Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993
Cell Growth and Division
Mechanics of Cell Division
In this final section, we will discuss the events of the M phase, which is the culmination of the Cell Cycle. During this relatively short period, the Chromosomes condense, and the Contents of the parent cell—having doubled due to synthetic activity in the preceding interphase—are distributed between two daughter Cells (Fig. 13-42).
Apparently, at THE MOLECULAR LEVEL, the M phase is initiated by a protein phosphorylation cascade triggered by the appearance of the M-phase-promoting factor (MPF), and it ends with dephosphorylation, which returns the Proteins to their interphase state (Sec. 13.2.5). In turn, protein phosphorylation during the M phase is likely responsible for many of the morphological changes accompanying mitosis, including chromosome Condensation, nuclear envelope breakdown, and the cytoskeletal changes described below. The first clearly visible manifestation of the onset of the M phase is the gradual compaction of dispersed interphase Chromatin into thread-like chromosomes. This chromosome condensation is essential for their subsequent orderly segregation into the daughter cells and is accompanied by the phosphorylation of numerous histone H1 molecules present in The Cell (up to six phosphate groups per H1 molecule). Because histone H1 is present at a ratio of approximately one molecule per nucleosome and is known to be involved in nucleosome packing (Sec. 13.2.5), its phosphorylation by MPF kinase (Sec. 9.1.12) at the beginning of the M phase must be a major driver of chromosome condensation. This molecular explanation, while still hypothetical, demonstrates the fundamental level at which the entire cell cycle must ultimately be described.
Someone once remarked that chromosomes in mitosis resemble a corpse at a funeral: they provide the occasion for action, but do not take an active part in it. The active role belongs to two special cytoskeletal structures that temporarily form during the M phase. The first to appear is the bipolar mitotic spindle, consisting of microtubules and associated proteins. Initially, it aligns the replicated chromosomes in the Cell Division plane; then, each chromosome splits into two daughter chromosomes, which are pulled by the spindle fibers to opposite sides of the cell. The second cytoskeletal Structure required in animal cell M phase is the contractile ring of Actin and Myosin filaments, which appears slightly later beneath The Plasma Membrane. This ring pinches the membrane inward, dividing the cell in two and thereby ensuring that each daughter cell receives not only a complete set of chromosomes, but also half of the parent cell's Cytoplasm and Organelles. These two cytoskeletal structures contain different sets of proteins and, in some specialized cells, can form independently of one another. However, their formation is usually closely coordinated, so that cytoplasmic division (cytokinesis) occurs immediately after the completion of nuclear division (mitosis)—the latter also applies to plant cells, although, as we will see, their rigid walls require a different mechanism of cytokinesis.
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Fig. 13-42. The M phase of the cell cycle begins after the G2 phase and ends by the onset of the G1 phase of the next cycle. It consists of five stages of nuclear division (mitosis) and cytoplasmic division (cytokinesis).
The description just given referred to Eukaryotic cells. Bacterial cells contain neither actin filaments nor microtubules; they usually have only a single chromosome, and following its Replication, the two copies are distributed between the daughter cells by a mechanism involving the attachment of the chromosome to the bacterial plasma membrane (see Sec. 13.5.18). The complex mitotic apparatus probably became necessary only with The Emergence of cells containing a much larger amount of DNA distributed across several distinct chromosomes. The primary function of this apparatus is to accurately distribute the replicated chromosomes between the two daughter cells. The fidelity of this distribution has been studied in Yeast cells, where it was found that only about one error occurs per 105 cell divisions.
13.5.1. The M phase is traditionally subdivided into six stages [35]
The basic strategy of cell division in eukaryotic organisms is remarkably constant. The first five Stages of the M phase comprise mitosis, and the sixth is cytokinesis. These six stages form a dynamic sequence whose complexity and beauty are difficult to appreciate from descriptions or a series of static images. The description of mitosis is based on Two Types of observations: light Microscopy of living cells (often combined with time-lapse cinematography) and light and Electron microscopy of fixed and stained cells. The various stages of cell division are briefly outlined in Scheme 13-1. The five stages of mitosis—prophase, prometaphase, metaphase, anaphase, and telophase—occur in a strictly defined order; cytokinesis begins during anaphase and continues until the end of the mitotic cycle (Fig. 13-43). Light micrographs of dividing typical animal and plant cells are shown in Figs. 13-44 and 13-45, respectively.
Countless variations of all the division stages summarized in Scheme 13-1 are found throughout the animal and plant kingdoms. We will mention some of these variations as we take a closer look at the MECHANISMS OF CELL division, as they can help us understand the operation of the various PARTS OF THE mammalian mitotic apparatus.
13.5.2. The formation of the mitotic spindle during the cell's M phase is accompanied by dramatic Changes in the dynamic properties of microtubules [36]
From Chapter 11, we know that the major microtubule-organizing center in most animal cells is the centrosome—an accumulation of amorphous material surrounding a pair of centrioles (Sec. 11.4.4). During interphase, centrosomal material initiates the growth of microtubules directed toward the cell periphery, while their initial segments (minus ends) remain anchored to the centrosome. This interphase array of microtubules radiating from the centrosome is a dynamic, constantly changing structure in which individual microtubules are continuously polymerizing and depolymerizing. New microtubules grow by The addition of tubulin molecules to their plus ends; sporadically and seemingly at random, individual microtubules become unstable and undergo rapid "catastrophic" depolymerization, returning their subunit molecules to the pool of unpolymerized tubulin contained in the cytoplasm (see Scheme 11-2).
Fig. 13-43. The time course of mitosis and cytokinesis typical of a mammalian cell. Exact figures vary for different cell types. Note that cytokinesis begins even before mitosis is complete. The onset of prophase (and thus of the M phase as a whole) is considered to be the point in the cell cycle when condensed chromosomes first become visible; this is a somewhat arbitrary criterion, as the degree of chromosome condensation progressively increases already in late G2 phase.
Fig. 13-44. These light micrographs of cultured marsupial cells (PtK cells) show the progression of mitosis in an animal cell. Microtubules are visualized by immunogold staining; chromatin is stained with toluidine blue. The main events of mitosis at the Light Microscope level have been known for over 100 years. In interphase, the centrosome, containing a pair of centrioles, serves as the center of the interphase microtubule array. In early prophase, the single centrosome contains two pairs of centrioles (not visible in the micrograph); in late prophase, the centrosome replicates, and the resulting asters move apart. In prometaphase, the nuclear envelope breaks down, allowing spindle microtubules to interact with the chromosomes. In metaphase, the bipolar STRUCTURE OF THE spindle is clearly visible, and all chromosomes are aligned at its equatorial plane. In early anaphase, all chromatids simultaneously separate and begin moving toward the poles driven by spindle fibers. During late anaphase, the spindle poles move further apart, pushing the two groups of chromatids even further apart. In telophase, daughter nuclei form, and in late telophase, cytokinesis is nearly complete; a midbody persists between the daughter cells. (Photographs kindly provided by M. de Brabander.)
Fig. 13-45. The progression of mitosis in a typical plant cell. Micrographs of a living *Haemanthus* (Liliaceae) cell obtained using differential Interference contrast microscopy (Sec. 4.1.5). The unusually large chromosomes in this cell are easy to observe.
Fig. 13-46. The centrosome cycle. In an interphase cell, the centrosome duplicates to form the two poles of the mitotic spindle. In most animal (but not plant) cells, a pair of centrioles (shown as a pair of short black segments) is embedded in the centrosomal material (highlighted in color) from which microtubules grow. At a certain point in the G1 phase, the two centrioles move apart by a few microns. During the S phase, a daughter centriole begins to form near each parental centriole at a right angle to it. The growth of daughter centrioles is usually completed by the G2 phase. Initially, both pairs of centrioles remain embedded in a single mass of centrosomal material forming one centrosome. In the early M phase, each pair of centrioles becomes part of a separate microtubule-organizing center from which a radial array of microtubules—an aster—radiates. The two asters, initially lying side by side near the nuclear envelope, now move apart. In late prophase, bundles of polar microtubules belonging to the two asters and interacting with one another selectively elongate as the two centers migrate to opposite sides of The Nucleus. In this manner, the mitotic spindle is rapidly formed.
