Overview of the Cell Cycle

T Overview of the cell cycle The most basic function of the cell cycle is to duplicate the vast amount of DNA in the chromosomes and then segregate the copies into two genetically identical daughter cells Chromosome duplication occurs during S phase (S for DNA synthesis), which requires 10 - 12 hours and occupies about half of the cell-cycle time in a typical mammalian cell M phase comprises two major events: nuclear division, or mitosis, during which the copied chromosomes are distributed into a pair of daughter nuclei; and cytoplasmic division, or cytokinesis, when the cell itself divides in two Early in mitosis, at a stage called prophase, the two DNA molecules are disentangled and condensed into pairs of rigid, compact rods called sister chromatids, which remain linked by sister-chromatid cohesion The eukaryotic cell cycle usually consists of four phases Most cells require much more time to grow and double their mass of proteins and organelles than they require to duplicate their chromosomes and divide Partly to allow time for growth, most cell cycles have gap phases - a G1 phase between M phase and S phase and a G2 phase between S phase and mitosis In a typical human cell proliferating in culture, interphase might occupy 23 hours of a 24-hour cycle, with 1 hour for M phase. Cell growth occurs throughout the cell cycle The two gap phases are more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations are complete before the cell commits itself to the complex events of S phase and mitosis If extracellular conditions become favorable or signals to grow and divide are introduced, cells in G(0) progress through a commitment point in G1 known as start (in yeasts) or the restriction point (in mammalian cells) Another important and common variation is the endocycle, also known as endoreduplication, in which multiple rounds of S phase occur without intervening m phases, resulting in cells with many copies of the genome - thereby enabling rapid increases in the production of numerous gene products Cell-cycle control is similar in all eukaryotes Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism The basic organization of the cycle, however, is essentially the same in all eukaryotic cells, and all eukaryotes appear to use similar machinery and control mechanisms to drive and regulate cell-cycle events The cell-cycle control system The cell-cycle control system triggers the major events of the cell cycle The cell-cycle control system operates much like a timer that triggers the events of the cell cycle in a set sequence. In its simplest form - as seen in the stripped-down cell cycles of early animal embryos, for example - the control system is rigidly programmed to provide a fixed amount of time for the completion of each cell-cycle event If some malfunction prevents the successful completion of DNA synthesis, for example, signals are sent to the control system to delay progression to M phase Such delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage - and segregated incompletely replicated chromosomes, for example The cell-cycle control system is based on a connected series of biochemical switches, each of which initiates a specific cell-cycle event. This system of switches possesses many important features that increase the accuracy and reliability of cell-cycle progression (1) first, the switches are generally binary (on/off) and launch events in a complete, irreversible fashion (2) second, the cell-cycle control system is remarkably robust and reliable, allowing the system to operate effectively under a variety of conditions and even if some components fail (3) finally, the control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals In most eukaryotic cells, the cell-cycle control system governs cell-cycle progression at three major regulatory transitions (1) the first is start (or the restriction point) in late G1, when the cell commits to cell-cycle entry and chromosome duplication (2) the second is the G2/M transition, when the control system triggers the early mitotic events that lead to chromosome alignment on the mitotic spindle in metaphase (3) the third is the metaphase-to-anaphase transition, when the control system stimulates sister chromatid separation, leading to the completion of mitosis and cytokinesis If the control system senses problems in the completion of DNA replication, for example, it will hold the cell at the G3/ M transition until those problems are solved The cell-cycle control system depends on cyclically activated cyclin-dependent protein kinases The activities of these kinases [Cdks] rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle An increase in Cdk activity at the G2/M transition, for example, increases the phosphorylation of proteins that control chromosome condensation, nuclear-envelope breakdown, spindle assembly, and other events that occur in early mitosis Cdks, as their name implies, depend on cyclins for their activity: unless they are bound lightly to a cyclin, they have no protein kinase activity Cyclins were originally named because they undergo a cycle of synthesis and degradation in each cell cycle. The levels of the Cdk proteins, by contrast, are constant Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin-Cdk complexes at specific stages of the cell cycle There are three major classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and carry out their functions: (1) G1/S-cyclins activate Cdks in late G1 and thereby help trigger progression through start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase (2) S - cyclins bind Cdks soon after progression through start and help stimulate chromosome duplication. S - cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events (3) M - cyclins activate Cdks that stimulate entry into mitosis at the G2/M transition. M - cyclin levels fall in mid-mitosis Cyclin binding alone does not fully activate the associated Cdk. Complete activation requires a separate kinase, the Cdk-activating kinase (CAK), which phosphorylates an amino acid near the entrance of the Cdk active site CAK activity is constant through the cell cycle, and this modification therefore occurs constitutively throughout the cycle At certain cell-cycle stages, phosphorylation at a pair of amino acids near the kinase active site, by a protein kinase known as Wee1, inhibits Cdk