Cell_Biology_Alberts

Cell_Biology_Alberts. The gradual juxtaposition of homologs occurs during a prolonged period called meiotic prophase (or prophase I), which can take hours in yeasts, days in mice, and weeks in higher plants. Like their mitotic counterparts, duplicated meiotic prophase chromosomes first appear as long threadlike structures, in which the sister chromatids are so tightly glued together that they appear as one. It is during early prophase I that the homologs begin to associate along their length in a process called pairing, which, in some organisms at least, begins with interactions between complementary DNA sequences (called pairing sites) in the two homologs. As prophase progresses, the homologs become more closely juxtaposed, forming a four-chromatid structure called a bivalent (Figure 17–54A). In most species, homolog pairs are then locked together by homologous recombination: DNA double-strand breaks are formed at several locations in each sister chromatid, resulting in large numbers of DNA recombination


Cell_Biology_Alberts

Cell_Biology_Alberts. homolog pairs are then locked together by homologous recombination: DNA double-strand breaks are formed at several locations in each sister chromatid, resulting in large numbers of DNA recombination events between the homologs (as described in Chapter 5). Some of these events lead to reciprocal DNA exchanges called crossovers, where the DNA of a chromatid crosses over to become continuous with the DNA of a homologous chromatid (Figure 17–54B; also see Figure 5–54).


Cell_Biology_Alberts

Cell_Biology_Alberts. Homolog pairing Culminates in the Formation of a synaptonemal Complex


Cell_Biology_Alberts

Cell_Biology_Alberts. The paired homologs are brought into close juxtaposition, with their structural axes (axial cores) about 400 nm apart, by a mechanism that depends in most species on the double-strand DNA breaks that occur in sister chromatids. What pulls the axes together? One possibility is that the large protein machine, called a recombination complex, which assembles on a double-strand break in a chromatid, binds the matching DNA sequence in the nearby homolog and helps reel in this partner. This so-called presynaptic alignment of the homologs is followed by synapsis, in which the axial core of a homolog becomes tightly linked to the axial core of its partner by a closely packed array of transverse filaments to create a synaptonemal complex, which bridges the gap, now only 100 nm, between the homologs (Figure 17–55). Although crossing-over begins before the synaptonemal complex assembles, the final steps occur while the DNA is held in the complex.


Cell_Biology_Alberts

Cell_Biology_Alberts. The morphological changes that occur during homolog pairing are the basis for dividing meiotic prophase into five sequential stages—leptotene, zygotene, pachytene, diplotene, and diakinesis (Figure 17–56). Prophase starts with leptotene, when homologs condense and pair and genetic recombination begins. At zygotene, the synaptonemal complex begins to assemble at sites where the homologs are closely associated and recombination events are occurring. At pachytene, the assembly process is complete, and the homologs are synapsed


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–54 Homolog pairing and crossing-over. (a) The structure formed by two closely aligned duplicated homologs is called a bivalent. as in mitosis, the sister chromatids in each homolog are tightly connected along their entire lengths, as well as at their centromeres. at this stage, the homologs are usually joined by a protein complex called the synaptonemal complex (not shown; see Figure 17–55). (B) a later-stage bivalent in which a single crossover has occurred between nonsister chromatids. It is only when the synaptonemal complex disassembles and the paired homologs separate a little at the end of prophase I, as shown, that the crossover is seen microscopically as a thin connection between the homologs called a chiasma. Figure 17–55 Simplified schematic drawing of a synaptonemal complex.


Cell_Biology_Alberts

Cell_Biology_Alberts. Each homolog is organized around a protein axial core, and the synaptonemal axes are linked by rod-shaped transverse filaments. The axial core of each homolog also interacts with the cohesin complexes axial cores that hold the sister chromatids together of the (see Figure 9–35). (modified from homologs k. nasmyth, Annu. Rev. Genet. 35:673– 745, 2001.) cohesin chromatin loops of sister chromatids of one homolog along their entire lengths (see Figure 9–35). The pachytene stage can persist for days or longer, until desynapsis begins at diplotene with the disassembly of the synaptonemal complexes and the concomitant condensation and shortening of the chromosomes. It is only at this stage, after the complexes have disassembled, that the individual crossover events between nonsister chromatids can be seen as inter-homolog connections called chiasmata (singular chiasma), which now play a crucial part in holding the compact homologs together (Figure 17–57). The homologs are now ready to begin


Cell_Biology_Alberts

Cell_Biology_Alberts. be seen as inter-homolog connections called chiasmata (singular chiasma), which now play a crucial part in holding the compact homologs together (Figure 17–57). The homologs are now ready to begin the process of segregation.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–56 Homolog synapsis and desynapsis during the different stages of prophase I. (a) a single bivalent is shown schematically. at leptotene, the two sister chromatids coalesce, and their chromatid loops extend out from a common axial core. assembly of the synaptonemal complex begins in early zygotene and is complete in pachytene. The complex disassembles in diplotene. (B) an electron micrograph of a synaptonemal complex from a meiotic cell at pachytene in a lily flower. (C and d) Immunofluorescence micrographs of prophase I cells of the fungus Sordaria. partially synapsed bivalents at zygotene are shown in (C) and fully synapsed bivalents are shown in (d). Red arrowheads in (C) point to regions where synapsis is still incomplete. (B, courtesy of Brian Wells; C and d, from a. storlazzi et al., Genes Dev. 17:2675–2687, 2003. With permission from Cold spring Harbor Laboratory press.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–58 Comparison of chromosome behavior in meiosis I, meiosis II, and mitosis. Chromosomes behave similarly in mitosis and meiosis II, but they behave very differently in meiosis I. (a) In meiosis I, the two sister kinetochores are located side-by-side on each homolog and attach to microtubules from the same spindle pole. The proteolytic cleavage of cohesin along the sister-chromatid arms unglues the arms and resolves the crossovers, allowing the duplicated homologs to separate at anaphase I, while the residual cohesin at the centromeres keeps the sisters together. Cleavage of centromeric cohesin allows the sister chromatids to separate at anaphase II. (B) In mitosis, by contrast, the two sister kinetochores attach to microtubules from different spindle poles, and the two sister chromatids come apart at the start of anaphase and segregate into separate daughter nuclei.


Cell_Biology_Alberts

Cell_Biology_Alberts. localized at the kinetochores in meiosis I, but we do not know in any detail how these proteins work. They are removed from kinetochores after meiosis I, so that in meiosis II the sister-chromatid pairs can be bi-oriented on the spindle as they are in mitosis. Second, crossovers generate a strong physical linkage between homologs, allowing their bi-orientation at the equator of the spindle—much like cohesion between sister chromatids is important for their bi-orientation in mitosis (and meiosis II). Crossovers hold homolog pairs together only because the arms of the sister chromatids are connected by sister-chromatid cohesion (see Figure 17–58A).


Cell_Biology_Alberts

Cell_Biology_Alberts. Third, cohesion is removed in anaphase I only from chromosome arms and not from the regions near the centromeres, where the kinetochores are located. The loss of arm cohesion triggers homolog separation at the onset of anaphase I. This process depends on APC/C activation, which leads to securin destruction, separase activation, and cohesin cleavage along the arms (see Figure 17–38).


Cell_Biology_Alberts

Cell_Biology_Alberts. Cohesins near the centromeres are protected from separase in meiosis I by a kinetochore-associated protein called shugoshin (from the Japanese word for “guardian spirit”). Shugoshin acts by recruiting a protein phosphatase that removes phosphates from centromeric cohesins. Cohesin phosphorylation is normally required for separase to cleave cohesin; thus, removal of this phosphorylation near the centromere prevents cohesin cleavage. Sister-chromatid pairs therefore remain linked through meiosis I, allowing their correct bi-orientation on the spindle in meiosis II. Shugoshin is inactivated after meiosis I. At the onset of anaphase II, APC/C activation triggers centromeric cohesin cleavage and sisterchromatid separation—much as it does in mitosis. Following anaphase II, nuclear envelopes form around the chromosomes to produce four haploid nuclei, after which cytokinesis and other differentiation processes lead to the production of haploid gametes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Crossing-over has two distinct functions in meiosis: it helps hold homologs together so that they are properly segregated to the two daughter nuclei produced by meiosis I, and it contributes to the genetic diversification of the gametes that are eventually produced. As might be expected, therefore, crossing-over is highly regulated: the number and location of double-strand breaks along each chromosome is controlled, as is the likelihood that a break will be converted into a crossover. On average, the result of this regulation is that each pair of human homologs is linked by about two or three crossovers (Figure 17–59). Although the double-strand breaks that occur in meiosis I can be located almost anywhere along the chromosome, they are not distributed uniformly: they cluster at “hot spots,” where the DNA is accessible, and occur only rarely in “cold spots,” such as the heterochromatin regions around centromeres and telomeres.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–59 Crossovers between homologs in the human testis. In these immunofluorescence micrographs, antibodies have been used to stain the synaptonemal complexes (red), the centromeres (blue), and the sites of crossing-over (green). note that all of the bivalents have at least one crossover and none have more than four. (modified from a. Lynn et al., Science 296:2222–2225, 2002. With permission from aaas.) At least two kinds of regulation influence the location and number of crossovers that form, neither of which is well understood. Both operate before the synaptonemal complex assembles. One ensures that at least one crossover forms between the members of each homolog pair, as is necessary for normal homolog segregation in meiosis I. In the other, called crossover interference, the presence of one crossover event inhibits another from forming close by, perhaps by locally depleting proteins required for converting a double-strand DNA break into a stable crossover.


Cell_Biology_Alberts

Cell_Biology_Alberts. The sorting of chromosomes that takes place during meiosis is a remarkable feat of intracellular bookkeeping. In humans, each meiosis requires that the starting cell keep track of 92 chromatids (46 chromosomes, each of which has duplicated), distributing one complete set of each type of autosome to each of the four haploid progeny. Not surprisingly, mistakes can occur in allocating the chromosomes during this elaborate process. Mistakes are especially common in human female meiosis, which arrests for years after diplotene: meiosis I is completed only at ovulation, and meiosis II only after the egg is fertilized. Indeed, such chromosome segregation errors during egg development are the most common cause of both spontaneous abortion (miscarriage) and mental retardation in humans.