PROPHASE
The transition from the G2 phase to the M phase, as observed under the microscope, occurs gradually. Chromatin, which appears diffuse during interphase, condenses into distinctly visible chromosomes. A characteristic and strictly constant number of chromosomes is found in each species. During the preceding S phase, each chromosome has duplicated and now consists of two sister chromatids. Each chromatid contains a specific DNA region called the centromere, which is essential for their proper segregation. At the end of prophase, the Cytoplasmic microtubules that make up part of the interphase Cytoskeleton disassemble, and the assembly of the spindle—the main component of the mitotic apparatus—begins. The spindle is a bipolar structure consisting of microtubules and associated proteins. Spindle assembly initially takes place outside the nucleus.
Scheme 13-1. The six stages of cell division.
PROMETAPHASE
Prometaphase begins with the rapid breakdown of the nuclear envelope into small membrane vesicles indistinguishable from fragments of The Endoplasmic reticulum. These vesicles remain visible near the spindle throughout mitosis. The spindle microtubules, which were previously outside the nucleus, can now invade the nuclear region. Specialized Protein Complexes called kinetochores form at the centromere of each chromosome; they attach to a subset of spindle microtubules, which are now referred to as kinetochore microtubules. The remaining spindle microtubules are called interpolar microtubules, and those lying outside the spindle are called astral microtubules. Kinetochore microtubules extend in opposite directions from the two sister chromatids of each chromosome and pull them in opposite directions, resulting in vigorous chromosome movement.
METAPHASE
Kinetochore microtubules ultimately align each chromosome in the equatorial plane, halfway between the spindle poles. Here, the chromosomes form the metaphase plate, where they are held in position by the tension exerted by kinetochore microtubules extending from them toward opposite spindle poles.
ANAPHASE
Triggered by a specific signal, anaphase begins with the sudden Separation of the paired kinetochores of each chromosome, after which its two chromatids slowly begin to move toward their respective poles. All chromatids move at a uniform rate of approximately 1 µm/min. Two distinct types of movement can be distinguished here. During anaphase A, kinetochore microtubules shorten as the chromosomes approach the poles. During anaphase B, the polar microtubules elongate, pushing the spindle poles even further apart. Anaphase typically lasts only a few minutes.
TELOPHASE
In telophase (from the Greek telos — end), the separated daughter chromatids reach the poles, and the kinetochore microtubules disappear. The polar microtubules continue to elongate, after which a new nuclear envelope forms around each group of daughter chromatids. The condensed chromatin begins to decondense, nucleoli (which disappeared during prophase) reappear, and mitosis comes to an end.
CYTOKINESIS
This is The process of cytoplasmic division, which typically begins sometime during anaphase. The diagram illustrates how this process occurs in animal cells. The plasma membrane in the equatorial region (between the two daughter nuclei) begins to invaginate inward toward the spindle axis, resulting in the formation of a Cleavage furrow. This furrow gradually deepens until it reaches the remnants of the spindle located between the nuclei. This bridge, known as the midbody, may persist for some time before ultimately breaking down, leading to the complete separation of the daughter cells.
Fig. 13-47. A model for the formation of a bipolar mitotic spindle through the selective stabilization of interacting microtubules.
New microtubules sprout in random directions from two centrosomes (represented by circles), to which they are anchored by their minus ends. Their plus ends are "dynamically unstable," rapidly transitioning between periods of steady growth and swift shortening, which frequently results in the complete depolymerization of the microtubule (Section 11.4.3). When two microtubules originating from opposite centrosomes interact within their overlap zone, microtubule-associated proteins cross-link them (shown in grey), thereby capping and stabilizing their plus ends and reducing the likelihood of depolymerization.
As shown in Fig. 13-46, the centrosome undergoes structural changes throughout1 the cell cycle. Sometime during S phase, the centriole pair replicates while remaining housed within a single aggregate of centrosomal material. In prophase, the centrosome splits, and each daughter centrosome becomes the organizing center for a separate aster—a microtubule-based structure whose ends are embedded in the centrosomal material. The microtubules of both asters elongate until they come into contact with one another, after which the two centrosomes move apart. Subsequently, in prometaphase, the nuclear envelope breaks down, allowing microtubules from each centrosome to penetrate the nucleus and interact with the chromosomes. The two daughter centrosomes are now referred to as the two spindle poles.
These events are thought to be driven by profound changes in microtubule stability and centrosomal properties that occur during prophase. As previously noted, evidence suggests that the MPF factor triggers the transition into M phase by initiating a phosphorylation cascade involving a multitude of proteins (Section 13.2.5).
This process results in the phosphorylation of several microtubule-interacting molecules. Consequently, upon the cell's entry into prophase, the half-life of an average microtubule drops roughly 20-fold (from about 5 min to 15 s, see Fig. 13-48). This is likely linked to a sharp increase in the probability that a typical growing microtubule will begin to shorten due to an alteration at its plus end (Section 11.4.3, Scheme 11-2), alongside prophase-induced changes in the centrosome that dramatically enhance its capacity to nucleate new microtubules (as can be observed in vitro). These two changes are sufficient to explain why the onset of M phase is marked by a rapid shift from a relatively small number of long microtubules radiating from the centrosome to the cell periphery (the interphase microtubule array) to a large population of short microtubules surrounding each centrosome (see prophase in Fig. 13-46).
During mitosis, the elongating ends of microtubules emanating from the spindle poles are thought to encounter structures that bind to them and protect them from catastrophic depolymerization. Because each pole emits microtubules in various directions, this selective stabilization generates the characteristic bipolar shape of the mitotic spindle, in which the majority of microtubules extend from the two poles toward the equatorial plate situated midway between them. Those microtubules that cross the equator can be selectively stabilized by accessory proteins that cross-link adjacent, parallel microtubules of opposite polarity (Fig. 13-47). By metaphase, the spindle in higher animal and plant cells may contain up to several thousand microtubules, whereas in certain Fungi there are only about 40.
Although some spindle microtubules are partially stabilized against spontaneous disassembly, the majority continuously exchange their subunits with the pool of soluble tubulin molecules in the Cytosol. This turnover can be directly measured using the technique illustrated in Fig. 13-48. It can also be demonstrated by exposing mitotic cells to conditions that reversibly shift the equilibrium between tubulin polymerization and depolymerization, and observing the birefringence of spindle microtubules under polarized light (Fig. 13-49). If mitotic cells are placed in heavy Water (D2O) or treated with taxol (treatments that suppress microtubule disassembly), the spindle fibers elongate. Such stabilized spindles are incapable of pulling chromosomes, and mitosis arrests. Mitosis is similarly blocked by the exact opposite Treatment—when spindle fibers are reversibly disrupted using one of three agents that inhibit tubulin assembly into microtubules: colchicine, cold Temperature, or high hydrostatic pressure. The fact that neither stabilized nor depolymerized spindle microtubules can successfully move chromosomes indicates that proper spindle function requires a delicate balance between assembly and disassembly. Before examining The Mechanism of these movements, we will describe in greater detail the Organization OF THE spindle and the arrangement of chromosomes during mitosis.
Fig. 13-48. Experimental results demonstrating that microtubules in M-phase cells are significantly more dynamic than those in interphase cells. Cultured mammalian cells were microinjected with tubulin covalently linked to a fluorescent dye. After allowing sufficient time for the fluorescent tubulin to be incorporated into microtubules, all fluorescence within a small localized region was bleached using an intense laser beam. The recovery of fluorescence in the irradiated microtubule region, driven by the replacement of bleached subunits with unbleached fluorescent tubulin from the soluble subunit pool, was subsequently plotted as a function of time. The time required for 50% fluorescence recovery, t1/2, is believed to equal the time needed for half of the microtubules in that region to depolymerize and recover. (Based on W. M. Saxton et al., J. Cell Biol. 99: 2175–2187, 1984; by copyright permission of the Rockefeller Univ. Press.)