activity. Dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity These small proteins [Cdk inhibitor proteins (CKIs)] wrap around the cyclin-Cdk complex, promoting A rearrangement in the Cdk active site that renders it inactive Protein phosphatases reverse the effects of Cdks Indeed, we will see that the activity of phosphatases tends to decline when Cdk activity increases, resulting in a more robust increase in the protein phosphorylation state Protein phosphatase 2A (PP2A) is a particularly critical regulator of Cdk substrates during the cell cycle. This three-subunit enzyme comes in multiple forms, depending on the identity of a subunit called the regulatory subunit, or B subunit The underlying mechanism is conceptually simple: M-Cdk turns off PP2A-B55 via the phosphorylation of an intermediary protein kinase called Greatwall. As a result, the kinase activity of M-Cdk goes relatively unopposed in early mitosis, contributing to the rapid phosphorylation of M-Cdk substrates When anaphase is initiated and M-Cdk activity declines after cyclin destruction, the system works in reverse: PP2A-B55 is reactivated to promote rapid dephosphorylation of Cdk substrates during anaphase and telophase Hundreds of Cdk substrates are phosphorylated in a defined order Clearly, however, these proteins are not all phosphorylated at the same time: proteins that trigger DNA replication in early S phase, for example, are phosphorylated much earlier than proteins that promote spindle assembly in early mitosis First, it is clear that cyclins do not simply activate the Cdk partner but also direct it to specific target proteins. The surface of each cyclin contains a binding site for short amino acid sequences that are found on certain Cdk substrates This ordering is likely to result from differences in the affinities of the interactions between the Cdk active site and the substrate: high-affinity substrates are phosphorylated earlier The total amount of enzyme activity is also important: M-Cdk activity, for example, continues to rise as the cell progresses through mitosis, and higher activity might be required for the phosphorylation of certain low-affinity targets that are phosphorylated later in mitosis Finally, we must not forget that the timing of protein phosphorylation also depends on the opposing phosphatases, each of which will have differing activation times, localization, and affinities for specific targets Positive feedback generates the switchlike behavior of cell-cycle transitions As mentioned earlier, a key feature of the cell-cycle control system is its ability to generate switchlike, binary decisions: progression through each major cell-cycle transition is a complete, irreversible commitment M-Cdk activation begins with the accumulation of M-cyclin during G2, which leads to a corresponding accumulation of M-Cdk complexes as the cell approaches mitosis Although the Cdk subunit in these complexes is phosphorylated at an activating site by the Cdkactivating kinase (CAK), as discussed earlier, the protein kinase Wee1 holds it in an inactive state by inhibitory phosphorylation at two neighboring sites Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that block the kinase active site The crucial event is activation of the protein phosphatase Cdc25, which removes the inhibitory phosphates that restrain M-Cdk. at the same time, the inhibitory activity of the kinase Wee1 is suppressed, further ensuring that M-Cdk activity increases Notably, Cdc25 is activated, at least in part, by its target, M-Cdk. M-Cdk also inhibits the inhibitory kinase Wee1 The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) results in positive feedback The results is that all M-Cdk complexes in the cell are rapidly and irreversibly activated, leading to rapid phosphorylation of the many proteins that drive the early events of mitosis As in all positive feedback systems, there must be some external trigger mechanism that first unleashes the feedback loop, in this case by causing a small increase in Cdc25 activity This mutual antagonism is essentially a form of positive feedback: when a small amount of greatwall activation is triggered by increasing M-Cdk, the resulting inhibition of PP2A-B55 increases greatwall phosphorylation and activation, and the system switches abruptly to a state of low PP2A-B55 activity A fascinating feature of this system is that PP2A-B55 is also an inhibitor of M-Cdk activation, because it dephosphorylates Cdc25 (causing its inactivation) and Wee1 (causing its activation) The positive feedback loops that promote phosphorylation in early mitosis can be flipped to the alternate dephosphorylation state in anaphase, when M-Cdk is abruptly inactivated by destruction of cyclins The anaphase-promoting complex/cyclosome (APC/C) triggers the metaphase-to-anaphase transition Whereas activation of specific cyclin-Cdk complexes drives progression through the start and G2/M transitions, progression through the metaphase-to-anaphase transition is triggered not by protein phosphorylation but by protein destruction, leading to The final stages of cell division Ubiquitin ligases are used in numerous cell processes to stimulate the proteolytic destruction of specific regulatory proteins. They polyubiquitylate specific target proteins, resulting in their destruction in proteasomes The APC/C catalyzes the ubiquitylation and destruction of two major proteins. The first is securin; its destruction in metaphase activates a protease that separates the sister-chromatid pairs and unleashes anaphase The S- and M-cyclins are the second major targets of the APC/C. Destroying these cyclins inactivates most Cdks in the cell As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by PP2A and other phosphatases in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase, including the final steps in mitosis and then cytokinesis Activation depends primarily on association with one of two activating subunits, Cdc20 or Cdh1. These subunits are essential for two reasons: (1) first, their binding causes a conformational change that enhances enzyme activity (2) second, they provide the main binding site for the enzymes protein substrates Cdc20 acts first, in metaphase, to trigger the destruction of securin and cyclins, resulting in chromosome segregation in anaphase. Cdc20 is then replaced by Cdh1, which maintains APC/C activity through late mitosis and G1, ensuring that cyclins and other