Cell_Biology_Alberts

Cell_Biology_Alberts. When homologs fail to separate properly—a phenomenon called nondisjunction—the result is that some of the resulting haploid gametes lack a particular chromosome, while others have more than one copy of it. Upon fertilization, these gametes form abnormal embryos, most of which die. Some survive, however. Down syndrome in humans, for example, which is the leading cause of mental retardation, is caused by an extra copy of chromosome 21, usually resulting from nondisjunction during meiosis I in the female ovary. Segregation errors during meiosis I increase greatly with advancing maternal age.


Cell_Biology_Alberts

Cell_Biology_Alberts. Haploid gametes are produced by meiosis, in which a diploid nucleus undergoes two successive cell divisions after one round of DNA replication. Meiosis is dominated by a prolonged prophase. At the start of prophase, the chromosomes have replicated and consist of two tightly joined sister chromatids. Homologous chromosomes then pair up and become progressively more closely juxtaposed as prophase proceeds. The tightly aligned homologs undergo genetic recombination, forming crossovers that help hold each pair of homologs together during metaphase I. Meiosis-specific, kinetochore-associated proteins help ensure that both sister chromatids in a homolog attach to the same spindle pole; other kinetochore-associated proteins ensure that the homologs remain connected at their centromeres during anaphase I, so that homologs rather than sister chromatids are segregated in meiosis I. After meiosis I, meiosis II follows rapidly, without DNA replication, in a process that resembles mitosis, in that


Cell_Biology_Alberts

Cell_Biology_Alberts. I, so that homologs rather than sister chromatids are segregated in meiosis I. After meiosis I, meiosis II follows rapidly, without DNA replication, in a process that resembles mitosis, in that sister chromatids are pulled apart at anaphase.


Cell_Biology_Alberts

Cell_Biology_Alberts. A fertilized mouse egg and a fertilized human egg are similar in size, yet they produce animals of very different sizes. What factors in the control of cell behavior in humans and mice are responsible for these size differences? The same fundamental question can be asked for each organ and tissue in an animal’s body. What factors determine the length of an elephant’s trunk or the size of its brain or its liver? These questions are largely unanswered, but it is nevertheless possible to say what the ingredients of an answer must be. The size of an organ or organism depends on its total cell mass, which depends on both the total number of cells and their size. Cell number, in turn, depends on the amounts of cell division and cell death. Organ and body size are therefore determined by three fundamental processes: cell growth, cell division, and cell survival. Each is tightly regulated—both by intracellular programs and by extracellular signal molecules that control these programs.


Cell_Biology_Alberts

Cell_Biology_Alberts. The extracellular signal molecules that regulate cell growth, division, and survival are generally soluble secreted proteins, proteins bound to the surface of cells, or components of the extracellular matrix. They can be divided operationally into three major classes: 1. Mitogens, which stimulate cell division, primarily by triggering a wave of G1/S-Cdk activity that relieves intracellular negative controls that otherwise block progress through the cell cycle. 2. Growth factors, which stimulate cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules and by inhibiting their degradation. 3. Survival factors, which promote cell survival by suppressing the form of programmed cell death known as apoptosis.


Cell_Biology_Alberts

Cell_Biology_Alberts. 3. Survival factors, which promote cell survival by suppressing the form of programmed cell death known as apoptosis. Many extracellular signal molecules promote all of these processes, while others promote one or two of them. Indeed, the term growth factor is often used inappropriately to describe a factor that has any of these activities. Even worse, the term cell growth is often used to mean an increase in cell number, or cell proliferation. In addition to these three classes of stimulating signals, there are extracellular signal molecules that suppress cell proliferation, cell growth, or both; in general, less is known about them. There are also extracellular signal molecules that activate apoptosis.


Cell_Biology_Alberts

Cell_Biology_Alberts. In this section, we focus primarily on how mitogens and other factors, such as DNA damage, control the rate of cell division. We then turn to the important but poorly understood problem of how a proliferating cell coordinates its growth with cell division so as to maintain its appropriate size. We discuss the control of cell survival and cell death by apoptosis in Chapter 18. Unicellular organisms tend to grow and divide as fast as they can, and their rate of proliferation depends largely on the availability of nutrients in the environment. The cells of a multicellular organism, however, divide only when the organism needs more cells. Thus, for an animal cell to proliferate, it must receive stimulatory extracellular signals, in the form of mitogens, from other cells, usually its neighbors. Mitogens overcome intracellular braking mechanisms that block progress through the cell cycle.


Cell_Biology_Alberts

Cell_Biology_Alberts. One of the first mitogens to be identified was platelet-derived growth factor (PDGF), and it is typical of many others discovered since. The path to its isolation began with the observation that fibroblasts in a culture dish proliferate when provided with serum but not when provided with plasma. Plasma is prepared by removing the cells from blood without allowing clotting to occur; serum is prepared by allowing blood to clot and taking the cell-free liquid that remains. When blood clots, platelets incorporated in the clot are stimulated to release the contents of their secretory vesicles (Figure 17–60). The superior ability of serum to support cell proliferation suggested that platelets contain one or more mitogens. This hypothesis was confirmed by showing that extracts of platelets could serve instead of serum to stimulate fibroblast proliferation. The crucial factor in the extracts was shown to be a protein, which was subsequently purified and named PDGF. In the body, PDGF liberated


Cell_Biology_Alberts

Cell_Biology_Alberts. serve instead of serum to stimulate fibroblast proliferation. The crucial factor in the extracts was shown to be a protein, which was subsequently purified and named PDGF. In the body, PDGF liberated from blood clots helps stimulate cell division during wound healing.


Cell_Biology_Alberts

Cell_Biology_Alberts. PDGF is only one of over 50 animal proteins that are known to act as mitogens. Most of these proteins have a broad specificity. PDGF, for example, can stimulate many types of cells to divide, including fibroblasts, smooth muscle cells, and neuroglial cells. Similarly, epidermal growth factor (EGF ) acts not only on epidermal cells but also on many other cell types, including both epithelial and nonepithelial cells. Some mitogens, however, have a narrow specificity; erythropoietin, for Figure 17–60 A platelet. platelets are miniature cells without a nucleus. They circulate in the blood and help stimulate blood clotting at sites of tissue damage, thereby preventing excessive bleeding. They also release various factors that stimulate wound healing. The platelet shown here has been cut in half to show its secretory vesicles, some of which contain platelet-derived growth factor (pdgF).


Cell_Biology_Alberts

Cell_Biology_Alberts. example, only induces the proliferation of red blood cell precursors. Many mitogens, including PDGF, also have actions other than the stimulation of cell division: they can stimulate cell growth, survival, differentiation, or migration, depending on the circumstances and the cell type. In some tissues, inhibitory extracellular signal proteins oppose the positive regulators and thereby inhibit organ growth. The best-understood inhibitory signal proteins are transforming growth factor-β (TGFβ) and its relatives. TGFβ inhibits the proliferation of several cell types, mainly by blocking cell-cycle progression in G1. In the absence of a mitogenic signal to proliferate, Cdk inhibition in G1 is maintained by the multiple mechanisms discussed earlier, and progression into a new cell cycle is blocked. In some cases, cells partly disassemble their cell-cycle control system and withdraw from the cycle to a specialized nondividing state called G0.


Cell_Biology_Alberts

Cell_Biology_Alberts. Most cells in our body are in G0, but the molecular basis and reversibility of this state vary in different cell types. Most of our neurons and skeletal muscle cells, for example, are in a terminally differentiated G0 state, in which their cell-cycle control system is completely dismantled: the expression of the genes encoding various Cdks and cyclins is permanently turned off, and cell division rarely occurs. Some cell types withdraw from the cell cycle only transiently and retain the ability to reassemble the cell-cycle control system quickly and re-enter the cycle. Most liver cells, for example, are in G0, but they can be stimulated to divide if the liver is damaged. Still other types of cells, including fibroblasts and some lymphocytes, withdraw from and re-enter the cell cycle repeatedly throughout their lifetime.


Cell_Biology_Alberts

Cell_Biology_Alberts. Almost all the variation in cell-cycle length in the adult body occurs during the time the cell spends in G1 or G0. By contrast, the time a cell takes to progress from the beginning of S phase through mitosis is usually brief (typically 12–24 hours in mammals) and relatively constant, regardless of the interval from one division to the next. For the vast majority of animal cells, mitogens control the rate of cell division by acting in the G1 phase of the cell cycle. As discussed earlier, multiple mechanisms act during G1 to suppress Cdk activity. Mitogens release these brakes on Cdk activity, thereby allowing entry into a new cell cycle.


Cell_Biology_Alberts

Cell_Biology_Alberts. As we discuss in Chapter 15, mitogens interact with cell-surface receptors to trigger multiple intracellular signaling pathways. One major pathway acts through the monomeric GTPase Ras, which leads to the activation of a mitogen-activated protein kinase (MAP kinase) cascade (see Figure 15–49). This leads to an increase in the production of transcription regulatory proteins, including Myc. Myc is thought to promote cell-cycle entry by several mechanisms, one of which is to increase the expression of genes encoding G1 cyclins (D cyclins), thereby increasing G1-Cdk (cyclin D–Cdk4) activity. Myc also has a major role in stimulating the transcription of genes that increase cell growth.


Cell_Biology_Alberts

Cell_Biology_Alberts. The key function of G1-Cdk complexes in animal cells is to activate a group of gene regulatory factors called the E2F proteins, which bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins involved in DNA synthesis and chromosome duplication. In the absence of mitogenic stimulation, E2F-dependent gene expression is inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family. When cells are stimulated to divide by mitogens, active G1-Cdk accumulates and phosphorylates Rb family members, reducing their binding to E2F. The liberated E2F proteins then activate expression of their target genes (Figure 17–61). This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that ensure that entry into the activation of transcription regulatory protein CYTOSOL


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–61 Mitogen stimulation of cell-cycle entry. as discussed in Chapter 15, mitogens bind to cell-surface receptors to initiate intracellular signaling pathways. One of the major pathways involves activation of the small gTpase Ras, which activates a map kinase cascade, leading to increased expression of numerous immediate early genes, including the gene encoding the transcription regulatory protein myc. myc increases the expression of many delayed-response genes, including some that lead to increased g1-Cdk activity (cyclin d– Cdk4), which triggers the phosphorylation of members of the Rb family of proteins. This inactivates the Rb proteins, freeing the gene regulatory protein E2F to activate the transcription of g1/s genes, including the genes for a g1/s-cyclin (cyclin E) and s-cyclin (cyclin a). The resulting g1/s-Cdk and s-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop. E2F proteins also stimulate the transcription of their own


Cell_Biology_Alberts

Cell_Biology_Alberts. (cyclin a). The resulting g1/s-Cdk and s-Cdk activities further enhance Rb protein phosphorylation, forming a positive feedback loop. E2F proteins also stimulate the transcription of their own genes, forming another positive feedback loop.