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13.5.3. During mitosis, chromosomes attach to microtubules via their kinetochores [37]
Replicated chromosomes attach to the mitotic spindle via specialized structures called kinetochores. At the onset of M phase, each chromosome consists of two sister chromatids paired along their entire length, though joined primarily near their centromeres—specialized DNA sequences essential for proper chromosome segregation. In late prophase, a single mature kinetochore forms on each centromere, meaning there are now two kinetochores (one on each sister chromatid) oriented in opposite directions. By metaphase, microtubules are attached to each kinetochore (Fig. 13-50). In most organisms, the kinetochore is a large, multi-protein complex that appears in electron micrographs as a trilaminar, plate-like structure (Fig. 13-51). The number of microtubules associated with each kinetochore varies considerably among species: for instance, human cells have between 20 and 40, whereas yeast and certain other microorganisms have only one, meaning a single microtubule is sufficient to pull a chromosome.
Fig. 13-49. Isolated metaphase spindle visualized using three different light Microscopy Techniques: differential interference contrast (A), phase contrast (B), and polarized light microscopy (C). (Courtesy of E. D. Salmon and R. R. Segall, J. Cell Biol. 86: 355–365, 1980. Repr. by copyright permission of the Rockefeller Univ. Press.)
Fig. 13-50. Schematic representation of a metaphase chromosome with its two sister chromatids, to which kinetochore microtubules are attached.
The information that dictates the specific Construction of a kinetochore at a particular chromosomal site must be encoded within the centromeric DNA sequence itself. In yeast, centromeric DNA can be identified genetically by its ability to ensure the stable inheritance of Plasmids; without it, plasmids are distributed unequally between daughter cells and are eventually lost. Molecular genetic techniques have demonstrated that all 17 chromosomes of the yeast Saccharomyces cerevisiae contain distinct centromeric sequences approximately 110 Base Pairs in length (Fig. 13-52). Nonetheless, all these sequences share significant homologous regions that can be inverted or translocated from one chromosome to another without loss of function. The yeast centromeric sequence binds specific proteins that presumably initiate the assembly of a multi-protein complex (the kinetochore), which in turn attaches to the plus end of a single microtubule. In mammals, centromeres are thought to consist of much longer and different DNA sequences, forming larger kinetochores capable of binding numerous microtubules.
An unexpected opportunity to study mammalian kinetochore proteins arose with the discovery that patients suffering from certain forms of scleroderma (a disease of unknown Etiology characterized by progressive fibrosis of the Connective Tissue of the Skin and other Organs) produce Antibodies that specifically react with kinetochores. When such fluorescently labeled antibodies are used to stain dividing cells, a characteristic pattern of fluorescent spots emerges, each marking THE POSITION OF a kinetochore. The same punctate pattern is observed in non-dividing cells, where the number of spots per cell corresponds to its chromosome number (Fig. 13-53), suggesting that a kinetochore precursor is associated with each centromere even in interphase nuclei. Scleroderma antibodies have also enabled the cloning of genes encoding several of the numerous proteins associated with kinetochores. Consequently, these typically rare proteins can now be produced in large quantities using Recombinant DNA technology and subsequently studied for their interactions with each other, with DNA, and with microtubules.
Fig. 13-51. Kinetochores. (A) A metaphase chromosome stained with human autoantibodies reacting with specific kinetochore proteins reveals two kinetochores, each associated with its respective chromatid (S). (B) An electron micrograph of an anaphase chromatid with microtubules attached to the kinetochore. Although most kinetochores are trilaminar, the one shown here (from a green alga) has an unusually complex structure with additional layers. (A and B courtesy of Bill Brinkley; C from J.D. Pickett-Heaps, L. C. Fowke, Aust. J. Biol. Sci. 23: 71-92, 1970. Repr. by permission of CSIRO.)
Fig. 13-52. DNA sequence of a typical centromere in the yeast Saccharomyces cerevisiae. The sequence shown here is sufficient to ensure proper chromatid segregation; it serves for the assembly of kinetochore proteins to which a single microtubule attaches.
Fig. 13-53. Immunofluorescence staining of kinetochores in interphase cells using antibodies that specifically bind to a kinetochore protein. The marsupial cells used have a relatively low chromosome number. (A) In cells at the G1 phase, one kinetochore per chromosome is stained. (B) In the G2 phase, two kinetochores per chromosome are stained. (S. L. Brenner, B. B. Brinkley, Cold Spring Harbor Symp. Quant. Biol. 46: 241-254, 1982.)
How do microtubules and kinetochores connect with one another? Their binding exhibits A number of unique features. If chemically labeled tubulin is injected into a mitotic cell in metaphase, it is continuously incorporated into microtubules near their attachment site at the kinetochore (Fig. 13-54). As we will see later, the reverse reaction takes place in anaphase: tubulin molecules dissociate from the microtubule at a region close to the kinetochore, causing the latter to move toward the spindle pole. It is somewhat puzzling that the kinetochore, despite the continuous addition and removal of tubulin molecules, maintains a strong mechanical attachment to microtubules—after all, it is via this attachment point that they pull chromosomes through the cytoplasm. Thus, the kinetochore appears to act somewhat like a sliding collar, maintaining lateral contact with the subunits of the polymerized tubulin near the microtubule end while simultaneously allowing tubulin molecules to be added or removed at that end (see Fig. 13-61 below).
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13.5.4. Kinetochores appear to capture the plus ends of microtubules emanating from the spindle pole [38]
The breakdown of the nuclear envelope, which marks the end of prophase and the beginning of prometaphase, allows the mitotic spindle to interact with the chromosomes. The ultimate outcome of this interaction is that each daughter nucleus receives precisely one chromatid of each chromosome. In this chromatid-segregation process, kinetochore-associated microtubules play a crucial role by: 1) orienting each chromosome relative to the spindle axis so that its two kinetochores face opposite poles of the cell, and 2) moving each chromosome to the equatorial plane of the cell, where they form the metaphase plate. In mammalian cells, this process takes 10–20 minutes and is completed by the end of prometaphase.
Fig. 13-54. Experiment demonstrating that metaphase kinetochore microtubules grow from the end attached to the kinetochore (the plus end). Mammalian metaphase cells in vitro were injected with tubulin covalently linked to a small organic molecule (biotin). After 1 min, the cells were fixed and stained with gold particle-linked antibodies against biotin, and then sectioned for electron microscopy. Regions of microtubules that incorporated biotinylated tubulin During the first minute after injection are densely studded with dark gold dots (colored arrows), whereas pre-existing regions remain unstained (black arrows). (Micrograph kindly provided by Luise Evans.)
Prometaphase is characterized by exceptionally high spindle activity, as the spindle seemingly strives to capture all chromosomes and align them into a metaphase plate. Indeed, chromosomes vigorously oscillate and move back and forth between the poles because their kinetochores are attached to microtubules growing from both opposite poles of the spindle, pulling them in opposing directions. Initial attachment of a chromosome typically occurs when it is located near one of the poles, at which point microtubules connect to only a single kinetochore; eventually, the second kinetochore also binds microtubules growing from the opposite pole. These random chromosomal movements during prometaphase and their eventual random orientation ensure the stochastic distribution of chromatids between daughter cells, which is essential for Gene reassortment during the analogous nuclear division in Meiosis (Section 15.2.7).
Only the plus ends of microtubules extend from the pole, and it is these ends that bind to the kinetochores. Thus, the kinetochore acts as a "cap" that to some extent protects the plus end of the microtubule from depolymerization, much in the same way that the centromere at the spindle pole protects the minus end from depolymerization. It is hardly surprising, therefore, that kinetochore-attached microtubules, capped at both ends, are remarkably stable. Other spindle microtubules (referred to as interpolar microtubules) are less stable.
Although kinetochore microtubules tend to pull the chromosome toward the corresponding pole (see below), some other force apparently repels chromosomes that approach a pole too closely. If chromosome arms are severed from the kinetochore by laser microsurgery, they exhibit a clear tendency to move away from the nearest spindle pole, even when detached from microtubules or any other cellular structure. One possible explanation is that the rapid polymerization of spindle microtubules directed away from each pole creates a "bulk flow" that sweeps any large unattached structure, such as chromosome arms, further away from the poles.