proteins are kept at low levels until the following cell cycle As a result, APC/C-Cdc20 is turned on when M-Cdk levels rise in early mitosis, whereas APC/C-Cdh1 activation occurs only after M-Cdk activity declines due to cyclin destruction in anaphase The cell-cycle control system also employs another ubiquitin ligase called SCF. Its major role in the cell cycle is to ubiquitylate certain CKI proteins in late G1, thereby helping to control the activation of S-Cdks and DNA replication Ubiquitylation by SCF is controlled instead by changes in the phosphorylation state of its target proteins, as F-box subunits recognize only specifically phosphorylated proteins The G1 phase is a stable state of Cdk inactivity In most cells, this state of Cdk inactivity generates a stable G1 gap phase, during which the cell grows and monitors its environment before committing to a new cell cycle Cells employ several mechanisms to suppress Cdk activity after mitosis. One mechanism, as we have just seen, depends on the late mitotic activation of APC/C-Cdh1, which ensures that cyclin destruction continues throughout G1 S Phase The linear chromosomes of eukaryotic cells are vast and dynamic assembles of DNA and protein, and their duplication is A complex process that takes up a major fraction of the cell cycle The central event of chromosome duplication - DNA replication - poses two problems for the cell: (1) first, replication must occur with extreme accuracy to minimize the risk of mutations in the next cell generation (2) second, every nucleotide in the genome must be copied once, but only once, to prevent the damaging effects of gene amplification S-Cdk initiates DNA replication once per cell cycle During S phase, DNA replication is initiated at these origins when a DNA helices unwinds the double helix and DNA replication enzymes are loaded onto the two single-stranded templates To ensure that chromosome duplication occurs only once per cell cycle, the initiation of DNA replication is divided into two distinct steps that occur at different times in the cell cycle The first step occurs only in late mitosis or early G1, when two inactive DNA helicases, called Mcm helicases, are loaded onto the DNA at the replication origin. This step is sometime: called licensing of replication origins because initiation of DNA synthesis is permitted only at origins that are preloaded with Mcm helicases The second step occurs in S phase, when the Mcm helicases are activated, primarily by S-Cdks, resulting in DNA unwinding and the initiation of DNA synthesis Once a replication origin has been activated in this way, it cannot be reused until new Mcm helicases are loaded at that origin, which can occur only when the cell reaches late mitosis or G1 A key player is a large multi-protein complex called the origin recognition complex (ORC), which binds to replication origins. In late mitosis and early G1, the proteins Cdc6 and Cdt1 collaborate with the ORC to load the Mcm helicases around the DNA next to the origin At the onset of S phase, S-Cdk triggers origin activation by phosphorylating specific accessory proteins, which bind and thereby activate the Mcm helicases at the origin At the same time as S-Cdk initiates DNA replication, it employs several mechanisms to prevent the loading of new Mcm helicases at origins. S-Cdk phosphorylates and thereby inhibits the ORC and Cdc6 proteins In late mitosis and early G1, APC/C-Cdh1 triggers the destruction of a Cdt1 inhibitor called geminin, thereby allowing Cdt1 to be active. When APC/C-Cdh1 is turned off in late G1, geminin accumulates and inhibits the Cdt1 that is not associated with DNA Also, the association of Cdt1 with a protein At active replication forks stimulates Cdt1 ubiquitylation by a ubiquitin ligase called CRL4-Cdt2, leading to Cdt1 degradation In late mitosis, APC/C activation leads to the inactivation of Cdks and the destruction of geminin. ORC and Cdc6 are dephosphorylated and Cdt1 is activated, allowing Mcm helicase loading to prepare the cell for the next S phase Chromosome duplication requires duplication of chromatin structure The production of chromatin proteins increases during S phase to provide the raw materials needed to package the newly synthesized DNA. Most important, SCdks stimulate a large increase in the synthesis of the four histone subunit that form the histone octamers at the core of each nucleosome Because these differences are important in gene regulation, it is crucial that chromatin structure, like the DNA within, is reproduced accurately during S phase During DNA synthesis, histone-modifying enzymes and various non-histone proteins are probably deposited onto the new DNA strands as they emerge from the replication fork, and these proteins are thought to reproduce the local chromatin structure of the parent chromosome Cohesins hold sister chromatids together At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids glued together along their length. This sister-chromatid cohesion sets the stage for a successful mitosis because it greatly facilitates the attachment of the two sister chromatids to opposite poles of the mitotic spindle Sister-chromatid cohesion depends on a large protein complex called cohesin, which forms a ring structure that surrounds the two chromatids During S phase, by mechanisms that remain obscure, the cohesin ring is held in place during passage of the replication fork, such that the cohesin ring encircles the pair of new sister chromatids as they are being synthesized Also during S phase, an acetyltransferase modifies a subpopulation of cohesins, locking them around the sisters to provide the stable sister-chromatid cohesion that is required to hold the sisters together until mitosis Sister-chromatid cohesion also results, at least in part, from the intertwining of sister DNA molecules that occurs when two replication forks meet during DNA synthesis Mitosis Mitosis is traditionally divided into five stages - prophase, prometaphase, metaphase, anaphase, and telophase - defined primarily on the basis of chromosome behavior as seen in a microscope From a regulatory point of view, mitosis can be divided into two major parts, each governed by distinct components of the cell-cycle control system (1) first, an increase in M-Cdk activity at the G2/M transition triggers the events of early mitosis (prophase, prometaphase, metaphase) (2) the second major part of