Cell_Biology_Alberts

Cell_Biology_Alberts. cell cycle is complete and irreversible. The liberated E2F proteins, for example, increase the transcription of their own genes. In addition, E2F-dependent transcription of G1/S-cyclin (cyclin E) and S-cyclin (cyclin A) genes leads to increased G1/S-Cdk and S-Cdk activities, which in turn increase Rb protein phosphorylation and promote further E2F release (see Figure 17–61).


Cell_Biology_Alberts

Cell_Biology_Alberts. The central member of the Rb family, the Rb protein itself, was identified originally through studies of an inherited form of eye cancer in children, known as retinoblastoma (discussed in Chapter 20). The loss of both copies of the Rb gene leads to excessive proliferation of some cells in the developing retina, suggesting that the Rb protein is particularly important for restraining cell division in this tissue. The complete loss of Rb does not immediately cause increased proliferation of retinal or other types of cells, in part because Cdh1 and CKIs also help inhibit progression through G1 and in part because other cell types contain Rb-related proteins that provide backup support in the absence of Rb. It is also likely that other proteins, unrelated to Rb, help to regulate the activity of E2F.


Cell_Biology_Alberts

Cell_Biology_Alberts. Additional layers of control promote an overwhelming increase in S-Cdk activity at the beginning of S phase. We mentioned earlier that the APC/C activator Cdh1 suppresses cyclin levels after mitosis. In animal cells, however, G1-and G1/S-cyclins are resistant to Cdh1–APC/C and can therefore act unopposed by the APC/C to promote Rb protein phosphorylation and E2F-dependent gene expression. S-cyclin, by contrast, is not resistant, and its level is initially restrained by Cdh1–APC/C activity. However, G1/S-Cdk also phosphorylates and inactivates Cdh1–APC/C, thereby allowing the accumulation of S-cyclin, further promoting S-Cdk activation. G1/S-Cdk also inactivates CKI proteins that suppress S-Cdk activity. The overall effect of all these interactions is the rapid and complete activation of the S-Cdk complexes required for S-phase initiation. dna damage Blocks Cell division: The dna damage Response


Cell_Biology_Alberts

Cell_Biology_Alberts. dna damage Blocks Cell division: The dna damage Response Progression through the cell cycle, and thus the rate of cell proliferation, is controlled not only by extracellular mitogens but also by other extracellular and intracellular signals. One of the most important influences is DNA damage, which can occur as a result of spontaneous chemical reactions in DNA, errors in DNA replication, or exposure to radiation or certain chemicals (discussed in Chapter 5). It is essential that damaged chromosomes are repaired before attempting to duplicate or segregate them. The cell-cycle control system can readily detect DNA damage and arrest the cycle at either of two transitions—one at Start, which prevents entry into the cell cycle and into S phase, and one at the G2/M transition, which prevents entry into mitosis (see Figure 17–16).


Cell_Biology_Alberts

Cell_Biology_Alberts. DNA damage initiates a signaling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chk1 and Chk2. These various kinases phosphorylate other target proteins that lead to cell-cycle arrest. A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding p21, a CKI protein; p21 binds to G1/S-Cdk and S-Cdk complexes and inhibits their activities, thereby helping to block entry into the cell cycle (Figure 17–62 and Movie 17.8).


Cell_Biology_Alberts

Cell_Biology_Alberts. DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphorylation of p53 after DNA damage reduces its binding to Mdm2. This decreases p53 degradation, which results in a marked increase in p53 concentration in the cell. In addition, the decreased binding to Mdm2 enhances the ability of p53 to stimulate gene transcription (see Figure 17–62).


Cell_Biology_Alberts

Cell_Biology_Alberts. The protein kinases Chk1 and Chk2 also block cell-cycle progression by phosphorylating members of the Cdc25 family of protein phosphatases, thereby inhibiting their function. As described earlier, these phosphatases are particularly important in the activation of M-Cdk at the beginning of mitosis (see Figure 17–20). Chk1 and Chk2 phosphorylate Cdc25 at inhibitory sites that are distinct from the phosphorylation sites that stimulate Cdc25 activity. The inhibition of Cdc25 activity by DNA damage helps block entry into mitosis (see Figure 17–16).


Cell_Biology_Alberts

Cell_Biology_Alberts. The DNA damage response can also be activated by problems that arise when a replication fork fails during DNA replication. When nucleotides are depleted, for example, replication forks stall during the elongation phase of DNA synthesis. To prevent the cell from attempting to segregate partially replicated chromosomes, the same mechanisms that respond to DNA damage detect the stalled replication forks and block entry into mitosis until the problems are resolved. A low level of DNA damage occurs in the normal life of any cell, and this damage accumulates in the cell’s progeny if the DNA damage response is not functioning. Over the long term, the accumulation of genetic damage in cells lacking the DNA damage response leads to an increased frequency of cancer-promoting mutations. Indeed, mutations in the p53 gene occur in at least half of all human cancers (discussed in Chapter 20). This loss of p53 function allows the cancer cell stable, active p53


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 17–62 How DNA damage arrests the cell cycle in G1. When dna is damaged, various protein kinases are recruited to the site of damage and initiate a signaling pathway that causes cell-cycle arrest. The first kinase at the damage site is either aTm or aTR, depending on the type of damage. additional protein kinases, called Chk1 and Chk2, are then recruited and activated, resulting in the phosphorylation of the transcription regulatory protein p53. mdm2 normally binds to p53 and promotes its ubiquitylation and destruction in proteasomes. phosphorylation of p53 blocks its binding to mdm2; as a result, p53 accumulates to high levels and stimulates transcription of numerous genes, including the gene that encodes the CkI protein p21. The p21 binds and inactivates g1/s-Cdk and s-Cdk complexes, arresting the cell in g1. In some cases, dna damage also induces either the phosphorylation of mdm2 or a decrease in mdm2 production, which causes a further increase in p53 (not shown).


Cell_Biology_Alberts

Cell_Biology_Alberts. to accumulate mutations more readily. Similarly, a rare genetic disease known as ataxia telangiectasia is caused by a defect in ATM, one of the protein kinases that are activated in response to x-ray-induced DNA damage; patients with this disease are very sensitive to x-rays and suffer from increased rates of cancer.


Cell_Biology_Alberts

Cell_Biology_Alberts. What happens if DNA damage is so severe that repair is not possible? The answer differs in different organisms. Unicellular organisms such as budding yeast arrest their cell cycle to try to repair the damage, but the cycle resumes even if the repair cannot be completed. For a single-celled organism, life with mutations is apparently better than no life at all. In multicellular organisms, however, the health of the organism takes precedence over the life of an individual cell. Cells that divide with severe DNA damage threaten the life of the organism, since genetic damage can often lead to cancer and other diseases. Thus, animal cells with severe DNA damage do not attempt to continue division, but instead commit suicide by undergoing apoptosis. Thus, unless the DNA damage is repaired, the DNA damage response can lead to either cell-cycle arrest or cell death. DNA damage-induced apoptosis often depends on the activation of p53. Indeed, it is this apoptosis-promoting function of p53 that


Cell_Biology_Alberts

Cell_Biology_Alberts. damage response can lead to either cell-cycle arrest or cell death. DNA damage-induced apoptosis often depends on the activation of p53. Indeed, it is this apoptosis-promoting function of p53 that is apparently most important in protecting us against cancer.


Cell_Biology_Alberts

Cell_Biology_Alberts. many Human Cells Have a Built-In Limitation on the number of Times They Can divide Many human cells divide a limited number of times before they stop and undergo a permanent cell-cycle arrest. Fibroblasts taken from normal human tissue, for example, go through only about 25–50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferation slows down and finally halts, and the cells enter a nondividing state from which they never recover. This phenomenon is called replicative cell senescence.


Cell_Biology_Alberts

Cell_Biology_Alberts. Replicative cell senescence in human fibroblasts seems to be caused by changes in the structure of the telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. As discussed in Chapter 5, when a cell divides, telomeric DNA sequences are not replicated in the same manner as the rest of the genome but instead are synthesized by the enzyme telomerase. Telomerase also promotes the formation of protein cap structures that protect the chromosome ends. Because human fibroblasts, and many other human somatic cells, do not produce telomerase, their telomeres become shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually, the exposed chromosome ends are sensed as DNA damage, which activates a p53-dependent cell-cycle arrest (see Figure 17–62). Rodent cells, by contrast, maintain telomerase activity when they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting


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Cell_Biology_Alberts. arrest (see Figure 17–62). Rodent cells, by contrast, maintain telomerase activity when they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting proliferation. The forced expression of telomerase in normal human fibroblasts, using genetic engineering techniques, blocks this form of senescence. Unfortunately, most cancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergo replicative cell senescence.


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Cell_Biology_Alberts. abnormal proliferation signals Cause Cell-Cycle arrest or apoptosis, Except in Cancer Cells Many of the components of mitogenic signaling pathways are encoded by genes that were originally identified as cancer-promoting genes, because mutations in them contribute to the development of cancer. The mutation of a single amino acid in the small GTPase Ras, for example, causes the protein to become permanently overactive, leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc stimulate excessive cell growth and proliferation and thereby promote the development of cancer (discussed in Chapter 20).


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Cell_Biology_Alberts. Surprisingly, however, when a hyperactivated form of Ras or Myc is experimentally overproduced in most normal cells, the result is not excessive proliferation but the opposite: the cells undergo either permanent cell-cycle arrest or apoptosis. The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division. Such responses help prevent the survival and proliferation of cells with various cancer-promoting mutations. Although it is not known how a cell detects excessive mitogenic stimulation, such stimulation often leads to the production of a cell-cycle inhibitor protein called Arf, which binds and inhibits Mdm2. As discussed earlier, Mdm2 normally promotes p53 degradation. Activation of Arf therefore causes p53 levels to increase, inducing either cell-cycle arrest or apoptosis (Figure 17–63).