13.5.5. Sister chromatids attach their kinetochores to opposite poles of the spindle [39]
In early prometaphase, both kinetochores of a single chromosome may attach to fibers originating from the same spindle pole. However, such abnormal configurations, which would lead to errors in chromosome segregation, are almost invariably corrected. Apparently, a balanced arrangement in which sister kinetochores attach to different spindle poles is the most stable state. The potential reason for this is indicated by experiments investigating the mechanism of chromosome attachment to the mitotic spindle.
Elegant experiments in which living mitotic cells had their chromosomes gently pulled or pushed using ultra-fine Glass needles demonstrated that a given kinetochore does not need to point toward a specific pole like a magnetic compass needle: if a chromosome is experimentally turned around, the same kinetochore can establish a connection with the opposite pole. Moreover, during prometaphase, both kinetochores of a chromosome can be forced by micromanipulation to bind to the same spindle pole. If such an abnormal attachment persists, the entire chromosome (as a pair of connected sister chromatids) will move toward the corresponding pole. Typically, however, such a connection is unstable—new microtubules from the other pole usually attach to the chromosome to establish a proper balanced configuration. Conversely, if the movement of an improperly attached chromosome is physically impeded by a glass needle, its connection to a single pole becomes stable: presumably, the association between microtubules and the kinetochore is reinforced by the poleward-directed pulling force. Therefore, only chromosomes connected to both poles maintain persistent microtubule attachments and thus interact stably with the spindle.
The tension generated by microtubules from opposing poles not only stabilizes the interaction between these microtubules and the kinetochores but ultimately drives each chromosome into the plane of the metaphase plate, as will be discussed next.
13.5.6. Balanced poleward-directed forces hold chromosomes at the metaphase plate [40]
Why do chromosomes align equidistant from both spindle poles during metaphase, forming the metaphase plate? Experiments involving the displacement of chromosomes with a glass needle indicate that the force exerted on a kinetochore is proportional to the length of the attached fibers—that is, it decreases as the kinetochore gets closer to the pole to which it is connected (Fig. 13-55). Each chromosome is connected as if by a "spring" to each of the two spindle poles, so that any displacement toward one pole generates an opposing restorative force in the opposite direction. The spindle configuration resulting from this balance of forces in metaphase is illustrated in Fig. 13-56.
These forces continue to act on chromosomes even after they have arranged themselves into the metaphase plate. Consequently, chromosomes undergo oscillatory movements in both directions while maintaining a force equilibrium. If the fibers attached to one of a pair of metaphase kinetochores are severed by a laser beam, the entire chromosome immediately begins to move toward the unperturbed pole. Similarly, if two chromatids are artificially separated during metaphase, they immediately start moving toward opposite poles, just as in anaphase. These findings suggest that as soon as the two kinetochores of each chromosome separate, the chromatids begin to segregate toward the poles under the Influence of the very same forces that previously established the metaphase plate.
Fig. 13-55. Chromosomes randomly enter the spindle during prometaphase and eventually align at the equatorial plane of the spindle, because the force acting on each kinetochore is smaller the closer it is to the pole. Therefore, chromosomes that end up at the equator are held there by balanced forces of attraction toward the two poles.
Fig. 13-56. Simplified diagram of a mitotic spindle in metaphase. The spindle is constructed from two half-spindles (shown in black and red), each of which includes kinetochores, polar microtubules, and astral microtubules. Microtubule polarity is indicated by the direction of the arrows. Polar spindle fibers extending from opposite poles have an overlap zone (shown in gray) where microtubule-associated proteins can cross-link them. Note that the microtubules are antiparallel in this zone.
Metaphase occupies a significant portion of the mitotic period (see Fig. 13-43), as if cells were waiting for all their chromosomes to properly position themselves at the equatorial plane. Several experiments support this view. Many cells arrest in mitosis for several hours or days if treated with microtubule-depolymerizing agents such as colchicine or vinblastine; in fact, this method of cell cycle arrest is widely used when A large number of mitotic cells are needed for cytological analysis of their condensed chromosomes (Section 9.2.3). Upon removal of the agent, the mitotic spindle rapidly regenerates, and normal mitosis frequently resumes as soon as the chromosomes are correctly arranged at the metaphase plate. It has been suggested that a chromosome with an unattached kinetochore serves as a source of a diffusible factor that normally delays the transition to anaphase, providing extra time for proper attachment. If such a factor exists, then treatment with spindle-disrupting agents would be expected to produce a potent signal leading to the prolongation of metaphase.
Fig. 13-57. Chromosome segregation in anaphase of a Haemanthus endosperm cell. Upon transition from metaphase (A) to anaphase (B), chromosomes are pulled apart by polar microtubules. Cells are stained with gold-labeled antibodies against tubulin. (Courtesy of Andrew Bajer.)
13.5.7. Sister chromatids suddenly separate in anaphase [41]
As we have just seen, metaphase is a relatively stable state, and under normal conditions, many cells remain in this stage for an hour or more while their chromosomes undergo only oscillatory movements within the metaphase plate. Anaphase begins with the sudden, synchronous splitting of all chromosomes into sister chromatids, each with its own kinetochore (Fig. 13-57). The signal for the onset of anaphase does not originate from the spindle itself, since even unattached chromosomes split into chromatids at the same time as attached ones. Judging by the results of certain experiments, this signal must be related to an increase in cytosolic Ca2+ concentration. First, continuous observation of cells containing a fluorescent calcium ion indicator (Section 4.2.3) shows that in some cells during anaphase, a rapid but transient tenfold increase in intracellular Ca2+ levels occurs. Second, microinjection of small amounts of calcium into cultured cells at the metaphase stage can lead to the premature onset of anaphase. Third, clusters of membrane vesicles are typically visible near the spindle poles, and special electron microscopy techniques make it possible to establish that these vesicles are rich in calcium. Thus, it is possible that spindle-associated vesicles release Ca2+ to initiate anaphase (Fig. 13-58), much like the sarcoplasmic reticulum releases Ca2+ to initiate skeletal Muscle contraction (Section 11.1.14).
Fig. 13-58. This electron micrograph shows a cluster of specially stained membrane vesicles (resembling the endoplasmic reticulum) near a spindle pole; the vesicles are elongated along the spindle microtubules. (Metaphase cell from a barley leaf; photo courtesy of Peter Hepler, from J. Cell Biol. 86: 490-499, 1980, by copyright permission of the Rockefeller Univ. Press.)
13.5.8. Anaphase chromosome movement consists of two processes [42]
As soon as each chromosome has split in response to the anaphase signal, its two chromatids begin to move toward opposite poles of the spindle, where they will be incorporated into the nuclei of the new cells. This movement appears to be the result of two independent processes occurring within the spindle (Fig. 13-59). The first consists of chromatid movement toward the poles and is associated with the shortening of kinetochore microtubules; this process is commonly called anaphase A. The second process is the separation of the poles themselves, associated with the elongation of polar microtubules, and is called anaphase B. These two processes can be distinguished by their selective sensitivity to certain poisons. For example, low concentrations of chloral hydrate prevent pole separation and polar microtubule elongation (anaphase B), but have no effect on either kinetochore microtubules or chromatid movement toward the poles (anaphase A). The relative contribution of each of these processes to the final chromosome segregation varies significantly depending on the Organism. For example, in mammalian cells, anaphase B begins shortly after the onset of chromatid movement toward the poles and ends when the spindle reaches 1.5 to 2 times its metaphase length. In some other cells, such as Yeasts, anaphase B begins only after the chromatids have reached their destination, whereas in certain Protozoa, anaphase B predominates and the spindle becomes 15 times longer than in metaphase.
13.5.9. Kinetochore microtubules depolymerize during anaphase A [43]
As chromosomes move from the region of the metaphase plate to the spindle poles, they are subjected to remarkably large forces. Measurements using fine glass needles yield an estimate of about 10-5 dyn per chromosome, which is 10,000 times greater than the force required simply to push a chromosome through the cytoplasm at the observed speed. Obviously, some powerful "motor" must exist to move the chromosomes, yet their movement speed must be limited by something other than medium viscosity. As already noted, this same motor could also pull chromosomes into the metaphase plate.