mitosis begins at the metaphase-to-anaphase transition, when the APC/C triggers the destruction of securin, liberating a protease that cleaves cohesin and thereby initiates separation of the sister chromatids The APC/C also promotes the destruction of cyclins, which leads to Cdk inactivation and the dephosphorylation of Cdk targets, which is required for the events of late M phase, including the completion of anaphase, the disassembly of the mitotic spindle, and the division of the cell by cytokinesis M-Cdk and other protein kinases drive entry into mitosis One of the most remarkable features of cell-cycle control is that a single protein kinase, M-Cdk, brings about so many of the diverse and complex cell rearrangements that occur in the early stages of mitosis At a minimum, M-Cdk must induce the assembly of the mitotic spindle and ensure that each sister chromatid in a pair is attached to the opposite pole of the spindle. It also triggers chromosome condensation, the large-scale reorganization of the intertwined sister chromatids into compact rodlike structures M-Cdk directly phosphorylates many of the proteins involved in mitotic processes, but it also acts indirectly by phosphorylating and thereby activation other protein kinases that carry out some mitotic functions Condensin helps configure duplicated chromosomes for separation At the end of S phase, the immensely long DNA molecules of the sister chromatids are tangled in a mass of partially intertwined DNA and proteins. Any attempt to pull the sisters apart in this state would undoubtedly lead to breaks in the chromosomes These chromosomal changes involve two overlapping processes: chromosome condensation, in which the chromatids are dramatically compacted; and sister-chromatid resolution, whereby the two sisters are resolved into distinct, separable units Resolution results from the disentangling of the sister DNAs, accompanied by the partial removal of cohesin molecules along the chromosome arms. As a result, when the cell reaches metaphase, the sister chromatids appear in the microscope as compact, rodlike structures that are joined tightly at their centromeric regions and only loosely among their arms The condensation and resolution of sister chromatids depend, at least in part, on a five-subunit protein complex called condensin, which is concentrated along the central axes of mitotic chromosomes The mitotic spindle is a dynamic microtobule-based machine The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase, thereby segregating the two sets of chromosomes to opposite ends of the cell, where they are packaged into daughter nuclei The metaphase spindle of an animal cell contains many thousands of microtubules radiating from two spindle poles, with minus ends oriented toward the pole and plus ends directed outward The kinetochore microtubules are the central players in chromosome segregation: their plus ends are attached to sister-chromatid pairs at large protein structures called kinetochores, which are located at the centromere of each sister chromatid These [non-kinetochore microtubules] relatively short and unstable microtubules are densely packed between the poles, cross-linked by various proteins to form a dynamic and adaptable scaffolding network that provides structural stability to the spindle In most animal cells, spindles also contain astral microtubules that radiate outward from the poles and contact the cell cortex, helping to position the spindle in the cell. Each of the two poles In these spindles is focused at a large protein organelle called the centrosome The pericentriolar material nucleates a radial array of microtubules, with their dynamic plus ends projecting outward and their minus ends associated with the centrism The assembly and function of a bipolar spindle depend on hundreds of different microtubule-associated proteins that can be placed in three general categories: nucleating factors that govern the formation of new microtubules, regulatory proteins that control rates of polymerization and depolymerization at both ends of the microtubules, and motor proteins that cross-link and more microtubules relative to each other Microtubules are nucleated in multiple regions of the spindle Mature metaphase spindles contain tens or hundreds of thousands of microtubules, most of which are turning over rapidly, resulting in the need for a constant supply of New microtubules. To meet this need, the spindle contains large numbers of microtubule nucleating factors In the many animal cell types that contain centrosomes, large numbers of gamma-TuRCs are anchored and activated in the pericentriolar material and drive the formation of microtabules that radiate outward from the centrosome Augmin binds to the microtubule in such a way that it orients the new microtubule with its plus end pointing in the same direction as the microtubule to which it binds; as a result, the new microtubule has the correct orientation when it is released and repositioned in the spindle microtubule network Spindle assembly also depends on increased microtubule synthesis in the vicinity of the chromosomes. Mitotic chromosomes generate local signals that activate gamma-TuRC and thereby promote microtubule formation Microtubule instability increases greatly in mitosis During prophase, and particularly in prometaphase and metaphase, the average lifetime of a microtubule decreases dramatically - particularly the lifetime of non-kinetochore microtubules, which exist for only 15-30 seconds This increase in microtubule instability, coupled with the increased ability of the spindle to nucleate microtubules as mentioned above, results in a remarkably dense and dynamic array of spindle microtubules Microtubule dynamics are controlled in the cell by a variety of regulatory proteins, including microtubuleassociated proteins (MAPs) that promote stability and depolymerization factors that destabilize microtubule plus ends Microtubule-based motor proteins govern spindle assembly and function Kinesin-5 is a large tetramer containing two dimeric motor domains at each end. The motor domains both move toward the plus end of a microtubule but can be oriented in opposite directions; as a result, they can associate with two antiparallel microtubules and slide them in opposite directions By attaching to the minus end of one microtubule and transporting it toward the minus end of a second