572 14

Cellular Reproduction

14.1 The Cycle According to the third tenet of the , new cells 14.2 M Phase: and Cytokinesis originate only from other living cells. The process by which this 14.3 occurs is called . For a multicellular , such as a or an oak tree, countless divisions of a single-celled THE HUMAN PER SP ECTIVE: Meiotic Nondisjunction produce an organism of astonishing cellular complexity and Its Consequences and organization. Cell division does not stop with the formation of EXPERIMENTAL PATHWAYS: The Discovery and the mature organism but continues in certain tissues throughout Characterization of MPF . Millions of cells residing within the marrow of your bones or the lining of your intestinal tract are undergoing division at this very moment. This enormous output of cells is needed to replace cells that have aged or died. Although cell division occurs in all , it takes place very differently in prokaryotes and . We will restrict discussion to the eukaryotic version. Two distinct types of eukaryotic cell division will be discussed in this chapter. Mitosis leads to production of cells that are genetically identical to their parent, whereas meiosis leads to production of cells with half the genetic content of the parent. Mitosis serves as the basis for producing new cells, meiosis as the basis for producing new

Fluorescence micrograph of a mitotic spindle that had assembled in a cell-free extract prepared from frog eggs, which are cells that lack a centrosome. The red spheres consist of chromatin-covered beads that were added to the extract. It is evident from this micrograph that a bipolar spindle can assemble in the absence of both and centrosomes. In this experiment, the chromatin-covered beads served as nucleating sites for the assembly of the microtubules that subsequently formed this spindle. The mechanism by which cells construct mitotic spindles in the absence of centrosomes is discussed on page 587. (F ROM R. H EALD , ET AL ., N ATURE VOL . 382, COVER OF 8/1/96; © 1996, REPRINTED WITH PERMISSION FROM MACMILLAN PUBLISHERS , L IMITED .) 573 sexually reproducing organisms. Together, these two types of cell and, in a broader sense, between living and the division form the links in the chain between parents and their earliest eukaryotic life forms present on Earth.

14.1 | The Cell Cycle thesized DNA. If [ 3H]thymidine is given to a culture of cells for a short period (e.g., 30 minutes) and a sample of the cell popula- In a of dividing cells, whether tion is fixed, dried onto a slide, and examined by autoradiogra- inside the body or in a culture dish, each phy, only a fraction of the cells are found to have radioactive cell passes through a series of defined nuclei. Among cells that were engaged in mitosis at the time of stages, which constitutes the cell cycle (Figure 14.1). The cell fixation (as evidenced by their compacted chromosomes) none is cycle can be divided into two major phases based on cellular ac- found to have a radioactively labeled nucleus.These mitotic cells tivities readily visible with a light microscope: M phase and in- have unlabeled chromosomes because they were not engaged in terphase. M phase includes (1) the process of mitosis , during DNA replication during the labeling period. which duplicated chromosomes are separated into two nuclei, If labeling is allowed to continue for one or two hours be- and (2) cytokinesis , during which the entire cell divides into fore the cells are sampled, there are still no cells with labeled two daughter cells. Interphase , the period between cell divi- mitotic chromosomes (Figure 14.2). We can conclude from sions, is a time when the cell grows and engages in diverse these results that there is a definite period of time between the metabolic activities. Whereas M phase usually lasts only an end of DNA synthesis and the beginning of M phase. This hour or so in mammalian cells, interphase may extend for days, period is termed G 2 (for second gap). The duration of G 2 is weeks, or longer, depending on the cell type and the conditions. revealed as one continues to take samples of cells from the cul- Although M phase is the period when the contents of a cell ture until labeled mitotic chromosomes are observed. The first are actually divided, numerous preparations for an upcoming cells whose mitotic chromosomes are labeled must have been mitosis occur during interphase, including replication of the at the last stages of DNA synthesis at the start of the incuba- cell’s DNA. One might guess that a cell engages in replication tion with [ 3H]thymidine. The length of time between the throughout interphase. However, studies in the early 1950s on start of the labeling period and the appearance of cells with la- asynchronous cultures (i.e., cultures whose cells are randomly beled mitotic figures corresponds to the duration of G 2. distributed throughout the cell cycle) showed that this is not the DNA replication occurs during a period of the cell cycle case. As described in Chapter 13, DNA replication can be mon- 3 termed S phase . S phase is also the period when the cell syn- itored by the incorporation of [ H]thymidine into newly syn- thesizes the additional histones that will be needed as the cell doubles the number of nucleosomes in its chromosomes (see Interphase Figure 13.23). The length of S phase can be determined di- rectly. In an asynchronous culture, the percentage of cells en- gaged in a particular activity is an approximate measure of the percentage of time that this activity occupies in the of cells. Thus, if we know the length of the entire cell cycle, the

G1: S: Cell grows and DNA replication carries out normal and ; duplication duplicate

G : s 2 ssiis too Cell grows Miit and prepares for mitosis

Cytokinesis Telophase


Metaphase Figure 14.1 An overview of the eukaryotic cell cycle. This diagram of the cell cycle Prometaphase Prophase indicates the stages through which a cell passes from one division to the next. The cell cycle is divided into two major phases: M phase and interphase. M phase includes the 14.1 M it successive events of mitosis and cytokinesis. Interphase is divided into G 1, S, and G 2

o Cycle Cell The ((M si M s phases, with S phase being equivalent to the period of DNA synthesis. The division of pph haass ee)) interphase into three separate phases based on the timing of DNA synthesis was first proposed in 1953 by Alma Howard and Stephen Pelc of Hammersmith Hospital, London, based on their experiments on meristem cells. Chapter 14 Cellular Reproduction 574 tured for 30 minutes in medium containing [ of the cell cycle. a defined period occurs during 2. 1. We three recognize can broad categories and divide. within a multicellular plant or is their cap ty various distinguishes that properties the of One Cell Cycles in Vivo [ with pulse brief age of the cells whose arenuclei radioactively lab fromdirectly t calculated be phasecan Slength of (Fplotted as shown. Figure 14.2 sis. When one adds up the periods of Gof periods the up adds one When sis. population that are cell seen to be engaged in mitosis o of percentage the from calculated be can phase 3 . be accounted for. This other phase, termed G termed phase, other This for. accounted be parent that there is an additional period in the ce percentage of those whose mitotic chromosomes cells that werefor cells inwer mitosis at the time they culture Each fixed and prepared for autoradiography. times in unlabeled m for various incubated (chased) is the period following mitosis and preceding DNA s proliferate by interaction with by anantiproliferate interaction appropriate be induced t can which and lymphocytes, of the liver, removalbe induced by the tosurgical proliferate o . and an divide DNA appropri synthesis when given gin do benot induce divide but can Cells that normally die.until they remain they in that have differentiated, these cells to divi are the ability and lack highly specialized or red blood muscle cells, cells, such as nerve Cells, shared by they most are cells; able to divide asymme Stem cells sion. have an important property that is roots and stems also exhibit rapid and continual ce ized cells of apical meristems located near the tip and the body surface (Figure The relatively 7.1). un the base of numerous epithelia that line the body c to red and white blood cells (Figure 17.6) and stem suchadult astissues, hematopoietic stem cells that activity. Cells that normally possess a relatively high level

Percentage of labeled mitoses 100 20 40 60 80 Included in this category are stem cells of various Experimental results demonstrating that replication results demonstrating Experimental Included in this group are liver cells, which can which in this group are liver cells, Included O TD BY STUDY A ROM 5 3 Hours after ]hmdn.Smlry h lnt o M of length the Similarly, H]thymidine. 015 10 3 H–thymidine addition .BR. 02 30 25 20 SRAANDASERGA 3 H]thymidine and then 2 xd and thee fixed, ϩ HeLa cells were cells cul-HeLa edium before being S dish was scanned 1 were labeled was ϩ (for first gap),first (for eled during a ll cycle ll yet cycle to acity acity to grow o mitotic of .WF. M, it is ap-is it M, he percent-he de. s of plant r cytokine- pes of cellsof pes cls that cells, give rise ll divi- avities d to be- n o t cells at of cells: special- ynthesis. gen. state trically. f part f part i the in s Once IEBEL o ate .) f h G the of mttc el a fsd ih n -hs cl,te S the 14.3 (Figure compacted cell, became also S-phase chromatin an with fused was cell mitotic a factors that could induce mitosis in a nonmitotic ( nonmitotic a in mitosis induce could that factors gested that the cytoplasm of a mitotic cell contain experiments these of results The chromosomes. pacted chromosomal fragments rather than intac “pulverized” format the to led nucleus s S-phase the in damage, compaction to sensitive especially is DNA replicating the fact that replication had already occurred (Fig somes (Figure 14.3 (Figure somes compaction fused, the G the fused, G ceed from Ggrowth-promotingareceive signalmustcell A phase. escent the Manymammalian cells liver. in the body are said cells are Qui often described as being in the G synthesis. DNA of initiation the preceding stage arr are dividing stopped have that cells exceptions, fe a With change. shouldconditions if divide to ity them but to theyan retainupcoming t cell division, them from the typical Gtypical the from them If a Ga If chromatin in the of nucleus the nonmitotic cell (Fi compacti induced always cell mitotic The cycle. cell s other in cells with cells mitotic fused they ment, In cycle. cell the of stages different in were that They approached the question by ties. fusing mammali ce affect that factors regulatory contains cells of is regulated.to understanding how cycle the cell t open helped Colorado of University the of Johnson and Rao Potu by out carried experiments fusion cell 1970 In division. own its regulate to breakdo ability cell’s a from results that disease a cancer, bating implication practical enormous has also but , i important only not is cycle cell the of study The Control of the Cell Cycle mosome compaction, but unlike those of a Ga of those unlike but compaction, mosome opce Gcompacted in a cleaving frog , whose cell cycles lack bo lack cycles cell whose embryo, frogcleaving a in 2 of the T cell in the Chapter 17 opening photo. and polar bodies in Figure 14.41 (or unequal) cell as illustrateddivisions, by the f Some types of nonstem cells can also engage in asym renewal and the formation of differentiated asymmetric divisions allow stem cells to engage in becoming a differentiated cell of In that ot tissue. ent and another daughter cell that has taken a step ter cell that remains an uncommitted stem cell like The asymmetric division of a stem cell produces one daughter cells have or different components, f sizes, An hss to several months in phases, slowly growing tissues, Rao Rao and Johnson wanted to know whether the cytoplas Cell cycles can range in length from as short as 30 , which means that they are in a state that will not will that state a in are they that means which , 1 asymmetric cell division paeada -hs elwr ue,thechromatin were -phaseandM-phasean cell fused, 1 -phase nucleus underwent underwent nucleus -phase o om st f lnae cmatd chromo- compacted elongated of set a form to 0 2 2 into G chromosomes also underwent premature chro-premature underwent also chromosomes hoooe eevsbyduld reflectingdoubled, visibly were chromosomes a 1 ). If a Ga If ). phase and thus reenter the cell cycle. 1 -phase cells that may soon enter Senter soon may that cells -phase 2 -phase and M-phase cell were cell M-phase and -phase is one in which the two rmtr chromosomal premature 0 b state to distinguish and the division 1 ll cycle activi- cycle ll b ormation of ure 14.3 ulu,thenucleus, ed diffusible .However,). , a series of series a , tages of theof tages one experi-one n basic cellbasic n he capabil- her words, gure 14.3). th Gth .. inter-i.e., to be cells. se i a in ested w notablew towards s in com-in s both self- its par- on of theof on ates. minutes n n a in wn to pro-to Robert he doorhe daugh- ,com-t, metric an cells such as -phase that o o ofion escent 1 sug- c qui- lead and .If). m 575

Mitotic Mitotic Chromosomes Chromosomes Pulverized chromosome fragments

G1 Chromosomes

Mitotic Chromosomes

G2 Chromosomes

(a) (b) (c) Figure 14.3 Experimental demonstration that cells contain factors the S-phase cell becomes pulverized. The elongated chromatids of the that stimulate entry into mitosis. The photographs show the results of G2-phase cell in c are doubled in comparison with those of the G 1 cell the fusion of an M-phase HeLa cell with a rat kangaroo PtK2 cell that in a. (F ROM KARL SPERLING AND POTU N. R AO , H UMANGENETIK had been in ( a) G 1 phase, ( b) S phase, or ( c) G 2 phase at the time of cell 23:437, 1974. WITH KIND PERMISSION OF SPRINGER SCIENCE + fusion. As described in the text, the chromatin of the G 1-phase and BUSINESS MEDIA .) G2-phase PtK2 cells undergoes premature compaction, whereas that of

phase) cell. This finding suggested that the transition from G 2 kinases (Cdks ). It has been found that Cdks are not only in- to M was under positive control; that is, the transition was in- volved in M phase but are the key agents that orchestrate ac- duced by the presence of some stimulatory agent. tivities throughout the cell cycle. Cdks carry out this

The Role of Kinases While the cell-fusion experi- Mitosis Mitosis Mitosis ments revealed the existence of factors that regulated the cell cycle, they provided no information about the biochemical properties of these factors. Insights into the of the agents that promote entry of a cell into mitosis (or meiosis) were first Interphase Interphase Interphase gained in a series of experiments on the oocytes and early of frogs and . These experiments are MPF activity High described in the Experimental Pathways at the end of this chapter. To summarize here, it was shown that entry of a cell into M phase is initiated by a protein called maturation- Low promoting factor (MPF ). MPF consists of two subunits: (1) a subunit with kinase activity that transfers phosphate groups Cyclin High from ATP to specific serine and threonine residues of specific protein substrates and (2) a regulatory subunit called cyclin . The Low term cyclin was coined because the concentration of this regula- Figure 14.4 Fluctuation of cyclin and MPF levels during the tory protein rises and falls in a predictable pattern with each cell cell cycle. This drawing depicts the cyclical changes that occur cycle (Figure 14.4). When the cyclin concentration is low, the during early frog development when mitotic divisions occur rapidly and kinase lacks the cyclin subunit and, as a result, is inactive. When synchronously in all cells of the embryo. The top tracing shows the the cyclin concentration rises, the kinase is activated, causing alternation between periods of mitosis and interphase, the middle tracing 14.1 the cell to enter M phase. These results suggested that (1) pro- shows the cyclical changes in MPF kinase activity, and the lower tracing gression of cells into mitosis depends on an whose sole shows the cyclical changes in the concentrations of cyclins that control the h Cl Cycle Cell The activity is to phosphorylate other , and (2) the activity relative activity of the MPF kinase. (F ROM A. W. M URRAY AND M. W. of this enzyme is controlled by a subunit whose concentration KIRSCHNER , S CIENCE 246:616, 1989; COPYRIGHT 1989, AAAS. S CIENCE varies from one stage of the cell cycle to another. BY MOSES KING , R EPRODUCED WITH PERMISSION OF AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE IN THE FORMAT Over the past two decades, a large number of laboratories REUSE IN A BOOK /TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .) have focused on MPF-like , called cyclin-dependent Chapter 14 Cellular Reproduction 576 ald TR,ocr i lt G late whic in occurs point, START, transition called first The 14.5. Figure in shown plified representation regulation of cell cycle in fi though in partnership with different cyclin mitment, sa the species, this poin boththrough passage responsiblefor is (cdc2) In cycle. cell complex least the other near the end of G cial event—either initiating replication or enterin cial event—either cell wherecycle a cell becomes committed to beginn primary stagesprimary of regulation occurs near the end of through its cell is cycle regulated at distinct sta supported the concept that the progression of a euk vertebrat different many as well as on search Subseq it was a cyclin-dependent kinase. other words, of subunit catalytic the to homologous be to found (and yeast fission cell cycle. The product of this , which was calle was which gene, this of product The cycle. cell of cells at elevated temperature to stop at certain laborat Both would cause the when mutated, yeast. identified a gene that, fission on working Oxford of sity ding yeast and subsequently that of Paul Nurse at the Univer-on working Washington of University the at Hartwell initially th began in the 1970s in two laboratories, low to grow cu a cell and reproduce in a laboratory activities major the of understanding comprehensive investigators ha and live-cell analyses, biochemical, gen combining By . including eukaryotes, higher genome the found—in usually aresought—and mologues cel in control involved has been identified in one gene of the two yeast a Once eukaryotes. of throughout conserved remarkably been has regulation o basis molecular The 14.6). Figure (see cells sized into splitting then and itself elongating by duces and the fission yeast,fission the and the formation of buds at one end of the cell (see F ding yeast yeast ding species yeast related distantly two on focused have ature of the to mutantstudy the effect gene produc to a higher (restrictive) temp temperature and then shifted (permissive)lower a at manner normal ca relatively a mutants in grown temperature-sensitive 549, page on processes cussed cycle cell various affect proteins normal of the availability of temperature-sensitive mutant least at cycle, cell the of studies in useful larly Yeast particula process cells involved have in cell division. a inhibiting or stimulating thereby cycle, durin point appropriate an at occurs event rylation by Each phosphorylating a diverse array of proteins. 1 restriction point cycle. cell the completing ultimately, and, it replicating to committed irrevocably is it START, continue through the remainder of the cell cycle wi in Afterthe cellthey cycle. have passed the restri quire the presence of growth factors in their cultu timately to completing Prior mitosis. to the restric Mammalian cells pass through a comparable point dur We whic will begin our discussion with fission yeast, Researchers studying the genetic control of the cel the of control genetic the studying Researchers Research into the genetic control of the cell cycle Saccharomyces cerevisiae Saccharomyces at which time, they become committed to DNA replica CDC28 Schizosaccharomyces pombeSchizosaccharomyces 2 These stages represent points in the. in yeast), was eventually was yeast), budding in , which reproduces throughreproduces which , 1 ne cl hs passed has cell a Once . to on,thesection same point, cells will re medium if they are to progress inpit mammaliantion point, cells re- thout external stimulation. ing G 1 asg throughPassage 1 referred to , as the e.One of theges. in part because part in , which repro- which , been particu- points in the ssion yeast is igure 1.18 at of Leland g mitosis. G s whose ab- pce,ho-species, ve gained a two equal- two lture dish. aryotic aryotic cell t. f cell cycle cell f ts of com-of ts e cells has cells e , the bud- the , phospho- ing a cru- g the cellthe g s dis- As . tion and ul- cellular r 1 .A sim-s. P;inMPF; d e Cdk me that al-that in yeast uent re- and an- s DN As growth cdc2 cyclel l cyclel bud- h has ories be n the etic, is h s ofs er- b in ), cyclins aecc iaeb ifrn lse fccis ei of cyclins, same cdc2 kinase by different classes junctures arrows) (black critical these two require ous cyclin–Cdk complexes indicate that cyclin bindi that cyclin complexes indicate ous cyclin–Cdk structu crystallographic X-ray site. enzyme’sactive conformation the in change major a causing Cdk, a of it binds to the cat cient concentration in the cell, reache cyclin a When time. over rise cyclins ticular Cyclin Binding vari These include:one another. a combinati in by operate that regulated “accelerators” and are “brakes” enzymes these of activities various its through cycle cell the drive that gines” page 592 in conjunction with other mitotic activiti d be will that event an 14.5), (Figure cyclins totic resultsthat fromconcentratio in plunge a activity 2 is also known as Cdk1.) ons START and the Gpoints, mitosis and entry mitosis into and Gentry triggered drop by a rapid of in mitot concentration A third occurs major transition at the end cyclins. a different group of cyclins—the plete cell division and reenter Greenter and division cell plete wil they whether determines which mitosis, of middle duri commitment third a make Cells mitosis. to phase somes and the that cytoskeleton shift characterize of both the in organization changes for the dynamic proteins are substrates the among cell Included the mitosis. for required are that substrates phorylate 61 page on described MPF (e.g., cyclin mitotic a ing START requires the activation of cdc2 by one or mor or one by cdc2 of activation STARTthe requires Cyclin-dependent kinases are often described as the as described often are kinases Cyclin-dependent PassagefromG , whose levels rise during late G during rise whose levels , cdc2 kinase Figure 14.5 in fission yeast. fission in G 1 cyclins G G 1 G START 1 /S 1 sidctdi iue1.,the As levels indicated of in par- Figure 14.5, /S cyclins 2 to mitosis requires activation of cdc2 byrequirescdc2 mitosis of to activation A simplified model for cell cycle regulationA simplified cycle model for cell 2 Mtasto.Passage through of a cell –M transition. The cell cycle is controlled primarily at two is controlled primarily cycle The cell M S 1 M S depends on a rapid decrease in Cdk 1 mitotic cyclins of the next cycle. Exit from Exit cycle. next the of cdc2 kinase cyclins G Mitotic G 2 1 2 (Figure 14.5).(Figure s the activation of the ther G cyclins Mitotic of mitosis and is cccis (ic cyclins. Cdks contain-. alytic alytic subunit 1 /S or mitotic n of the mi-the of n sae.Thestages. iscussed oniscussed from inter- es. res of vari-of res ng causes chromo- o enter to s a suffi-a s 1) phos-1) Note: required n withon e l com-l of theof ng theng t of ety G “en- cdc 1 / S 577 the movement of a flexible loop of the Cdk polypeptide chain other residue. In other words, the effect of Wee1 overrides the away from the opening of the active site, allowing the Cdk to effect of CAK, keeping the Cdk in an inactive state. Line 2 of phosphorylate its protein substrates. Figure 14.6 c shows the phenotype of cells with a mutant wee1 gene. These mutants cannot maintain the Cdk in an inactive Cdk Phosphorylation/dephosphorylation We have already seen state and divide at an early stage in the cell cycle producing in other chapters that many events that take place in a cell are smaller cells, hence the name “wee.” In normal (wild-type) regulated by the addition and removal of phosphate groups cells, Wee1 keeps the Cdk inactive until the end of G 2. Then, from proteins. The same is true for the events that lead to the at the end of G 2, the inhibitory phosphate at Tyr 15 is removed onset of mitosis. We can see from Figure 14.5 that the level of by the third enzyme, a phosphatase named Cdc25 (step 2, Fig- mitotic cyclins rises through S and G 2. The mitotic cyclins ure 14.6 b). Removal of this phosphate switches the stored cy- present in a yeast cell during this period bind to the Cdk to clin–Cdk into the active state, allowing it to form a cyclin–Cdk complex, but the complex shows little evi- phosphorylate key substrates and drive the yeast cell into mito- dence of kinase activity. Then, late in G 2, the cyclin–Cdk be- sis. Line 3 of Figure 14.6 c shows the phenotype of cells with a comes activated and mitosis is triggered. To understand this mutant cdc25 gene. These mutants cannot remove the in- change in Cdk activity, we have to look at the activity of three hibitory phosphate from the Cdk and cannot enter mitosis. other regulatory enzymes—two kinases and a phosphatase. We The balance between Wee1 kinase and Cdc25 phosphatase ac- will look briefly at the events that occur in fission yeast (Figure tivities, which normally determines whether the cell will re- 14.6 a). The roles of these enzymes in the fission yeast cycle, main in G 2 or progress into mitosis, is regulated by still other which is illustrated in Figure 14.6 b, was revealed through a kinases and phosphatases. As we will see shortly, these path- combination of genetic and biochemical analyses. In step 1, ways can stop the cell from entering mitosis under conditions one of the kinases, called CAK (Cdk-activating kinase), phos- that might lead to an abnormal cell division. phorylates a critical threonine residue (Thr 161 of cdc2 in Fig- ure 14.6 b). Phosphorylation of this residue is necessary, but not Cdk Inhibitors Cdk activity can be blocked by a variety of in- sufficient, for the Cdk to be active. A second protein kinase hibitors. In budding yeast, for example, a protein called Sic1 shown in step 1, called Wee1, phosphorylates a key tyrosine acts as a Cdk inhibitor during G 1. The degradation of Sic1 al- residue in the ATP-binding pocket of the enzyme (Tyr 15 of lows the cyclin–Cdk that is present in the cell to initiate DNA cdc2 in Figure 14.6 b). If this residue is phosphorylated, the en- replication. The role of Cdk inhibitors in mammalian cells is zyme is inactive, regardless of the phosphorylation state of any discussed on page 581.