Fig. 13-59. Different forces acting during anaphase as sister chromatids segregate. A. Chromatids are pulled toward opposite poles As a result of the shortening of kinetochore microtubules (a movement termed anaphase A). B. At the same time, the two spindle poles move further apart from each other (a movement termed anaphase B). It is possible that the forces driving anaphase B are similar to those that lead to centrosome splitting and the separation of daughter centrosomes to form the two spindle poles in prophase (see Fig. 13-46). There is evidence that two separate forces are responsible for anaphase B: 1) the elongation and sliding of polar microtubules push the two poles apart, while at the same time 2) other forces acting on the asters pull the poles in opposite directions.
Fig. 13-60. The behavior of kinetochore microtubules changes during the transition from metaphase to anaphase. A. In metaphase, tubulin subunits are added at the plus end of the microtubule at the kinetochore and removed at the minus end at the pole. Thus, subunits continuously move toward the pole, so that microtubules maintain a constant length and remain under tension. B. In anaphase, the tension is released and the kinetochore begins to move rapidly along the microtubule while removing subunits from its plus end (left); as a result, the attached chromatid moves toward the spindle pole. In at least some organisms, chromosome movement is partly driven by the simultaneous shortening of microtubules at the pole as well (right).
As chromosomes move toward the poles, the microtubules attached to their kinetochores depolymerize, so that they are scarcely visible in telophase. The region where subunit loss occurs can be determined by injecting labeled tubulin into the cell during metaphase. It has been found that labeled subunits are first added to the end of the microtubule associated with the kinetochore and are subsequently lost during anaphase A. This indicates that the kinetochore, as it were, "chews" its way toward the poles along its microtubules. This Conclusion is also supported by the fact that anaphase kinetochores move toward a stationary mark made on the microtubules. Microtubule depolymerization at the kinetochores, poles, or both sites is likely necessary for chromosome movement to the poles (Fig. 13-60), since their movement halts if microtubule depolymerization is blocked by the addition of taxol or D2O.
The mechanism by which the kinetochore—and with it, the chromosome—moves along the spindle during anaphase A remains unknown. Two possible models for this are schematically presented in Fig. 13-61. According to the first model, the kinetochore hydrolyzes ATP as it moves along the attached microtubule, while the plus end of the microtubule depolymerizes as it becomes exposed. In another model, microtubule depolymerization itself drives the passive movement of the kinetochore while optimizing its binding energy with the microtubule. A third possibility, not shown in Fig. 13-61, is that microtubules are not directly responsible for generating the force that drives the kinetochore toward the poles, but simply regulate movement caused by some other structure. For example, it has been suggested that a system of elastic protein filaments exists (perhaps similar to the very long elastic filaments of striated muscle—see Section 11.1.13) that connects the kinetochore to the pole and gradually pulls it inward.
Fig. 13-61. Generation of force by the kinetochore to move a chromosome to the pole in anaphase: two alternative models. A. The kinetochore contains "motor" proteins similar to dynein or kinesin; they move along the microtubule using energy from ATP Hydrolysis (Section 10.4.9). B. Chromosome movement is driven by microtubule disassembly: as tubulin subunits dissociate, the kinetochore must slide toward the pole to maintain attachment to the microtubule. The same mechanisms may operate at the spindle pole, which also appears capable of maintaining attachment to microtubules while permitting their controlled depolymerization (see Fig. 13-60).
Regardless of The Nature of the force-generating mechanism, one must still account for the dramatic change in microtubule polymerization at the kinetochore during the transition from metaphase to anaphase (polymerization predominates in metaphase, disassembly in anaphase; see Fig. 13-60). This may simply be related to a sharp decrease in the pulling force exerted on the kinetochore in anaphase; the relaxation of tension could directly alter microtubule polymerization dynamics or lead to chemical modifications within the kinetochore.
13.5.10. Two distinct forces may operate in anaphase B [44]
In anaphase B, the distance between the two spindle poles increases; unlike anaphase A, this process is accompanied by microtubule assembly. As the poles move apart, the interpolar microtubules lengthen, apparently through assembly at their distal plus ends.
Both the separation of the spindle poles from one another during anaphase and the extent of interpolar microtubule overlap in the equatorial zone vary greatly from species to species. The zone of spindle microtubule overlap is particularly large in many diatoms (Fig. 13-62), in which mitosis takes place within the nuclear envelope (Section 13.5.18). As painstaking three-dimensional reconstructions of entire diatom spindles from hundreds of serial electron microscopy sections have shown, the polar microtubules of the two half-spindles overlap in a central zone near the spindle equator. During anaphase, these two groups of antiparallel microtubules apparently slide past one another as they move in opposite directions.
Anaphase movements can also be studied using lysed diatom cells. In such a model system, the mitotic spindle is readily accessible to macromolecules, making it possible to test the effects of various macromolecular agents, including specific antibodies. Inhibitors that bind to actin or myosin (such as anti-myosin antibodies) have no effect on the movement of anaphase chromosomes, ruling out an Actomyosin-based system like the one operating in muscle. Instead, the force here could be generated by proteins similar to dynein, which is associated with microtubules in Cilia and flagella (Section 11.3.7), or kinesin, which is involved in fast axonal transport (Section 10.4.9). Both of these proteins bind to microtubules and drive directed movement via ATP hydrolysis, though it remains unclear whether they play an essential role in mitosis.
Fig. 13-62. These electron micrographs illustrate how the spindle elongates and the degree of interpolar microtubule overlap decreases during mitosis in a diatom. A. Metaphase. B. Late anaphase. (Courtesy of Jeremy D. Pickett-Heaps.)
In higher eukaryotic cells, the nuclear envelope breaks down before the spindle forms; consequently, astral microtubules (those projecting away from the mitotic spindle, see Fig. 13-56) may play a more prominent role in anaphase B than they do in diatoms. For example, in the eggs of certain marine invertebrates, the spindle microtubules can be destroyed without blocking anaphase B. This suggests that the spindle poles are pushed or pulled apart by tensile forces—likely resulting from interactions between astral microtubules and the cell cortex. Similar interactions could also operate in cases of asymmetric cell division (Section 13.5.13).
13.5.11. In Telophase, the Nuclear Envelope Re-forms Initially Around Individual Chromosomes [45]
By the end of anaphase, the chromosomes have completely segregated into two identical groups, one at each spindle pole. In The final stage of mitosis—telophase—a nuclear envelope re-forms around each group of chromosomes, yielding two daughter interphase nuclei. To understand the breakdown and subsequent reassembly of the nuclear envelope, we must examine at least three of its components:
1) the outer and inner nuclear membranes, which are continuous with the membranes of the endoplasmic reticulum;
2) the underlying nuclear lamina—a thin, two-dimensional meshwork of Intermediate filaments composed of nuclear lamins, which interacts with the inner nuclear membrane, chromatin, and nuclear pores (Section 11.5.5);
3) nuclear pores, formed by large Complexes of Proteins that are not yet fully characterized (Section 8.3.1).
During prophase, many proteins undergo phosphorylation. While the phosphorylation of histone H1 molecules appears to promote chromosome condensation (Section 13.1.10), the phosphorylation of nuclear lamins helps regulate the breakdown and reassembly of the nuclear envelope. Lamin phosphorylation occurs at multiple sites across each polypeptide chain, leading to their disassembly and, consequently, the destruction of the nuclear lamina. Subsequently—likely in response to a distinct signal—the nuclear envelope itself fragments into small membrane vesicles.
The abrupt transition from metaphase to anaphase apparently triggers the dephosphorylation of many proteins (including histone H1 and lamins) that were phosphorylated during prophase. Shortly thereafter, in telophase, the nuclear membrane vesicles bind to The surface of individual chromosomes and fuse, re-forming nuclear membranes that initially enclose groups of chromosomes only partially before the complete nuclear envelope is restored (Fig. 13-63). Concurrently, nuclear pores are re-established, and the dephosphorylated lamins re-aggregate to form the nuclear lamina. One lamin protein (lamin B) remains associated with nuclear membrane fragments throughout mitosis, potentially facilitating their rejoining in telophase. Once the nuclear envelope has re-formed, RNA Synthesis resumes, leading to nucleolar reappearance (Section 9.4.19), while the chromatin decondenses and returns to the dispersed state characteristic of interphase.