microtubule, dynein functions to connect new microtubules formed in the body of the spindle to microtubules nucleated at a centrosome, and to focus the spindle poles By moving toward the minus end of astral microtubules, dynein motors pull the spindle poles toward the cell cortex and away from each other It [kinesin-14] contains a dimeric motor domain and a second domain that can bind to a neighboring microtubule in a specific orientation, enabling it to cross-link antiparallel microtubules at the center of the spindle and pull the poles together Bipolar spindle assembly in most animal cells begins with centrosome duplication The mitotic spindle must have two poles if it is to pull the two sets of sister chromatids to opposite ends of the cell in anaphase A typical animal cell enters mitosis with a pair of centrosomes, each of which nucleates a radial array of microtubules The other mechanisms depend on the ability of mitotic chromosomes to nucleate and stabilize microtubules and on the ability of motor proteins to organize microtubules into a bipolar array Centrosome duplication begins at about the same time as the cell enters S phase. The G1/S-Cdk that triggers cell-cycle entry also helps initiate centrosome duplication, together with another protein kinase called PIK4 Each of the two centrioles in the centrosome nucleates the formation of a new centriole, which is gradually constructed in S phase to generate a closely linked pair of centrosomes There are interesting parallels between centrosome duplication and chromosome duplication. Both use a semiconservative mechanism of duplication, in which the two halves separate and serve as templates for construction of a new half Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters mitosis with precisely two copies: an incorrect number of centrosomes can lead to defects in spindle assembly and thus errors in chromosome segregation These mechanisms are reminiscent of those that restrict DNA replication to once per cell cycle: after duplication has occurred in S phase, passage through mitosis is required for the "licensing" of centrosome duplication in the next cell cycle Spindle assembly in animal cells requires nuclear-envelope breakdown In cells that contain centrosomes, spindle assembly begins when the two centrosomes move apart along the nuclear envelope in prophase, pulled by dynein motor proteins that link astral microtubules to the cell cortex The plus ends of the microtubules between the centrosomes interdigitate to form an array of antiparallel microtubules, and kinesin-5 motor proteins cross-link these microtubules and push the centrosomes apart Centrosomes and microtubules are located in the cytoplasm, where they have no access to the sisterchromatid pairs inside the nucleus In addition, many of the motor proteins and microtubule regulators that promote spindle assembly are associated with the chromosomes inside the nucleus, and they require nuclear-envelope breakdown to carry out their functions Nuclear-envelope breakdown is a complex, multistep process, which is thought to begin when M-Cdk phosphorylates several subunits of the nuclear pore complexes in the nuclear envelope. This phosphorylation initiates the disassembly of nuclear pore complexes and their dissociation from the envelope In parallel, phosphorylation of several inner-nuclear-envelope proteins leads to detachment of lamin proteins and chromosomes from the nuclear envelope, which is then incorporated into the membranes of the endoplasmic reticulum Mitotic chromosomes promote bipolar spindle assembly Centrosomes are very efficient microtubule nucleators that also act to organize the spindle poles, rapidly generating a bipolar microtubule array that rains down on the chromosomes when the nuclear envelope is removed By creating A local environment that favors both microtubule nucleation and microtubule stabilization, they play an active part in spindle formation For some cells in metaphase, if a single chromosome is tugged out of alignment, a mass of new spindle microtubules rapidly appears around the newly positioned chromosome, while the spindle microtubules at the chromosomes former position depolymerize This property of the chromosomes seems to depend, at least in part, on a guanine nucleotide exchange factor (GEF) that is bound to chromatin; the GEF stimulates a small GTPase in the cytosol called Ran to bind GTP in place of GDP The activated Ran-GTP, which is also involved in nuclear transport, releases microtubule-regulatory proteins from protein complexes in the cytosol, thereby stimulating the local nucleation and stabilization of microtubules around chromosomes In the absence of centrosomes, spindle assembly is thought to begin with the formation of microtubules around the chromosomes Dynein, by transporting one microtubule minus end toward the minus end of another, is required for the focusing of minus ends in a discrete pole at each end of the bipolar array Cells that normally lack centrosomes, such as those of higher plants and many animal oocytes, use chromosome-based self-organization mechanisms to form spindles Although the resulting acentrosomal spindle can segregate chromosomes normally, it lacks astral microtubules, which are responsible for positioning the spindle in animal cells; as a result, the spindle can be mispositioned in the cell Kinetochores attach sister chromatids to the spindle Spindle microtubules become attached to each chromatid at its kinetochore, a giant, multilayered protein structure that is built at the centromeric region of the chromatid In metaphase, the plus ends of kinetochore microtubules are embedded head-on in specialized microtubule-attachment sites within the outer region of the kinetochore, furthest from the DNA Attachment of each microtubule depends on multiple copies of a rod-shaped protein complex called the Ndc80 complex, which is anchored in the kinetochore at one end and interacts with the sides of the microtubule at the other, thereby linking the microtubule to the kinetochore while still allowing the addition or removal of tubulin in subunits at this end At the end of prophase in animal cells, the centrosomes of the growing spindle generally lie on opposite sides of the nuclear envelope. Thus, when the envelope breaks down, the sister-chromatid pairs are bombarded by dynamic microtubule plus ends coming from two directions Bi-orientation is