1 2 Mitosis 3

Interphase (G2) Interphase (G1) Thr161– P Tyr15– P Thr161– P CAK Wee1 Cdc25 cdc2 cdc2 cdc2 cdc2 kinase kinase kinase kinase

G Post-mitotic fission Cyclin Cyclin 2 Cyclin Inactive Inactive yeast cell yeast cells Inactive Active Cyclin (b) (a) Degradation

Figure 14.6 Progression through the fission yeast cell cycle 1 Wild type requires the phosphorylation and dephosphorylation of critical G2 M cdc2 residues. (a) Colorized scanning electron micrograph of wild type – fission yeast cells. (b) During G 2, the cdc2 kinase interacts with a 2 wee1 mitotic cyclin but remains inactive as the result of phosphorylation of a G2 M key tyrosine residue (Tyr 15 in fission yeast) by Wee1 (step 1). A sepa- rate kinase, called CAK, transfers a phosphate to another residue (Thr – 161), which is required for cdc2 kinase activity later in the cell cycle. 3 cdc25 When the cell reaches a critical size, an enzyme called Cdc25 phosphatase is activated, which removes the inhibitory phosphate on the Tyr 15 residue. The resulting activation of the cdc2 kinase drives the cell (c) into mitosis (step 2). By the end of mitosis (step 3), the stimulatory phosphate group is removed from Thr 161 by another phosphatase. The mutant cdc25 gene; the cell does not divide but continues to grow. The free cyclin is subsequently degraded, and the cell begins another cycle. red arrow marks the time when the temperature is raised to inactivate 14.1 (The mitotic Cdk in mammalian cells is phosphorylated and the mutant protein. ( A: S TEVE GSCHMEISSNER /P HOTO dephosphorylated in a similar manner.) ( c) Identification of Wee1 kinase RESEARCHERS , I NC . B: A FTER T. R. C OLEMAN AND W. G. D UNPHY , Cycle Cell The and Cdc25 phosphatase was made by studying mutants that behaved as CURR . O PIN . C ELL BIOL . 76:877, 1994. C URRENT OPINION IN CELL shown in this figure. Line 1 shows the G 2 and M stages of a wild-type BIOLOGY BY ELSEVIER LTD . R EPRODUCED WITH PERMISSION OF cell. Line 2 shows the effect of a mutant wee1 gene; the cell divides ELSEVIER LTD . IN THE FORMAT REUSE IN A BOOK /TEXTBOOK VIA prematurely, forming small (wee) cells. Line 3 shows the effect of a COPYRIGHT CLEARANCE CENTER .) Chapter 14 Cellular Reproduction 578 pcfi,ad ny eti cmiain ae on (Fi found are combinations 14.8 certain only and specific, a cyclins individual between pairing The cycle. cell points different at substrates of groups complexes different cyclin–Cdk Different kinase. protein this versi different several produce cells mammalian Cdk, have which cells, yeast Unlike next. the to stage one ce mammalian driving in role key a play cyclins ent differ-of degradation and synthesis of waves successive yeast, among conserved remarkably are cycle cell cont that processes and proteins the above, noted As cells fail to initiattion of the cyclin is blocked, if nuclear Regardless ac of the machinery. mechanism, nuclea the ofcomponentsactivating and phorylating cyclin proposal, Cdk1 alternate stimulates it own an translocation into to the nucle According cytoplasm. phorylation blocks subsequent export of themodel, cyclin this In 492). page (NES, signal export nuclear res that residues serine more or one of phorylation nuclear accumulation of cyclin B1 isposal, facilita to According 14.7). (Figure mitosis of onset the to ln –d2 ope die te el no pae wh of activity B1–Cdk1 a complex cyclin (the mammalian phase, S into cell the drives complex E–Cdk2 clin ioi (e Fgr 14.26 Figure (see mitosis G ysis of key proteins, such as G suchas proteins, keyofysis Mutationscell cycle. that inhibit SCFs from mediati Cdks)that phorylatedtheproteinby kinases (i.e., Theseproteins become targets for an SCF after they complexes)functionasthat requires two classes of multisubunit complexes (SCF Regulationof singledirection. a in cyclecell the Cdk degradationactivity, is an irreversible event t Unlikemechanismsotherthat 541. pagedescribedon ubiquitin–proteasomethe ofaccomplishedmeans p by the at different points in the cell cycle. justingboth the rate ofsynthesis and the rate ofd prot cycle cell keyother and cyclins, centrationof leads to changes in Cellsthe activity regu of Cdks. that cyclin concentrations oscillate during each ce ControlledProteolysis esm.The teasome. SCF complex is active from late Gdestructionensurestheir which polyubiquitinchain, recognizedegradedtheseprproteinslinkbeand to toplasm until G in animal cells (cyclin B1) shuttles between the nu majormitottheof one Forexample, differentstages. regulatorscellcycle aremoved intodifferent comp in phenomenon dynamic a is localization Subcellular separatedproteinsintthefromtheyorunitedwith eith can molecules regulatory which in compartments LocalizationSubcellular to exit mitosis and enter a new cell cycle (page 59 Destructionmitotictheofcyclins mitoticcyclins. inclu proteins, mitotickey ofnumber degrades a and complexAPCm Theactsin replicatingand theirDNA. canprevent Sic1mentionedcells from above, enterin 1 S yln,Ck niios n ohr el yl proteins. cycle cell other and inhibitors, Cdk cyclins, /S a ). In mammalian cells, for example, the activity of a of activity the example, for cells, mammalian In ). 2 when it accumulates in , the nucleus just prior Itis evident from Figures 14.4 and 14.5 Cells contain a number of differentof number a contain Cells a ad eits h dsrcin of destruction the mediates and ) ubiquitinligases 1 /S cyclins or the Cdk inhibitorCdkthecyclinsor /S . These complexes These . e cell division. 1 Degradation is hat helps drive lcce whichll cycle, 2). through early the cell cyclecellthe cleus and cy- ted by phos- estructionof late the con- allowscella eins, by ad- by eins, regulatethe artmentsat us by phos- back to the ng proteol- nd Cdks isCdks nd ihn thewithin eract with.eract oteins to aoteinsto and APC are phos- in a pro-a in gSphase d i its in ide ic cyclinsic n pro-one r importr a singlea ig theding cumula- l fromlls s. As in As s. which control athway MPF) o therol n ofons target phos- r be er B1- itosis ereas gure cy- yln 1Ck atvt drn G during activity B1–Cdk1 cyclin but can also inhibit inappropriate tivities, events. sti always not do Cdks mitosis. into cell the drives prising results (Figure 14.8 withso teinshavebeenreexamined knockoutin mice, the of roles the years, few past the Over decades. two chemical studies carried out on mammalian cells for iue 14.8Figure the genome once is replicatedper cel once and only This helps ensure the that cell cycle ea (page 560). replicated been already has that DNA rereplicating ls.The micrograph in plasm. almost B1 is localized labeledfluorescently cyclin this change in localization is discussed in the tex n in the cell B1 is concentrated the labeled cyclin AND YPRISO FROM PERMISSION BY shown The cell in 273). B1 linked to the green fluorescinjected with cyclin cycle. cell the during Figure 14.7 (a) (Figure 14.8(Figure survive not does animal the although organs, formed and6) is capable of developing to a stage 4, Cdks2, encoding the mouse a embryo Incontrast, fornormala cellcycle. ar genesthese by encodedproteinsthe thatgesting die as early embr or cyclin cyclinsA2, E1 and E2, B1, Cdk1, synthesize to unable are that Mice eliminated. particularknockout mouse depends onthe gene that stage of the cell cycle. This is a classical case of caseclassical a is This cycle. cellthe stageof phosphoryla are substratesrequired the of all that absence their for “cover” to able is Cdk1 cycle, cell mamm the during times specific at expressed normally though even theother other In words, thecellcycle. required the is Cdk1 yeast, in as that, indicatesfinding This though more slowlyproliferating than no in culture, h bd.I adto,cci D-ecet nml dis D1-deficient cyclin addition, In body. the stemsfromreduction a theinlevelcellof divisio are smaller for example, thancyclin control D1, anim at leastmalities, in certain types Mice of la cells. cyclins or Cdkstial” typically results in distinct theabsence of one of these Still, normallyperform. which a protein is able toout carry functions that The roles of the various cyclin–Cdk complexes showncomplexescyclin–Cdk various the of roles The J ON A THAN to drive a mammalianadrive tothroughcellstages the of all a Experimental demonstration of subcellular localizat of subcellular demonstration Experimental b ). Cells taken from such embryos are capable ofcapableare embryos such fromtaken Cells ). have been determined by a wide range of bio-of range wide a by determined been have P INES Micrographs of a living HeLa cell that has been cell Micrographs of a living HeLa M N, all a ACMILLAN is in the G b ATURE of the other cell cycle Cdks (namely,Cdks cycle cell other the of hw h eli rpaeo ioi,andshows in prophase the cell of mitosis, b .A xetd the phenotype of a As expected, ). C ELL 2 P hs fiscl yl,and thephase cycle, of its cell UBLISHERS (b) B IOL 2 rvns cl from cell a prevents :3 99 R 1999. 1:83, . .(Ft. ces The basis forucleus. entirely in the cyto-entirely ent protein (page L ROM IMITED cell cycle abnor- cking a gene for

redundancy n throughoutn For example, it would not P ch ch region of ted at eachat ted AUL “nonessen- more than mulate ac-mulate with fully e essentiale rmal cells. l cycle. ensuring, l,whichals, Cdksare thatlacks only Cdkonly .) EPRINTED earlier in earlier o,sug-yos, hasbeen me sur-me to birthto e pro-se C ly aplay cyclin LUTE alian in, ion in of Decreased 579 No cell division haematopoietic precursors and (two-cell embryo) Decreased cardiomyocytes haematopoietic precursors Cyclin Decreased –/– B/A + Cdk1 M Cdk1 cardiomyocytes Cdk2+/+ G Stop +/+ 2 Cdk4 +/+ +/+ Cdk1 Cdk6 –/– Cdk2 Male and Stop –/– Cdk4 sterility G1 Cdk6 –/– Cdk1+/+ Cdk2+/+ Cyclin Stop Cdk4 –/– S D's + Cdk4 Cdk6 –/– Cdk1+/+ Cyclin Cdk2–/– Cdk6 Stop –/– A + Cdk2 Cdk4 Cdk6+/+ Cdk1+/+ Cdk2 –/– Cdk4+/+ Cdk6 –/– Cyclin E + Cdk2 E1.5 E12.5 E16.5 PO (a) (b) Figure 14.8 Cyclin-Cdks in the mammalian cell cycle. that indicated in Figure 14.6.) (b) The effects on mouse development of (a) Combinations between various cyclins and cyclin-dependent the deletion of genes (shown in red) encoding various Cdks. Of the four kinases at different stages in the mammalian cell cycle. Cdk activity dur- primary mammalian Cdks, only Cdk1 is absolutely required for cell di- ing early G 1 is very low, which promotes the formation of prereplication vision. Embryos that express only Cdk1 die during the course of embry- complexes at the origins of replication (see Figure 13.20). By mid-G 1, onic development. Mice expressing both Cdk1 and Cdk4 develop into Cdk activity is evident due to the association of Cdk4 and Cdk6 with that are sterile, owing to defects in the meiotic cell cycles. E, the D-type cyclins (D1, D2, and D3). Among the substrates for these embryonic day number; P, postnatal day number. (A: C. G. S HERR , C ELL Cdks is an important regulatory protein called pRb (Section 16.3, Fig- 73:1060, 1993; C ELL BY CELL PRESS . R EPRODUCED WITH PERMISSION ure 16.12). The phosphorylation of pRb leads to the transcription of a OF CELL PRESS IN THE FORMAT REUSE IN A BOOK / TEXTBOOK VIA number of genes, including those that code for cyclins E and A, Cdk1, COPYRIGHT CLEARANCE CENTER . H. A. C OLLER , N ATURE REVS . M OL . and proteins involved in replication. The G 1–S transition, which CELL BIOL . 8:667, 2007. N ATURE REVIEWS MOLECULAR includes the initiation of replication, is driven by the activity of the cy- BY NATURE PUBLISHING GROUP . R EPRODUCED WITH PERMISSION OF clin E–Cdk2 and cyclin A–Cdk2 complexes. The transition from G 2 to NATURE PUBLISHING GROUP IN THE FORMAT REUSE IN A BOOK / M and passage through early M is driven by the sequential activity of TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER . B: M ALUMBERS AND cyclin A–Cdk1 and cyclin B1–Cdk1 complexes, which phosphorylate BARBACID , N AT REVS CANCER 9, 160, 2009 F IGURE 2. N ATURE such diverse substrates as cytoskeletal proteins, histones, and proteins of REVIEWS CANCER BY NATURE PUBLISHING GROUP . R EPRODUCED WITH the nuclear envelope. (The mammalian Cdk1 kinase is equivalent to the PERMISSION OF NATURE PUBLISHING GROUP IN THE FORMAT REUSE IN fission yeast cdc2 kinase, and its inhibition and activation are similar to A BOOK /TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .)