Fig. 13-63. Schematic diagram of the cyclic changes in the nuclear envelope during mitosis. In prometaphase, the nuclear membranes break down into small vesicles and are subsequently re-formed in telophase. Between these two phases, while the nuclear envelope is disrupted and the nuclear pores and lamina have dissociated into subunits, all the processes driving the two sets of chromosomes to opposite poles take place. As illustrated, the new nuclear envelope for each daughter cell forms through the fusion of membrane vesicles around the clustering individual chromosomes, thereby excluding most cytoplasmic components from the new nucleus.
Both the breakdown and the re-formation of nuclear architecture can be reproduced in crude Xenopus egg extracts, provided these extracts are prepared from cells at the appropriate stages of the cell cycle (mitotic extracts for breakdown and interphase extracts for reassembly). In such extracts, the entire process—involving lamins, nuclear pores, and nuclear membranes—proceeds seemingly normally in response to phosphorylation and dephosphorylation cycles. Thus, such in vitro systems can serve as valuable assay systems for identifying and purifying the proteins that catalyze nuclear envelope breakdown and re-formation within the cell, including regulatory proteins such as MPF. To achieve nuclear reassembly, DNA must be added to these extracts; complete nuclear envelopes will form around purified DNA molecules from any organism, even from a bacterial virus. Therefore, although DNA-binding proteins must be involved, it is unlikely that specific nucleotide sequences are recognized in the process.
Interestingly, nuclear envelope breakdown is not an absolute requirement for mitosis. Indeed, as we will see later, lower eukaryotes do not break down their nuclear envelope during mitosis; these organisms are traditionally described as having a "closed" rather than an "open" spindle.
13.5.12. Metaphase and Interphase Can Be Viewed as Alternative "Stable" States of the Cell [46]
Figure 13-64 presents a schematic outline of a modern hypothesis regarding the mitotic cycle. It adopts a somewhat chemical perspective on mitosis, treating interphase and metaphase as two alternative "stable" states of the cell, with the other mitotic stages serving merely as necessary transitional states between them. According to this view, at the end of interphase, a mechanism (the "M-phase switch") is triggered, driving the cell through prophase and prometaphase into the more stable metaphase state. At the end of metaphase, this mechanism is abruptly switched off, and the cell passes through anaphase and telophase to return to interphase, which is the most stable state when the regulator is turned off.
"on" and "off." Under this hypothesis, switching on leads to the phosphorylation of numerous proteins, a process restricted to mitotic cells. The sequence of structural changes associated with spindle assembly requires no special triggers; rather, it represents a series of energetically favorable steps leading toward a stable metaphase state. This state persists until an anaphase trigger flips the switch to the "off" position, and protein dephosphorylation restores the original global parameters. This initiates a fresh cascade of structural changes (including chromosome segregation), once again returning the cell to a stable interphase state.
This perspective on mitosis is supported by evidence regarding cytoskeletal microtubule dynamics (see Fig. 13-48), as well as abrupt shifts in the activity and phosphorylation state of certain mitosis-related proteins at the interphase/prophase boundary (switch-on) and the metaphase/anaphase boundary (switch-off) (see Section 13.1.10). The position of the "switch" could directly correspond to the level of MPF activity within the cell (see the Discussion of the MPF cycle in Section 13.1.11 and Fig. 13-15).
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13.5.13. The Mitotic Spindle Determines the Plane of Cytoplasmic Cleavage During Cytokinesis
During cytokinesis, the cytoplasm is partitioned. Although nuclear division and cytoplasmic cleavage are generally coupled, this linkage is not absolute. Even under normal conditions, nuclear division is not always followed by cytokinesis. For example, in the early Drosophila embryo, numerous cycles of nuclear division occur without cytoplasmic cleavage, resulting in a single large syncytial cell containing 6,000 nuclei arranged in a monolayer near its surface. Mononucleate cells are formed later when the cytoplasm subsequently partitions around all these nuclei (Section 16.5.2).
Although cytokinesis is not always immediately accompanied by cytokinesis, the mitotic spindle plays a crucial role in determining when and how it will occur. Cytokinesis typically begins during anaphase, continues through telophase, and extends into part of the subsequent interphase period. The first visible sign of cytokinesis in animal cells is the formation of a small depression in the plasma membrane, which appears in anaphase and is called the cleavage furrow (Fig. 13-65). This furrow always forms in the plane of the metaphasic plate, at a right angle to the long axis of the mitotic spindle. If the spindle is displaced using a micromanipulator early enough in anaphase, the nascent furrow will disappear and a new one will form in accordance with the new position of the spindle. Elegant experiments on eggs of the sea urchin Echinarachnius demonstrate that the cleavage furrow will form midway between the asters generated by the two centrosomes, even if the centrosomes are not connected by a mitotic spindle (Fig. 13-66). Later, once the process is well underway, cytokinesis will continue even if the spindle and its asters are removed with a pipette or disrupted with colchicine.
Fig. 13-65. Initiation of frog egg cleavage. Scanning electron micrographs. The Formation of the furrow is driven by The activity of a submembranous contractile ring. A. Low-magnification view of the cell surface. B. High-magnification view of a region of the furrow. (H.W. Beams, R.G. Kessel, Am. Sci. 64: 279-290, 1976.)
Fig. 13-66. Experiment demonstrating The Effect of spindle position on the plane of division. If the mitotic spindle is mechanically displaced to one side of the cell, the cleavage furrow does not reach the opposite side. Subsequent divisions will occur not only along the equators of the two mitotic spindles (as normally happens), but also between two adjacent asters not connected by a mitotic spindle. Apparently, the contractile bundle of actin filaments that produces the cleavage furrow always forms in the region lying midway between the two asters. This implies that the asters somehow modify the surrounding region of the cell cortex.
Most cells divide symmetrically. The cleavage furrow forms at the equator of the parent cell, so that the daughter cells are equal in size and have approximately identical properties. During embryonic development, however, cells frequently divide asymmetrically: the furrow separates two distinct cells that will develop along different pathways. Divisions of this type are often spatially strictly determined. For instance, they may occur in specific planes relative to the surface of an epithelial sheet or lead to the segregation of cytoplasmic domains with different sets of organelles. Regardless of whether division is symmetric or asymmetric, the position of the furrow—and hence of the plane of division—is always determined by the position of the mitotic spindle. If necessary, the spindle can rotate in a programmed manner, taking up the required position within the cell and orienting the plane of division accordingly (Fig. 13-67). It is likely that these spindle movements are governed by changes in specific areas of the cell cortex, which shifts the spindle poles via astral microtubules. A similar mechanism presumably determines the position of the centrosome in a polarized cell (Section 11.4.5). The structure of the actin-rich cortex is discussed in Chapter 11 (Section 11.2).
Microtubules and bundles of actin filaments present in the interphase cytoplasm are disassembled during mitosis. However, cytoplasmic intermediate filaments remain intact in many cells. In such cells, the network of intermediate filaments surrounding the interphase nucleus stretches during mitosis to encompass both daughter nuclei, and is ultimately severed into two parts by the cleavage furrow (Fig. 13-68).
13.5.14. Actin and Myosin Generate the Force Required for Cytokinesis
Cytoplasmic division results from the contraction of a ring composed chiefly of actin filaments. This filament bundle, termed the contractile ring (Fig. 13-69), attaches to the inner face of the plasma membrane via unidentified proteins. The contractile ring forms at the onset of anaphase, and the mechanism of its assembly remains unknown; the force it generates is sufficient to bend a fine glass needle inserted into the cell. There is little doubt that the source of this force, as in muscle, is the sliding of actin and myosin filaments past one another. For instance, in lysed mitotic cells, the addition of inactivated myosin subfragments blocks the myosin-binding sites on actin, thereby arresting cytoplasmic division. Similarly, microinjection of antibodies against myosin into sea urchin eggs causes the cleavage furrow to flatten but does not affect nuclear mitosis. Nevertheless, it remains unclear precisely how the interaction between actin and myosin draws the plasma membrane inward to form the cleavage furrow.
