II (green). Whereas all of the visible myosin II has redistributed to the contractile ring, only some of the actin has done so; the rest remains in the cortex of the nascent daughter cells. (B, from H.W. Beams and


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Cell_Biology_Alberts. R.g. kessel, Am. Sci. 64:279–290, 1976. With permission from sigma Xi; C, courtesy of Yoshio Fukui.) be placed between the two sets of daughter chromosomes, thereby ensuring that each daughter cell receives a complete set. The correct timing and positioning of cytokinesis in animal cells are achieved by mechanisms that depend on the mitotic spindle. During anaphase, the spindle generates signals that initiate furrow formation at a position midway between the spindle poles, thereby ensuring that division occurs between the two sets of separated chromosomes. Because these signals originate in the anaphase spindle, this mechanism also contributes to the correct timing of cytokinesis in late mitosis. Cytokinesis also occurs at the correct time because dephosphorylation of Cdk substrates, which depends on cyclin destruction in metaphase and anaphase, initiates cytokinesis. We now describe these regulatory mechanisms in more detail, with an emphasis on cytokinesis in animal cells.


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Cell_Biology_Alberts. Studies of the fertilized eggs of marine invertebrates first revealed the importance of spindle microtubules in determining the placement of the contractile ring. After fertilization, these embryos cleave rapidly without intervening periods of growth. In this way, the original egg is progressively divided into smaller and smaller cells. Because the cytoplasm is clear, the spindle can be observed in real time with 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—supporting the idea that signals generated by the spindle induce local furrow formation. How does the mitotic spindle specify the site of division? Three general mechanisms have been proposed, and most cells appear to employ a combination of these (Figure 17–45). The first is termed the astral stimulation model, in which the


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Cell_Biology_Alberts. Figure 17–43 The midbody. (a) a scanning electron micrograph of a cultured animal cell dividing; the midbody still joins the two daughter cells. (B) a conventional electron micrograph of the midbody of a dividing animal cell. Cleavage is almost complete, but the daughter cells remain attached by this thin strand of cytoplasm containing the remains of the central spindle. (a, courtesy of guenter albrecht-Buehler; B, courtesy of J.m. mullins.) assembly and contraction of actin–myosin ring Figure 17–44 Regulation of the contractile ring by the GTPase RhoA.


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Cell_Biology_Alberts. Figure 17–44 Regulation of the contractile ring by the GTPase RhoA. Like other Rho family gTpases, Rhoa is activated by a RhogEF protein and inactivated by a Rho gTpase-activating protein (Rhogap). The active gTpbound form of Rhoa is focused at the future cleavage site. By binding formins, activated Rhoa promotes the assembly of actin filaments in the contractile ring. By activating Rho-activated protein kinases, such as Rock, it stimulates myosin II filament formation and activity, thereby promoting contraction of the ring. astral microtubules carry furrow-inducing signals, which are somehow focused in a ring on the cell cortex, halfway between the spindle poles. Evidence for this model comes from ingenious experiments in large embryonic cells, which demonstrate that a cleavage furrow forms midway between two asters, even when the two centrosomes nucleating the asters are not connected to each other by a mitotic spindle (Figure 17–46).


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Cell_Biology_Alberts. A second possibility, called the central spindle stimulation model, is that the spindle midzone, or central spindle, generates a furrow-inducing signal that specifies the site of furrow formation at the cell cortex (see Figure 17–45). The overlapping interpolar microtubules of the central spindle associate with numerous signaling proteins, including proteins that may stimulate RhoA (Figure 17–47). Defects in the functions of these proteins (in Drosophila mutants, for example) result in failure of cytokinesis.


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Cell_Biology_Alberts. A third model proposes that, in some cell types, the astral microtubules promote the local relaxation of actin–myosin bundles at the cell cortex. According to this astral relaxation model, the cortical relaxation is minimal at the spindle equator, thus promoting cortical contraction at that site (see Figure 17–45). In the early embryos of Caenorhabditis elegans, for example, treatments that result in the loss of astral microtubules lead to increased contractile activity throughout the cell cortex, consistent with this model.