particular lack of cell proliferation during development of the normal cell is irradiated during the G 1 phase of the cell cycle, retina. Mice lacking Cdk4 develop without insulin-producing it delays progression into S phase. Similarly, cells irradiated in cells in their pancreas. Mice lacking Cdk2 appear to develop S phase delay further DNA synthesis, whereas cells irradiated normally but exhibit specific defects during meiosis (Figure in G 2 delay entry into mitosis. 14.8 b), which reinforces the important differences in the regu- Studies of this type carried out in yeast gave rise to a con- lation of mitotic and meiotic divisions. cept, formulated by Leland Hartwell and Ted Weinert in 1988, that cells possess checkpoints as part of their cell cycle. Checkpoints, Cdk Inhibitors, and Cellular Responses Checkpoints are surveillance mechanisms that halt the Ataxia-telangiectasia (AT ) is an inherited recessive disorder progress of the cell cycle if (1) any of the chromosomal DNA characterized by a host of diverse symptoms, including a is damaged, or (2) certain critical processes, such as DNA 2 greatly increased risk for certain types of cancer. During the replication during S phase or chromosome alignment during late 1960s—following the of several individuals under- M phase, have not been properly completed. Checkpoints en- going radiation therapy—it was discovered that patients with sure that each of the various events that make up the cell cycle AT are extremely sensitive to ionizing radiation (page 567). occurs accurately and in the proper . Many of the proteins So too are cells from these patients, which lack a crucial pro- of the checkpoint machinery have no role in normal cell cycle tective response found in normal cells. When normal cells are events and are only called into action when an abnormality ap- subjected to treatments that damage DNA, such as ionizing pears. In fact, the genes encoding several checkpoint proteins 14.1 radiation or DNA-altering drugs, their progress through the were first identified in mutant yeast cells that continued their cell cycle stops while the damage is repaired. If, for example, a progress through the cell cycle, despite suffering DNA damage h Cl Cycle Cell The or other abnormalities that caused serious defects. 2 Other symptoms of the disease include unsteady posture (ataxia) resulting from Checkpoints are activated throughout the cell cycle by a degeneration of nerve cells in the cerebellum, permanently dilated blood vessels (telangiectasia) in the face and elsewhere, susceptibility to infection, and cells system of sensors that recognize DNA damage or cellular ab- with an abnormally high number of chromosome aberrations. The basis for the normalities. If a sensor detects the presence of a defect, it trig- first two symptoms has yet to be determined. gers a response that temporarily arrests further cell cycle Chapter 14 Cellular Reproduction 580 1. in response to DNA damage. pathways available to mammalian cells to well- arrest the two examine briefly will We next? the to cycle that participate in cell cycle checkpoints and DNA r p of remarkablevariety a phosphorylate ATRcan and ATMbound, Once DNA. damagedcontainschromatin that a ATMATR aremultiproteinofpart Both complexes capableb of irradiation. UV or DNA replicated pletely frresultingthose including lesions, of types other as breaks DNA by activated also isATR called nase Arelated causingATMp cell cycle arrest. molecules, large-scalea rapid, cause sufficientto moleculesis c the of one in breaksingle a ofpresence the ably, particularly double-strandedsions, breaks (page 567 certaactivatedby is proteinthatkinase encodesa 2 . Thegene responsible for ataxia-telangiectasia (the d to path this of example another provides response portance in cell and molecular Thebiology. cell’s D study of a rare human disease has led to a discover arrest (known cycle as se cell a state of permanent leads either to (1) the orof (2)the it cell sign a transmit can mechanism checkpoint the repair, If transformed the into DNAa cancer is cell. damage of risk the run damage genetic with division dergo This is especially important because mammalian cell the to continuing than rather defect thecorrect or The then cell can use the delay to repair progress. ope emdMN(tpa iue1.) MRN can be Figure 14.9). complex termed MRN (step a, radiation as serve sites for the recruitment of a p the breaks in DNA thatresponse, are caused by ioniz In this particular DNA-damagecheckpoint mechanism. ATMstrates isand involved from i entering S phase. This prevents the cells from phosphorylating key su a key rolea key in the G pla Cdc25 normally As discussed on page 577, nucleus. it from and prevents being into reimported activity This inhibits interaction Cdc25’s phosp 5). (steps 4, adaptor protein that binds to Cdc25 in the cytoplas making the Cdc25 molecule a target for a special 3), residue serine Cdc25 on a particular phosphorylates in t which Chk1 (step 2), called kinase, a checkpoint ATR and activa phosphorylates paired 13.25). (Figure such as those present as UV-damaged DNA is re-14.9), Figur DNA single-stranded (step 1, of protein-coated, ATR kinase molecules are thought toto be recruited ito,ATR kinase arrest is activated and the cell diation, preparing to enter mitosis is subjected Ift a cell tion in G cells exposed to ionizin For example, the cell cycle. that directly inhibit the cyclin–Cdk complex that d Damage to DNA also leads to the synthesis of protei of 21 kDa) that inhibits the kinase activity of the and the cell arrested cell the and Gin leaves the Cdk in an inactive state (step 6)the nucleus the absence of Cdc25 from Thus, phosphates from Cdk1. How does a cell stop its progress from one stage of We have seen in numerous places in this text where text this in placesnumerous in seen haveWe 1 synthesize a protein called p21 (molecular mass 2 /M transition by removing/M transition inhibitory 2 . s conversion to y of basic im-

in DNA le-DNAin NA damage nescence). the damage ATM ctivationof m incom-om next stage.next ir cell cycle o UV irra- .Remark-). rotein epair. l’ DNAell’s G becoming s that un- roteinki- d beyond indingto g radia- iscovery. s in G rives the cell the studied 1 b- el aswell m roteins hatase al thatal (step ns n this ing Cdk. urn sites gene) tes e the ys nd 2 . G st phosphorylated leaves the Cdk in its inactivated, into th be reimported (step 5) and cannot cytoplasm Cdc25 is bound by an adaptor pr Once phosphorylated, and cyt the nucleus shuttles between normally which and inactivates the phosphatas phosphorylates which ATR phosphorylates and activates the checkpoint kin ur pn Cell Biol. Opin. Curr. mediating the response to DNA damage but are not di and histone variants chromatin remodeling complexes, histone-modi including Many other proteins, ). (step f p21 direct (step e), and translated Once transcribed to activate enhancing its ability protein, by Ch but phosphorylation short-lived, very normally p53 phosphorylates which point kinase Chk2 (step b), eardatrvrostpso aae In the G of damage. types repaired various after when replicationforms forks or the become stalled becomes activated by protein-coated ssDN other hand, arewhich detected by the MRN protein complex (step ATM becomes activated in response to double- arrest. th signaling pathways teins acts through checkpoint Eac of DNAactivated following specific types damage. damage checkpoints. 2009; Figure 14.9 1 aha hw ee ATM and activates phosphorylates pathway shown here, turn phosphorylates a transcription factor (p53) (s Chk2other i checkpoint kinase called Chk2 (step b). which phosphorylates and activatesactivates ATM, an considered MRN recruits as aa sensor of DNA breaks. 5 Nature Cell Biol. Inactive complex ssDNA-protein (14–3–3 Adaptor radiation Ultraviolet protein Cdc25 P Chk1 Cdc25 Models for the mechanism of action of two DNA- two of action of mechanism the for Models σ Cytoplasm ) 4 Active 3 2 ATR Cdc25 125 2009; 21:245, G ATM and ATR are protein kinases that become 316,21;and 2011; 13:1161, CELL CYCLE P 2 Chk1 ARREST Inactive 1 Cdk P Nucleus 6 P Nature Revs. Mol. Cell Biol. Cell Mol. Nature Revs. mRNA P p21 P P p53 Chk2 p53 p21 Genes Develop. d transcription (step d).transcription p21 Stable a c Active ATM G 2 p53 gene 1 pathway shown here, p21 t se ) In theate (step 6). e DNA is being ly inhibits the Cdkly CYCLE ARREST CELL at lead to cell cycleat lead to cell b ulu,whiche nucleus, Unstable oplasm (step 4). e Cdc25 (step 3), ase Chk1 (step 2), k2 stabilizes thek2 stabilizes strand breaks,strand fying enzymes,fying (tpc.p53 is (step c). are in involved Active a.AR on the ATR, a). MRN complex scussed (see A (step 1) that h of these pro- Chk2 otein in the Cdk radiation Ionizing 549 2011.25:409, f DNA the check- Inactive Inactive tep c), Cdk p21 10:243, n - nd 581

(a) (b) (c) Figure 14.10 p27: a Cdk inhibitor that arrests cell cycle from a normal (left) and a p27-/- mouse (right). The gland from the progression. (a) Three-dimensional of a complex between p27 knockout mouse is much larger owing to an increased number of p27 and cyclin A–Cdk2. Interaction with p27 alters the conformation cells. ( A: F ROM ALICIA A. R USSO ET AL ., N ATURE 382:327, 1996, of the Cdk catalytic subunit, inhibiting its protein kinase activity. ( b) A FIG . 2 A. COURTESY OF NIKOLA PAVLETICH , H OWARD HUGHES pair of littermates at 12 weeks of age. In addition to possessing differ- MEDICAL INSTITUTE ; REPRINTED BY PERMISSION OF MACMILLAN ent genes for coat color, the mouse with dark fur has been genetically PUBLISHERS LIMITED ; B,C: FROM KEIKO NAKAYAMA ET AL ., -/- engineered to lack both copies of the p27 gene (denoted as p27 ), COURTESY OF KEI -ICHI NAKAYAMA , C ELL 85:710, 711, 1996; WITH which accounts for its larger size. ( c) Comparison of the thymus glands PERMISSION FROM ELSEVIER .)

which leads to the transcription and translation of the p21 2. Describe how [ 3H]thymidine and autoradiography can gene (steps d and e) and subsequent inhibition of Cdk be used to determine the length of the various periods (step f). Approximately 50 percent of all human tumors of the cell cycle. show evidence of in the gene that encodes p53, 3. What is the effect of fusing a G 1-phase cell with one in which reflects its importance in the control of . M; of fusing a G 2- or S-phase cell with one in M? The role of p53 is discussed at length in Chapter 16. 4. How does the activity of MPF vary throughout the cell p21 is only one of at least seven known Cdk inhibitors. cycle? How is this correlated with the concentration of The interaction between a related Cdk inhibitor (p27) and cyclins? How does the cyclin concentration affect MPF one of the cyclin–Cdk complexes is shown in Figure 14.10 a. activity? In this structural model, the p27 molecule drapes itself across 5. What are the respective roles of CAK, Wee1, and both subunits of the cyclin A–Cdk2 complex, changing the Cdc25 in controlling Cdk activity in fission yeast cells? conformation of the catalytic subunit and inhibiting its kinase What is the effect of mutations in the wee1 or cdc25 activity. In many cells, p27 must be phosphorylated and then genes in these cells? degraded before progression into S phase can occur. 6. What is meant by a cell cycle checkpoint? What is its Cdk inhibitors, such as p21 and p27, are also active in cell importance? How does a cell stop its progress at one of differentiation. Just before cells begin to differentiate— these checkpoints? whether into muscle cells, liver cells, blood cells, or some other type—they typically withdraw from the cell cycle and stop di- viding. Cdk inhibitors are thought to either allow or directly induce cell cycle withdrawal. Just as the functions of specific Cdks and cyclins have been studied in knockout mice, so too 14.2 | M Phase: Mitosis have their inhibitors. Knockout mice that lack the gene

p27 14.2 show a distinctive phenotype: they are larger than normal and Cytokinesis (Figure 14.10 b), and certain organs, such as the thymus gland Whereas our understanding of cell cycle Cytokinesisand Mitosis Phase: M and spleen, contain a significantly greater number of cells than regulation rests largely on genetic studies in those of a normal animal (Figure 14.10 c). In normal mice, the yeast, our knowledge of M phase is based cells of these particular organs synthesize relatively high levels on more than a century of microscopic and biochemical research of p27, and it is presumed that the absence of this protein in on animals and .The name “mitosis” comes from the Greek the p27-deficient animals allows the cells to divide several word mitos , meaning “thread.” The name was coined in 1882 by more times before they differentiate. the German Walther Flemming to describe the thread- like chromosomes that mysteriously appeared in animal cells just R E V I E W before they divided in two. The beauty and precision of cell di- 1. What is the cell cycle? What are the stages of the cell vision is best appreciated by watching a time-lapse video of the cycle? How does the cell cycle vary among different process (e.g., www.bio.unc.edu/faculty/salmon/lab/mitosis/ types of cells? mitosismovies.html) rather than reading about it in a textbook. Chapter 14 Cellular Reproduction 582 (M Figure 14.11 5. Daughtercellsformedbycytokinesis. 4. GolgicomplexandERreforms. chromosome clusters. 3. Nuclearenvelopeassemblesaround 2. Chromosomes becomedispersed. spindlepoles. 1. Chromosomes clusteratopposite apart.3. Spindlepolesmovefarther poles. 2. Chromosomes movetooppositespindle separate. 1. Centromeres split,andchromatids microtubules tobothpoles. metaphaseplate,attachedbychromosomal 1. Chromosomes are alignedalong spindleequator. 2. Chromosomes are movedto kinetochores ofchromosomes. 1. Chromosomal microtubules attachto Nuclearenvelopedisperses. 3. GolgicomplexandERfragment. spindleisassembled. 2. Cytoskeletonisdisassembled,andmitotic centromere. oftwochromatids attachedtogetheratthe Chromosomes are seentobecomposed formcompactmitoticchromosomes. 1. Chromosomal materialcondensesto CORPSCUTS OF COURTESY ICROGRAPHS The stages of mitosis in an animal cell (left drawi stages of (left mitosis in an animal cell The A NDREW B AJER .) Prometaphase Metaphase Telophase Anaphase Prophase ngs) and a plant cell (right photos). (right ngs) and a plant cell 583 Mitosis is a process of nuclear division in which the repli- cated DNA molecules of each chromosome are faithfully seg- regated into two nuclei. Mitosis is usually accompanied by cytokinesis , a process by which a dividing cell splits in two, partitioning the cytoplasm into two cellular packages. The two daughter cells resulting from mitosis and cytokinesis pos- sess a genetic content identical to each other and to the mother cell from which they arose. Mitosis, therefore, main- tains the chromosome number and generates new cells for the growth and maintenance of an organism. Mitosis can take place in either haploid or diploid cells. Haploid mitotic cells are found in fungi, plant gametophytes, and a few animals (in- cluding male known as drones). Mitosis is a stage of the cell cycle when the cell devotes virtually all of its to a single activity—chromosome segregation. As a result, most metabolic activities of the cell, including transcription and translation, are curtailed during mitosis, and the cell becomes relatively unresponsive to external stimuli. We have seen in previous chapters how much can be Figure 14.12 learned about the factors responsible for a particular process Prophase nuclear morphology. Light-optical section through two mouse cell nuclei in prophase, recorded with super- by studying that process outside of a living cell (page 276). resolution 3D-structured illumination microscopy (3D-SIM). Our understanding of the of mitosis has been Condensed chromosomes are shown in red, the nuclear envelope in greatly aided by the use of extracts prepared from frog eggs. blue and microtubules, in green. Scale bar is 5 ␮m. (C OURTESY OF These extracts contain stockpiles of all the materials (his- LOTHAR SCHERMELLEH .) tones, tubulin, etc.) necessary to support mitosis. When chromatin or whole nuclei are added to the egg extract, the chromatin is compacted into mitotic chromosomes, which are segregated by a mitotic spindle that assembles sponta- Although there is debate on this issue, mitotic chromosomes neously within the cell-free mixture. In many experiments, are thought to be composed of similar types of fibers as seen the role of a particular protein in mitosis can be studied by re- by electron microscopic examination of whole chromosomes moving that protein from the egg extract by addition of an isolated from mitotic cells (Figure 14.13 a). According to this antibody (immunodepletion) and determining whether the viewpoint, chromosome compaction does not alter the nature process can continue in the absence of that substance (see of the chromatin fiber, but rather the way that the chromatin Figure 14.21 for an example). fiber is packaged. Treatment of mitotic chromosomes with so- Mitosis is generally divided into five stages (Figure 14.11), lutions that solubilize the histones and the majority of the prophase, prometaphase, metaphase, anaphase , and telophase , each nonhistone proteins reveals a structural framework or scaffold characterized by a particular series of events. Keep in mind that that retains the basic shape of the intact chromosome (Figure each of these stages represents a segment of a continuous 14.13b). Loops of DNA are attached at their base to the non- process; the division of mitosis into arbitrary phases is done histone proteins that make up this chromosome scaffold only for the sake of discussion and experimentation. (shown at higher magnification in Figure 12.15). Research on chromosome compaction has focused on an abundant multiprotein complex called . The proteins Prophase condensin of condensin were discovered by incubating nuclei in frog egg During the first stage of mitosis, that of prophase , the dupli- extracts and identifying those proteins that associated with cated chromosomes are prepared for segregation and the mi- the chromosomes as they underwent compaction. Removal of 14.2 totic machinery is assembled. condensin from the extracts prevented normal chromosome

compaction. How is condensin involved in such dramatic Cytokinesisand Mitosis Phase: M Formation of the Mitotic Chromosome The nucleus of changes in chromatin architecture? There is very little data an interphase cell contains tremendous lengths of chromatin available from in vivo studies to answer this question, but fibers. The extended state of interphase chromatin is ideally there is considerable speculation. suited for the processes of transcription and replication but Supercoiled DNA occupies a much smaller volume than not for segregation into two daughter cells. Before segregating relaxed DNA (see Figure 10.12), and studies suggest that its chromosomes, a cell converts them into much shorter, DNA supercoiling plays a key role in compacting a chromatin thicker by a remarkable process of chromosome fiber into the tiny volume occupied by a mitotic chromosome. compaction (or chromosome condensation ), which occurs dur- In the presence of a topoisomerase and ATP,condensin is able ing early prophase (Figures 14.11 and 14.12). to bind to DNA in vitro and curl the DNA into positively su- As described on page 496, the chromatin of an interphase percoiled loops. This finding fits nicely with the observation cell is organized into fibers approximately 30 nm in diameter. that chromosome compaction at prophase requires topoiso- Chapter 14 Cellular Reproduction 584 which is similar to that found in interphase chromo is similar towhich that found in interphase is seenstructure to be composed of a knobby fiber 3 a whole-mount preparation of a human mitotic chromo W 14.13 (Figure scaffold chromosome mitotic which along with merase condensin II, is present as L BF. Figure 14.13 r hw oeceryi iue1.5.( 12.15). in Figure are shown more clearly from loops of DNA which scaffold are seen to emerge The residual pro histone proteins have been removed. the histonesance of a and mitotic chromosome after model for condensin action is shown in Figure 14.14 Figure in shown is action condensin for model AEMMLI (b) ASHINGTON (a) AHR A, C, RMED ELL The mitotic chromosome. mitotic The D.C.;, 280 1977, 12:820, F OR CE S B Scaffold : FROM I SIUEOFNSTITUTE IHPRISO FROM PERMISSION WITH J AMES .PR. A ( P C: a USNANDAULSON ATHOLOGY ) Electron micrograph of UTS OFOURTESY oe.(somes. 0 nm in diameter, b most of the non- ). A speculative A ). teins form ateins form (the DNA loops oe Thesome. , U E part part of the b G LSEVIER LRICH ) Appear- UNTHE R DNA Con-. K. .) 14.13 previous interphase. opsdo w irriae “sister”mirror-image, two of composed them of each reveals chromosomes mitotic of nation C structures. rod-like distinct, as appear cell totic continuously throughcontinuously Gchromatids sister two the holds cohesin replication, ecule is shown in the right inset of Figure 14.14.ecule is shown inset of Figure in the right The subunit structure activities. of a V-shaped cond trigger to able are Cdks which through targets the tiprotein complex called called complex tiprotein w length its along sites at associated becomes some driving cells from Gfrom cells respon driving cyclin–Cdk the by subunits its of several of phos by mitosis of onset the at activated is densin densin also have two or threedensin also have two non-SMC subunits that Coh where the N- and come C-termini together. coiled coil with an ATP-bind elongated antiparallel, on itself t folds back of the SMC polypeptides Each Both complexes are built around a pair o spectively. subunit of structure an individual cohesin and cond and rig The top left into a mitotic chromosome fiber. wh the supercoiled loops into larger coils, organize condensin mol between interactions that cooperative It is proposed (but not sho ter chromatids together. Cohesin molecules would continue to hold chromatin. aroundcompaction supercoiled a ring by forming loo about condensin brings In this model, of the drawing. as shown i aided by condensin molecules, would begin, the entered As the cell mitosis, of the drawing. left the sister DNAhesin molecules that encircled helic helices of a pair of sister chromatids would be hel chromosomes. mitotic of formation ring-like structure of these structure proteins.ring-like Figure 14.14 Smc3 ro orpiain the DNAPrior of to each replication, interphase chr As the result of compaction, the chromosomes of a mi a of chromosomes the compaction, of result the As a nepaeProphase Interphase .Sse crmtd ae rsl o rpiain in replication of result a are chromatids Sister ). Cohesin Scc3 Condensin Model for the roles of condensin and cohesin in the Scc1 Smc1 Cohesin 2 into mitosis. Thus condensin is one ofone is condensin Thus mitosis. into 2 and into mitosis when they are ulti- cohesin utatrrpiain the DNA replication, Just after Fgr 1.4.Following 14.14). (Figure chromatids d in association by co- ich areich then folded compaction process wn in this drawing), f SMC subunits. ni ope,re-ensin complex, s as shown at thees, o form a highlyo form ing globular Smc4 ecules would then ht insets show the ps of DNA within chromosome complete the n the right partn the right the DNA of sis- Condensin esin and con- phorylation lose exami-lose ensin mol- ith a mul-a ith together cell cycle cell il forsible (Figure Smc2 omo- o be to the - 585