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Cell_Biology_Alberts. How do cancer cells ever arise if these mechanisms block the division or survival of mutant cells with overactive proliferation signals? The answer is that the protective system is often inactivated in cancer cells by mutations in the genes that encode essential components of the blocking mechanisms, such as Arf or p53 or the proteins that help activate them. Cell proliferation is accompanied by Cell growth If cells proliferated without growing, they would get progressively smaller and there would be no net increase in total cell mass. In most proliferating cell populations, therefore, cell growth accompanies cell division. In single-celled organisms stable, active p53 such as yeasts, both cell growth and cell division require only nutrients. In animals, by contrast, both cell growth and cell proliferation depend on extracellular signal molecules, produced by other cells, which we call growth factors and mitogens, respectively.


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Cell_Biology_Alberts. Like mitogens, the extracellular growth factors that stimulate animal cell growth bind to receptors on the cell surface and activate intracellular signaling pathways. These pathways stimulate the accumulation of proteins and other macromolecules, and they do so by both increasing their rate of synthesis and decreasing their rate of degradation. They also trigger increased uptake of nutrients and production of the ATP required to fuel the increased protein synthesis. One of the most important intracellular signaling pathways activated by growth factor receptors involves the enzyme phosphoinositide 3-kinase (PI 3-kinase), which adds a phosphate from ATP to the 3ʹ position of inositol phospholipids in the plasma membrane (discussed in Chapter 15). The activation of PI 3-kinase leads to the activation of a kinase called TOR, which lies at the heart of cell growth regulatory pathways in all eukaryotes. TOR activates many targets in the cell that stimulate metabolic processes , including


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Cell_Biology_Alberts. activation of a kinase called TOR, which lies at the heart of cell growth regulatory pathways in all eukaryotes. TOR activates many targets in the cell that stimulate metabolic processes , including protein synthesis. One target is a protein kinase called S6 kinase (S6K), which phosphorylates ribosomal protein S6, increasing the ability of ribosomes to translate a subset of mRNAs that mostly encode ribosomal components. TOR also indirectly activates a translation initiation factor called eIF4E and directly activates transcription regulators that promote the increased expression of genes encoding ribosomal subunits (Figure 17–64).


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Cell_Biology_Alberts. PI(3,4,5)P3 PI(4,5)P2 growth factor amino acids activated growth factor receptor activated PI 3-kinase TOR S6K 4E-BP S6 eIF4E transcription regulatory proteins P P P P P PP Figure 17–63 Cell-cycle arrest or apoptosis induced by excessive stimulation of mitogenic pathways. abnormally high levels of myc cause the activation of arf, which binds and inhibits mdm2 and thereby increases p53 levels (see Figure 17–62). depending on the cell type and extracellular conditions, p53 then causes either cell-cycle arrest or apoptosis.


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Cell_Biology_Alberts. Figure 17–64 Stimulation of cell growth by extracellular growth factors and nutrients. The occupation of cell-surface receptors by growth factors leads to the activation of pI 3-kinase, which promotes protein synthesis through a complex signaling pathway that leads to the activation of the protein kinase TOR; extracellular nutrients such as amino acids also help activate TOR. TOR phosphorylates multiple proteins to stimulate protein synthesis, as shown; it also inhibits protein degradation (not shown). growth factors also stimulate increased production of the transcription regulatory protein myc (not shown), which activates the transcription of various genes that promote cell metabolism and growth. 4E-Bp is an inhibitor of the translation initiation factor eIF4E. pI(4,5)p2, phosphatidylinositol 4,5-bisphosphate; pI(3,4,5)p3, phosphatidylinositol 3,4,5-trisphosphate.


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Cell_Biology_Alberts. Figure 17–65 Potential mechanisms for coordinating cell growth and division. In proliferating cells, cell size is maintained by mechanisms that coordinate rates of cell division and cell growth. numerous alternative coupling mechanisms are thought to exist, and different cell types appear to employ different combinations of these mechanisms. (a) In many cell types—particularly yeast—the rate of cell division is governed by the rate of cell growth, so that division occurs only when growth rate achieves some minimal threshold; in yeasts, it is mainly the levels of extracellular nutrients that regulate the rate of cell growth and thereby the rate of cell division. (B) In some animal cell types, growth and division can each be controlled by separate extracellular factors (growth factors and mitogens, respectively), and cell size depends on the relative levels of the two types of factors.


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Cell_Biology_Alberts. (C) some extracellular factors can stimulate both cell growth and cell division by simultaneously activating signaling pathways that promote growth and other pathways that promote cell-cycle progression. For proliferating cells to maintain a constant size, they must coordinate their • progression through the cell cycle growth with cell division to ensure that cell size doubles with each division: if cells depends on the phosphorylation grow too slowly, they will get smaller with each division, and if they grow too fast, of hundreds of different proteins by they will get larger with each division. It is not clear how cells achieve this coordi cyclin–Cdk complexes. What are the nation, but it is likely to involve multiple mechanisms that vary in different organ- isms and even in different cell types of the same organism (Figure 17–65).


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Cell_Biology_Alberts. these proteins are phosphorylated at Animal cell growth and division are not always coordinated, however. In many precisely the right time and place? cases, they are completely uncoupled to allow growth without division or division without growth. Muscle cells and nerve cells, for example, can grow dramatically • during s phase, how are histones after they have permanently withdrawn from the cell cycle. Similarly, the eggs of and their modifying enzymes many animals grow to an extremely large size without dividing; after fertilization, controlled to replicate chromatin however, this relationship is reversed, and many rounds of division occur without structure on the duplicated dna? growth. Compared to cell division, there has been surprisingly little study of how cell • What is the structural basis of size is controlled in animals. As a result, it remains a mystery how cell size is deter-chromosome condensation, and how is the process stimulated during mined and why different cell


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Cell_Biology_Alberts. basis of size is controlled in animals. As a result, it remains a mystery how cell size is deter-chromosome condensation, and how is the process stimulated during mined and why different cell types in the same animal grow to be so different in mitosis?


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Cell_Biology_Alberts. size. One of the best-understood cases in mammals is the adult sympathetic neuron, which has permanently withdrawn from the cell cycle. Its size depends on the • What are the mechanisms by which amount of nerve growth factor (NGF) secreted by the target cells it innervates; the greater the amount of NGF the neuron has access to, the larger it becomes. It seems are sensed at the kinetochore by the likely that the genes a cell expresses set limits on the size it can be, while extracel components of the spindle assembly lular signal molecules and nutrients regulate the size within these limits. The chal checkpoint? lenge is to identify the relevant genes and signal molecules for each cell type. • How is cell growth coordinated with cell division to ensure that cell size remains constant?


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Cell_Biology_Alberts. lenge is to identify the relevant genes and signal molecules for each cell type. • How is cell growth coordinated with cell division to ensure that cell size remains constant? In multicellular animals, cell size, cell division, and cell survival are carefully controlled to ensure that the organism and its organs achieve and maintain an appropriate size. Mitogens stimulate the rate of cell division by removing intracellular molecular brakes that restrain cell-cycle progression in G1. Growth factors promote cell growth (an increase in cell mass) by stimulating the synthesis and inhibiting the degradation of macromolecules. To maintain a constant cell size, proliferating cells employ multiple mechanisms to ensure that cell growth is coordinated with cell division. number of cells Which statements are true? explain why or why not. 17–1 Since there are about 1013 cells in an adult human, and about 1010 cells die and are replaced each day, we become new people every three years.


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Cell_Biology_Alberts. 17–1 Since there are about 1013 cells in an adult human, and about 1010 cells die and are replaced each day, we become new people every three years. 17–2 In order for proliferating cells to maintain a relatively constant size, the length of the cell cycle must match the time it takes for the cell to double in size. 17–3 While other proteins come and go during the cell cycle, the proteins of the origin recognition complex remain bound to the DNA throughout. 17–4 Chromosomes are positioned on the metaphase plate by equal and opposite forces that pull them toward the two poles of the spindle. 17–5 Meiosis segregates the paternal homologs into sperm and the maternal homologs into eggs. 17–6 If we could turn on telomerase activity in all our cells, we could prevent aging. Discuss the following problems.


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Cell_Biology_Alberts. 17–6 If we could turn on telomerase activity in all our cells, we could prevent aging. Discuss the following problems. 17–7 Many cell-cycle genes from human cells function perfectly well when expressed in yeast cells. Why do you suppose that is considered remarkable? After all, many human genes encoding enzymes for metabolic reactions also function in yeast, and no one thinks that is remarkable. 17–8 Hoechst 33342 is a membrane-permeant dye that fluoresces when it binds to DNA. When a population of cells is incubated briefly with Hoechst dye and then sorted in a flow cytometer, which measures the fluorescence of each cell, the cells display various levels of fluorescence as shown in Figure Q17–1. A. Which cells in Figure Q17–1 are in the G1, S, G2, and M phases of the cell cycle? Explain the basis for your answer.


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Cell_Biology_Alberts. A. Which cells in Figure Q17–1 are in the G1, S, G2, and M phases of the cell cycle? Explain the basis for your answer. b. Sketch the sorting distributions you would expect for cells that were treated with inhibitors that block the cell cycle in the G1, S, or M phase. Explain your reasoning. Figure Q17–1 analysis of Hoechst 33342 fluorescence in a population of cells sorted in a flow cytometer (problem 17–8). 17–9 The yeast cohesin subunit Scc1, which is essential for sister-chromatid cohesion, can be artificially regulated for expression at any point in the cell cycle. If expression is turned on at the beginning of S phase, all the cells divide satisfactorily and survive. By contrast, if Scc1 expression is turned on only after S phase is completed, the cells fail to divide and they die, even though Scc1 accumulates in the nucleus and interacts efficiently with chromosomes. Why do you suppose that cohesin must be present during S phase for cells to divide normally?


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Cell_Biology_Alberts. 17–10 High doses of caffeine interfere with the DNA damage response in mammalian cells. Why then do you suppose the Surgeon General has not yet issued an appropriate warning to heavy coffee and cola drinkers? A typical cup of coffee (150 mL) contains 100 mg of caffeine (196 g/ mole). How many cups of coffee would you have to drink to reach the dose (10 mM) required to interfere with the DNA damage response? (A typical adult contains about 40 liters of water.) 17–11 How many kinetochores are there in a human cell at mitosis? 17–12 A living cell from the lung epithelium of a newt is shown at different stages in M phase in Figure Q17–2. Order these light micrographs into the correct sequence and identify the stage in M phase that each represents.