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Cell_Biology_Alberts. In some cell types, the site of ring assembly is chosen before mitosis. In budding yeasts, for example, a ring of proteins called septins assembles in late G1 at the future division site. The septins are thought to form a scaffold onto which other components of the contractile ring, including myosin II, assemble. In plant cells, an organized band of microtubules and actin filaments, called the preprophase band, assembles just before mitosis and marks the site where the cell wall will assemble and divide the cell in two, as we now discuss. both nuclei cleavage occurs both between the centrosomes enter mitosis linked by mitotic spindles and between the two centrosomes that are simply adjacent, and four daughter cells are formed


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Cell_Biology_Alberts. both nuclei cleavage occurs both between the centrosomes enter mitosis linked by mitotic spindles and between the two centrosomes that are simply adjacent, and four daughter cells are formed Figure 17–45 Three current models of how the microtubules of the anaphase spindle generate signals that influence the positioning of the contractile ring. no single model explains all the observations, and furrow positioning is probably determined by a combination of these mechanisms, with the importance of the different mechanisms varying in different organisms. see text for details.


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Cell_Biology_Alberts. Figure 17–46 An experiment demonstrating the influence of the position of microtubule asters on the subsequent plane of cleavage in a large egg cell. If the mitotic spindle is mechanically pushed to one side of the cell with a glass bead, the membrane furrowing is incomplete, failing to occur on the opposite side of the cell. subsequent cleavages occur not only at the midzone of each of the two subsequent mitotic spindles (yellow arrowheads), but also between the two adjacent asters that are not linked by a mitotic spindle—but in this abnormal cell share the same cytoplasm (red arrowhead). apparently, the contractile ring that produces the cleavage furrow in these cells always forms in the region midway between two asters, suggesting that the asters somehow alter the adjacent region of cell cortex to induce furrow formation between them. The phragmoplast guides Cytokinesis in Higher plants


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Cell_Biology_Alberts. The phragmoplast guides Cytokinesis in Higher plants In most animal cells, the inward movement of the cleavage furrow depends on an increase in the surface area of the plasma membrane. New membrane is added at the inner edge of the cleavage furrow and is generally provided by small membrane vesicles that are transported on microtubules from the Golgi apparatus to the furrow.


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Cell_Biology_Alberts. Membrane deposition is particularly important for cytokinesis in higher-plant cells. These cells are enclosed by a semirigid cell wall. 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 17–48). The assembly of the cell plate begins in late anaphase and is guided by a structure called the phragmoplast, which contains microtubules derived from the mitotic spindle. Motor proteins transport small vesicles along these microtubules from the Golgi apparatus to the cell center. These vesicles, filled with polysaccharide and glycoproteins required for the synthesis of the new cell wall, fuse to form a disc-like, membrane-enclosed structure called the early cell plate. The plate expands outward by further


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Cell_Biology_Alberts. Figure 17–48 Cytokinesis in a plant cell in telophase. In this light micrograph, the early cell plate (between the two arrowheads) has formed in a plane perpendicular to the plane of the page. The microtubules of the spindle are stained with gold-labeled antibodies against tubulin, and the dna in the two sets of daughter chromosomes is stained with a fluorescent dye. note that there are no astral microtubules, because there are no centrosomes in higher-plant cells. (Courtesy of andrew Bajer.)


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Cell_Biology_Alberts. Figure 17–47 Localization of cytokinesis regulators at the central spindle of the human cell. (a) at center is a cultured human cell at the beginning of cytokinesis, showing the locations of the gTpase Rhoa (red) and a protein called Cyk4 (green), which is one of several regulatory proteins that form complexes at the overlapping plus ends of interpolar microtubules. These proteins are thought to generate signals that help control Rhoa activity at the cell cortex. (B) When the same three-dimensional image is viewed in the plane of the contractile ring, as shown here, Rhoa (red) is seen as a ring beneath the cell surface, while the central spindle protein Cyk4 (green) is associated with microtubule bundles scattered throughout the equatorial plane of the cell. (Courtesy of alisa piekny and michael glotzer.) 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


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Cell_Biology_Alberts. michael glotzer.) 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 17–49).


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Cell_Biology_Alberts. 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. When a eukaryotic cell divides, however, each daughter cell must also inherit all of the other essential cell components, including the membrane-enclosed organelles. As discussed in Chapter 12, organelles such as mitochondria and chloroplasts cannot be assembled de novo from their individual components; they can arise only by 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.