(a) (b)

Figure 14.15 Each mitotic chromosome is comprised of a pair of human cell. The DNA is stained blue, the kinetochores are green, and sister chromatids connected to one another by the protein complex cohesin is red. At this stage of mitosis, cohesin has been lost from the cohesin. (a) Colorized scanning electron micrograph of several arms of the sister chromatids but remains concentrated at the centromeres metaphase chromosomes showing the paired identical chromatids asso- where the two sisters are tightly joined. ( A: A NDREW SYRED /P HOTO ciated loosely along their length and joined tightly at the centromere. RESEARCHERS , I NC .; B: B Y S. H AUF AND JAN -M ICHAEL PETERS , The chromatids are not split apart from one another until anaphase. NATURE CELL BIOL . 3: E17, 2001 FIG . 1 C. R EPRINTED BY (b) Fluorescence micrograph of a metaphase chromosome in a cultured PERMISSION FROM MACMILLAN PUBLISHERS LIMITED .)

mately separated. As indicated in the insets of Figure 14.14, mitotic chromosome reveals the presence of a proteinaceous, condensin and cohesin have a similar structural organization. button-like structure, called the kinetochore , at the outer sur- A number of experiments support the hypothesis that the co- face of the centromere of each chromatid (Figure 14.16 a,b). hesin ring encircles two sister DNA molecules as shown in Most of the proteins that make up the kinetochore assemble both the left and right portions of Figure 14.14. at the centromere at early prophase. Kinetochore proteins are In , cohesin is released from the chromosomes thought to be recruited to the centromere because of the pres- in two distinct stages. Most of the cohesin dissociates from ence there of the novel nucleosomes containing the histone the arms of the chromosomes as they become compacted dur- variant CENP-A (page 509). As will be apparent shortly, the ing prophase. Dissociation is induced by phosphorylation of kinetochore functions as (1) the site of attachment of the cohesin subunits by two important mitotic enzymes called chromosome to the dynamic microtubules of the mitotic spin- Polo-like kinase and Aurora B kinase. In the wake of this dle (as in Figure 14.30), (2) the residence of several motor event, the chromatids of each mitotic chromosome are held proteins involved in chromosome motility (Figure 14.16 c), relatively loosely along their extended arms, but much more and (3) a key component in the signaling pathway of an im- tightly at their centromeres (Figure 14.13 a and Figure 14.15). portant mitotic checkpoint (see Figure 14.31).

Cohesin remains at the centromeres because of the presence A question of great interest to scientists studying kineto- 14.2 there of a phosphatase that removes any phosphate groups chores is how these structures are able to maintain their at- added to the protein by the kinases. Release of cohesin from tachment to microtubules that are continually growing and Cytokinesisand Mitosis Phase: M the centromeres is normally delayed until anaphase as de- shrinking at their plus end. To maintain this type of “floating scribed on page 592. If the phosphatase is experimentally in- grip,” the coupler would have to move with the end of the mi- activated, sister chromatids separate from one another crotubule as subunits were added or removed. Figure 14.16 c prematurely prior to anaphase. depicts two types of proteins that have been implicated as possible linkers of a kinetochore to dynamic microtubules, Centromeres and Kinetochores The most notable landmark on namely, motor proteins and a rod-shaped protein complex a mitotic chromosome is an indentation or primary constric- called Ndc80. Ndc80 is an essential kinetochore component tion , which marks the position of the centromere (Figure that forms fibrils that appear to reach out and bind the surface 14.13a). The centromere is the residence of highly repeated of the adjacent microtubule. Cells lacking any of the four pro- DNA sequences (see Figure 10.19) that serve as the binding teins that make up the Ndc80 complex exhibit severe spindle sites for specific proteins. Examination of sections through a attachment defects. Chapter 14 Cellular Reproduction 586 ope,micro-sized machine called complex, the componen as reassembly their for preparation in bly microtubulescytoskeletontheofundergo sweeping d cert with the cell cycle (Figure 14.17(Figure cycle cell the with cert need to first examine the and the activation of proteins that destabilize the microtubule-associatedmicrotubules(e.g., proteins, tha proteinsinactivation of the accomplishedby be rapiddisassemblyinterphasethecytoskeleton of is proteins cytoplasmi associated with the kinetochore, teins found at the outer surface of the kinetochore schematic model showing a proposed disposition of s which binds motor proteins involved in chromosome m Associated with the outer platechromosome. is the fi variety of proteins attached to the centromeric het inner and outer plates are indicated in part Proposedseparated by a lightly staining interzone. which contains an electron-densekinetochore, inner otriaea h ieohr.(to terminate at the kinetochore. Microtubules of the mitotic(trilaminar) structure. showingmammalian its three-la metaphase chromosome, its “replication” in the cytoplasm. The The process begin i in the cytoplasm. its centrosome “replication” the of centriole each phase, S of onset DNA as replication beginsnucle thein Later, gether. 592) and cleaves a proteinaceous link holding the c mitos in lateactivated becomes which separase, zyme (they are said to This “disengage”). event is trigge of each daughter cell lose their close association Even before cytokinesis has the beentwo completed, to angles right at situatedcentrioles two taining thecytoplasm contains singlea centr exitsmitosis, Formation of the Mitotic Spindle (page 339). As a cell progresses past Gprogressespastcell a As the 339). (page structure, microtubule-organizing special ter 9 how microtubule assembly in animal cells is i To understand the formation of the mitotic spindle, mitotic the of formation the understandTo (a) Microtubules kinetochore formation chromatin interface centromere replication Inner kinetochore signal transduction microtubule motoractivity microtubule binding Outer kinetochore of a section through one of the kinetochores of a Figure 14.16 The kinetochore.The centrosome cycle 0.2 b ) Schematic representation of the µ m (b) a a The inner plate contains a. ). When an animal cellanimal an When ). We discussed in Chap- ( 2 as it progresses in con- a ) Electron micrograph n nomtss themitosis, into and mitotic spindle Among the motor. erochromatin of the spindle can be seen functions of the Heterochromatin Pericentromeric Inner plate c dynein moves and outer plate everal of the pro- heterochromatin brous corona, Centromeric to one another vmn.(ovement. se polymers. red by the en- yered one another.one centrosome nitiated by a entrioles to- osomecon- thoughtto or MAPs)or s s with the stabilizet centrioles Interzone us at theatus isassem- is (pageis nitiates s f a of ts c The. ) A Kinetochore we Outer plate UCSD, is a protein complex consisting pr of four different are in an inactive state (the microtubule is not de of microtubules In this drawin rather than motility. member of the kinesin superfamily that functions in Themicrotubule protein labeledto the kinetochore. These motorstoward may also the play plus a end. ro toward whereas the CENP- minus end of a microtubule, tiation of centrosome duplication at the Geachcontaining apair of mother–daughter centriole theof centrosomemitosis, splits into two adjacent centriole into a full-length daughter At centriole. enl cnroe n oine a rgt nls o i to angles 14.17 right at oriented and centriole ternal) appearance of a small FROM contribute to the development of cancer (Figure 14. divisabnormalcellto lead ditionalcentrioles can daughter centriole The during formati each cell cycle. trolled process so that each mother centriole produ Centrosometiduplicationa is cation(Figure14.8). thesame agent responsible forthe onsetCdk2, ofDN mally triggered by phosphorylation of a centrosomal n fadnmcmcouue (end of a dynamic microtubule. fibrils have been implicated as couplers of the kine mediate attachment to the microtubule and kinetocho Globular domains bodyat either of the kinetochore. rod-shaped molecule extendingform a 57 out nm-long, uue drn pohs.Te rcs o atr format aster of process The prophase. during tubules spi of nucleation stimulating in role key a play to rylation of proteins of the PCM by Polo-like kinase Ph olar material (PCM) of the centrosome (page 339). remainends p minus the their with while associated Chapt their to subunits of addition by grow in microtubules discussed As prophase. early during trosome us”arneet or arrangement, burst” typical animal cell is the appearance of microtubul Microtubule Fibrous corona The first stage in the formation of the mitotic spin b E .Subsequent microtubule). elongation converts each p TALET LSEVIER ,C., (c) I. ELL Microtubule MAGE 1:0,2003 F112:408, subunits are addedorlost Plus endofmicrotubules where + procentriole Ndc80 C Outer kinetochore plate aster UTS OFOURTESY Depolymerase A F: Fgr 41) around each cen- (Figure 14.18), ROM next to each preexisting (ma- CENP-E IG 1. D K EVIN ON C , IHPERMISSIONWITH Corona fiber .CW. 1 oyeiig.Ndc80polymerizing). S –S transition is nor- otein subunits that tochore to the plus ULLIVAN ,the depolymerasesg, depolymerization dplmrs”is a“depolymerase” end of the complex le in tethering the L E V E L A N D e These Ndc80re. ward from the the beginning E moves centrosomes, ion and mayand ion ces only one ndle micro- ndle es in a “sun- 17 .) is thought protein by .Theini- s. ghtly con-ghtly Dynein plus ends,plus ericentri- (Figuret on of ad- _ c A repli-A ). dle dle in a ospho- , o is ion r 9,er ro- 587

Early G1 phase S phase


Mitosis (a)

Figure 14.17 The centrosome cycle of an animal cell. (a) During G 1, the centrosome contains a single pair of centrioles that are no longer as tightly associated as they were during mitosis. During S phase, daughter procentrioles form adjacent to maternal centrioles so that two pairs of centrioles become visible within the centrosome (see b). The daughter procentrioles continue to elongate during G 2 phase, and at the beginning of mitosis, the centrosome splits, with each pair of centrioles becoming part of its own centrosome. As they separate, the centrosomes organize (c) the microtubule fibers that make up the mitotic spindle. ( b) The centro- some of this cell is seen to contain two mother centrioles, each with an as- sociated daughter procentriole (arrows). ( c) This mouse mammary cancer cell contains more than the normal complement of two centrosomes (red) and has assembled a multipolar spindle apparatus (green). Additional cen- trosomes lead to chromosome missegregation and abnormal numbers of chromosomes (blue), which are characteristic of malignant cells. (A: A FTER Astral microtubules D. R. K ELLOGG ET AL ., REPRODUCED WITH PERMISSION FROM THE ANNUAL REVIEW OF BIOCHEMISTRY , V OL . 63; COPYRIGHT 1994, A NNUAL REVIEW OF BIOCHEMISTRY BY ANNUAL REVIEWS . R EPRODUCED WITH PERMISSION OF ANNUAL REVIEWS IN THE FORMAT REPUBLISH IN A BOOK VIA COPYRIGHT ; B: FROM JEROME B. R ATTNER AND STEPHANIE G. P HILLIPS , J. C ELL BIOL . 57:363, 1973, F IG . 4. R EPRODUCED WITH PERMISSION OF THE ROCKEFELLER Centrosome UNIVERSITY PRESS ; C: COURTESY OF THEA GOEPFERT AND W. R. B RINKLEY , BAYLOR COLLEGE OF MEDICINE , H OUSTON , T X.) followed by separation of the centrosomes from one another and their subsequent movement around the nucleus toward opposite ends of the cell. Centrosome separation is driven by 14.2 Figure 14.18 motor proteins associated with the adjacent microtubules. As Formation of the mitotic spindle. During prophase, as the chromosomes are beginning to condense, the the centrosomes separate, the microtubules stretching be- Cytokinesisand Mitosis Phase: M centrosomes move apart from one another as they organize the bundles tween them increase in number and elongate (Figure 14.18). of microtubules that form the mitotic spindle. This micrograph shows Eventually, the two centrosomes reach points opposite one a cultured newt lung cell in early prophase that has been stained with another, thus establishing the two poles of a bipolar mitotic fluorescent antibodies against tubulin, which reveals the distribution of spindle (as in Figure 14.17 a). Following mitosis, one centro- the cell’s microtubules (green). The microtubules of the developing some will be distributed to each daughter cell. mitotic spindle are seen to emanate as asters from two sites within the A number of different types of animal cells (including cell. These sites correspond to the locations of the two centrosomes those of the early mouse embryo) lack centrosomes, as do the that are moving toward opposite poles at this stage of prophase. The cells of higher plants, yet all of these cells construct a bipolar centrosomes are situated above the cell nucleus, which appears as an mitotic spindle and undergo a relatively typical mitosis. Func- unstained dark region. (F ROM JENNIFER WATERS , R ICHARD W. tional mitotic spindles can even form in mutant Drosophila cells COLE , AND CONLY L. R IEDER , J. C ELL BIOL . 122:364, 1993, F IG . 2. REPRODUCED WITH PERMISSION OF THE ROCKEFELLER that lack centrosomes or in mammalian cells in which the cen- UNIVERSITY PRESS . C OURTESY OF CONLY L. R IEDER .) trosome has been experimentally removed. In all of these cases, Chapter 14 Cellular Reproduction 588 C may be absorbed into the membranes of the ER. the Alternatively, membranes toticof thecell. nucle throughodisperse that vesicles small of population fragmented are membranes nuclear the view, classical envelope has been the Accord subject of controversy. The subsequent fate of the membranous portion of th dynein molecules associated with the outer nuclear cally as holes are torn into the nuclear envelope b integrity of the nuclear membranes is first disrupte disassembled by depolymerization of the lamin filame lam nuclear The medium. surrounding the into sociate oporinsubcomplexes aredisruptedsubcomplethe and complexes are disassembled as the interactions betw nucl The B–Cdk1. cyclin particularly mitotickinases, processe these of All thought to be initiated by phosphorylation processes. of key s separate in sembled and nuclear d nuclear complexes, , membranes—are envelope—thenuclearnuclea the componentsofmajor down of the nuclear envelope at the end of prophase the by possible made chromosomesis and spindle the Interaction somesare compacted in the nucleoplasm. mitotic spindle is assembled in the cytoplasm and t OrganellesCytoplasmic of The Dissolution of the Nuclear Envelope and Partiti of their spindle microtubules at the chromosomes. cells with functional centrosomes nucleate a signifi and cellsame simultaneously operatethemation in studiescentindicatedhave pathwayss boththat to centrosome-independent—toachieve the same end resul ferent mechanisms—one centrosome-dependent and the experiments suggested that cells possess two fundame tract through the activity of microtubule Th motors. fr a formedinbipolarhas thatspindleshows a 572 teins The(Figure chapter-opening 14.19). photograph spindlethrougheachpolecused)activityatthe of microtubulesthebrought togetherofareends minus centroso oncethey wherehave polymeriz Then, would poles normally reside. the at than rather chromosomes microtubulesthemitotic thenucleatspindleareof BOOKA Figure 14.19 somes. role when centrosomes are present. tion of spindle poles in the absence of centrosomes This type of mechanism is thoughtspindle topole. f causes the minus ends of the microtubules to conver bound The movement to different of the microtubules. R POUE IHPRISO OF PERMISSION WITH EPRODUCED ELL _ _ 446 1996;84:406, _ / nti oe,each motor protein has multipleIn this heads model, ETOKVIATEXTBOOK + protein Motor Formation of a spindle pole in the absence of centr YPRISO OF PERMISSION BY + + _ C OPYRIGHT + + _ (F C C ELL C LEARANCE ROM ELL nms uaytccls thecells, eukaryotic most In P .A H A. A. ESI H OMTRUEIN REUSE FORMAT THE IN RESS P RESS C C. MNANDYMAN ENTER L BYELL but may also play a ge to form a distinct acilitate the forma- se motor proteins .) y cytoplasmic C cant fraction + motorpro- ubstrates by he chromo- d mechani- ELL membrane. ed near theneared ar envelope ese types of .KE. pindle for-pindle The three. og egg ex-egg og ut the mi-the ut een nucle- which are, ntally dif- ing to the that eventhat e nuclear (.. fo-(i.e., on page between + t.Thents. ear poreear P ARSENTI xes dis-xes break- no a into d theed, RESS oning porer .Re-t. + n isina other are s + + isas- o- mes . , undergoes extensive fragmentation during prophase.undergoes extensive during fragmentation t suggested that embryos and eggs on largely formed This view challenges earlier intact during mitosis. re remains network ER the that suggest cells cultured malian living, on studies Recent changed. also have Our ideas nisms about of the Golgi fate inheritance. diffe utilize organisms or cells of le types may different we Ultimately, structure. original re the cell of daughter half each with two, in splitting complex view the hasprotists third and in studies on primarily A cells. daughter between partitioned are fragmented to form a distinct population of small v the Golgi According membranes to an alternate view, distinct a as briefly exist to ceases complex Golgi and prophase, during ER the into incorporated become the contents of the Golgi According to one view, sis. duri partitioned are reticulum endoplasmic and plex Gol the which by mechanism the over years recent in been has debate Considerable cell. plantc a of plasts the as well as peroxisomes, and lysosomes, chondria, incl these mitosis; through intact relatively remain ld hy eoe noprtd no h mttc spin mitotic the into the two sister chromati of howRegardless it occurs, incorporated become they bled, onc and, kinetochore the at subunits tubulin of tion incorp by chromosome the from out grow microtubules microtubule of assembly the for sites nucleating as servekinetochores unattached that reported been also has It pole spindle opposite the from microtubules own its chromatid sister the on kinetochore unattached the un an Ev represents coursestagetheprometaphase. inof intermediate pole spindle one only from tubules to attached is that chromosome A 2). (step poles dle end of one or more spindle microtubules from one of with associated stably become to tends kinetochore howe Soon, motor proteins located in the kinetochore. pow microtubule, the of wall the along actively move otc ieohr r cpue”and stabilized. contact a kinetochore are microtubules “captured” Those chromatin. containing site a ward preferentgrow may microtubules that suggests dence ran entirely is searching whether certain not is It chro a for were“searching” they if as fashion, namic f the growmicrotubulesto areshrin theseen and of ends cell, the of region central the into penetrate 14.20 (step end its than rather microtubule a of sidewall ein Fgr 14.20 (Figure region somes are scattered throughout the space that was t many variations on these events have prom been reported. of steps the of picture generalized a provides intoposition Thefollowiat the center of the cell. spindle assembly is completed and the chromosomes a secondmitosis,phaseof dissolutionThenuclear envelopetheof marks s the Prometaphase Some of the membranous organelles of the cytoplasm cytoplasm the of organelles membranous the of Some A kinetochore typically makes initial contact with with contact initial makes typically kinetochore A At the beginning of prometaphase, compacted chromo- compacted prometaphase, of beginning the At b .Oc iiil otc i md,sm chromosomes some made, is contact initial Once ). a .A te irtbls f h spindle the of microtubules the As ). prometaphase duringwhichmitotic , ngdiscussion rent mecha-rent dom, as evi- as dom, entireGolgi studies per- esicles esicles that generated he nuclear ude mito-ude of the ER 1 Figure1, tart of theoftart ds of each . e (plus)ree k in a dy-a in k the spin- mosome. etaphase; ng mito-ng complex (step 3).(step re moved e assem-e .Theses. entually, captures r that arn the plusthe ially to- ially gi com-gi become micro- ered byered e,thever, latively ceiving mam- he ERhe based hloro- stable that ora- the dle. the 589 mitotic chromosome ultimately become connected by their kinetochores to microtubules that extend from opposite poles. Observations in living cells indicate that prometaphase chromosomes associated with spindle microtubules are not moved directly to the center of the spindle but rather oscillate back and forth in both a poleward and antipoleward direction. Ultimately, the chromosomes of a prometaphase cell are moved by a process called congression toward the center of the mitotic spindle, midway between the poles (step 4, Figure 14.20 b). The forces required for chromosome movements during prometaphase are generated by motor proteins associ- ated with both the kinetochores and arms of the chromo- somes (depicted in Figure 14.33 a and discussed in the legend). Figure 14.21 shows the consequences of a deficiency of a chromosomal motor protein whose activity pushes chro- (a) mosomes away from the poles.

