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In Chapter 21, we consider in detail how progression through the cell cycle, and hence cell replication, is regulated. In this section, we focus on the mechanics of mitosis in a "typical" animal cell. Mistakes in mitosis can lead to missing or extra chromosomes, causing abnormal patterns of development when they occur during embryogenesis and pathologies when they occur after birth. To ensure that mitosis proceeds without errors in the trillions of cell divisions that take place in the life span of an organism, a highly redundant mechanism has evolved in which each crucial step is carried out concurrently by microtubule motor proteins and micro-tubule assembly dynamics.

The Mitotic Apparatus Is a Microtubule Machine for Separating Chromosomes

The structure of the mitotic apparatus changes constantly during the course of mitosis (Figure 20-30). For one brief moment at metaphase, however, the chromosomes are aligned at the equator of the cell. We begin our discussion by examining the structure of the mitotic apparatus at metaphase and then describe how it captures and organizes chromosomes during prophase, how it separates chromosomes during anaphase, and how it determines where cells divide during telophase.

▲ EXPERIMENTAL FIGURE 20-30 Fluorescence microscopy reveals changes in the organization of chromosomes and microtubules at four mitotic stages.

Cultured fibroblasts were stained with a fluorescent anti-tubulin antibody (green) and the DNA-binding dye ethidium (purple). Thus in these fluorescence micrographs, green reveals microtubules; purple, chromatin; and blue, regions with both structures. (a) During early prophase, the nucleus is surrounded by an array of interphase microtubules and the chromatin is diffuse. (b) By prometaphase, the nuclear membrane has broken down and the replicated centrosomes (centrioles) have migrated to the poles from which microtubules radiate. (c) At metaphase, the fully condensed chromosomes have aligned midway between the poles to form the metaphase plate. Dense bundles of microtubules connect the chromosomes to the poles. (d) In late anaphase, the chromosomes are pulled to the poles along the radiating microtubules. [From J. C. Waters, R. W. Cole, and C. L. Rieder, 1993, J. Cell Biol. 122:361; courtesy of C. L. Rieder.]

At metaphase, the mitotic apparatus is organized into two parts: a central mitotic spindle and a pair of asters (Figure 20-31a; see also Figure 20-2c). The spindle is a bilaterally symmetric bundle of microtubules and associated proteins with the overall shape of a football; it is divided into opposing halves at the equator of the cell by the metaphase chromosomes. An aster is a radial array of microtubules at each pole of the spindle.

In each half of the spindle, a single centrosome at the pole organizes three distinct sets of microtubules whose (— ) ends all point toward the centrosome (Figure 20-31b). One set, the astral microtubules, forms the aster; they radiate outward from the centrosome toward the cortex of the cell, where they help position the mitotic apparatus and later help to determine the cleavage plane in cytokinesis. The other two sets of microtubules compose the spindle. The kinetochore microtubules attach to chromosomes at specialized attachment sites on the chromosomes called kinetochores. Polar microtubules do not interact with chromosomes but instead overlap with polar microtubules from the opposite pole. Two types of interactions hold the spindle halves together to form the bilaterally symmetric mitotic apparatus: (1) lateral interactions between the overlapping (+) ends of the polar microtubules and (2) end-on interactions between the kine-tochore microtubules and the kinetochores of the sister chro-matids. The large protein complexes, called cohesins, that link sister chromatids together are discussed in Chapter 21.

The mitotic apparatus is basic to mitosis in all organisms, but its appearance and components can vary widely. In the budding yeast Saccharomyces cerevisiae, for instance, the mi-totic apparatus consists of just a spindle, which itself is constructed from a minimal number of kinetochore and polar microtubules. These microtubules are organized by spindle pole bodies, trilaminated structures located in the nuclear membrane, which do not break down during mitosis. Furthermore, because a yeast cell is small, it does not require well-developed asters to assist in mitosis. Although the spindle pole body and centrosome differ structurally, they have proteins such as ^-tubulin in common that act to organize the mitotic spindle. Like yeast cells, most plant cells do not contain visible centrosomes. We consider the unique features of the mi-totic apparatus in plant cells at the end of this section.

(b) Zone of interdigitation

Aster Spindle Aster

Aster Spindle Aster

▲ EXPERIMENTAL FIGURE 20-31 High-voltage electron microscopy visualizes components of the mitotic apparatus in a metaphase mammalian cell. (a) Microtubules were stained with biotin-tagged anti-tubulin antibodies to increase their size in this electron micrograph. The large cylindrical objects are chromosomes. (b) Schematic diagram corresponding to the metaphase cell in (a). Three sets of microtubules (MTs) make up the mitotic apparatus. All the microtubules have their (—) ends at the poles (centrosomes). Astral microtubules project toward the cortex and are linked to it. Kinetochore microtubules are connected to chromosomes (blue). Polar microtubules project toward the cell center with their distal (+) ends overlapping. [Part (a) courtesy of J. R. McIntosh.]

The Kinetochore Is a Centromere-Based Protein Complex That Captures and Helps Transport Chromosomes

The sister chromatids of a metaphase chromosome are transported to each pole bound to kinetochore microtubules. In regard to their attachment to microtubules and movement, chromosomes differ substantially from the vesicle and organelle cargoes transported along cytosolic microtubules. The linkage of metaphase chromosomes to the (+) ends of kinetochore microtubules is mediated by a large protein complex, the kinetochore, which has several functions: to trap and attach microtubule ends to the chromosomes, to generate force to move chromosomes along microtubules, and to regulate chromosome separation and translocation to the poles. In an animal cell, the kinetochore forms at the centromere and is organized into an inner and outer layer embedded within a fibrous corona (Figure 20-32).

In all eukaryotes, three components participate in attaching chromosomes to microtubules: the centromere, kine-tochore and spindle proteins, and the cell-cycle machinery. The location of the centromere and hence that of the kine-tochore is directly controlled by a specific sequence of chromosomal DNA termed centromeric DNA (Chapter 10). Although the sequences of centromeric DNA and of DNA-binding proteins in the kinetochore are not well conserved through evolution, the cell-cycle proteins and many of the proteins that link the kinetochore to the spindle are homologous in humans and yeast. Microtubule-binding proteins (e.g., CLIP170, CENP-E) and microtubule motor proteins (e.g., the mitotic kinesin MCAK and cytosolic dynein) cooperate in attaching the kinetochore to a microtubule end while tubulin subunits are added or released. The presence of these motor proteins indicates that kinetochores play a role in transporting chromosomes to opposite ends of the cell in mitosis.

Duplicated Centrosomes Align and Begin Separating in Prophase

Because each half of the metaphase mitotic apparatus emanates from a polar centrosome, its assembly depends on duplication of the centrosome and movement of the daughter centrosomes to opposite halves of the cell. This process, known as the centriole cycle (or centrosome cycle) marks the first steps in mitosis, beginning during G1 when the centrioles and other centrosome components are duplicated (Figure 20-33). By G2, the two "daughter" centrioles have reached full length, but the duplicated centrioles are still present within a single centrosome. Early in mitosis, the two pairs of centrioles separate and migrate to opposite sides of the nucleus, establishing the bipolarity of the dividing cell. In some respects, then, mitosis can be understood as the migration of duplicated centrosomes, which along their journey pick up chromosomes, pause in metaphase, and during

Sister chromatids

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