Fig. 13-67. Programmed Rotation of the mitotic spindle at the two-cell stage of the nematode Caenorhabditis elegans embryo in preparation for division into four precisely positioned cells. (Courtesy of John White.)
During normal cell division, the contractile ring does not thicken as the furrow deepens. This suggests that it gradually decreases in volume through the loss of some of its filaments. Upon the completion of cytokinesis, the contractile ring disassembles entirely, and the plasma membrane at the furrow site constricts to surround the midbody, which still connects the two daughter cells. The midbody contains remnants of two sets of polar microtubules tightly packed together with a dense matrix material (Fig. 13-70).
Cytokinesis, which yields two cells from one, substantially increases the total surface area of the cell. Consequently, the two daughter cells require more plasma membrane material than the original cell. In animal cells, the Biosynthesis of membrane components is upregulated immediately prior to division. Excess membrane in cells preparing to divide is apparently stored in the form of surface blebs.
13.5.15. Cytokinesis Occurs by a Radically Different Mechanism in Higher Plants [49]
Most higher plant cells are surrounded by a rigid Cell wall, and therefore the mechanism of cytokinesis differs substantially from that just described for animal cells. Instead of forming two daughter cells by pinching them off via a sub-surface contractile ring, the cytoplasm is partitioned here by the formation of a new wall at the boundary between the daughter cells. The resulting partition precisely determines the relative position of the two new cells within the plant. It follows that the orientation of cell division planes and cell enlargement determine the plant's overall form (see Chapter 20).
Fig. 13-68. During mitosis, a bundle of intermediate filaments surrounds the nuclear region. Micrographs obtained by staining permeabilized cells with fluorescent antibodies that bind to intermediate filaments. A—anaphase; B—early telophase (arrows indicate the position of the contractile ring); C—late telophase. (S.H. Blose, Proc. Natl. Acad. Sci. USA 76: 3372-3376, 1979.)
Fig. 13-69. Electron micrograph of the base of a furrow forming during animal cell division. A diagram of the cleavage furrow is shown at the top for clarity. (H.W. Beams, R.G. Kessel, Am. Sci. 64: 279-290, 1976.)
The new transverse partition, or cell plate, begins to form in the plane between the two daughter nuclei in association with residual polar spindle microtubules that assemble into a cylindrical structure called the phragmoplast. This structure, analogous to the midbody microtubules of animal cells, consists of two sets of oppositely oriented microtubules arranged parallel to one another (see Fig. 20-42). The microtubules are presumably anchored to the nuclear surface, such that their plus ends terminate in an electron-dense disk at the equatorial plane. As shown in Fig. 13-71, small membrane-bounded vesicles, derived primarily from the Golgi apparatus and laden with cell wall precursors, contact the microtubules on either side of the phragmoplast and are transported along them toward the equatorial region of the cell. Here they fuse to form a disk-shaped, membrane-enclosed structure—the early cell plate. Polysaccharide molecules released by these vesicles cross-link within the early cell plate to form pectin, hemicellulose, and Other components of the primary cell wall. This disk must then expand until its margins reach the parental cell wall. To enable this, the microtubules of the early phragmoplast undergo reorganization at the periphery of the nascent cell plate. New vesicles contact them here and subsequently fuse at the equator, enlarging the plate. This process repeats until the growing cell plate reaches the plasma membrane of the mother cell, and the membranes fuse, completely separating the two new daughter cells (see Figs. 20-41 and 20-42). Cellulose microfibrils are then deposited within the cell plate, completing the assembly of the new cell wall (Figs. 13-71 and 13-72).
Fig. 13-70. A. Cultured animal cell at the end of division: the midbody remains connected to both daughter cells. Scanning electron micrograph. B. Electron micrograph of the midbody of a dividing animal cell. Division is virtually complete, but the daughter cells are still joined by a thin cytoplasmic bridge. (Courtesy of Guenter Albrecht-Buehler [A] and J.M. Mullins [B].)
Fig. 13-71. The course of cytokinesis in higher plant cells possessing a rigid cell wall.
Elements of the endoplasmic reticulum associate with the vesicles of the forming cell plate and are frequently incorporated into it. Subsequently, they give rise to plasmodesmata—complex pores that traverse the mature cell wall and interconnect the cytoplasm of all plant cells (see Section 20.2.1 and Fig. 20-20).
Fig. 13-72. Cytokinesis in a plant cell. The cell plate (between the two arrows) forms in a plane perpendicular to the plane of the drawing. One cell (A) is photographed using differential interference contrast microscopy; the other is stained with antibodies linked to gold particles, which label two clusters of microtubules that are part of the phragmoplast. In both cases, the arrows indicate the plane of the cell plate. [Courtesy of Jeremy D. Pickett-Heaps (A) and Andrew Bajer (B).]
Fig. 13-73. Organization of actin filaments in a plant cell during cytokinesis. Actin filaments (highlighted in dark red) form a radial network that extends from the ends of the phragmoplast to the cell cortex, forming a ring around the cell. This network apparently determines the plane of cell plate formation. Another group of actin filaments lies parallel to the microtubules involved in forming the new cell plate within the phragmoplast. Yet another group of actin filaments (not shown in the figure) approaches the cortex from the region of the two daughter nuclei across the large central vacuole characteristic of plant cells (Section 20.40.7); these filaments help maintain the thin cytoplasmic bridges that cross the vacuole.
Just as in animals, mitosis and cytokinesis in plants can be uncoupled. For example, in seed endosperm, mitoses occur without cytokinesis, leading to the formation of a Giant multinucleated cell. Much later, long after the mitotic spindle has disintegrated, new cell walls are built between individual nuclei, resulting in separate cells.
A mitotic spindle alone is usually not sufficient to determine the precise position and shape of the cell plate. The junction site of the future plate with the parent cell wall appears to be determined very early, even before the onset of mitosis, by a narrow bundle of microtubules—the preprophase band—located just beneath the plasma membrane (see Section 20.5.5 and Fig. 20-64). Although these microtubules disappear at THE START OF mitosis, they dictate the cortical region where the radial network of actin filaments will attach; this network persists throughout M phase and guides the growing edge of the cell plate to the proper cortical zone (Fig. 13-73). Thus, actin plays a crucial role in the division of cells with rigid walls, where active contraction apparently plays no part. Because actin is also involved in forming cell septa in fungi, it is possible that it directs cytokinesis in all eukaryotes.
13.5.16. Cytokinesis must ensure the correct distribution of cytoplasmic organelles [50]
The nucleus is only one of many cellular organelles whose doubling requires a pre-existing organelle of the same type. For example, Ribosomes can assemble spontaneously from their components, but their construction requires other ribosomes to synthesize the necessary proteins. On the other hand, Mitochondria and METABOLISM/14.html">Chloroplasts are incapable of spontaneous self-assembly and can form only through the growth and division of pre-existing organelles (Section 7.5.1). Similarly, the growth mechanisms of several other organelles, such as the Golgi apparatus and the endoplasmic reticulum, are such that it is difficult to conceive of their de novo formation in the absence of at least fragments of the corresponding structures (see Chapter 8). In some Algae that possess only a single chloroplast or a single Golgi apparatus, this sole organelle splits into two halves prior to cytokinesis, which are then distributed between the daughter cells (see Fig. 7-67). An example of the same phenomenon is the duplication and segregation of the centrosome in animal cells (see Fig. 13-46).
How are the various membrane-bounded organelles (excepting the nucleus) partitioned during the division of higher eukaryotic cells? In most cases, the number of these organelles is large enough (see Table 8-1) that, even with random distribution during cytokinesis, each daughter cell receives a more or less representative set. Thus, although a mammalian cell will not survive without receiving, for example, at least one mitochondrion, it is quite possible that no special mechanism is required for their reliable inheritance by daughter cells. Of course, organelles present in large numbers in cells will always be successfully inherited as long as their average number doubles in each cell generation. Other organelles, such as the Golgi apparatus and the endoplasmic reticulum, break down into smaller fragments and vesicles during mitosis. Such fragmentation likely facilitates their equal distribution between the daughter cells.