achieved by trial and error The success of mitosis demands that sister chromatids in a pair attach to opposite roles of the mitotic spindle, so that they move to opposite ends of the cell when they separate in anaphase Incorrect attachments are corrected by a system of trial and error that is based on a simple principle: most incorrect attachments are highly unstable and do not last, whereas correct detachments are held in place When a sister-chromatid pair is properly bi-oriented on the spindle, the two kinetochores are pulled in opposite directions by strong poleward forces. Sister-chromatid cohesion resists these poleward forces, creating high levels of tension within the kinetochore When chromosomes are incorrectly attached - when both sister chromatids are attached to the same spindle pole, for example - tension is low and the kinetochore generates an inhibitory signal that loosens the group of its microtubule attachment site, allowing detachment to occur The tension-sensing mechanism depends on the protein kinase Aurora-B, which is associated with the kinetochore and is thought to generate the inhibitory signal that reduces the strength of microtubule attachment in the absence of tension After their attachment of the two spindle poles, the chromosomes are tugged back and forth, eventually assuming a position equidistant between the two poles, a position called the metaphase plate Multiple forces act on chromosomes in the spindle The spindle is a highly dynamic assembly that exists at a steady state that depends on the precise balance of numerous forces generated by various motor proteins and by microtubule polymerization and depolymerization The first major force pulls the kinetochore and its associated chromatid along the kinetochore microtubule toward the spindle pole. This force is generated by depolymerization at the plus end of the microtubule The energy that drives the movement is stored in the microtubule and is released when the microtubule depolymerizes; it ultimately comes from the hydrolysis of GTP that occurs after a tubulin subunit adds to the end of a microtubule Because the low affinity attachments are constantly breaking and re-forming at new sites, the kinetochore remains attached to a microtubule even as the microtubule depolymerizes A second poleward force is provided in some cell types by microtubule flux, where by the microtubules themselves are pulled toward the spindle poles and dismantled at their minus ends In metaphase, the addition of new tubulin at the plus end of a microtubule compensates for the loss of tubulin at the minus end, so that microtubule length remains constant despite the movement of microtubules toward the spindle pole Plus end-directed kinesin-4 and kinesin-10 motors on chromosome arms interact with microtubules and transport the chromosomes away from the spindle poles The APC/C triggers sister-chromatid separation and the completion of mitosis Although M-Cdk activity sets the stage for this event, the anaphase-promoting complex, or cyclosome, discussed earlier throws the switch that initiates sister-chromatid separation by ubiquitylating several mitotic regulatory proteins and thereby triggering their destruction When the cell reaches metaphase, cohesins remain primarily at the centromeric regions of the chromosomes, adjacent to the kinetochores, where they serve to resist the poleward forces that pull the sister chromatids apart The APC/C initiates the process by targeting the inhibitory protein securin for destruction. Before anaphase, securin binds to and inhibits the activity of a protease called seperase Dephosphorylation of Cdk targets depends in part on the inactivation of most Cdks in the cell, which results when the APC/C targets S- and M-cyclins for destruction When Cdk activity declines after cyclin destruction, PP2A and other phosphatases are activated, further driving protein dephosphorylation and the completion of mitosis Unattached chromosomes block sister-chromatid separation: the spindle assembly checkpoint The spindle assembly checkpoint mechanism ensures that cells do not enter anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle Any kinetochore that is not properly attached to microtubules sends out a diffusible negative signal that blocks APC/C-Cdc20 activation throughout the cell and thus blocks the metaphase-to-anaphase transition The unattached kinetochore acts as an enzyme that catalyzes a change in the conformation of Mad2, so then Mad2 then interacts with other proteins to form a large multi-protein complex that binds and thereby inhibits APC/C-Cdc20 When proper microtubule attachment is achieved, these inhibitory complexes are disassembled, and APC/C-Cdc20 inhibition is thereby relieved Chromosomes segregate in anaphase A and B The sudden loss of sister-chromatid cohesion at the onset of anaphase leads to separation, which allows the forces of the mitotic spindle to pull the sisters to opposite poles of the cell - called chromosome segregation The first, anaphase A, is the initial poleward movement of the chromosomes, which is accompanied by shortening of the kinetochore microtubules The second, anaphase B, is the separation of the spindle poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apart Spindle-pole separation during anaphase B depends on motor-driven mechanisms similar to those that separate the two centrosomes in early mitosis Dynein motor proteins that anchor astral microtubule plus ends to the cell cortex pull the poles apart. Kinesin-5, which cross-links antiparallel microtubules at the center of the spindle, pushes the poles apart Cytokinesis In most animal cells, cytokinesis begins in anaphase and ends shortly after the completion of mitosis in telophase Cytokinesis begins in an animal cell with the appearance of a cleavage furrow on the cell surface. The furrow rapidly deepens and spreads around the cell until it completely divides the cell in two The structure underlying this process is the contractile ring - a dynamic assembly composed of actin filaments, myosin II filaments, and many structural and regulatory proteins Actin and myosin II in the contractile ring guide the process of cytokinesis As the sister chromatids separate in anaphase, actin and myosin II begin to accumulate in the rapidly assembling contractile ring, which also contains numerous other proteins that provide structural support or assist