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Cell_Biology_Alberts. Figure Q17–2 Light micrographs of a single cell at different stages of m phase (problem 17–12). (Courtesy of Conly L. Rieder.) 17–13 Down syndrome (trisomy 21) and Edwards syndrome (trisomy 18) are the most common autosomal trisomies seen in human infants. Does this fact mean that these chromosomes are the most difficult to segregate properly during meiosis? 17–14 The human genome consists of 23 pairs of chromosomes (22 pairs of autosomes and one pair of sex chromosomes). During meiosis, the maternal and paternal sets of homologs pair, and then are separated into gametes, so that each contains 23 chromosomes. If you assume that the chromosomes in the paired homologs are randomly assorted to daughter cells, how many potential combinations of paternal and maternal homologs can be generated during meiosis? (For the purposes of this calculation, assume that no recombination occurs.)


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Cell_Biology_Alberts. Overview of the Cell Cycle morgan dO (2007) The Cell Cycle: principles of Control. London: new science press. murray aW & Hunt T (1993) The Cell Cycle: an Introduction. new York: WH Freeman and Co. The Cell-Cycle Control System Evans T, Rosenthal ET, Youngblom J et al. (1983) Cyclin: a protein specified by maternal mRna in sea urchin eggs that is destroyed at each cleavage division. Cell 33, 389–396. Hartwell LH, Culotti J, pringle JR et al (1974) genetic control of the cell division cycle in yeast. Science 183, 46–51. Holt LJ, Tuch BB, Villen J et al. (2009) global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325, 1682–1686. nurse p, Thuriaux p & nasmyth k (1976) genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146, 167–178.


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Cell_Biology_Alberts. nurse p, Thuriaux p & nasmyth k (1976) genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146, 167–178. pavletich np (1999) mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and CIp and Ink4 inhibitors. J. Mol. Biol. 287, 821–828. primorac I & musacchio a (2013) panta rhei: The apC/C at steady state. J. Cell Biol. 201, 177–189. Wittenberg C & Reed sI (2005) Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes. Oncogene 24, 2746–2755. arias EE & Walter JC (2007) strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev. 21, 497–518. Bell sp & kaguni Jm (2013) Helicase loading at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. 5, a010124. groth a, Rocha W, Verreault a et al. (2007) Chromatin challenges during dna replication and repair. Cell 128, 721–733.


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Cell_Biology_Alberts. groth a, Rocha W, Verreault a et al. (2007) Chromatin challenges during dna replication and repair. Cell 128, 721–733. masai H, matsumoto s, You Z et al. (2010) Eukaryotic chromosome dna replication: where, when, and how? Annu. Rev. Biochem. 79, 89–130. siddiqui k, On kF & diffley JF (2013) Regulating dna replication in eukarya. Cold Spring Harb. Perspect. Biol. 5, a012930. Tanaka s & araki H (2013) Helicase activation and establishment of replication forks at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. 5, a010371. alushin g & nogales E (2011) Visualizing kinetochore architecture. Curr. Opin. Struct. Biol. 21, 661–669. Cuylen s & Haering CH (2011) deciphering condensin action during chromosome segregation. Trends Cell Biol. 21, 552–559. gonczy p (2012) Towards a molecular architecture of centriole assembly. Nat. Rev. Mol. Cell Biol. 13, 425–435.


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Cell_Biology_Alberts. gonczy p (2012) Towards a molecular architecture of centriole assembly. Nat. Rev. Mol. Cell Biol. 13, 425–435. Hirano T (2012) Condensins: universal organizers of chromosomes with diverse functions. Genes Dev. 26, 1659–1678. Joglekar ap, Bloom ks & salmon Ed (2010) mechanisms of force generation by end-on kinetochore-microtubule attachments. Curr. Opin. Cell Biol. 22, 57–67. Lampson ma & Cheeseman Im (2011) sensing centromere tension: aurora B and the regulation of kinetochore function. Trends Cell Biol. 21, 133–140. magidson V, O’Connell CB, Loncarek J et al. (2011) The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly. Cell 146, 555–567. musacchio a & salmon Ed (2007) The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393. nasmyth k & Haering CH (2009) Cohesin: its roles and mechanisms. Annu. Rev. Genet. 43, 525–558.


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Cell_Biology_Alberts. nasmyth k & Haering CH (2009) Cohesin: its roles and mechanisms. Annu. Rev. Genet. 43, 525–558. nigg Ea & stearns T (2011) The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries. Nat. Cell Biol. 13, 1154–1160. Rago F & Cheeseman Im (2013) The functions and consequences of force at kinetochores. J. Cell Biol. 200, 557–565. Wadsworth p & khodjakov a (2004) E pluribus unum: towards a universal mechanism for spindle assembly. Trends Cell Biol. 14, 413–419. Walczak CE, Cai s & khodjakov a (2010) mechanisms of chromosome behaviour during mitosis. Nat. Rev. Mol. Cell Biol. 11, 91–102. Fededa Jp & gerlich dW (2012) molecular control of animal cell cytokinesis. Nat. Cell Biol. 14, 440–447. green Ra, paluch E & Oegema k (2012) Cytokinesis in animal cells. Annu. Rev. Cell Dev. Biol. 28, 29–58. Jurgens g (2005) plant cytokinesis: fission by fusion. Trends Cell Biol. 15, 277–283.


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Cell_Biology_Alberts. green Ra, paluch E & Oegema k (2012) Cytokinesis in animal cells. Annu. Rev. Cell Dev. Biol. 28, 29–58. Jurgens g (2005) plant cytokinesis: fission by fusion. Trends Cell Biol. 15, 277–283. Oliferenko s, Chew Tg & Balasubramanian mk (2009) positioning cytokinesis. Genes Dev. 23, 660–674. pollard Td (2010) mechanics of cytokinesis in eukaryotes. Curr. Opin. Cell Biol. 22, 50–56. Rappaport R (1986) Establishment of the mechanism of cytokinesis in animal cells. Int. Rev. Cytol. 105, 245–281. schiel Ja & prekeris R (2013) membrane dynamics during cytokinesis. Curr. Opin. Cell Biol. 25, 92–98. Bhalla n & dernburg aF (2008) prelude to a division. Annu. Rev. Cell Dev. Biol. 24, 397–424. gerton JL & Hawley Rs (2005) Homologous chromosome interactions in meiosis: diversity amidst conservation. Nat. Rev. Genet. 6, 477–487.


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Cell_Biology_Alberts. gerton JL & Hawley Rs (2005) Homologous chromosome interactions in meiosis: diversity amidst conservation. Nat. Rev. Genet. 6, 477–487. Hall H, Hunt p & Hassold T (2006) meiosis and sex chromosome aneuploidy: how meiotic errors cause aneuploidy; how aneuploidy causes meiotic errors. Curr. Opin. Genet. Dev. 16, 323–329. Jordan p (2006) Initiation of homologous chromosome pairing during meiosis. Biochem. Soc. Trans. 34, 545–549. Lake Cm & Hawley Rs (2012) The molecular control of meiotic chromosomal behavior: events in early meiotic prophase in Drosophila oocytes. Annu. Rev. Physiol. 74, 425–51. Watanabe Y (2012) geometry and force behind kinetochore orientation: lessons from meiosis. Nat. Rev. Mol. Cell Biol. 13, 370–382. Control of Cell Division and Cell Growth adhikary s & Eilers m (2005) Transcriptional regulation and transformation by myc proteins. Nat. Rev. Mol. Cell Biol. 6, 635–645.


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Cell_Biology_Alberts. Control of Cell Division and Cell Growth adhikary s & Eilers m (2005) Transcriptional regulation and transformation by myc proteins. Nat. Rev. Mol. Cell Biol. 6, 635–645. Bertoli C, skotheim Jm & de Bruin Ra (2013) Control of cell cycle transcription during g1 and s phases. Nat. Rev. Mol. Cell Biol. 14, 518–528. dick Fa & Rubin sm (2013) molecular mechanisms underlying RB protein function. Nat. Rev. Mol. Cell Biol. 14, 297–306. Jackson sp & Bartek J (2009) The dna-damage response in human biology and disease. Nature 461, 1071–1078. Jorgensen p & Tyers m (2004) How cells coordinate growth and division. Curr. Biol. 14, R1014–R1027. shimobayashi m & Hall mn (2014) making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat. Rev. Mol. Cell Biol. 15, 155–162. Turner JJ, Ewald JC & skotheim Jm (2012) Cell size control in yeast. Curr. Biol. 22, R350–R359.


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Cell_Biology_Alberts. Turner JJ, Ewald JC & skotheim Jm (2012) Cell size control in yeast. Curr. Biol. 22, R350–R359. van den Heuvel s & dyson nJ (2008) Conserved functions of the pRB and E2F families. Nat. Rev. Mol. Cell Biol. 9, 713–724. Vousden kH & Lu X (2002) Live or let die: the cell’s response to p53. Nat. Rev. Cancer 2, 594–604. Zoncu R, Efeyan a & sabatini dm (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35.


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Cell_Biology_Alberts. The growth, development, and maintenance of multicellular organisms depend not only on the production of cells but also on mechanisms to destroy them. The maintenance of tissue size, for example, requires that cells die at the same rate as they are produced. During development, carefully orchestrated patterns of cell death help determine the size and shape of limbs and other tissues. Cells also die when they become damaged or infected, ensuring that they are removed before they threaten the health of the organism. In these and most other cases, cell death is not a random process but occurs by a programmed sequence of molecular events, in which the cell systematically destroys itself from within and is then eaten by other cells, leaving no trace. In most cases, this programmed cell death occurs by a process called apoptosis—from the Greek word meaning “falling off,” as leaves from a tree.