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13.5.17. In special cases, certain cellular components may be inherited by only one daughter cell
So far, we have considered cell division as a mechanism designed to produce two identical cells from one. However, during the development and maintenance of Tissues in complex Multicellular Organisms, division is in many cases clearly asymmetrical. This is especially true for early cleavage divisions, when a large fertilized egg divides into smaller cells that will develop into different parts of the organism (Section 16.2.2). We have already discussed how an asymmetric position of the spindle during mitosis can lead to The production of two cells of unequal size (Section 13.5.13); however, the generation of biochemically distinct daughter cells poses a separate problem.
A striking example of this process is the behavior of a group of peculiar granules in the egg of the nematode Caenorhabditis elegans. In the cytoplasm of the unfertilized egg, these "P granules" are distributed uniformly, but immediately before the First Division, they migrate to the posterior end of the cell and thus end up in only one of the two blastomeres (Fig. 13-74). This unequal distribution is repeated in subsequent cell divisions, so that eventually the granules are found only in the cells that will form the germ line (i.e., the precursors of eggs and sperm). It is possible that P granules play a role in the further differentiation of these cells.
P granules will move to the posterior end of the cell even in mutants where the mitotic spindle is rotated at a right angle to its normal position. Furthermore, results from experiments with cytoskeleton inhibitors suggest that the directed movement of P granules depends not on microtubules, but on actin filaments (it is blocked by cytochalasin D). Although the uneven distribution of the granules is apparently determined by some asymmetrical property of the actin cytoskeleton, the Molecular Mechanism of their directed movement remains unknown. It is tempting to think that similar mechanisms might govern the movement of many normally invisible cell components, thereby programming the resulting unequal daughter cells for further differentiation (Section 16.4.1).
Fig. 13-74. Programmed asymmetric transmission of a cytoplasmic component to one of the daughter cells during the first two Divisions of the fertilized egg of the nematode Caenorhabditis elegans. Left: live cells photographed using differential interference contrast microscopy; right: the same cells stained with antibodies against P granules. These small granules of unknown function (0.5–1 µm in diameter) are distributed randomly in the cytoplasm of the unfertilized egg. (Courtesy of Susan Strome.)
13.5.18. The complex mitotic process of higher organisms evolved gradually from prokaryotic division mechanisms [52]
In Prokaryotic Cells, the separation of DNA and cytoplasm is a single continuous process. During DNA replication, the two copies of the chromosome are attached to specific sites on The cell membrane, which gradually move apart as the membrane grows between them. Cytoplasmic division occurs between the two DNA attachment points so that each daughter cell receives a single chromosome (Fig. 13-75). With the advent of eukaryotes, The Genome became more complex, and the size and number of chromosomes increased. This necessitated the evolution of a more sophisticated mechanism for distributing chromosomes between daughter cells.
Clearly, the mitotic apparatus could not have evolved all at once. In many primitive eukaryotes, such as the dinoflagellate Crypthecodinium cohnii, mitosis remained a membrane-associated process, with the nuclear envelope taking on The Role of the prokaryotic plasma membrane. The intermediate position of this large unicellular alga is also reflected in the biochemistry of its chromosomes, which, like prokaryotic chromosomes, contain relatively few associated proteins. The nuclear envelope in C. cohnii persists throughout mitosis, and the spindle microtubules lie entirely outside the nucleus. Where the microtubules press against the nuclear envelope, it invaginates, forming a series of parallel through-channels (Fig. 13-75). The chromosomes attach to the inner membrane of the nuclear envelope in the region of these channels, and chromosome separation occurs entirely on the inner surface of this membrane. Thus, the extranuclear "spindle" (which is a rigid rod lacking any dynamic properties) is used simply to give the nuclear envelope a specific shape that determines the plane of division.
Fig. 13-75. Various organisms employ different mechanisms for chromosome segregation. Some of these may represent transitional stages in the Evolution of the mitotic spindle of higher organisms. In all Examples except Bacteria, only the central, nuclear region of the cell is shown.
A slightly more advanced, yet still extranuclear spindle is characteristic of hypermastigotes, in which the nuclear envelope also persists during mitosis. These large Protozoans from the gut of insects illustrate particularly well the independence of spindle elongation and chromosome movement leading to chromatid separation, since sister kinetochores move apart even before attaching to the spindle, driven by the growth of the nuclear envelope to which they are anchored. Only when the kinetochores come close to the spindle poles do they gain The ability to attach to the spindle via kinetochore fibers. Since the spindle fibers remain separated from the chromosomes by the nuclear envelope, the kinetochore fibers forming outside the nucleus must somehow attach to the chromosomes through the nuclear membrane. Following this attachment, the kinetochores are pulled toward the poles in the usual manner (13-75).
The next stage in the evolution of mitotic mechanisms is represented by a group of organisms with a spindle located inside an intact nucleus. In yeast, the spindle consists of a continuous intranuclear bundle of microtubules that stretches from one pole to the other and elongates during mitosis (see Figs. 13-75 and 13-16). In other species, such as diatoms, the continuous spindle is replaced by two conventional half-spindles whose microtubule ends intertwine to form an overlap zone. In both yeast and diatoms, chromosomes are connected to the spindle by their kinetochores, and chromosome segregation occurs much as it does in mammalian cells, except that the entire process takes place within the nuclear envelope. It remains unexplained why Higher Plants and animals instead evolved a mitotic apparatus that requires the controlled and reversible breakdown of the nuclear envelope.
Conclusion
The process of cell division consists of nuclear division (mitosis) followed by cytoplasmic division (cytokinesis). Mitosis begins with prophase, a transitional period when the splitting of the centrosome leads to the formation of two spindle poles, which subsequently organize the distribution of nuclear material. At the same time, the onset of M phase is accompanied by a marked increase in the phosphorylation of specific proteins. As a result, an unusually dynamic system of microtubules is apparently established in the mitotic cell. Following the breakdown of the nuclear envelope in prometaphase, the kinetochores of condensed chromosomes can be captured and stabilized by groups of microtubules emanating in large numbers from both spindle poles. These microtubules pull the kinetochores toward opposite poles, causing the chromosomes to align along the spindle equator during metaphase. In anaphase, this tension is suddenly released when sister chromatids separate from one another and move to opposite poles. In addition, the two poles often move apart. In the final stage of mitosis, telophase, a nuclear envelope re-forms around each group of separated chromosomes as the proteins phosphorylated at the start of M phase are dephosphorylated.
Cell division culminates in the division of the cytoplasm (cytokinesis); chromosomes decondense, and RNA synthesis resumes on them. Apparently, cytokinesis in such diverse eukaryotic organisms as animals, plants, and fungi is directed by organized bundles of actin filaments. Large membrane-bounded organelles, such as the Golgi apparatus and the endoplasmic reticulum, break down into smaller fragments and vesicles during the M phase, ensuring their even distribution between daughter cells. However, cytokinesis can also involve a programmed asymmetric distribution of material. For instance, a cell may divide to produce daughter cells of unequal size, or a cytoplasmic component may accumulate on one side of the cell prior to cytokinesis and be inherited by only one of the two otherwise identical daughter cells.
General
Baserya R. The Biology of Cell Reproduction. Cambridge, MA, Harvard University Press, 1985.
Beach D., Basilica C., Newport J., eds. Cell Cycle Control in Eukaryotes. Cold Spring Harbor NY, Cold Spring Harbor Laboratory, 1988.
John P.C.I., ed. The Cell Cycle. Cambridge U.K., Cambridge University Press, 1981.
Mitchison J. M. The Biology of the Cell Cycle. Cambridge U. K., Cambridge University Press, 1971.
Pollack R. Readings in Mammalian Cell Culture, 2nd ed. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1981. (An anthology, including many important papers on cell growth and division.)
Prescott D.M. Reproduction of Eukaryotic Cells. New York, Academic Press, 1976.
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