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Cell_Biology_Alberts. How, then, do the various membrane-enclosed organelles segregate 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 and breakdown of the nuclear envelope releases the ER. In most cells, the ER remains largely intact and is cut in two during cytokinesis. The Golgi apparatus is reorganized and fragmented during mitosis. Golgi fragments associate with the spindle poles and are thereby distributed to opposite ends of the spindle, ensuring that each daughter cell inherits the materials needed to reconstruct the Golgi in telophase. some Cells Reposition Their spindle to divide asymmetrically


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Cell_Biology_Alberts. some Cells Reposition Their spindle to divide asymmetrically Most animal cells divide symmetrically: the contractile ring forms around the equator of the parent cell, producing two daughter cells of equal size and with the same components. This symmetry results from the placement of the mitotic spindle, which in most cases tends to center itself in the cytoplasm. Astral micro-tubules and motor proteins that either push or pull on these microtubules contribute to the centering process. 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 different daughter cells are destined to develop


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Cell_Biology_Alberts. Figure 17–49 The special features of cytokinesis in a higher-plant cell. The division plane is established before m phase by a band of microtubules and actin filaments (the preprophase band) at the cell cortex. at the beginning of telophase, after the chromosomes have segregated, a new cell wall starts to assemble inside the cell at the equator of the old spindle. The interpolar microtubules of the mitotic spindle remaining at telophase form the phragmoplast. The plus ends of these microtubules no longer overlap but end at the cell equator. golgi-derived vesicles, filled with cell-wall material, are transported along these microtubules and fuse to form the new cell wall, which grows outward to reach the plasma membrane and original cell wall. The plasma membrane and the membrane surrounding the new cell wall fuse, separating the two daughter cells.


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Cell_Biology_Alberts. along different pathways. To create daughter cells with different fates in this way, the mother cell must first segregate certain components (called cell 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 17–50). 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 changes in local regions of the cell cortex direct such spindle movements and that motor proteins localized there pull one of the spindle poles, via its astral microtubules, to the appropriate region. Genetic analyses in C. elegans and Drosophila have identified some of the proteins required for such asymmetric divisions, and some of these proteins seem to have a similar role in vertebrates.


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Cell_Biology_Alberts. 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 several thousand nuclei, arranged in a monolayer near the surface. A cell in which multiple nuclei share the same cytoplasm is called a syncytium. 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, membranes are created around each nucleus in one round of coordinated cytokinesis called cellularization. The plasma membrane extends inward and, with the help of an actin– myosin ring, pinches off to enclose each nucleus (Figure 17–51).


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Cell_Biology_Alberts. Nuclear division without cytokinesis also occurs in some types of mammalian cells. Megakaryocytes, which produce blood platelets, and some hepatocytes and heart muscle cells, for example, become multinucleated in this way. After cytokinesis, most cells enter G1, in which Cdks are mostly inactive. We end this section by discussing how this state is achieved at the end of M phase. The g1 phase Is a stable state of Cdk Inactivity A key regulatory event in late M phase is the inactivation of Cdks, which is driven primarily by APC/C-dependent cyclin destruction. As described earlier, the inactivation of Cdks in late M phase has many functions: it triggers the events of late mitosis, promotes cytokinesis, and enables the synthesis of prereplicative complexes at DNA replication origins. It also provides a mechanism for resetting the Figure 17–50 An asymmetric cell division segregating cytoplasmic components to only one daughter cell.


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Cell_Biology_Alberts. Figure 17–50 An asymmetric cell division segregating cytoplasmic components to only one daughter cell. These light micrographs illustrate the controlled asymmetric segregation of specific cytoplasmic components to one daughter cell during the first division of a fertilized egg of the nematode


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Cell_Biology_Alberts. C. elegans. The fertilized egg is shown in the left micrographs and the two daughter cells in the right micrographs. The cells above have been stained with a blue, dna-binding, fluorescent dye to show the nucleus (and polar bodies); they are viewed by both differential-interference-contrast and fluorescence microscopy. The cells below are the same cells stained with an antibody against p-granules and viewed by fluorescence microscopy. These small granules are made of Rna and proteins and determine which cells become germ cells. They are distributed randomly throughout the cytoplasm of the unfertilized egg (not shown) but become segregated to the posterior pole of the fertilized egg. The cleavage plane is oriented to ensure that only the posterior daughter cell receives the p-granules when the egg divides. The same segregation process is repeated in several subsequent cell divisions, so that the p-granules end up only in cells that give rise to eggs and sperm. (Courtesy of susan


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Cell_Biology_Alberts. when the egg divides. The same segregation process is repeated in several subsequent cell divisions, so that the p-granules end up only in cells that give rise to eggs and sperm. (Courtesy of susan strome.)