in ring assembly Assembly of the contractile ring results in part from the local formation of new actin filaments, which depends on formin proteins that nucleate the assembly of parallel arrays of linear, branched actin filaments As the ring constricts, it maintains the same thickness, suggesting that its total volume and the number of filaments it contains decrease steadily The contractile ring is finally dispensed with altogether when cleavage ends and the plasma membrane of the cleavage furrow narrows to form the mid-body The mid-body persists as a tether between the two daughter cells and contains the remains of the central spindle, a large protein structure derived from the antiparallel microtubules of the spindle midzone, packed tightly together within a dense matrix material Local activation of RhoA triggers assembly and contraction of the contractile ring RhoA, a small GTPase of the Ras superfamily, controls the assembly and function of the contractile ring at the site of cleavage It stimulates actin filament formation by activating formins, and it promotes myosin II assembly and contractions by activating multiple protein kinases, including the Rho-associated kinase (ROCK) RhoA is activated by a guanine nucleotide exchange factor (GEF) called Ect2, which stimulates the release of GDP and binding of GTP to RhoA. Ect2 is localized to the division site and activated by complex mechanisms involving spindle microtubules The microtubules of the mitotic spindle determine the plane of animal cell division Cytokinesis must occur only after the two sets of chromosomes are fully segregated from each other, and the site of division must be placed between the two sets of daughter chromosomes, thereby ensuring that each daughter cell receives a complete set During anaphase, the spindle generates signals that initiate furrow formation at a position midway between the spindle poles, thereby ensuring that division occurs between the two sets of separated chromosomes If the spindle is tugged into a new position with a fine glass needle in early anaphase, the incipient cleavage furrow disappears, and a new one develops in accord with the new spindle site - supporting the idea that signals generated by the spindle induce local furrow formation The key mechanism appears to be that the mid-zone of the anaphase spindle generates a signal that promotes furrow formation at the cell cortex Centralspindlin assembly at the mid-zone is stimulated by the protein kinase Aurora-B, which also localizes to the spindle mid-zone in anaphase Aurora-B localization to the central spindle depends on dephosphorylation of Cdk substrates, providing one mechanism that delays cytokinesis until anaphase The focusing of centralspindlin and Ect2 at the equator depends in part on the ability of astral microtubules to somehow inhibit centralspindlin localization outside the equatorial region In plant cells, an organized band of microtubules and actin filaments, called the pre-prophase band, assembles just before mitosis and marks the side where the cell wall will assemble and divide the cell in two The phragmoplast guides cytokinesis in higher plants New membrane is added primarily at the cleavage furrow and is generally provided by small membrane vesicles that are transported on microtubules from the Golgi apparatus to the furrow Rather than a contractile ring dividing the cytoplasm from the outside in, the cytoplasm of the plant cell is partitioned from the inside out by the construction of a new cell wall, called the cell plate, between the two daughter nuclei The assembly of the cell plate begins in late anaphase and is guided by a structure called the phragmoplast, which contains microtubules derived from the mitotic spindle The plate expands outward by further vesicle fusion until is reaches the plasma membrane and the original cell wall and divides the cell in two. Later, cellulose microfibrils are laid down within the matrix of the cell plate to complete the construction of the new cell wall Membrane-enclosed organelles must be distributed to daughter cells during cytokinesis When a eukaryotic cell divides, however, each daughter cell must also inherit all of the other essential cell components, including the membrane-enclosed organelles Organelles such as mitochondria and chloroplasts are usually present in large enough numbers to be safely inherited if, on average, their numbers roughly double once each cycle In most cells, the Er remains largely intact and is cut in two during cytokinesis. The Golgi apparatus is reorganized and fragmented during mitosis Golgi fragments associate with the spindle poles and are thereby distributed to opposite ends of the spindle, ensuring that each daughter cell inherits the materials needed to reconstruct the Golgi in telophase Some cells reposition their spindle to divide asymmetrically To create daughter cells with different fates in this way, the mother cell must first segregate certain components to one side of the cell and then position the plane of division so that the appropriate daughter cell inherits these components It seems likely that changes in local regions of the cell cortex direct such spindle movements and that motor proteins localized there pull one of the spindle poles, via its astral microtubules, to the appropriate region Mitosis can occur without cytokinesis After these rapid nuclear divisions, membranes are created around each nucleus in one round of coordinated cytokinesis called cellularization. The plasma membrane extends inward and, with the help of an actin-myosin ring, pinches off to enclose each nucleus Meiosis Most eukaryotic organisms reproduce sexually: the genomes of two parents mix to generate offspring that are genetically distinct from either parent The cells of these organisms are generally diploid; that is, they contain two slightly different copies, or homologs, of each chromosome, one from each parent Meiosis includes two rounds of chromosome segregation As in the mitotic cell cycle, the cell begins the meiotic program by duplicating its chromosomes in meiotic S phase, resulting in pairs of sister chromatids that are tightly linked along their entire lengths by cohesin complexes The duplicated paternal and maternal homologs pair up alongside each other and become physically linked by the process of genetic recombination In the first meiotic anaphase, duplicated homologs