Syntelic attachment 4 (no tension) Tension applied by microtubules


Bi-oriented chromosome 3 (under tension)



Mono-oriented chromosome (no tension) (b)

Figure 14.20 Prometaphase. (a) Fluorescence micrograph of a cultured newt lung cell at the early prometaphase stage of mitosis, just after the nuclear envelope has broken. The microtubules of the mitotic spindle are now able to interact with the chromosomes. The mitotic spindle appears green after labeling with a monoclonal antibody against tubulin, whereas the chromosomes appear blue after labeling with a fluorescent dye. (b) Schematic view of some of the successive steps in

chromosome-microtubule interactions during prometaphase. In step 1, 14.2 a kinetochore has made contact with the sidewall of a microtubule and is capable of utilizing kinetochore-bound motors to slide in either M Phase: Mitosis and Cytokinesisand Mitosis Phase: M direction along the microtubule. In step 2, a chromosome has become Figure 14.21 The consequence of a missing motor protein on chro- attached to the plus end of a microtubule from one spindle pole (an mosome alignment during prometaphase. The top micrograph shows end-on attachment forming a mono-oriented chromosome). In step 3, a mitotic spindle that has assembled in a complete frog egg extract. the chromosome has become attached in an end-on orientation to mi- The lower micrograph shows a mitotic spindle that has assembled in a crotubules from both poles (forming a bi-oriented chromosome). In frog egg extract that has been depleted of a particular kinesin-related step 4, the bi-oriented chromosome has been moved to the center of protein called Kid that is present along the arms of prometaphase the cell and will become part of the metaphase plate. Chromosomes at chromosomes. In the absence of this motor protein, the chromosomes this stage are under tension (as indicated by the space between the fail to align at the center of the spindle and instead are found stretched chromatids) due to the opposing pulling forces exerted by the along spindle microtubules and clustered near the poles. Kid normally microtubules from opposite poles. The chromosome in step 3a has both provides force for moving chromosomes away from the poles (see of its kinetochores attached to microtubules from the same spindle Figure 14.33 a). (F ROM CELIA ANTONIO ET AL ., CELL VOL . 102, pole. This abnormal syntelic attachment is discussed on page 596. COVER #4, 2000; WITH PERMISSION FROM ELSEVIER . C OURTESY OF (A: C OURTESY OF ALEXEY KHODJAKOV , W ADSWORTH CENTER , NY.) ISABELLE VERNOS .) Chapter 14 Cellular Reproduction 590 pnl eutrwt oe hoai o ec chromoso each of chromatid at one equator—with aligned spindle become have chromosomes the of all Once Metaphase shown in the series of photos in Figure 14.23.shown of photos in Figure in the series prometa during spindle mitotic the of center the to ward chromosome from a peripheral site near one of of movement The length. in equivalent are pole each microtub that so spindle, the of center the at plane microtubuleeach remains to a kinetochore. attached this dynamic activity occurs Remarkably, while the p (Figure microtubule the of end plus the at subunits loss by primarily occur microtubules of elongation Shor force (tension) on the two sister kinetochores. in differences by governed be to thought are length micr in changes These elongated. are kinetochore ter kinetochore one microtubules t while theattached shorter to shortened, attached microtubules longer the s mitotic the of center the toward congress mosomes chr the As prometaphase. during movements chromosome Eventually, each each chromosome movesEventually, into position alon Microtubule dynamics also play a key role in facili in role key a play also dynamics Microtubule tening and or gain ofgain or lus end of ls fromules o the sis- the poles hs isphase 14.22). pulling otubule pindle, a way-a tating are the me g a o- be divided into three 14.24): groups (Figure animal an of spindle metaphase the of microtubules s Functionallyand chromatid cell. the of center the at tioned duplicated the separating of task the for idea is that microtubules of array organized highly plate chromosomesthe referredmetaphaseis at the as to stage of reac has cell pole—the opposite the from crotubules kinetocho its by connected chromatid sister its and o from microtubules to kinetochore its by connected 3. 2. 1. important events occur. events important t a is metaphase that reveals analysis experimental hal a to come suddenly activities mitotic all if as pears as a stage during which the cell pauses for a kinetochore toward the pole. This This pole. the toward kinetochore chromosomalthesubunitsalongmicrotubulesmove fr loss, net a microtubulesexperiencethe of ends nus net addition of subunits at the Meanwhikinetochore. more subunits are added to the plus end than are lo butnal rathersubunits, it is the site of dynamic a the end blocking of the microtubule, the entry or ex the the kinetochorekinetochore. Thus, does not act l evenmosomal though microtubules, these ends are att Subunits are rapidly lost and dyna added highly at athe plus in end exist microtubules the that dicate at aligned are studiesusing metaphasefluorescently plate, chromosomes labeled the as microtubules mal Spindle Metaphase the thoughthereobvious nois change lengthin the of in Flux Microtubule of the spindle. int basket that maintains the mechanical structural The polar microtubules a form opposite centrosome. from one with centrosome their counterparts overlap Polar microtubules fro trosome past the chromosomes. Polar movement of the chromosomes toward the poles. chromosomal microtubules are required for anaphase, Duringchromosomes situated at the metaphase plate. tions within the kinetochore oscillations and cause forces These pulling deform generate opposite poles. fibers forcespulling by chromosomal exerted spindle balan between plane by a the equatorial “tug-of-war” the chromosomes are maintained As a result, tochores. mosomal force microtubulesa pulling on the exert k a spindle a spindle fo which of 20–30 microtubules, to a bundle attached kinetochore each is In mammalian cells, mosomes. tween the centrosome and the kinetochores of the ch Chromosomal may help the determine plane of cytokinesis. in the cel apparatus help position the spindle They some into the region outside the body of the spindl microtubulesAstral soewthsfim rvdo fmtss metaphase mitosis, of videos or films watches one As h mttc pnl o te eahs cl contains cell metaphase the of spindle mitotic The . metaphase (or interpolar fiber (or (or Fgr 42) The plane of alignment of (Figure 14.24). kinetochore k ) that radiate outward fromthat outward radiate the centro- - fiber microtubules .Drn eahs,the chro- metaphase, During ). ) microtubules that extend from the cen- oead flux poleward that extend be- tvt.Becausectivity. s of the chro- brief period, .However,t. t there is ast, aily thepatially, it of termi- tubulinin- ike a cap at chromoso- f tubulin of m whenime e the mi-le, i state.mic metaphase l suited lly re to mi-to re n thusand ached to e thehed of the cell can cell e polene egrity l and m theom posi- s e. ine- from a- ced the Even ro- rms the m in ap- the a 591



Figure 14.23 The engagement of a chromosome during from both poles. The wayward chromosome has become attached to prometaphase and its movement to the metaphase plate. This series spindle fibers from opposite poles in B and then moves toward the spin- of photographs taken from a video recording shows the movements of dle equator with variable velocity until it reaches a stable position in F. the chromosomes of a newt lung cell over a period of 100 seconds during The position of one pole is indicated by the arrowhead in A. (F ROM prometaphase. Although most of the cell’s chromosomes were nearly STEPHEN P.A LEXANDER AND CONLY L. R IEDER , J. C ELL BIOL . aligned at the metaphase plate at the beginning of the sequence, one of 113:807, 1991 F IG . 1. R EPRODUCED WITH PERMISSION OF THE the chromosomes (arrow) had failed to become attached to spindle fibers ROCKEFELLER UNIVERSITY PRESS . C OURTESY CONLY . L. R IEDER .)

Astral spindle microtubules Astral spindle microtubules Centriole Chromosomes Centriole 14.2 M Phase: Mitosis and Cytokinesisand Mitosis Phase: M

Pericentriolar material Chromosomal (kinetochore) spindle fibers Polar spindle microtubules Pericentriolar material Figure 14.24 The mitotic spindle of an animal cell. Each spindle text. All of the spindle microtubules, which can number in the pole contains a pair of centrioles surrounded by amorphous thousands, have their minus ends pointed toward the centrosome. pericentriolar material at which the microtubules are nucleated. Three Although not shown here, spindles may also contain shorter types of spindle microtubules—astral, chromosomal, and polar spindle microtubules that do not make contact with either a kinetochore or a microtubules—are evident, and their functions are described in the spindle pole. Chapter 14 Cellular Reproduction 592 uig nepae n otat the contrast, In interphase. during ization rather than movement (page 336). proteins whose function is to promote microtubule d kinesin-13thememberof famil a likelyaided by is ex the illustrated in indicatedin Loss Figureof is tubulin14.25. spindlesubunit mitotic a in subunits o at a rate the microtubules of a metaphase spindle Figure 14.25 C ad P cmlxs r atv ae hw i Figur in shown are 14.26 active are complexes APC and whi SCF in cycle cell the during periods The proteasome. targeting them for des ent stages of the cell cycle, add ubiquitin to proteins SCF at and APC, complexes, It was pointed out that earlier two distinct cycle. of cell cycle regulatory proteins at precise times derliness depends to a large degree on the selectiv cyc cell the throughout order proper their in place activ specific that is it important how seen Wehave The Role of Proteolysis in Progression Through Mito op poles. toward movement their start and apart split some Anaphase Anaphase rtblsi h eino h oe.Tubulin subun crotubules in the region of the poles. are and they lost preferentially from the m tubules, chromosomal microtubules ends of and the equatorial preferentially at the are incorporated Subunits flux. are that in the components of the spindle indicates injection of fluorescently labeled tub at this stage, at metaphase.spindle a s lutae i Fgr 14.26 Figure in illustrated As . begins when the sister chromatids of each chromo- Tubulin through flux microtubules the mitotic the of uui lxTubulin flux Tubulin flux Tubulin flux Tubulin flux Even though the microtubules appear stationary npae rmtn com- promoting anaphase a C at primarily acts SCF , ulin subunits inus ends of the mi- a dynamic state of a dynamic f about 1 kinetochores of the its move through the polar micro- during during the cell e destruction truction truction by a multiprotein s at the poles le. This or- This le. y of motorof y epolymer- ␮ ities takeities periment m/min. ch the ch differ- posite sis e a bgn y APC by begun was the enzyme completesAPC, the ubiquitination of cycl and progression of the cell out of mitosis and into precipitous drop in activity of the mitotic Cdk (cy plex known as APCadap these of other the or one A containing complexes mitosis. during selection substrate Cdh1—determine protein—Cdc2 adaptor this of versions alternate substrate.Two APC the as serve proteins which determining in plays that an “adaptorto protein” addition in units, core dozen a about contains APC The mitosis. during eahs pae r slt n ycrn a te onset the at synchrony in and anaphase, split the chromatids (now are referred to as plate chr metaphase Anaphase of Events The the normal cell irreversible cycle in a direcsingle, dramatically illustrates the importance of proteoly T inhibitor. its removalof byactivity Cdk1 of tion theseeventsofaretriggeredAll inappropri the by Figurshownin as chr metaphasethetosomes backplate, the of movement and spindle, mitotic a of sembly envelopnuclear the breakdownof chromosomes, the of Thisreversal backischaracterizedinto mitosis. by actually progressactive Cdk1, in the reverse directcyclin bothcontain now which cells, Such 14.27). ure ready exited mitosis are washed free of the the Cdk1 when in that obtained cells Cdk1-inhibited is proteasome-inhibited and all of finding striking most the haps experimentalactivatorbycyclin itsorinh of tion the cessation of activity of Cdk1 (either by the no tosis and The cytokinesis. completion of mitosis cle cell will return to its normal activities and conti withcompounda that inhibits the kinase activity o subsequmitosisarearrested inare that cells such of stagelate a arrestedinremain cells proteasome, inhibi an withprevented is B cyclin of destruction isbest revealed with the use of inhibitors (Figure regulating the events of mitosis and the reentry of of the next The importancecell cycle. of protein de eto (iue 14.26 (Figure lection takes control of the Cdh1, ternate APC’s adaptor, sub ment towards the poles. to the separation of chromatids and their anaphase- Cleavage of cohesin by metaphase. such enzymes leads a been had that cells into injected been have zymes sister chromatids comes from studies in which prote att the maintaining in cohesin of role the for port Experiment chromatids to mark the onset of anaphase. oeue ht od sse crmtd tgte (Fig together chromatids sister 14.26 holds that molecule separase atcurin the end of metaphase release an active pro destructio and ubiquitination The chromatids. sister securin— 14.26 or , APC Near the end of mitosis, Cdc20 is inactivated, and th and inactivated, Cdc20is mitosis, ofendNear the b a ). Cleavage of cohesin triggers the separation of si of separation the triggers cohesin of Cleavage ). ad bqiiae a e aahs ihbtr called inhibitor anaphase key a ubiquitinates and ) . Separase Separase then cleaves the Scc1 subunit of the cohe . APC so named because it secures the attachment between securesattachment it the because named so Cdc20 , plays a key role in regulating events that occurthat events regulating in role key a plays , Cdc20 becomes activated prior to metaphase (Figuremetaphase to prior activated becomes Cdc20 or APC a .We Ch i ascae wt the with associated is Cdh1 When ). . Destruction of the cyclin leads to ato leadscyclin the Destructionof . Cdh1 l te hoooe o the of chromosomes the All (Figure 14.26(Figure nue through mi- a tion. 1.7.Ifthe 14.27). clin B–Cdk1) the G rmal destruc- sis in moving cells into G bto) Per-ibition). ). ion and head ently treatedently hibitor (Fig- compaction ate reactiva-ate gradation in arly requires achment of achment d1 theCdk1, f tease called omosomes, mtss Ifmitosis. his findinghis like move- a key role key a o o the of tor rrested inrrested olytic en-olytic strate se- in B that ae al-have o se- of n e 14.27.e os aretors rapidly B andB 1 al sup- and0 phase omo- ,as-e, al-e sub- ster of PC ure sin 1


SCF/ APCCdh1 APC APC activities SAC SCF Cdc20 Cdh-1 APCCdc20 Pro Meta Ana Telo Time


cycle G1 SG2 M Securin Mitotic cyclins phase

Chromosome status Cohesin G Metaphase Anaphase 1 (a) (b) Figure 14.26 SCF and APC activities during the cell cycle. substrates promotes the metaphase– anaphase transition. APC Cdh1 is SCF and APC are multisubunit complexes that ubiquitinate sub- responsible for ubiquitinating proteins, such as mitotic cyclins, that in- strates, leading to their destruction by proteasomes. ( a) SCF is active hibit exit from mitosis. Destruction of these substrates promotes the Cdh1 primarily during interphase, whereas APC (anaphase promoting com- mitosis–G 1 transition. APC activity during early G 1 helps maintain plex) is active during mitosis and G 1. Two different versions of APC are the low cyclin–Cdk activity (Figure 14.8) required to assemble prerepli- indicated. These two APCs differ in containing either a Cdc20 or a cation complexes at the origins of replication (Figure 13.20). (Although Cdh1 adaptor protein, which alters the substrates recognized by the not discussed here, the activation of phosphatases that remove the phos- APC. APC Cdc20 is active early in mitosis, at a time when Cdh1 is phate groups added by Cdk1 also plays a substantial part in driving the inhibited by Cdk1-mediated phosphorylation. As Cdk1 activity drops events that occur during the latter stages of mitosis and reentry into G 1; sharply in late mitosis, Cdh1 is activated, leading to the activation of see Nature Revs. Mol. Cell Biol. 12:469, 2011.) ( A: A FTER J-M P ETERS , Cdh1 APC . The label SAC stands for spindle assembly checkpoint, CURR . O PIN . C ELL BIOL . 10:762, 1998; COPYRIGHT 1998. C URRENT Cdc20 which is discussed on page 596. The SAC prevents APC from OPINION IN CELL BIOLOGY BY ELSEVIER LTD . R EPRODUCED WITH triggering anaphase until all the chromosomes are properly aligned at PERMISSION OF ELSEVIER LTD . IN THE FORMAT REUSE IN A BOOK / Cdc20 the metaphase plate. ( b) APC is responsible for destroying TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER . S EE ALSO proteins, such as securin, that inhibit anaphase. Destruction of these NATURE REVS . M OL . C ELL BIOL . 7:650, 2006.) because they are no longer attached to their sisters) begin their edge with the arms of the chromosome trailing behind (Figure poleward migration (see Figure 14.11). As each chromosome 14.28 a). The movement of chromosomes toward opposite moves during anaphase, its centromere is seen at its leading poles is very slow relative to other types of cellular movements, 14.2 M Phase: Mitosis and Cytokinesisand Mitosis Phase: M