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Cell_Biology_Alberts. Cells dying by apoptosis undergo characteristic morphological changes. They shrink and condense, the cytoskeleton collapses, the nuclear envelope disassembles, and the nuclear chromatin condenses and breaks up into fragments (Figure 18–1A). The cell surface often bulges outward and, if the cell is large, it breaks up into membrane-enclosed fragments called apoptotic bodies. The surface of the cell or apoptotic bodies becomes chemically altered, so that a neighboring cell or a macrophage (a specialized phagocytic cell, discussed in Chapter 22) rapidly engulfs them, before they can spill their contents (Figure 18–1B). In this way, the cell dies neatly and is rapidly cleared away, without causing a damaging inflammatory response. Because the cells are eaten and digested so quickly, there are usually few dead cells to be seen, even when large numbers of cells have died by apoptosis. This is probably why biologists overlooked apoptosis for many years and still might underestimate its


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Cell_Biology_Alberts. are usually few dead cells to be seen, even when large numbers of cells have died by apoptosis. This is probably why biologists overlooked apoptosis for many years and still might underestimate its extent.


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Cell_Biology_Alberts. In contrast to apoptosis, animal cells that die in response to an acute insult, such as trauma or a lack of blood supply, usually do so by a process called cell necrosis. Necrotic cells swell and burst, spilling their contents over their neighbors and eliciting an inflammatory response (Figure 18–1C). In most cases, necrosis is likely to be caused by energy depletion, which leads to metabolic defects and loss of the ionic gradients that normally exist across the cell membrane. One form of necrosis, called necroptosis, is a form of programmed cell death that is triggered by a specific regulatory signal from other cells, although we are only just beginning to understand the underlying mechanisms. Some form of programmed cell death occurs in many organisms, but apoptosis is found primarily in animals. This chapter focuses on the major functions of apoptosis, its mechanism and regulation, and how excessive or insufficient apoptosis can contribute to human disease.


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Cell_Biology_Alberts. The amount of apoptotic cell death that occurs in developing and adult animal tissues is astonishing. In the developing vertebrate nervous system, for example, more than half of many types of nerve cells normally die soon after they are formed. It seems remarkably wasteful for so many cells to die, especially as the vast majority are perfectly healthy at the time they kill themselves. What purposes does this massive cell death serve?


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Cell_Biology_Alberts. In some cases, the answer is clear. Cell death helps sculpt hands and feet during embryonic development: they start out as spade-like structures, and the individual digits separate only as the cells between them die, as illustrated for a mouse paw in Figure 18–2. In other cases, cells die when the structure they form is no longer needed. When a tadpole changes into a frog at metamorphosis, the cells in the tail die, and the tail, which is not needed in the frog, disappears. Apoptosis also functions as a quality-control process in development, eliminating cells that are abnormal, misplaced, nonfunctional, or potentially dangerous to the animal. Striking examples occur in the vertebrate adaptive immune system, where apoptosis eliminates developing T and B lymphocytes that either fail to produce potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous (discussed in Chapter 24); it also eliminates most of the lymphocytes


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Cell_Biology_Alberts. potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous (discussed in Chapter 24); it also eliminates most of the lymphocytes activated by an infection, after they have helped destroy the responsible microbes.


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Cell_Biology_Alberts. In adult tissues that are neither growing nor shrinking, cell death and cell division must be tightly regulated to ensure that they are exactly in balance. If part of the liver is removed in an adult rat, for example, liver cell proliferation increases to make up the loss. Conversely, if a rat is treated with the drug phenobarbital— which stimulates liver cell division (and thereby liver enlargement)—and then the phenobarbital treatment is stopped, apoptosis in the liver greatly increases until the liver has returned to its original size, usually within a week or so. Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate. The control mechanisms responsible for such regulation are largely unknown.


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Cell_Biology_Alberts. Animal cells can recognize damage in their various organelles and, if the damage is great enough, they can kill themselves by undergoing apoptosis. An important example is DNA damage, which can produce cancer-promoting mutations if not repaired. Cells have various ways of detecting DNA damage, and undergo apoptosis if they cannot repair it. Apoptosis is triggered by members of a family of specialized intracellular proteases, which cleave specific sequences in numerous proteins inside the cell, thereby bringing about the dramatic changes that lead to cell death and engulfment. These proteases have a cysteine at their active site and cleave their target proteins at specific aspartic acids; they are therefore called caspases (c for cysteine and asp for aspartic acid). Caspases are synthesized in the cell as inactive precursors and are activated only during apoptosis. There are two major classes of apoptotic caspases: initiator caspases and executioner caspases.


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Cell_Biology_Alberts. Figure 18–1 Two distinct forms of cell death. these electron micrographs show cells that have died by apoptosis (A and B) or by necrosis (C). the cells in (A) and (C) died in a culture dish, whereas the cell in (B) died in a developing tissue and has been engulfed by a phagocytic cell. Note that the cells in (A) and (B) have condensed but seem relatively intact, whereas the cell in (C) seems to have exploded. the large vacuoles visible in the cytoplasm of the cell in (A) are a variable feature of apoptosis. (Courtesy of Julia Burne.) Figure 18–2 Sculpting the digits in the developing mouse paw by apoptosis. the paw in this mouse fetus has been stained with a dye that specifically labels cells that have undergone apoptosis. the apoptotic cells appear as bright green dots between the developing digits.


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Cell_Biology_Alberts. the paw in this mouse fetus has been stained with a dye that specifically labels cells that have undergone apoptosis. the apoptotic cells appear as bright green dots between the developing digits. the interdigital cell death has eliminated the tissue between the developing digits, as seen one day later, when there are very few apoptotic cells. (From W. Wood et al., Development 127:5245–5252, 2000. With permission from the Company of Biologists.) initiator caspase (caspases 8,9) DIMERIZATION, ACTIVATION, AND CLEAVAGE executioner caspase (caspases 3,6,7) CLEAVAGE OF MULTIPLE SUBSTRATES


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Cell_Biology_Alberts. DIMERIZATION, ACTIVATION, AND CLEAVAGE executioner caspase (caspases 3,6,7) CLEAVAGE OF MULTIPLE SUBSTRATES Initiator caspases, as their name implies, begin the apoptotic process. They normally exist as inactive, soluble monomers in the cytosol. An apoptotic signal triggers the assembly of large protein platforms that bring multiple initiator caspases together into large complexes. Within these complexes, pairs of caspases associate to form dimers, resulting in protease activation (Figure 18–3). Each caspase in the dimer then cleaves its partner at a specific site in the protease domain, which stabilizes the active complex and is required for the proper function of the enzyme in the cell.


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Cell_Biology_Alberts. The major function of the initiator caspases is to activate the executioner caspases. These normally exist as inactive dimers. When they are cleaved by an initiator caspase at a site in the protease domain, the active site is rearranged from an inactive to an active conformation. One initiator caspase complex can activate many executioner caspases, resulting in an amplifying proteolytic cascade. Once activated, executioner caspases catalyze the widespread protein cleavage events that kill the cell.


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Cell_Biology_Alberts. Various experimental approaches have led to the identification of over a thousand proteins that are cleaved by caspases during apoptosis. Only a few of these proteins have been studied in any detail. These include the nuclear lamins, the cleavage of which causes the irreversible breakdown of the nuclear lamina (discussed in Chapter 12). Another target is a protein that normally holds a DNA-degrading endonuclease in an inactive form; its cleavage frees the endonuclease to cut up the DNA in the cell nucleus (Figure 18–4). Other target proteins include components of the cytoskeleton and cell–cell adhesion proteins that attach cells to their neighbors; the cleavage of these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easier for a neighboring cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet. The caspase cascade is not only destructive and self-amplifying but also


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Cell_Biology_Alberts. cell to engulf it, or, in the case of an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet. The caspase cascade is not only destructive and self-amplifying but also irreversible, so that once a cell starts out along the path to destruction, it cannot turn back.


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Cell_Biology_Alberts. How is the initiator caspase first activated in response to an apoptotic signal? The two best-understood activation mechanisms in mammalian cells are called the extrinsic pathway and the intrinsic, or mitochondrial, pathway. Each uses its own initiator caspase and activation system, as we now discuss.


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Cell_Biology_Alberts. Figure 18–3 Caspase activation during apoptosis. An initiator caspase contains a protease domain in its carboxy-terminal region and a small protein interaction domain near its amino terminus. It is initially made in an inactive, monomeric form, sometimes called procaspase. Apoptotic signals trigger the assembly of adaptor proteins carrying multiple binding sites for the caspase amino-terminal domain. Upon binding to the adaptor proteins, the initiator caspases dimerize and are thereby activated, leading to cleavage of a specific site in their protease domains. Each protease domain is then rearranged into a large and small subunit. In some cases (not shown), the adaptor-binding domain of the initiator caspase is also cleaved (see Figure 18–5). Executioner caspases are initially formed as inactive dimers. Upon cleavage at a site in the protease domain by an initiator caspase, the executioner caspase dimer undergoes an activating conformational change. the executioner caspases then


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Cell_Biology_Alberts. as inactive dimers. Upon cleavage at a site in the protease domain by an initiator caspase, the executioner caspase dimer undergoes an activating conformational change. the executioner caspases then cleave a variety of key proteins, leading to the controlled death of the cell.


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Cell_Biology_Alberts. Figure 18–4 DNA fragmentation during apoptosis. (A) In healthy cells, the endonuclease CAD associates with its inhibitor, iCAD. Activation of executioner caspases in the cell leads to cleavage of iCAD, which unleashes the nuclease. Activated CAD cuts the chromosomal DNA between nucleosomes, resulting in the production of DNA fragments that form a ladder pattern (see B) upon gel electrophoresis. (B) Mouse thymus lymphocytes were treated with an antibody against the cell-surface death receptor Fas (discussed in the text), inducing the cells to undergo apoptosis. DNA was extracted at the times indicated above the figure, and the fragments were separated by size by electrophoresis in an agarose gel and stained with ethidium bromide. Because the cleavages occur in the linker regions between nucleosomes, the fragments separate into a characteristic ladder pattern on these gels. Note that in gel electrophoresis, smaller molecules are more widely separated in the lower part of the gel, so


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Cell_Biology_Alberts. nucleosomes, the fragments separate into a characteristic ladder pattern on these gels. Note that in gel electrophoresis, smaller molecules are more widely separated in the lower part of the gel, so that removal of a single nucleosome has a greater apparent effect on their gel mobility. (C) Apoptotic nuclei can be detected using a technique that adds a fluorescent label to DNA ends. In the image shown here, this technique was used in a tissue section of a developing chick leg bud; this cross section through the skin and underlying tissue is from a region between two developing digits, as indicated in the underlying drawing. the procedure is called the tUNEL (tdt-mediated dUtpnick end labeling) technique because the enzyme terminal deoxynucleotidyl transferase (tdt) adds chains of labeled deoxynucleotide (dUtp) to the 3ʹ-Oh ends of DNA fragments. the presence of large numbers of DNA fragments therefore results in bright fluorescent dots in apoptotic cells. (B, from D. McIlroy et al.,


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Cell_Biology_Alberts. deoxynucleotide (dUtp) to the 3ʹ-Oh ends of DNA fragments. the presence of large numbers of DNA fragments therefore results in bright fluorescent dots in apoptotic cells. (B, from D. McIlroy et al., Genes Dev. 14:549–558, 2000. With permission from Cold Spring harbor Laboratory press; C, from V. Zuzarte-Luís and J.M. hurlé, Int. J. Dev. Biol. 46:871–876, 2002. With permission from UBC press.)