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Cell_Biology_Alberts. Figure 17–51 Mitosis without cytokinesis in the early Drosophila embryo. (a) The first 13 nuclear divisions occur synchronously and without cytoplasmic division to create a large syncytium. most of the nuclei migrate to the cortex, and the plasma membrane extends inward and pinches off to surround each nucleus to form individual cells in a process called cellularization. (B) Fluorescence micrograph of multiple mitotic spindles in a Drosophila embryo before cellularization. The microtubules are stained green and the centrosomes red. note that all the nuclei go through the cycle synchronously; here, they are all in metaphase, with the unlabeled chromosomes seen as a dark band at the spindle equator. (B, courtesy of kristina Yu and William sullivan.) cell-cycle control system to a state of Cdk inactivity as the cell prepares to enter a new cell cycle. In most cells, this state of Cdk inactivity generates a G1 gap phase, during which the cell grows and monitors its environment before


Cell_Biology_Alberts

Cell_Biology_Alberts. of Cdk inactivity as the cell prepares to enter a new cell cycle. In most cells, this state of Cdk inactivity generates a G1 gap phase, during which the cell grows and monitors its environment before committing to a new cell cycle.


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Cell_Biology_Alberts. In early animal embryos, the inactivation of M-Cdk in late mitosis is due almost entirely to the action of Cdc20–APC/C, discussed earlier. Recall, however, that M-Cdk stimulates Cdc20–APC/C activity. Thus, the destruction of M-cyclin in late mitosis soon leads to the inactivation of all APC/C activity in an embryonic cell. This APC/C inactivation immediately after mitosis is especially useful in rapid embryonic cell cycles, as it allows the cell to quickly begin accumulating new M-cyclin for the next cycle (Figure 17–52A).


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Cell_Biology_Alberts. Rapid cyclin accumulation immediately after mitosis is not useful, however, for cells in which a G1 phase is needed to allow control of entry into the next cell cycle. These cells employ several mechanisms to prevent Cdk reactivation after mitosis. One mechanism uses another APC/C-activating protein called Cdh1, mentioned earlier as a close relative of Cdc20 (see Table 17–2). Although both Cdh1 and Cdc20 bind to and activate the APC/C, they differ in one important respect. Whereas M-Cdk activates the Cdc20–APC/C complex, it inhibits the Cdh1–APC/C complex by directly phosphorylating Cdh1. As a result of this relationship, Cdh1– APC/C activity increases in late mitosis after the Cdc20–APC/C complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20–APC/C activity has declined, Cdh1–APC/C activity is high (Figure 17–52B).


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Cell_Biology_Alberts. A second mechanism that suppresses Cdk activity in G1 depends on the increased production of CKIs, the Cdk inhibitor proteins discussed earlier. Budding yeast cells, in which this mechanism is best understood, contain a CKI protein called Sic1, which binds to and inactivates M-Cdk in late mitosis and G1 (see Figure 17–52 The creation of a G1 phase by stable Cdk inhibition after mitosis. (a) In early embryonic cell cycles, Cdc20–apC/C activity rises at the end of metaphase, triggering m-cyclin destruction. Because m-Cdk activity stimulates Cdc20–apC/C activity, the loss of m-cyclin leads to apC/C inactivation after mitosis, which allows m-cyclins to begin accumulating again. (B) In cells that have a g1 phase, the drop in m-Cdk activity in late mitosis leads to the activation of Cdh1–apC/C (as well as to the accumulation of Cdk inhibitor proteins; not shown). This ensures a continued suppression of Cdk activity after mitosis, as required for a g1 phase.


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Cell_Biology_Alberts. (A) embryonic cells with no G1 phase Cdc20–APC/C activity (B) cells with G1 phase Cdc20–APC/C activity Table 17–2). Like Cdh1, Sic1 is inhibited by M-Cdk, which phosphorylates Sic1 during mitosis and thereby promotes its ubiquitylation by SCF. Thus, Sic1 and M-Cdk, like Cdh1 and M-Cdk, inhibit each other. As a result, the decline in M-Cdk activity that occurs in late mitosis causes the Sic1 protein to accumulate, and this CKI helps keep M-Cdk activity low after mitosis. A CKI protein called p27 (see Figure 17–14) may serve similar functions in animal cells.


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Cell_Biology_Alberts. In most cells, decreased transcription of M-cyclin genes also inactivates M-Cdks in late mitosis. In budding yeast, for example, M-Cdk promotes the expression of these genes, resulting in a positive feedback loop. This loop is turned off as cells exit from mitosis: the inactivation of M-Cdk by Cdh1 and Sic1 leads to decreased M-cyclin gene transcription and thus decreased M-cyclin synthesis. Gene regulatory proteins that promote the expression of G1/Sand S-cyclins are also inhibited during G1.


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Cell_Biology_Alberts. Thus, Cdh1–APC/C activation, CKI accumulation, and decreased cyclin gene expression act together to ensure that the early G1 phase is a time when essentially all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the use of multiple regulatory mechanisms allows the system to operate with reasonable efficiency even if one mechanism fails. So how does the cell escape from this stable G1 state to initiate a new cell cycle? The answer is that G1/S-Cdk activity, which rises in late G1, releases all the braking mechanisms that suppress Cdk activity, as we describe later, in the last section of this chapter.