Figure 14.27 Experimental demonstration of the importance of last five frames of the video. The upper row shows phase-contrast proteolysis in a cell’s irreversible exit from mitosis. This illustration images of the cell at various times, and the lower row shows the corre- shows frames from a video of a cell that had been arrested in mitosis by sponding fluorescence micrographs with times indicated. Video 3, from the presence of a proteasome inhibitor (MG132). At time 0, a Cdk1 which these frames were taken, can be seen at the online version of this inhibitor (flavopiridol) was added to the medium, which caused the cell paper. Bar in lower right image equals 10 ␮m. (F ROM TAMARA A. to complete mitosis and initiate cytokinesis. At 25 min, the cell was POTAPOVA , E T AL., N ATURE 440:954, 2006. © 2006, REPRINTED washed free of the Cdk1 inhibitor. Because the cell still contained WITH PERMISSION FROM MACMILLAN PUBLISHERS LIMITED . cyclin B (which would normally have been destroyed by proteasomes), COURTESY OF GARY J. G ORBSKY .) the cell reentered mitosis and progressed to metaphase as seen in the Chapter 14 Cellular Reproduction 594 referred to as eutn i sotnn o te hoooa fies (F fibers 14.28 chromosomal the of shortening in resulting 14 whereas subunits are (Figure lost from the plus ends during constant fibers chromosomal the of length keep metaphase, during microtubules of ends plus the a are subunits that is anaphase and metaphase tween an dynami 14.22 microtubule in difference primary The (Figures 14.25). metaphase and prometaphase during that subunits tubulin of flux poleward continued the a as microtubules these of ends minus the from lost proceeding at approximately 1 at approximately proceeding uue drn aahs (iue 14.28 (Figure anaphase during mi tubules chromosomal of ends (kinetochore-based) plus the are subunits tubulin that appreciated been long has microtubu chromosomal of shortening obvious by nied follo the section. in discussed are chromosome anaphase power during to movement thought forces The entanglement. and accurately segregate the sures chromosomes that movement chromosome of rate slow The years. approximate million take would that Italy to Carolina North to to be by researcher equivalent lated one mitosis chores following of the sister chromatid separation is thought to be triggered by the loss of tension o htocr uigaahs .( anaphase B. that occurs during are thought to be responsible the separ for causing Relative movements ofseparating the po chromosomes. visible in the int are clearly however, microtubules, edges of the chromosomes and the poles. the forward anaphase stage are and are extremely short no longe fi The chromosomal spindle ward the respective poles. areThe arms behind as the seenkinetochor to trail showing compacted of the chromosome arms the highly .Mawie tubulin subunits are added to the plus Meanwhile, A. movement of the chromosomes toward the poles during resulting of chromosomal in shortening microtubules, Tubulin subunits are lost from both ends o anaphase. The movement of the chromosomes toward the poles is poles the toward chromosomes the of movement The h plwr mvmn o crmsms s accompa- is chromosomes of movement poleward The anaphase. Figure 14.28 b (a) .This change in behavior at the microtubule plus ). e anaphase A ( a ) Fluorescence micrograph at late of anaphas a cell ) Fluorescence The mitotic spindle and chromosomes at spindle mitotic The to distinguish it from a separate but b ) Microtubule dynamics during) Microtubule dynamics ␮ m per minute, a value calcu- a value m minute, per b .Sbnt ae also are Subunits ). ation of the poles erzone between the between erzone es lead the way to- r evident betweenr evident f the chromosomal fibers and The polar spindle ends of polar bers at this late lar microtubules anaphase s at this stage. n the kineto- a fromtrip s. anaphase, lost fromlost result ofresult without dd todded occurs ing theing s be-cs e.Itles. y 14 ly igure wing .25), cro- nds en- e, d (b) B simultaneous movement, called called simultaneous movement, totic spindle (Figure 14.28 (Figure totic spindle th of regions different in time same the at tubules mic chromosomal from removed the and microtubules ad of polar preferentially be ends can subunits plus the Thus, to microtubules. subunits tubulin of dition totic spindle during anaphase B is accompanied by t of elongation The apart. farther move poles spindle rto o slbe uui,wih n un rmts t promotes turn in which of types These microtubules. the of tubulin, depolymerization soluble of tration thereduces Dilution medium. the of lowingdilution a microtubule-bound chromosome (arrow) occurs in vi the mov In ments this is case, shown in Figure 14.29. these of one of example An microtubules. attached of depolyme the of result the as movement considerable underw chromosomes which in studies from came model some forward. a pull to force mechanical sufficient generate could spin a comprise that microtubules the of merization chromosome d that suggested Inoué of it. consequence of cause the during a but movement simply microtubules not was chromosomal anaphase t of that proposed Hole depolymerization Woods at Laboratory at Biological Movements Chromosome Anaphase for Required Forces N fteplsdrn npaeB ( anaphase B. of the poles during le also slide across which one another, microtubules, EMSINFROMPERMISSION ERMÚDAEZ ATURE Anaphase continues Anaphase begins al eprmna spot o te disassembly-forc the for support experimental Early C ELL D, B In the early 1960s, Shinya Inoué of the Marinethe of Inoué Shinya 1960s, early the In ANIEL IOL M ,#,20;©20,R ©2007, 2007; #7, 9, . ACMILLAN G ERLICH b ). A , F: P AND UBLISHERS anaphase B ROM J AN F ELIPE E PITDBYEPRINTED L L E NBE R G L IMITED in which the two, M ading to separation ORA .) , - e same mi-same e OE OFCOVER he net ad- chromo- ement of dle fiber dle concen- experi- h mi-the rization experi- tro fol- e toded epoly- polar ent ro- he he he e 595

a 0 b 51 c 75

Figure 14.29 Experimental demonstration that microtubule chromosome attached to the end of a bundle of microtubules. The con- depolymerization can move attached chromosomes in vitro. The centration of soluble tubulin within the chamber was then decreased by structure at the lower left is the remnant of a lysed protozoan. In the dilution, causing the depolymerization of the microtubules. As shown presence of tubulin, the basal bodies at the surface of the protozoan in this video sequence, the shrinkage of the microtubules was accompa- were used as sites for the initiation of microtubules, which grew nied by the movement of the attached chromosome. Bar equals 5 ␮m. outward into the medium. Once the microtubules had formed, (F ROM MARTINE COUE , V IVIAN A. L OMBILLO , AND J. R ICHARD condensed mitotic chromosomes were introduced into the chamber MCINTOSH , J. C ELL BIOL . 112:1169, 1991, F IG . 3. R EPRODUCED and allowed to bind to the ends of the microtubules. The arrow shows a WITH PERMISSION OF THE ROCKEFELLER UNIVERSITY PRESS .) ments, as well as direct force-measuring studies, indicate that toward the poles due to poleward flux, reminiscent of a person microtubule depolymerization alone can generate sufficient standing on a “moving walkway” in an airport. In contrast, de- force to pull chromosomes through a cell. polymerization at the microtubule plus ends serves to “chew Figure 14.30 a depicts a model of the events proposed to up” the fiber that is towing the chromosomes. Some cells rely occur during chromosome movement at anaphase in a verte- more on poleward flux, others more on plus-end depolymer- brate cell. As indicated in Figure 14.28 b, the microtubules ization. Studies of animal cells in anaphase have revealed that that comprise the chromosomal spindle fibers undergo de- both the plus and minus ends of chromosomal fibers are sites polymerization at both their minus and plus ends during where depolymerizing kinesins (members of the kinesin-13 anaphase. These combined activities lead to the movement of family, page 336) are localized. These depolymerases are indi- chromosomes toward the pole. Depolymerization at the mi- cated at the opposite ends of the microtubule in Figure 14.30 a. crotubule minus ends serves to transport the chromosomes If either of these microtubule “depolymerases” is specifically

Ndc80 complex Spindle pole

Microtubule of Poleward chromosomal fiber microtubule flux

؉ ؊

Depolymerase Depolymerase 14.2 M Phase: Mitosis and Cytokinesisand Mitosis Phase: M Outer plate of kinetochore Figure 14.30 Proposed mechanism for the movement of kinetochore plate acts as the device that couples microtubule chromosomes during anaphase in animal cells. In the model depicted depolymerization to chromosome segregation. The force required for here, chromosome movement toward the poles is accomplished by a chromosome movement is provided by the release of strain energy as combination of poleward flux, which moves the body of each the microtubule depolymerizes. The released energy is utilized by the microtubule toward one of the poles, and simultaneous depolymeriza- curled ends of the depolymerizing protofilaments to bias the movement tion of the microtubule at both ends. Depolymerizing kinesins of the of the bound heads of the Ndc80 complex (dashed black arrow) toward kinesin-13 family have been localized at both the plus (kinetochore) the minus end of the microtubule. Motor proteins of the kinetochore, and minus (polar) ends of chromosomal microtubules and are such as cytoplasmic dynein, may also have a force-generating role in postulated to be responsible for depolymerization at their respective chromosome movement during anaphase. sites. In this model, the Ndc80 protein complex of the outer Chapter 14 Cellular Reproduction 596 ons prts t h tasto bten metaphase between The anaphase. transition the at operates points t of One cycle. cell the during events of status the m that mechanisms checkpoint possess cells 579, page chromoso chromosomal fiber. attached the result, the bytowed is it as pole spindle thetoward moves a As tip. disassembling protofilaments curling the by along pushed end, minus N the of heads terminal complexes are able to travel along the microtubule the 14.30, Figure in dicated tha suggest studies of number A tethers. Ndc80 these microtubule is contacted around its circumference b estimate is It end. plus its behind just microtubule an with linkages weak relatively form to out reach molecular as present are kinetochore the of plexes Ndc The subunits. tubulin losing are that crotubules which kinetochores are able to hold on to mec the plus the is mitosis of field the in interest greatest forchromosome segregation basis during mitosis. the forms depolymerization kinesin-mediated ent, ThesefindingssuggestATP- that disrupted. partially h Side seby Checkpoint Assembly Spindle The en s og s h cl cnan uaind chromos unaligned contains cell the as long As tein. M the contain still to seen ischromosome unaligned Unlike all of the other kinetochores some. in this c unaligned single a to due metaphase to prior rested niie,chromosome segregation during anaphase is inhibited, increased risk of developing cancer.increased of developing risk and cells aneuploid of percentage high a by terized individuals exhibit a which is disorder (named MVA), protei checkpoint spindle the of one in deficiencies confirm been has with the of identification a number of children with expectation This (aneuploidy). somes of number abnormal an receiving cells daughter the the elevate greatly would it segregation, chromosome t able not were cell a If equator. spindle the along pos proper its assumed has chromosome misplaced the anapha of onset the delays mechanism checkpoint the h this When plate. metaphase the at properly aligned to fails chromosome a when revealed best is called, Figure Figure 14.23 a the by chromosomewaywardindicated the of stance the probably is which pole, spindle one from tubules t attached only is that chromosome a p consider Let’s metaphase the at aligned properly are chromosomes complex leaves the kinetochore, which turns off the the off signal and allows to progress the cell into anaphas turns which kinetochore, the leaves complex s the plate, metaphase the at a aligned properly poles comes spindle both from fibers spindle to attached become chromosome wayward the Once anaphase. into on the cell cycle machinery that prevents the cell fro an at proteins o presence The checkpoint. assembly spindle the ates t the best studied of which is Mad2, called proteins, It was mentioned on page 585 that one of the questi How does the cell determine whether or not all of t of all not or whether determine cell the does How Figure Figure 14.31 shows the mitotic spindle of a cell th a . Unattached Unattached kinetochores contain a complex of. unattached spindle assembly checkpoint checkpoint assembly spindle kinetochore sends a “wait” signal to signal “wait” a sends kinetochore s icse on discussed As ( SAC m continuing hese check-hese e. ell, only only theell, ends of mi- d that eachthat d o postponeo towards its aim byhanism fibrils that fibrils shrinking y 6 to 9 of hat medi- inherited s Thesens. chromo- a greatlya ), as it isit as ), o micro-o chromo- ad2 pro-ad2 circum- 80 com-80 attached depend- ignaling charac- become t, as in- as t, appens, at is ar- at least risk of risk se untilse rrowin d be- nd thesef of theof “wait” onitor ons of omes, and dc80 ition late? me he ed s .CJ. tachment (step 3a, Figure 14.20 Figure 3a, (step tachment a as referredtocondition a pole, spindle same the microtubu to attached become will chromatids sister kinetochor two the corr occasion, on require Forexample, also measures. that metaphase to progression the aris that abnormalities chromosomal other are there butis activated by the presence of an unattached kinetochore, tached to one tached another by their cohesin “glue.” ch sister the of all keeping thus securin, inhibitor Mad2 to bound is Cdc20 APC complexes would be that unable to ubiquitinate activat the a period APC the During the and Cdc20. Mad2 between t interaction achieved is direct inhibition model, favored a Ac- to cording progress. cycle cell inhibit to able are molecules Mad2 Figure 14.20Figure pulled by microtubules from opposite spindle poles kinetoch ar chromatids sister chromosome’s when develops Tension normally the on tension of lack the by att syntelic a with chromosome a of presence the to The cell is like daughter devoid of this chromosome. leaving cells, daughter the of one to chromatids ter to lead to likely the movement o is attachment very of the Ndc80 complex and the kinesin depolymerase o mem including attachment, kinetochore–microtubule in rora B kinase are several of the proteins thought t of substrates the Among metaphase. and prometaphase centromethe residesat that complexprotein mobile p is which kinase, B Aurora called enzyme an of tion through connections) microtubule abnormal of types in opeetteiiito faahs.(Fcient the toinitiation prevent of anaphase. The presence of Mad2 on the kinetochore of this chr this chromosome has not yet become aligned at the m one of the chromosomesOnly is of seen this to cell The chromosomes tubulin of the microtubules (green). protein Mad2 (pink) and checkpoint antibodies against the spindle micrograph of a mammalian in late prometaphase cell Figure 14.31 EMSINOFPERMISSION R EY ELL -H It is well established that the spindle assembly ch Cells are able to correct syntelic attachments (and attachments syntelic correct to able are Cells UE I B IOL C HEN 141,. b The spindle assembly checkpoint. assembly spindle The ). T A, HE COVER NDREW R OCKEFELLER 5 98 R 1998; #5, .MW. URRA Y b U ). If not corrected, a syntelic a corrected, not If ). POUE WITHEPRODUCED NIVERSITY , AND ROM .D S D. E. J cnanMd,and contain Mad2, Fluorescence ENNIFER P omosome is suffi- RESS labeled with etaphase plate. appear blue. o be involved ALMON romatidsat- .) syntelic at- syntelic (steps 3–4, f both sis- the otherthe reduring ly ly alerted W eckpoint achment duringe e fromles naphase f Figure beinge art of aof art hrough the ac-the ATERS other , ective es ofes ores. Au- bers or , , 597 14.30. Studies suggest that Aurora B kinase molecules of an let’s look back and consider the motor proteins involved in incorrectly attached chromosome phosphorylate these protein several of the major chromosome movements that take place substrates, which destabilizes microtubule attachment to both during mitosis. kinetochores. Once freed of their bonds, the kinetochores of each sister chromatid have a fresh opportunity to become attached to microtubules from opposite spindle poles. Inhibi- Motor Proteins Required for Mitotic Movements tion of Aurora B kinase in cells or extracts leads to misalign- Mitosis is characterized by extensive movements of cellular ment and missegregation of chromosomes (see Figure 18.51). structures. Prophase is accompanied by movement of the spindle poles to opposite ends of the cell, prometaphase by Telophase movement of the chromosomes to the spindle equator, anaphase A by movement of the chromosomes from the spin- As the chromosomes near their respective poles, they tend to dle equator to its poles, and anaphase B by the elongation of collect in a mass, which marks the beginning of the final stage the spindle. Over the past decade, a variety of different molec- of mitosis, or telophase (Figures 14.11 and 14.32). During ular motors have been identified in different locations in mi- telophase, daughter cells return to the interphase condition: totic cells of widely diverse species. The motors involved in the mitotic spindle disassembles, the nuclear envelope re- mitotic movements are primarily microtubule motors, includ- forms, and the chromosomes become more and more dis- ing a number of different kinesin-related proteins and cyto- persed until they disappear from view under the microscope. plasmic dynein. Some of the motors move toward the plus end The actual partitioning of the cytoplasm into two daughter of the microtubule, others toward the minus end. As discussed cells occurs by a process to be discussed shortly. First, however, above, one group of kinesins does not move anywhere, but promotes microtubule depolymerization. Motor proteins have been localized at the spindle poles, along the spindle fibers, and within both the kinetochores and arms of the chromo- somes. Although firm conclusions about the functions of spe- cific motor proteins cannot yet be drawn, a general picture of the roles of these molecules is suggested (Figure 14.33): I Motor proteins located along the polar microtubules probably contribute by keeping the poles apart (Figure 14.33a,b). I Motor proteins residing on the chromosomes are probably important in the movements of the chromosomes during prometaphase (Figure 14.33 a), in maintaining the chro- mosomes at the metaphase plate (Figure 14.33 b), and in separating the chromosomes during anaphase (Figure Furrow 14.33c). (Cytokinesis) I Motor proteins situated along the overlapping polar mi- Nuclear envelope crotubules in the region of the spindle equator are proba- reforming bly responsible for cross-linking antiparallel microtubules and sliding them over one another, thus elongating the spindle during anaphase B (Figure 14.33 c).

Cytokinesis 14.2 Mitosis accomplishes the segregation of duplicated chromo-

somes into daughter nuclei, but the cell is divided into two Cytokinesisand Mitosis Phase: M daughter cells by a separate process called cytokinesis . The first hint of cytokinesis in most animal cells appears during anaphase as an indentation of the cell surface in a narrow band around the cell. As time progresses, the indentation deepens to form a furrow that moves inward toward the center of the cell. The plane of the furrow lies in the same plane previously occupied by the chromosomes of the metaphase plate, so that the two sets of chromosomes are ultimately partitioned into Figure 14.32 Telophase. Electron micrograph of a section through a different cells (as in Figure 14.32). As one cell becomes two human lymphocyte in telophase. (D AVID M. P HILLIPS / P HOTO cells, additional plasma membrane is delivered to the cell sur- RESEARCHERS , I NC .) face via cytoplasmic vesicles that fuse with the advancing Chapter 14 Cellular Reproduction 598 ioi pnl.Mcouue r re,actin is red Microtubules are green, mitotic spindle. with remnants that is packed of the cent mic bridge furrow the cleavage cuts throughwhich the midbody, between between the daughter cells called the cytoplasm a forms which spindle, mitotic the of tion advancing the stages, passes through latter the tightly packed remnants of the its c In furrow. cleavage BOOK A IN REUSE FORMAT E Figure 14.33 S The final step of cytokinesis is called called is cytokinesis of step final The ( surfaces surfaces of the cleavage furrow fuse with one anoth (see (Figure 14.21). ing at the kinetochore and especially along the chr the poles are mediated by plus-end-directed motors residing at the kinetochore Chromosome mov(step 2). are mediated by minus-end-directed cyt motors (i.e., midway Poleward between spindle, the chromosom poles. the chromosomes Ultimately, aretubules. moved to the microtubules and can be seen oscillating back and f the chromosomes have become attached to the chromos associated with the centrosomes and cortex are not opposite poles and cause them to slide apart (step bind by their heads toThese antiparalle motors can (4-headed) plus-end-directed motors (members of the due to the from one anotherapart to opposite poles, siding at the kinetochore ( (step 5). balanced activity of plus end-and minus end-directe somes are thought to be maintained at the equatoria within these microtubules that is depicted in Figur sociated with step 4 is also suspected of promoting ity associated with the polar microtubules (step 4) their separation as the result of continued plus-en directed motors of the polar microtubules (step 7). thought to result from the continuing activity of t ends of microtubules The (step separation 6). of the depolymerases that catalyze depolymerization at bot chromosomes toward the poles is thought to require a CHOLEY LSEVIER ) Prometaphase. The two halves of the mitotic spindl halves The two ) Prometaphase. are undergoing the final step in cytokinesis, called called are undergoing the final step in cytokinesis, Figure 14.34 T, L TD RENDS (a) R. Proposed activity of motor proteins during mitosis. Proposed of motor proteins during activity POUE IHPRISO OF PERMISSION WITH EPRODUCED C ELL b ) TheMetaphase. two halves of the spindle maintain Cytokinesis B / IOL ETOKVIATEXTBOOK :2,19.T 1991. 1:122, . c ) The Anaphase. movement of the ( a ) These cultured mammalian cells C OPYRIGHT ED NCL ILG BY BIOLOGY CELL IN RENDS midbody E he bipolar plus-end- d-directed motor activ- The motor . activity as- omosome arms (step 3) the flux of subunits 42.Thee chromo-14.25. ) (Additional motors1). K .S E. (K. LSEVIER poles (anaphase B) is orth along the micro- d motor proteins re- l plane by the ie,kinesins)(i.e., resid- h the plus and minus C shown.) Meanwhile, the activity of kinesin oplasmic dynein) and DNA is blue., l microtubules from ral portion of the portion ral action of bipolar LEARANCE (Figure (Figure 14.34 kinesin-5 family). ements away from a thin cytoplas- center of the omal e are moving WNANDAWIN e movements bcsin inabscission, L Midbody TD , when the the when , r splittinger, entral entral por- . ic bridgeic NTHEIN C furrow ENTER .M.J. a ). .) (b) ( yoiei.(Fcytokinesis. OF F (c) (b) (a) + b IG ) sea urchin egg has just been spli This fertilized + + + + T 1. R Y GGVE A + R. + PITDWT EMSINFROM PERMISSION WITH EPRINTED + – – – + – – – – G ROM – – – – – – UST AFSON – – – – – – 6 A 2 HNA + + + .) .SR. + 5 + + 1 + O TAL ET KOP + 7 4 + + + + + 6 + + ,S., 3 CIE NCE – – – – – – + – – – – AAAS. – – – – t into two cells by cells t into two – – – + – – + 0:1 2004, 305:61, B : + + COURTESY + + + + + 599