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Cell_Biology_Alberts. Cell-Surface Death receptors Activate the Extrinsic pathway of Apoptosis Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis. Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domain, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosis factor (TNF) receptor family, which includes a receptor for TNF itself and the Fas death receptor. The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNF family of signal proteins.


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Cell_Biology_Alberts. A well-understood example of how death receptors trigger the extrinsic pathway of apoptosis is the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (cytotoxic) lymphocyte. When activated by the binding of Fas ligand, the death domains on the cytosolic tails of the Fas death receptors bind intracellular adaptor proteins, which in turn bind initiator caspases (primarily caspase-8), forming a death-inducing signaling complex (DISC). Once dimerized and activated in the DISC, the initiator caspases cleave their partners and then activate downstream executioner caspases to induce apoptosis (Figure 18–5). In some cells, the extrinsic pathway recruits the intrinsic apoptotic pathway to amplify the caspase cascade and kill the cell.


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Cell_Biology_Alberts. Many cells produce inhibitory proteins that act to restrain the extrinsic pathway. For example, some cells produce the protein FLIP, which resembles an initiator caspase but has no protease activity because it lacks the key cysteine in its active site. FLIP dimerizes with caspase-8 in the DISC; although caspase-8 appears to be active in these heterodimers, it is not cleaved at the site required for its stable activation, and the apoptotic signal is blocked. Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis. the Intrinsic pathway of Apoptosis Depends on Mitochondria


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Cell_Biology_Alberts. the Intrinsic pathway of Apoptosis Depends on Mitochondria Cells can also activate their apoptosis program from inside the cell, often in response to stresses, such as DNA damage, or in response to developmental signals. In vertebrate cells, these responses are governed by the intrinsic, or mitochondrial, pathway of apoptosis, which depends on the release into the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles (see Figure 12–19). Some of the released proteins activate a caspase proteolytic cascade in the cytoplasm, leading to apoptosis.


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Cell_Biology_Alberts. A key protein in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. When released into the cytosol (Figure 18–6), it takes on a new function: it binds to an adaptor protein called Apaf1 (apoptotic protease activating factor-1), causing the Apaf1 to oligomerize into a wheel-like heptamer called an apoptosome. The Apaf1 proteins in the apoptosome then recruit initiator caspase-9 proteins, which are thought to be activated by proximity in the apoptosome, just as caspase-8 is activated in the DISC. The activated caspase-9 molecules then activate downstream executioner caspases to induce apoptosis (Figure 18–7). Bcl2 proteins regulate the Intrinsic pathway of Apoptosis


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Cell_Biology_Alberts. Bcl2 proteins regulate the Intrinsic pathway of Apoptosis The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill themselves only when it is appropriate. A major class of intracellular regulators of the intrinsic pathway is the Bcl2 family of proteins, which, like the caspase family, has been conserved in evolution from worms to humans; a human Bcl2 protein, for example, can suppress apoptosis when expressed in the worm Caenorhabditis elegans.


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Cell_Biology_Alberts. Figure 18–5 The extrinsic pathway of apoptosis activated through Fas death receptors. trimeric Fas ligands on the surface of a killer lymphocyte interact with trimeric Fas receptors on the surface of the target cell, leading to clustering of several ligand-bound receptor trimers (only one trimer is shown here for clarity). receptor clustering activates death domains on the receptor tails, which interact with similar domains on the adaptor protein FADD (FADD stands for Fas-associated death domain). Each FADD protein then recruits an initiator caspase (caspase-8) via a death effector domain on both FADD and the caspase, forming a death-inducing signaling complex (DISC). Within the DISC, two adjacent initiator caspases interact and cleave one another to form an activated protease dimer, which then cleaves itself in the region linking the protease to the death effector domain. this stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases


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Cell_Biology_Alberts. then cleaves itself in the region linking the protease to the death effector domain. this stabilizes and releases the active caspase dimer into the cytosol, where it activates executioner caspases by cleaving them.


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Cell_Biology_Alberts. Mammalian Bcl2 family proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cytochrome c and other intermembrane mitochondrial proteins into the cytosol. Some Bcl2 family proteins are pro-apoptotic and promote apoptosis by enhancing the release, whereas others are antiapoptotic and inhibit apoptosis by blocking the release. The pro-apoptotic and anti-apoptotic proteins can bind to each other in various combinations to form heterodimers in which the two proteins inhibit each other’s function. The balance between the activities of these two functional classes of Bcl2 family proteins largely determines whether a mammalian cell lives or dies by the intrinsic pathway of apoptosis. As illustrated in Figure 18–8, the anti-apoptotic Bcl2 family proteins, including Bcl2 itself (the founding member of the Bcl2 family) and BclXL, share four distinctive Bcl2 homology (BH) domains (BH1–4). The pro-apoptotic Bcl2 family proteins


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Cell_Biology_Alberts. Figure 18–6 Release of cytochrome c from mitochondria in the intrinsic pathway of apoptosis. Fluorescence micrographs of human cancer cells in culture. (A) the control cells were transfected with a gene encoding a fusion protein consisting of cytochrome c linked to green fluorescent protein (cytochromec-GFp); they were also treated with a red dye that accumulates in mitochondria. the overlapping distribution of the green and red indicates that the cytochromec-GFp is located in mitochondria.


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Cell_Biology_Alberts. (B) Cells expressing cytochrome-c-GFp were irradiated with ultraviolet (UV) light to induce the intrinsic pathway of apoptosis and were photographed 5 hours later. the six cells in the bottom half of this micrograph have released their cytochrome c from mitochondria into the cytosol, whereas the cells in the upper half of the micrograph have not yet done so (Movie 18.1). (From J.C. Goldstein et al., Nat. Cell Biol. 2:156–162, 2000. With permission from Macmillan publishers Ltd.) activation of caspase-9, which cleaves and thereby activates executioner caspases


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Cell_Biology_Alberts. Figure 18–7 The intrinsic pathway of apoptosis. Intracellular apoptotic stimuli cause mitochondria to release cytochrome c, which interacts with Apaf1. the binding of cytochrome c causes Apaf1 to unfold partly, exposing a domain that interacts with the same domain in other activated Apaf1 molecules. Seven activated Apaf1 proteins form a large ring complex called the apoptosome. Each Apaf1 protein contains a caspase recruitment domain (CArD), and these are clustered above the central hub of the apoptosome. the CArDs bind similar domains in multiple caspase-9 molecules, which are thereby recruited into the apoptosome and activated. the mechanism of caspase-9 activation is not clear: it probably results from dimerization and cleavage of adjacent caspase-9 proteins, but it might also depend on interactions between caspase-9 and Apaf1. Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases. Note that the CArD is related in structure and function to the


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Cell_Biology_Alberts. on interactions between caspase-9 and Apaf1. Once activated, caspase-9 cleaves and thereby activates downstream executioner caspases. Note that the CArD is related in structure and function to the death effector domain of caspase-8 (see Figure 18–5). Some scientists use the term “apoptosome” to refer to the complex containing caspase-9.


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Cell_Biology_Alberts. (e.g., Bcl2, BclXL) (e.g., Bax, Bak) (e.g., Bad, Bim, Bid, Puma, Noxa) consist of two subfamilies—the effector Bcl2 family proteins and the BH3-only proteins. The main effector proteins are Bax and Bak, which are structurally similar to Bcl2 but lack the BH4 domain. The BH3-only proteins share sequence homology with Bcl2 in only the BH3 domain.


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Cell_Biology_Alberts. When an apoptotic stimulus triggers the intrinsic pathway, the pro-apoptotic effector Bcl2 family proteins become activated and aggregate to form oligomers in the mitochondrial outer membrane, inducing the release of cytochrome c and other intermembrane proteins by an unknown mechanism (Figure 18–9). In mammalian cells, Bax and Bak are the main effector Bcl2 family proteins, and at least one of them is required for the intrinsic pathway of apoptosis to operate: mutant mouse cells that lack both proteins are resistant to all pro-apoptotic signals that normally activate this pathway. Whereas Bak is bound to the mitochondrial outer membrane even in the absence of an apoptotic signal, Bax is mainly located in the cytosol and translocates to the mitochondria only after an apoptotic signal activates it. As we discuss below, the activation of Bax and Bak usually depends on activated pro-apoptotic BH3-only proteins.


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Cell_Biology_Alberts. The anti-apoptotic Bcl2 family proteins such as Bcl2 itself and BclXL are also located on the cytosolic surface of the outer mitochondrial membrane, where they help prevent inappropriate release of intermembrane proteins. The antiapoptotic Bcl2 family proteins inhibit apoptosis mainly by binding to and inhibiting pro-apoptotic Bcl2 family proteins—either on the mitochondrial membrane or in the cytosol. On the outer mitochondrial membrane, for example, they bind to Bak and prevent it from oligomerizing, thereby inhibiting the release of cytochrome c and other intermembrane proteins. There are at least five mammalian anti-apoptotic Bcl2 family proteins, and every mammalian cell requires at least one to survive. Moreover, a number of these proteins must be inhibited for the intrinsic pathway to induce apoptosis; the BH3-only proteins mediate the inhibition.