rather than sister chromatids are pulled apart and segregated into the two daughter nuclei Only in the second division (meiosis II), which occurs without further DNA replication, are the sister chromatids pulled apart and segregated (as in mitosis) to produce haploid daughter nuclear Duplicated homologs pair during meiotic prophase During meiosis I, however, it is crucial that homologs recognize each other and associate physically in order for the maternal and paternal homologs to be bi-oriented on the first meiotic spindle 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 In most species, homolog pairs are then locked together by homologous recombination: DNA doublestrand breaks are formed at several locations in each sister chromatid, resulting in large numbers of DNA recombination events between the homologs 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 Homolog pairing culminates in the formation of a synaptonemal complex 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 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 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 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 along their entire lengths The pachytene stage can persist for days or longer, until desynapsis begins at diplotene with the disassembly of the synaptonemal complex and the concomitant condensation and shortening of the chromosomes It is only at this stage, after the complex has disassembled, that the individual crossover events between non-sister chromatids can be seen as inter-homolog connections called chiasmata (singular, chiasma), which now play a crucial part in holding the compact homologs together Homolog segregation depends on several unique features of meiosis I A fundamental difference between meiosis I and mitosis (and meiosis II) is that in meiosis I, homologs rather than sister chromatids separate and then segregate In meiosis I, however, the two sister kinetochores are fused into a single microtubule-binding unit that attaches to just one pole. The fusion of sister kinetochores is achieved by a complex of proteins that is localized at the kinetochores in meiosis I 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 biorientation in mitosis Third, cohesin is removed in anaphase I only from chromosome arms and not from the regions near the centromeres, where the kinetochores are located Cohesin phosphorylation is normally required for separase to cleave cohesin; thus, removal of this phosphorylation near the centromere prevents cohesin cleavage After 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 Crossing-over is highly regulated 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 doublestrand breaks along each chromosome are controlled, as is the likelihood that a break will be converted into a crossover 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 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 Control of Cell Division and Cell Growth 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 rates 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 The extracellular signal molecules that regulate cell growth, division, and survival are generally soluble secreted proteins bound to the surface of cells, or components of the extracellular matrix (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 by suppressing the form of programmed cell death known as apoptosis In addition to these three classes of stimulating signals, there are extracellular signal molecules that suppress cell proliferation, cell growth, or both Mitogens stimulate cell division 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 Thus, for an animal cell to proliferate, it must receive stimulator extracellular signals, in the form of mitogens, from other cells, usually its neighbors 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 The best-understood inhibitory signal proteins are transforming growth factor beta (TGFB) and its relatives. TGFB inhibits the proliferation of several cell types, mainly by blocking cell-cycle progression in G1 Cells can enter a specialized non-dividing state 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 non-dividing state called G0 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 recenter the cycle 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 Mitogens stimulate G1-Cdk and G1/S-Cdk activities One major pathway acts through the monomeric GTPase Ras, which leads to the activation of a mitogen-activated protein kinase (MAP kinase) Myc is thought to promote cell-cycle entry by several mechanisms, one of which is to increase the expression of genes encoding G1-cyclins, thereby increasing G1-Cdk activity 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 G1Cdk accumulates and phosphorylates Rb family members, reducing their binding to E2F This transcriptional control system, like so many other control systems that regulate the cell cycle, includes feedback loops that ensure that entry into the cell cycle is complete and irreversible 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 Rbrelated proteins that provide backup support in the absence of Rb In animal cells, however, G1- and G1/S-cyclins are resistant to APC/C-Cdh1 and can therefore act unopposed by the APC/ C to promote Rb protein phosphorylation and E2F-dependent gene expression However, G1/S-Cdk also phosphorylates and inactivates APC/C-Cdh1, thereby allowing the accumulation of S-cyclin, further promoting S-Cdk activation DNA damage blocks cell division 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 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 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 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 DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations Phosphorylation of p53 after DNA damage reduces its binding to Mdm2. This decreases p53 degradation, which results in a marked increase in p 53 concentration in the cell 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 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 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 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 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 Many human cells have a built-in limitation on the number of times they can divide 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 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