Actin filament

Bipolar myosin filament


Figure 14.35 The formation and operation of the contractile Contractile ring ring during cytokinesis. (a) Actin filaments become assembled in a ring at the cell equator. Contraction of the ring, which requires the action of myosin, causes the formation of a furrow that splits the cell in two. ( b) Confocal fluorescence micrograph of a fly spermatocyte undergoing cytokinesis at the end of the first meiotic division. Actin filaments, which have been stained with the mushroom toxin phalloidin, are seen to be concentrated in a circular equatorial band within the cleavage furrow: ( B: F ROM DANIEL SAUL , ET AL ., J. C ELL SCIENCE 117:3893, 2004, #17 COVER , COURTESY OF JULIE A. B RILL , BY PERMISSION OF THE COMPANY OF LTD . Cleavage furrow http://jcs.biologists.org/content/117/17.cover-expansion)

as evidenced by their binding of anti-myosin II antibodies (Figure 14.36 b). The importance of myosin II in cytokinesis is evident from the fact that (1) anti-myosin II antibodies cause the rapid cessation of cytokinesis when injected into a divid- ing cell (Figure 14.36 c), and (2) cells lacking a functional myosin II gene carry out nuclear division by mitosis but can- Daughter cells not divide normally into daughter cells. The assembly of the actin-myosin contractile machinery in the plane of the future (a) cleavage furrow is orchestrated by a G protein called RhoA. In its GTP-bound state, RhoA triggers a cascade of events that leads to both the assembly of actin filaments and the activa- the cell in two (Figure 14.34 b). Abscission requires the action tion of myosin II’s motor activity. If RhoA is depleted from of ESCRT complexes—the same proteins responsible for cells or inactivated, a cleavage furrow fails to develop. severing the intraluminal vesicles that form within endosomes The force-generating mechanism operating during cy- (page 312). tokinesis is thought to be similar to the actin and myosin-

Our present concept of the mechanism responsible for based contraction of muscle cells. In fact, the cytokinesis 14.2 cytokinesis stems from a proposal made by Douglas Marsland furrow of a primitive single-celled eukaryote is the likely evo- in the 1950s known as the contractile ring theory (Figure lutionary ancestor of the contractile machinery of animal Cytokinesisand Mitosis Phase: M 14.35a). Marsland proposed that the force required to cleave a muscle cells. Whereas the sliding of actin filaments of a mus- cell is generated in a thin band of contractile cytoplasm lo- cle cell brings about the shortening of the muscle fiber, sliding cated in the cortex , just beneath the plasma membrane of the of the filaments of the contractile ring pulls the cortex and at- furrow. Microscopic examination of the cortex beneath the tached plasma membrane toward the center of the cell. As a furrow of a cleaving cell reveals the presence of large numbers result, the contractile ring constricts the equatorial region of of actin filaments (Figure 14.35 b and 14.36 a). These un- the cell, much like pulling on a purse string narrows the open- branched actin filaments are assembled in the cell cortex by ing of a purse. the action of a formin protein (page 372), which may also an- It is generally agreed that the position of the cleavage fur- chor the filaments to the overlying plasma membrane. row is determined by the position of the anaphase mitotic Interspersed among the actin filaments are short, bipolar spindle, which induces the activation of RhoA in a narrow myosin filaments. These filaments are composed of myosin II, ring within the cortex. However, there has been considerable Chapter 14 Cellular Reproduction 600 (as revealed by the mitotic spindles) continues una (as revealed by the mitotic spindles) suppressed has been completely by the a cytokinesis due to the presencebackground microtub of oriented to appear either bright the mitotic spindles causes under pol as observed myosin, tibody against starfish studies suggest that the site of actin-filament asse actin-filament of site the that suggest studies 14.3 Figure of experiment the in shown is plane age poles spindle the of position the between tionship of example An cell. the into inserted microneedle a even if tween one the of spindle poles, the poles is plane a in forms ring contractile the that demon strated York New in College Union of Rappaport Ray by invertebr marine on studies pioneering Early lected. co the within zone particular this how to as debate U M Figure 14.36 ti muoursec.(stain immunofluorescence. Dictyostelium elmvmn Scin97.( 9.7). movementcell (Section where th furrowthe cleavage periphery and the cell myosin in cytokinesis. C h qao.(the equator. where r of a contractile it is part furrow, cleavage R POUE IHPRISO OF PERMISSION WITH EPRODUCED UTS OFOURTESY NIVERSITY ABUCHI (c) a (b) (a) , AND c P ameba during cytokinesis as demonstrated by as double- demonstrated cytokinesis ameba during ) A starfish egg that had been microinjected) A starfish with an RESS Experimental demonstration of the importance of importance the of demonstration Experimental Y OSHIO S HINY A .) ( Furrow a F , I UKUI b NOUÉ ) Localization of actin and myosin II in a) Localization a ) Actin filaments (red) are at both located b ) Myosin II (green) is localized at the) Myosin II (green) is localized ; C .C J. , : FROM ELL T HE D B ANIEL IOL R OCKEFELLER 417 92 F 1982, 94:167, . ing that encompasses er or darker than the fce.(ffected. .KP. ey play a key role play a key ey in tbde,mitosisntibodies, arized light (whicharized ls.Whileules). IE HAR T displaced by midway be-midway Furrow A and cleav-and by andmbly, rtex is se-is rtex , B the rela-the .These7. ate eggsate : I, IG SSEI an- 1.. - rm h side oe t te el otx ln the along cortex cell the to poles spindle the from thought is signal The poles. spindle the from nating si a by determined is cytokinesis, of plane the thus ( polesdle that have formed prior to the second divi The two dark spotsassume in eaa cylindrical shape. one of the cells was drawn into a micropitwo cells, once the mitotic spindle appea Then, two-cell embryo. spindle. time at which cleavage occurs depends on the positi T B Figure 14.37 equals 80 and (2) cleavage occurs more rapidly in th position, (1) the cleavage plane forms between the spindle po the spherical Thes cell has not yet begun to cleave. ( shortening the time it takes the cleavage signal to from the poles (blue spheres) to the site of cleava and (2) cleavage occurs earlier in the cylindrical cleavage plane (brown bar) forms where the astral m A b IOL HE (b) ) Nine minutes later the cylindrical cell has compl (a) , B F: 4:8,19,F 1999, 146:987, . R ROM OCKEFELLER ( ␮ a ) This echinoderm egg was allowed to divide once to .(m. C HARLES The siteThe of formation of the cleavage plane and the c ) These results can be explained by assuming that ( U .SB. NIVERSITY IG .R 5. . UTRANDHUSTER POUE IHPRISO OF PERMISSION WITH EPRODUCED P RESS .) D AVID cell because the distance ei eue,therebyge is reduced, reach the surface. .BR. e photos indicate that sion of each cell. e,regardless les, of their et,causing it topette, yidia el Bare cylindrical cell. tdcevg,whileeted cleavage, ch cell are the spin- icrotubules overlap, on of the mitotic URGESS red in each of the gnal ema-gnal to travelto .C J. , form a astral 1) the ELL 601 microtubules. When the distance between the poles and the The plane in which the cell plate forms is perpendicular cortex is modified experimentally, the timing of cytokinesis to the axis of the mitotic spindle but, unlike the case for ani- can be dramatically altered (Figure 14.37). In contrast, re- mal cells, the plane is not determined by the position of the searchers conducting studies on smaller mammalian cells have spindle nor is it determined late in mitosis. Rather, the orien- found evidence that the site of cleavage furrow formation is tation of both the mitotic spindle and cell plate are deter- defined by a signal that originates in the central part of the mined by a belt of cortical microtubules—the preprophase mitotic spindle rather than its poles. Researchers have strug- band— that forms in late G 2 (see Figure 9.21). Even though gled to reconcile these opposing findings. The challenge is the preprophase band has disassembled by prometaphase, it made greater by the fact that, unlike mitosis, cytokinesis has leaves an invisible imprint that determines the future division not been reconstituted in egg extracts, where it would be eas- site. The first sign of cell plate formation is seen in late iest to study. The simplest explanations are that (1) different anaphase with the appearance of the in the cen- cell types utilize different mechanisms or (2) both mecha- ter of the dividing cell. The phragmoplast consists of clusters nisms operate in the same cell. In fact, recent studies provide of interdigitating microtubules oriented perpendicular to the evidence for this latter possibility. future plate (Figure 9.21), together with actin filaments, membranous vesicles, and electron-dense material. The mi- Cytokinesis in Plant Cells: Formation of the Cell Plate crotubules of the phragmoplast, which arise from remnants of Plant cells, which are enclosed by a relatively inextensible cell the mitotic spindle, serve as tracks for the movement of small wall, undergo cytokinesis by a very different mechanism. Unlike Golgi-derived secretory vesicles into the region. The vesicles animal cells, which are constricted by a furrow that advances in- become aligned along a plane between the daughter nuclei ward from the outer cell surface, plant cells must construct an (Figure 14.38 a). Electron micrographs of rapidly frozen to- extracellular wall inside a living cell. Wall formation starts in the bacco cells have revealed the steps by which the Golgi-derived center of the cell and grows outward to meet the existing lateral vesicles become reorganized into the cell plate. To begin the walls. The formation of a new begins with the con- process (step 1, Figure 14.38 b), the vesicles send out finger- struction of a simpler precursor, which is called the cell plate . like tubules that contact and fuse with neighboring vesicles to

+ Microtubule

_ Vesicle


2 (a) 14.2 Plasma Figure 14.38 The formation of a cell plate between two daughter plant membrane Cytokinesisand Mitosis Phase: M nuclei during cytokinesis. (a) A low-magnification electron micrograph showing the formation of the cell plate between the future daughter cells. Secretory vesicles derived from nearby Golgi complexes have become aligned along the equatorial plane (arrow) and are beginning to fuse with one another. The membrane of the vesicles forms the plasma membranes of the two daugh- ter cells, and the contents of the vesicles will provide the material that forms the cell plate separating the cells. ( b) Steps in the formation of the cell plate as Parent cell wall described in the text. (A: D AVID PHILLIPS /P HOTO RESEARCHERS , I NC .; B: A. L. S AMUELS , T. H. G IDDINGS , J R. & L. A. S TAEHELIN , J OURNAL CELL BIOLOGY 130:1354, 1995. T HE JOURNAL OF CELL BIOLOGY BY ROCKEFELLER 3 INSTITUTE ; A MERICAN SOCIETY FOR CELL BIOLOGY COPYRIGHT 1995 REPRODUCED WITH PERMISSION OF ROCKEFELLER UNIVERSITY PRESS IN THE (b) FORMAT REPUBLISH IN A TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .) Chapter 14 Cellular Reproduction 602 gle are split apart and separate into two daughter nucl daughter two into separate and apart split are chromo each of chromatids the chro two mitosis, During of matids. consisting chromosome each with mosomes, aee.Ti i acmlse by accomplished is This . tion in chromosome number at a stage prior to forma numberat fertilization is compensated by an equiva chrothedoubling of the Chapterdiscussed10, inAs meiosis, diploid G diploid meiosis, mi both Priorto chromatids. the of fate the examine and generation, sexual each reproduction would with not be possible. double would number mosome meiosis, Without phase. diploidtilizationensuresa Meiosi “reduction.” ensures production meaning of a haploid phase word in the life c Greek the from 1905 14.3 the mature wall. cell vesi the cellulose and other materials are added t completed, within carried c the Once been plate. cell intervening the to contribute had that products tory t whereas cells, daughter adjacent two the of branes plasma the become network tubular the of membranes partiti flattened continuous, a into matures and gaps c the of c its loses network tubular the Ultimately, boundary the 3). (step at membrane plasma parent the growingnetwork the of edge leading the Eventually, ( direction outward an in network the extends which a formation tubule of process the continue vesicles new The alon network. the of edges lateral the to tubules directed then are vesicles Additional 2). (step of center the in network tubular interwoven an form .Contrast the events that occur during cytokinesis 6. .Describe the similarities and differences in micr 4. Describe the events that occur in a cell during3. What aresomeoftheactivitieskinetochore 2. Howdotheeventsofmitoticprophaseprepare 1. .What types of force-generating mechanisms might b 5. W E I V E R division. As a result, cells cells produced As by a mitosis result, cont division. To we nee compare the events of mitosis and meiosis, anaphase? responsible forchromosome movementduring typical plant and animal cells. prometaphase andduringanaphase. during mitosis? chromatids forlaterseparationatanaphase? movements? the differences relatedtoanaphaseA andB dynamics betweenmetaphaseandanaphase.Howare | Meiosis 2 each with a haploid set of chromosomes.of sethaploid a with each production includes the union of two cells, productionTheoffspringsexual re-ofby cells contain pairs of homologous chro-homologous of pairs contain cells meiosis tr cie in coined term a , ce and fer-ycle, lentreduc- ytoplasmic ei in a in ei nd fusion,nd ell plate isplate ell otubule tion of the o produce the chro-the ly arrived ly he secre-he micro-g tosis andtosis in mosome ain pairs contacts the cell the n Theon. step 2).step mem- some e d to sin- cles ell ell - s uted among four daughter nuclei. Meiosis accomplishe Meiosis nuclei. daughter four among uted are chromosomes homologous replicated of pair a of the four c in contrast, meiosis, During their parents. ident genetically are and chromosomes homologous of Figure 14.39 Metaphase l Telophase l Anaphase l Prophase l Interphase The stages of meiosis.The haploid cells Metaphase Telophase Anaphase Prophase 4 ll ll ll ll hromatids distrib- ical to ical s thiss 603 feat by incorporating two sequential divisions without an in- maternal and paternal (see metaphase I, Figure 14.39). tervening round of DNA replication (Figure 14.39). In the In the second meiotic division, the two chromatids of each first meiotic division, each chromosome (consisting of two chromosome are separated from one another (anaphase II, chromatids) is separated from its homologue. As a result, each Figure 14.39). daughter cell contains only one member of each pair of ho- A survey of various eukaryotes reveals marked differences mologous chromosomes. For this to occur, homologous chro- with respect to the stage within the life cycle at which meiosis mosomes are paired during prophase of the first meiotic occurs and the duration of the haploid phase. The following division (prophase I, Figure 14.39) by an elaborate process three groups (Figure 14.40) can be identified on these bases: that has no counterpart in mitosis. As they are paired, homol- 1. Gametic or terminal meiosis. In this group, which includes ogous chromosomes engage in a process of genetic recombi- all multicellular animals and many , the meiotic nation that produces chromosomes with new combinations of divisions are closely linked to the formation of the ga- metes (Figure 14.40, left). In male vertebrates (Figure 14.41a), for example, meiosis occurs just prior to the dif- Gametic Sporic Zygotic ferentiation of the spermatozoa. Spermatogonia that are or or or committed to undergo meiosis become primary spermato- terminal intermediate initial cytes , which then undergo the two divisions of meiosis to Gametes (n) produce four relatively undifferentiated spermatids . Each spermatid undergoes a complex differentiation to become Zygote (2n ) the highly specialized cell ( ). In fe- male vertebrates (Figure 14.41 b), oogonia become primary Meiosis oocytes , which then enter a greatly extended meiotic prophase. During this prophase, the primary grows and becomes filled with yolk and other materials. It is only after differentiation of the oocyte is complete (i.e., Diploid Animal Sporophyte the oocyte has reached essentially the same state as when generation (2n ) (2n ) (2n ) it is fertilized) that the meiotic divisions occur. eggs are typically fertilized at a stage before the comple- tion of meiosis (usually at metaphase II). Meiosis is com- pleted after fertilization, while the sperm resides in the egg cytoplasm. Meiosis 2. Zygotic or initial meiosis. In this group, which includes only protists and fungi, the meiotic divisions occur just after fertilization (Figure 14.40, right) to produce haploid spores. The spores divide by mitosis to produce a haploid Spores (n) adult generation. Consequently, the diploid stage of the life cycle is restricted to a brief period after fertilization when the individual is still a zygote.