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Cell_Biology_Alberts. The BH3-only proteins are the largest subclass of Bcl2 family proteins. The cell either produces or activates them in response to an apoptotic stimulus, and they are thought to promote apoptosis mainly by inhibiting anti-apoptotic Bcl2 family Figure 18–8 The three classes of Bcl2 family proteins. Note that the Bh3 domain is the only Bh domain shared by all Bcl2 family members; it mediates the direct interactions between pro-apoptotic and anti-apoptotic family members. Figure 18–9 The role of pro-apoptotic effector Bcl2 family proteins (mainly Bax and Bak) in the release of mitochondrial intermembrane proteins in the intrinsic pathway of apoptosis. When activated by an apoptotic stimulus, the effector Bcl2 family proteins aggregate on the outer mitochondrial membrane and release cytochrome c and other proteins from the intermembrane space into the cytosol by an unknown mechanism.


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Cell_Biology_Alberts. proteins. Their BH3 domain binds to a long hydrophobic groove on anti-apoptotic Bcl2 family proteins, neutralizing their activity. This binding and inhibition enables the aggregation of Bax and Bak on the surface of mitochondria, which triggers the release of the intermembrane mitochondrial proteins that induce apoptosis (Figure 18–10). Some BH3-only proteins may bind directly to Bax and Bak to help stimulate their aggregation.


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Cell_Biology_Alberts. BH3-only proteins provide the crucial link between apoptotic stimuli and the intrinsic pathway of apoptosis, with different stimuli activating different BH3only proteins. Some extracellular survival signals, for example, block apoptosis by inhibiting the synthesis or activity of certain BH3-only proteins (see Figure 18–12B). Similarly, in response to DNA damage that cannot be repaired, the tumor suppressor protein p53 accumulates (discussed in Chapters 17 and 20) and activates the transcription of genes that encode the BH3-only proteins Puma and Noxa. These BH3-only proteins then trigger the intrinsic pathway, thereby eliminating a potentially dangerous cell that could otherwise become cancerous.


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Cell_Biology_Alberts. As mentioned earlier, in some cells the extrinsic apoptotic pathway recruits the intrinsic pathway to amplify the caspase cascade to kill the cell. The BH3-only protein Bid is the link between the two pathways. Bid is normally inactive. However, when death receptors activate the extrinsic pathway in some cells, the initiator caspase, caspase-8, cleaves Bid, producing an active form of Bid that trans-locates to the outer mitochondrial membrane and inhibits anti-apoptotic Bcl2 family proteins, thereby amplifying the death signal.


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Cell_Biology_Alberts. Figure 18–10 How pro-apoptotic BH3only and anti-apoptotic Bcl2 family proteins regulate the intrinsic pathway of apoptosis. (A) In the absence of an apoptotic stimulus, anti-apoptotic Bcl2 family proteins bind to and inhibit the effector Bcl2 family proteins on the mitochondrial outer membrane (and in the cytosol—not shown). (B) In the presence of an apoptotic stimulus, Bh3-only proteins are activated and bind to the anti-apoptotic Bcl2 family proteins so that they can no longer inhibit the effector Bcl2 family proteins; the latter then become activated, aggregate in the outer mitochondrial membrane, and promote the release of intermembrane mitochondrial proteins into the cytosol. Some activated Bh3only proteins may stimulate mitochondrial protein release more directly by binding to and activating the effector Bcl2 family proteins. Although not shown, the antiapoptotic Bcl2 family proteins are bound to the mitochondrial surface.


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Cell_Biology_Alberts. Because activation of a caspase cascade leads to certain death, the cell employs multiple robust mechanisms to ensure that these proteases are activated only when appropriate. One line of defense is provided by a family of proteins called inhibitors of apoptosis (IAPs). These proteins were first identified in certain insect viruses (baculoviruses), which encode IAP proteins to prevent a host cell that is infected by the virus from killing itself by apoptosis. It is now known that most animal cells also make IAP proteins. All IAPs have one or more BIR (baculovirus IAP repeat) domains, which enable them to bind to and inhibit activated caspases. Some IAPs also polyubiquitylate caspases, marking the caspases for destruction by proteasomes. In this way, the IAPs set an inhibitory threshold that caspases must overcome to trigger apoptosis.


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Cell_Biology_Alberts. In Drosophila at least, the inhibitory barrier provided by IAPs can be neutralized by anti-IAP proteins, which are produced in response to various apoptotic stimuli. There are numerous anti-IAPs in flies, including Reaper, Grim, and Hid, and their only structural similarity is their short, N-terminal, IAP-binding motif, which binds to the BIR domain of IAPs, preventing the domain from binding to a caspase. Deletion of the three genes encoding Reaper, Grim, and Hid blocks apoptosis in flies. Conversely, inactivation of one of the two genes that encode IAPs in Drosophila causes all of the cells in the developing fly embryo to undergo apoptosis. Clearly, the balance between IAPs and anti-IAPs is tightly regulated and is crucial for controlling apoptosis in the fly.


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Cell_Biology_Alberts. The role of mammalian IAP and anti-IAP proteins in apoptosis is less clear. Anti-IAPs are released from the mitochondrial intermembrane space when the intrinsic pathway of apoptosis is activated, blocking IAPs in the cytosol and thereby promoting apoptosis. However, mice appear to develop normally if they are missing either the major mammalian IAP (called XIAP) or the two known mammalian anti-IAPs (called Smac/Diablo and Omi). Worms do not even contain a caspaseinhibiting IAP protein. Apparently, the tight control of caspase activity is achieved by different mechanisms in different animals.


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Cell_Biology_Alberts. Intercellular signals regulate most activities of animal cells, including apoptosis. These extracellular signals are part of the normal “social” controls that ensure that individual cells behave for the good of the organism as a whole—in this case, by surviving when they are needed and killing themselves when they are not. Some extracellular signal molecules stimulate apoptosis, whereas others inhibit it. We have discussed signal proteins such as Fas ligand that activate death receptors and thereby trigger the extrinsic pathway of apoptosis. Other extracellular signal molecules that stimulate apoptosis are especially important during vertebrate development: a surge of thyroid hormone in the bloodstream, for example, signals cells in the tadpole tail to undergo apoptosis at metamorphosis. In mice, locally produced signal proteins stimulate cells between developing fingers and toes to kill themselves (see Figure 18–2). Here, however, we focus on extracellular signal molecules that


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Cell_Biology_Alberts. In mice, locally produced signal proteins stimulate cells between developing fingers and toes to kill themselves (see Figure 18–2). Here, however, we focus on extracellular signal molecules that inhibit apoptosis, which are collectively called survival factors.


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Cell_Biology_Alberts. Most animal cells require continuous signaling from other cells to avoid apoptosis. This surprising arrangement apparently helps ensure that cells survive only when and where they are needed. Nerve cells, for example, are produced in excess in the developing nervous system and then compete for limited amounts of survival factors that are secreted by the target cells that they normally connect to (see Figure 21–81). Nerve cells that receive enough survival signals live, while the others die. In this way, the number of surviving neurons is automatically adjusted so that it is appropriate for the number of target cells they connect with (Figure 18–11). A similar competition for limited amounts of survival factors produced by neighboring cells is thought to control cell numbers in other tissues, both during development and in adulthood.


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Cell_Biology_Alberts. Survival factors usually bind to cell-surface receptors, which activate intracellular signaling pathways that suppress the apoptotic program, often by regulating members of the Bcl2 family of proteins. Some survival factors, for example, stimulate the synthesis of anti-apoptotic Bcl2 family proteins such as Bcl2 itself or BclXL (Figure 18–12A). Others act by inhibiting the function of pro-apoptotic BH3-only proteins such as Bad (Figure M18–12B). In Drosophila, some survival factors act by phosphorylating and inactivating anti-IAP proteins such as Hid, thereby enabling IAP proteins to suppress apoptosis (Figure 18–12C). Some developing neurons, like those illustrated in Figure 18–11, use an ingenious alternative approach: survival-factor receptors stimulate apoptosis—by an unknown mechanism—when they are not occupied, and then stop promoting death when survival factor binds. The end result in all these cases is the same: cell survival depends on survival factor binding.


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Cell_Biology_Alberts. phagocytes remove the Apoptotic Cell Apoptotic cell death is a remarkably tidy process: the apoptotic cell and its fragments do not break open and release their contents, but instead remain intact as they are efficiently eaten—or phagocytosed—by neighboring cells, leaving no trace and therefore triggering no inflammatory response (see Figure 18–1B and Movie 13.5). This engulfment process depends on chemical changes on the surface of the apoptotic cell, which displays signals that recruit phagocytic cells. An especially important change occurs in the distribution of the negatively charged phospholipid phosphatidylserine on the cell surface. This phospholipid is normally located exclusively in the inner leaflet of the lipid bilayer of the plasma membrane (see Figure 10–15), but it flips to the outer leaflet in apoptotic cells. The


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Cell_Biology_Alberts. Figure 18–11 The role of survival factors and cell death in adjusting the number of developing nerve cells to the amount of target tissue. More nerve cells are produced than can be supported by the limited amount of survival factors released by the target cells. therefore, some nerve cells receive an insufficient amount of survival factors to avoid apoptosis. this strategy of overproduction followed by culling helps ensure that all target cells are contacted by nerve cells and that the extra nerve cells are automatically eliminated.


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Cell_Biology_Alberts. Figure 18–12 Three ways that extracellular survival factors can inhibit apoptosis. (A) Some survival factors suppress apoptosis by stimulating the transcription of genes that encode antiapoptotic Bcl2 family proteins such as Bcl2 itself or BclXL. (B) Many others activate the serine/threonine protein kinase Akt, which, among many other targets, phosphorylates and inactivates the pro-apoptotic Bh3-only protein Bad (see Figure 15–53). When not phosphorylated, Bad promotes apoptosis by binding to and inhibiting Bcl2; once phosphorylated, Bad dissociates, freeing Bcl2 to suppress apoptosis. Akt also suppresses apoptosis by phosphorylating and inactivating transcription regulatory proteins that stimulate the transcription of genes encoding proteins that promote apoptosis (not shown). (C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti-IAp protein hid. When not phosphorylated, hid promotes cell death by inhibiting IAps. Once


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Cell_Biology_Alberts. (C) In Drosophila, some survival factors inhibit apoptosis by stimulating the phosphorylation of the anti-IAp protein hid. When not phosphorylated, hid promotes cell death by inhibiting IAps. Once phosphorylated, hid no longer inhibits IAps, which become active and block apoptosis. MAp kinase, mitogenactivated protein kinase.