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Cell_Biology_Alberts. After mitosis completes the formation of a pair of daughter nuclei, cytokinesis finishes the cell cycle by dividing the cell itself. Cytokinesis depends on a ring of actin and myosin filaments that contracts in late mitosis at a site midway between the segregated chromosomes. In animal cells, the positioning of the contractile ring is determined by signals emanating from the microtubules of the anaphase spindle. Dephosphorylation of Cdk targets, which results from Cdk inactivation in anaphase, triggers cytokinesis at the correct time after anaphase. After cytokinesis, the cell enters a stable G1 state of low Cdk activity, where it awaits signals to enter a new cell cycle.


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Cell_Biology_Alberts. Most eukaryotic organisms reproduce sexually: the genomes of two parents mix to generate offspring that are genetically distinct from either parent. The cells of these organisms are generally diploid: that is, they contain two slightly different copies, or homologs, of each chromosome, one from each parent. Sexual reproduction depends on a specialized nuclear division process called meiosis, which produces haploid cells carrying only a single copy of each chromosome. In many organisms, the haploid cells differentiate into specialized reproductive cells called gametes—eggs and sperm in most species. In these species, the reproductive cycle ends when a sperm and egg fuse to form a diploid zygote, which has the potential to form a new individual. In this section, we consider the basic mechanisms and regulation of meiosis, with an emphasis on how they compare with those of mitosis. meiosis Includes Two Rounds of Chromosome segregation


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Cell_Biology_Alberts. Meiosis reduces the chromosome number by half using many of the same molecular machines and control systems that operate in mitosis. As in the mitotic cell cycle, the cell begins the meiotic program by duplicating its chromosomes in meiotic S phase, resulting in pairs of sister chromatids that are tightly linked along their entire lengths by cohesin complexes. Unlike mitosis, however, two successive rounds of chromosome segregation then occur (Figure 17–53). The first of these divisions (meiosis I) solves the problem, unique to meiosis, of segregating the homologs. The duplicated paternal and maternal homologs pair up alongside each other and become physically linked by the process of genetic recombination. These pairs of homologs, each containing a pair of sister chromatids, then line up on the first meiotic spindle. In the first meiotic anaphase, duplicated homo-logs rather than sister chromatids are pulled apart and segregated into the two daughter nuclei. Only in the second


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Cell_Biology_Alberts. up on the first meiotic spindle. In the first meiotic anaphase, duplicated homo-logs rather than sister chromatids are pulled apart and segregated into the two daughter nuclei. Only in the second division (meiosis II), which occurs without further DNA replication, are the sister chromatids pulled apart and segregated (as in mitosis) to produce haploid daughter nuclei. In this way, each diploid nucleus that enters meiosis produces four haploid nuclei, each of which contains either the maternal or paternal copy of each chromosome, but not both (Movie 17.7).


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Cell_Biology_Alberts. Figure 17–53 Comparison of meiosis and mitosis. For clarity, only one pair of homologous chromosomes (homologs) is shown.


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Cell_Biology_Alberts. (a) meiosis is a form of nuclear division in which a single round of chromosome duplication (meiotic s phase) is followed by two rounds of chromosome segregation. The duplicated homologs, each consisting of tightly bound sister chromatids, pair up and are segregated into different daughter nuclei in meiosis I; the sister chromatids are segregated in meiosis II. as indicated by the formation of chromosomes that are partly red and partly gray, homolog pairing in meiosis leads to genetic recombination (crossing-over) during meiosis I. Each diploid cell that enters meiosis therefore produces four genetically different haploid nuclei, which are distributed by cytokinesis into haploid cells that differentiate into gametes. (B) In mitosis, by contrast, homologs do not pair up, and the sister chromatids are segregated during the single division. Thus, each diploid cell that divides by mitosis produces two genetically identical diploid daughter nuclei, which are distributed by cytokinesis into


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Cell_Biology_Alberts. are segregated during the single division. Thus, each diploid cell that divides by mitosis produces two genetically identical diploid daughter nuclei, which are distributed by cytokinesis into a pair of daughter cells.


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Cell_Biology_Alberts. During mitosis in most organisms, homologous chromosomes behave independently of each other. During meiosis I, however, it is crucial that homologs recognize each other and associate physically in order for the maternal and paternal homologs to be bi-oriented on the first meiotic spindle. Special mechanisms mediate these interactions.