Haploid Gameto– 3. Sporic or intermediate meiosis. In this group, which in- generation (n) phyte (n) cludes plants and some algae, the meiotic divisions take (n) place at a stage unrelated to either formation or fertilization (Figure 14.40, center). If we begin the life cycle with the union of a male gamete (the pollen grain) and a female gamete (the egg), the diploid zygote under- Meiosis goes mitosis and develops into a diploid sporophyte . At some stage in the development of the sporophyte, sporoge- nesis (which includes meiosis) occurs, producing spores that germinate directly into a haploid gametophyte . The gametophyte can be either an independent stage or, as in the case of plants, a tiny structure retained within the Gametes (n) ovules. In either case, the gametes are produced from the haploid gametophyte by mitosis . Figure 14.40 A comparison of three major groups of organisms based on the stage within the life cycle at which meiosis occurs and the duration of the haploid phase. (T HE CELL IN DEVELOPMENT AND The Stages of Meiosis 14.3 HEREDITY BY WILSON , E DMUND B. C OPYRIGHT 1987. REPRODUCED As with mitosis, the prelude to meiosis includes DNA repli- WITH PERMISSION OF TAYLOR & F RANCIS GROUP LLC - B OOKS IN THE cation. The premeiotic S phase generally takes several times Meiosis FORMAT TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .) longer than a premitotic S phase. Prophase of the first meiotic Chapter 14 Cellular Reproduction 604 sis questions: On what basis do the homologues recognize homologues the do basis what On questions: another. This process of chromosome pairing is calle is process chromosome This of pairing another. homologues of association visible the by marked is to be composed of pairedchromatids. rev arechromosomes the however, microscope, electron chromatids identical of pair a of composed actually chro each that indication no is there stage, earlier a replicated have chromosomes the Although croscope. lig the in visible and compacted become chromosomes sexually reproducing eukaryotes (Figure 14.42). (Figure reproducingsexually eukaryotes si are that stages several into divided customarily The first meiotic prophase is decades. also very comp arrested in the same approximate stage of prophase rem oocytes human many Consequently, . reaches an after so or days 28 every occurs which ovulated, time the to prior just meiosis resume Oocytes rest. prophase initiate oocytes meiosis prior to t and then enter a period of In p example, for prophase. female, mitotic human to compared when fashion nary lengthened typically is I) prophase (i.e., division (spermatogonia or oogonia) from the which gametes d a population by proliferates mitosis to of form go the embryo g population a relatively of small primordial sexes, n i a itiun eet ih motn unanswere important with event intriguing an is and The second stage of prophase I, which is called called is which I, prophase of stage second The The first stage of prophase I is (a) comparison between the formation of sperm and eggs. of sperm the formation between comparison Figure 14.41 Spermatogenesis

Meiosis Spermatogonia divisions Meiotic h tgso aeoeei nvrerts a in vertebrates: stages of gametogenesis The Growth Primary spermatocyte leptotene Secondary spermatocytes Differentiation cells sperm Four Spermatids erm cells present cells erm in , during during which the, feetae Inifferentiate. in extraordi-in rolonged ar- milar in allin milar mosome ismosome for several individual lex and is Mitoses nial cells with onewith d zygotene hy are they n the In . In both synap- ht mi-ht ealed one ant of I ain he he d , homologous chromosomes is found to share a joint te joint a share to found is chromosomeshomologous cells about to enter meiotic prophase are examined, randomly dispersed Whe throughout the nuclear space. ch somesoccupydiscrete individual regionsratherwithin nuclei that 510 page on saw We cells. meiotic particular DNA sequences within the nuclei of preme mosomes are paired. visibly well before gest the DNA breaks occur in leptotene, sug-mice and yeast both in Studies DNAmolecules. aligned b double-stranded of introduction deliberate the is the will first be step discussed in below, genetic re microsc the under visible arrangement this make ply lept during zygotene another during synapsis and Chromosomecompaction one with associated already are homologous regions of DNA from homologous chromosom demonstrat University Harvard at colleagues her initiat and Kl Nancy by cells chromosomes yeast on studies as However, begins synapsis. first chromosomes gous had been assumed for years that interaction between cent studies have shed considerable light on these alig perfectly so occur first homologuesrecognition doesbetween When become pair the does How another? female (female the male ( or three polar bodies. one fertiliz only forms oocyte whereas primary each to four v gives rise generally spermatocyte primary (b) Oogenesis

Thesefindings are supported studiesby aimedloc at Meiosis b ), both meiotic divisions occur after differentiatio both meiotic divisions occur after ), a ,missocr eoedfeetain whereas in meiosis occurs before differentiation, ), Oogonia Body Polar oocyte Primary Growth anddifferentiation oocyte Secondary Body Polar Fertilization iable gametes, able egg and two able egg and two Mitoses combination usin.Itquestions. n. Each Each n. each pair of than beingthan Egg homolo- the chro- iotic and the ek in reaks p.Asope. ed thated n yeast eckner rritory romo- otene. ? Re-? ating ned? sim- es e 605



Synaptonemal complex Leptotene Zygotene Pachytene


Diplotene Diakinesis Metaphase 1 Figure 14.42 The stages of prophase I. The events at each stage are described in the text. distinct from the territories shared by other pairs of homo- telomeres (terminal segments) of leptotene chromosomes are logues. This finding suggests that homologous chromosomes distributed throughout the nucleus. Then, near the end of lep- are paired to some extent before meiotic prophase begins. The totene, there is a dramatic reorganization of chromosomes in many species so that the telomeres become localized at the in- ner surface of the nuclear envelope at one side of the nucleus. Clustering of telomeres at one end of the nuclear envelope oc- curs in a wide variety of eukaryotes and causes the chromo- somes to resemble the clustered stems of a bouquet of flowers 3 (Figure 14.43). Mice carrying mutations that prevent the asso- 4 ciation of chromosomes with the nuclear envelope exhibit de- fects in synapsis, , and gamete formation. These experimental results suggest that the nuclear envelope plays an important role in the interaction between homologous chromosomes during meiosis. Electron micrographs indicate that chromosome synapsis is accompanied by the formation of a complex structure called 5 6 the synaptonemal complex. The synaptonemal complex (SC ) is a ladder-like structure with transverse protein filaments X connecting the two lateral elements (Figure 14.44). The chro- matin of each homologue is organized into loops that extend from one of the lateral elements of the SC (Figure 14.44 b). The lateral elements are composed primarily of cohesin (page 7 584), which presumably binds together the chromatin of the 8 sister chromatids. For many years, the SC was thought to hold each pair of homologous chromosomes in the proper position Figure 14.43 Association of the telomeres of meiotic chromosomes to initiate genetic recombination between strands of homolo- with the nuclear envelope. The chromosomes of a stage of meiotic gous DNA. It is now evident that the SC is not required for prophase of the male grasshopper in which homologous chromosomes genetic recombination. Not only does the SC form after ge- are physically associated as part of a bivalent. The bivalents are netic recombination has been initiated, but mutant yeast cells arranged in a well-defined “bouquet” with their terminal regions unable to assemble an SC can still engage in the exchange of clustered near the inner surface of the nuclear envelope, at the base of genetic information between homologues. It is currently 14.3 the photo. (F ROM B. J OHN , M EIOSIS , © 1990. C AMBRIDGE thought that the SC functions primarily as a scaffold to allow UNIVERSITY PRESS . R EPRINTED WITH PERMISSION . C OURTESY OF interacting chromatids to complete their crossover activities, Meiosis BERNARD JOHN .) as described below. Chapter 14 Cellular Reproduction 606 osse hoais sson ( as shown. nonsister chromatids, (crossing-over) is presumed to occur between the DN Genetic recombi configuration by cohesin (not shown). The mainta loops are likely chromosome are depicted. paired loops of DNA from sister chromatids the two stage i is thought to begin atwhich a much earlier required to complete geneti machinery the enzymatic in the center of the SC (indicated by the arrowheadin the center of the SC (indicated The (recombination dense granules chromosomal fibers. ( kin K, chromosomes ordered array. held in a tightly parallel of bivalent a human pachytene showing a pair of hom Figure 14.44 FROM SJ. chromosomes is called a (Figure 14.44 (Figure paral into DNAextended The is chromatidssister of length their along together closely held are logues the ho pachytene, During formed synaptonemal complex. n te einn o te et tg o pohs I c I, prophase of stage pachytene next the of beginning the and zyg of end the marks synapsis of end The chromatids. pres the to attention calls term latter the whereas homo two contains complex the that fact the reflects (a) (contains cohesin) Lateral element (b) b ) Schematic diagram of the synaptonemal complex and of the synaptonemal diagram ) Schematic OLARI The complex formed by a pair of synapsed homologous synapsed of pair a by formed complex The S PRINGER C, , (Figure 14.44 (Figure , HROMOSOMA homologous chromosome chromatids ofone DNA ofsister The synaptonemal complex. synaptonemal The b ). Under the electron microscope, a number ofnumber a microscope, electron the Under ). S CIE NCE ϩ 130 90 W 1980. 81:330, bivalent a ), which is characterized by a fullya by characterized is which ), B USINESS A C: UTS OFOURTESY or a M EDIA tetrad T IDPERMISSION KIND ITH ( a ) Electron micrograph .) rpaeI Closelyn prophase I. The former term. A in part in part of each of each LBERTO c recombination, A loops from ologous ined in a paired nation nodules) seen its associated ence of fourof ence K nodule Recombination Crossover by the SC.the by etochore. a ) contain lel loopslel logues, otene alled mo- mata te tseicpit yXsae tutrs term other at specific points by X-shaped structures, one to attached chromosomes the leaves which SC, the diss the by recognized is 14.42), (Figure I prophase bivalent in the crossoversindicates thatoccur have presumably The accomp chromatids homologous of each chromosome. bivalents from showing the grasshopper the chiasmat ewe a hoai fo oe ooou ad nonsister These chromatidpoints of from the a other homologue. and homologue one from chromatid a between jun covalent by formed are Chiasmata occurred. ously tween DNA molecules from the two chromosomes had pr crossing- where chromosomes the on sites at located combination, which is completed by which the end of pachytene.combination, ge facilitates that machinery enzymatic the nod contain Recombination 610). (page recombination of steps inte during occurs DNAthat of synthesis associated eviden as place, taking is crossing-over where sites ihn h cne o te C hs srcue hv b have structures These SC. named the of center are the diameter within in nm 100 about bodies electron-dense poe xeta h hamt.(apposed except at the chiasmata. J W P Figure 14.45 bivalent from the locust with three chiasmat F OHN ROM RESS (c) OLF (b) (a) The beginning of of beginning The W R. B, B (singular (singular ERNARD ILEY recombination nodulesrecombination PITDWT PERMISSION WITH EPRINTED IO a E The chrsmatids of each diplotene chromosome. are cl SS & S Visible evidence of crossing-over. evidence Visible 618 94 R 1994. 16:108, . J ONS OHN chiasma .) M, EIOSIS Crossoversites diplotene (chiasmata) (iue 44) hamt are Chiasmata 14.45). (Figure ) PITDWT EMSINOF PERMISSION WITH EPRINTED c © 1990 C, ) Scanning electron micrograph of a because they correspond to theto correspond they because h nx sae f meiotic of stage next the , . C : FROM AMBRIDGE K arw) (a (arrows). ( red within the a LAUS , a formed betweena formed b ) Diplotene U W NIVERSITY anying insetanying ced by theby ced olution ofolution ERNER ed rmediate ei re-netic A over be-over attach- , B chias- ctions : osely seen an- ules evi- een 607 ment provide a striking visual portrayal of the extent of ge- netic recombination. The chiasmata are made more visible by a tendency for the homologues to separate from one another at the diplotene stage. In vertebrates, diplotene can be an extremely extended phase of oogenesis during which the bulk of oocyte growth occurs. Thus diplotene can be a period of intense metabolic activity. Transcription during diplotene in the oocyte provides the RNA utilized for protein synthesis during both oogenesis and early embryonic development following fertilization. Metaphase I Anaphase I During the final stage of meiotic prophase I, called diaki- (a) (b) nesis , the meiotic spindle is assembled and the chromosomes are prepared for separation. In those species in which the Cohesin Kinetochore chromosomes become highly dispersed during diplotene, the chromosomes become recompacted during diakinesis. Diaki- nesis ends with the disappearance of the nucleolus, the break- down of the nuclear envelope, and the movement of the tetrads to the metaphase plate. In vertebrate oocytes, these events are triggered by an increase in the level of the protein Kinetochore kinase activity of MPF (maturation-promoting factor). As discussed in the Experimental Pathways at the end of the chapter, MPF was first identified by its ability to initiate these Metaphase II Anaphase II events, which represent the maturation of the oocyte (page (c) (d) 611). Figure 14.46 In most eukaryotic species, chiasmata can still be seen in Separation of homologous chromosomes during meiosis I and separation of chromatids during meiosis II. homologous chromosomes aligned at the metaphase plate of (a) Schematic diagram of a pair of homologous chromosomes at meiosis I. In fact, chiasmata are required to hold the homo- metaphase I. The chromatids are held together along both their arms logues together as a bivalent during this stage. In humans and and centromeres by cohesin. The pair of homologues are maintained as other vertebrates, every pair of homologues typically contains a bivalent by the chiasmata. Inset micrograph shows that the at least one chiasma, and the longer chromosomes tend to kinetochores (arrowheads) of sister chromatids are situated on one side have two or three of them. It is thought that some mechanism of the chromosome, facing the same pole. The black dots are gold exists to ensure that even the smallest chromosomes form a particles bound to the motor protein CENP-E of the kinetochores (see chiasma. If a chiasma does not occur between a pair of homol- Figure 14.16 c). ( b) At anaphase I, the cohesin holding the arms of the ogous chromosomes, the chromosomes of that bivalent tend chromatids is cleaved, allowing the homologues to separate from one to separate from one another after dissolution of the SC. This another. Cohesin remains at the centromere, holding the chromatids premature separation of homologues often results in the for- together. ( c) At metaphase II, the chromatids are held together at the centromere, with microtubules from opposite poles attached to the two mation of nuclei with an abnormal number of chromosomes. kinetochores. Inset micrograph shows the kinetochores of the sister The consequences of such an event are discussed in the chromatids are now on opposite sides of the chromosome, facing accompanying Human Perspective. opposite poles. ( d ) At anaphase II, the cohesin holding the chromatids At metaphase I, the two homologous chromosomes of together has been cleaved, allowing the chromosomes to move to each bivalent are connected to the spindle fibers from opposite opposite poles. (I NSETS : F ROM JIBAK LEE ET AL ., M OL . R EPROD . poles (Figure 14.46 a). In contrast, sister chromatids are con- DEVELOP . 56:51, 2000. R EPRINTED WITH PERMISSION OF JOHN nected to microtubules from the same spindle pole, which is WILEY & S ONS .) made possible by the side-by-side arrangement of their kine- tochores as seen in the inset of Figure 14.46 a. The orientation of the maternal and paternal chromosomes of each bivalent on the metaphase I plate is random; the maternal member of a tween sister chromatids in regions that flank these sites of re- particular bivalent has an equal likelihood of facing either combination (Figure 14.46 a). The chiasmata disappear at the pole. Consequently, when homologous chromosomes separate metaphase I–anaphase I transition, as the arms of the chro- during anaphase I, each pole receives a random assortment of matids of each bivalent lose cohesion (Figure 14.46b). Loss of maternal and paternal chromosomes (see Figure 14.39). Thus, cohesion between the arms is accomplished by proteolytic anaphase I is the cytological event that corresponds to cleavage of the cohesin molecules in those regions of the chro- Mendel’s law of independent assortment (page 388). As a re- mosome. In contrast, cohesion between the joined cen- sult of independent assortment, organisms are capable of gen- tromeres of sister chromatids remains strong, because the erating a nearly unlimited variety of gametes. cohesin situated there is protected from proteolytic attack 14.3 Separation of homologous chromosomes at anaphase I (Figure 14.46 b). As a result, sister chromatids remain firmly requires the dissolution of the chiasmata that hold the biva- attached to one another as they move together toward a spin- Meiosis lents together. The chiasmata are maintained by cohesion be- dle pole during anaphase I. Chapter 14 Cellular Reproduction 608 nondisjunction haploid cells are affected. (More complex types of n (More complex types haploidare cells affected. division, which is called is called which division, If nondisjunction the sec occurs during chromosomes. wh the first meiotic division, occurs during separate shown here also occur.) can the two meiotic divisions is called called is divisions meiotic two the The stage may or may not reform during telophase I. nuclear The nucleus. interphase the of state tended extreme the reach not do they dispersion, some dergo ofte chromosomes Although mitosis. of telophase than condition known as zyg the into an infant whose have cells an chromosabnormal however, cases, few a In birth. st and conception some at dies that embryo abnormal an into velops cases, most In arise. consequences serious and forms, a with zygote an abnormal number of c normal gamete, t happens gametes these of one If 1). (Figure mosome miss a or chromosome extra an chromosomes—either of gametes are formed that contain an ab ations occurs, eithe When meiosis II. may during fail to come apart fail to separate from o each other during meiosis I, autosomes. abno an of effects the considering by Webegin will chromosome 21, for example, has trisomy 21. A missing produces A chromosomes) 45 of total a (producing some 21. trisomy has example, for 21, chromosome a as to con a creates chromosomes) 47 of total a (producing extra An chromosomes. sex of pairone autosomes and pend chromosome on which or chromosomes are affecte oe bcm rcmatd n ln u a te metapha the at up line and recompacted become somes c The again. down broken is it I, telophase in formed envelo nuclear the If predecessor. its than prophase a twice and has a 1C DNA which content.much cell, as a sperm content, DNA 4C a has which spermatocyte, half as much as a p said to have a 2C amount of DNA, by spermato Secondary a pair of attached chromatids. rep still is chromosome each because gamete haploid EvenDNA much as twice have they chromosomes. haploid, are they though homologous of pair each of member contain they because haploid being as characterized to as stage fleeting this in cells animals, In short-lived. E V I T C E P S R E P N A M U H E H T Telophase I of meiosis I produces less dramatic cha dramatic less produces I meiosis of TelophaseI h omlhmncrmsm opeeti 6 22 pai The normal human chromosome complement is 46: Interkinesis is followed by prophase II, a much simp much a II, prophase by followed is Interkinesis secondary spermatocytessecondary Figure 1 alt eaaefo ahohrdrn eoi.If fail meiosis. from to separate other during each trisomy , all of the haploid cells have an abnormal number o have an abnormal of the haploid cells all , Meiotic nondisjunction occurs when chromosomes Fgr 2.A esn hs cls oti a extra an contain cells whose person A 2). (Figure aneuploidy omn oooos hoooe may chromosomes Homologous common. meiotic and mistakes in humans appear to be surprisingly process, complex a is Meiosis secondary nondisjunctionsecondary The consequences of aneuploidy de-. or secondary oocytessecondary Meiotic NondisjunctionMeiotic and Its Consequences interkinesis , only two of the four two only , ich is called is called ich ondisjunction than r sister chromatids and is generally is and These cells are. ond meiotic rmal number ofnumber rmal normal normal number m ubr aome number, the failure to dition referreddition r of these situ- the zygote de- zygote the o fuse with awith fuse o chromosome a hromosomes g betweenage are referredare ote developsote d. pe had re-had pe only one only monosomy envelope n chro-ing cytes cytes are resented between chromo- hromo- primary rimary ly ex- ly un-n f as aas nges rs of ler se s . of Caof Fertilizationto leads fertilized. is egg) an called oocyt the when only released is arrest II Metaphase and the cells cannot progress to i the next tained, meiotic stage. activity Cdk oocyte, the within high remain levels as long As degradation. B cyclin preventing thereby tg uigebynco ea eeomn.Consequ development. fetal or embryonic during stage invariably proves to b which chromosome is affected, o oe oad poie oe o te el Fgr 1 (Figure cell the of poles opposite toward move to allow chromatidssistertogether, the held had which cen the of splitting synchronous the with begins II A changes by completing the second meiotic division. responds egg fertilized The B. cyclin of destruction meiosis are with a 1C amount haploid of nucle cells are once again enclosed by The a p nuclear envelope. in thewhich chro Meiosis II ends with telophase II, ruh aot y atr ta ihbt APC inhibit metaphase that at factors by meiosis about of brought arrest The II. metaphase at 14.46 tached to opposing sets of beco chromosomal spindle fiber and poles opposite sister face II of metaphase kinetochores of matids the I, metaphase Unlike plate. chromosome with extra Gametes The absence of one autosomal chromosome, regardless regardless chromosome, autosomal one of absence The omlNormal Normal 2 c .The progression of ). meiosis in vertebrate oocytes ϩ odsucinNormal Nondisjunction ions, the activation of APCof activation the ions, chromosome missing a Gametes division division Second meiotic meiotic First gametes Normal omlNondisjunction Normal Cdc20 (page 592), and the and 592), (page with extra mosome Gamete Cdc20 chro- e lethal at some a rapid influx rapid a activation, ently, a zy- a ently, roducts of ar DNA. tromeres, mosomes ing theming mosome to theseto s (Figure Gamete missing a chro- cyclin B cyclin naphase main-s 4.46 (nowe e at-me chro- stops I is II d of ).