on the kinetochore (Figure 20-36). Whether a chromosome attaches to the (+) end of a spindle microtubule by a direct hit or by the side capture/sliding process, the kinetochore "caps" the (+) end of the microtubule. Eventually, the kinetochore of each sister chromatid in a chromosome is captured by micro-tubules arising from the nearest spindle poles. Each chromosome arm becomes attached to additional microtubules as mitosis progresses toward metaphase.

During late prophase (prometaphase), the newly condensed chromosomes attached to the (+) ends of kinetochore microtubules move to the equator of the spindle. Along the way, the chromosomes exhibit saltatory behavior, oscillating between movements toward and then away from the pole or equator. These oscillations result from alternating depolymer-ization and polymerization at the (+ ) ends of kinetochore mi-crotubules. In addition, motor proteins associated with both ends of kinetochore microtubules and with the distal ends of polar microtubules on chromosome arms generate opposing forces that are thought to position captured chromosomes equally between the two spindle poles (Figure 20-37a).

Although the lengths of kinetochore and polar micro-tubules eventually become stable, there continues to be a flow, or treadmilling, of subunits through the microtubules toward the poles. At metaphase, the loss of tubulin subunits at the (—) ends of spindle microtubules is balanced by the addition of subunits at the (+) ends (Figure 20-37b). The flow of tubulin subunits from kinetochores to the poles can be visualized after a very small "pulse" of fluorescently labeled tubulin subunits has been microinjected into a cell (Figure 20-38). Microtubules appear speckled because very few of the subunits are fluores cent. By comparing the positions of each speckle, one can see whether the tubulin subunits are moving in a specific direction. The images show that the mitotic spindle at metaphase is a finely balanced, yet dynamic, structure that holds chromosomes at the equatorial plate. By mechanisms discussed in Chapter 21, the cell cycle is held in check until all chromosomes have been captured and aligned. A single unattached kinetochore is sufficient to prevent entry into anaphase.

Anaphase Chromosomes Separate and the Spindle Elongates

The same forces that form the spindle during prophase and metaphase also direct the separation of chromosomes toward opposite poles at anaphase. Anaphase is divided into two distinct stages, anaphase A and anaphase B (early and late anaphase). Anaphase A is characterized by the shortening of kinetochore microtubules at their (+) ends, which pulls the chromosomes toward the poles. In anaphase B, the two poles move farther apart, bringing the attached chromosomes with them into what will become the two daughter cells.

Microtubule Shortening in Anaphase A The results of in vitro studies have indicated that the depolymerization of microtubules in Xenopus eggs is sufficient to move chromosomes toward the poles. In one such study, purified micro-tubules were mixed with purified anaphase chromosomes; as expected, the kinetochores bound preferentially to the (+) ends of the microtubules. To induce depolymerization of the

Pole (MTOC)

Kinetochore microtubules containing fluorescent tubulin

Target region for laser light

Kinetochore microtubules containing fluorescent tubulin

Target region for laser light

Expose to laser light; kinetochore microtubules depolymerize at (+) ends and kinetochores move toward poles

Expose to laser light; kinetochore microtubules depolymerize at (+) ends and kinetochores move toward poles

Region of depolymerization

▲ EXPERIMENTAL FIGURE 20-39 Shortening at the (+)

end of kinetochore microtubules moves chromosomes poleward in anaphase A. Fibroblasts are injected with fluorescent tubulin and then allowed to enter metaphase so that all the microtubules are fluorescent. Only the kinetochore microtubules are shown. In early anaphase, a band of microtubules (yellow box) is subjected to a laser light, which bleaches the fluorescence but leaves the microtubules continuous and functional across the bleached region. The bleached segment of each microtubule thus provides a marker for the position of that part of the microtubule. In anaphase, the distance between the bleached zone and the adjacent pole (marked by red double-headed arrows) does not change, whereas the distance to the adjacent chromatid (marked by black double-headed arrows) becomes shorter. This finding indicates that during anaphase microtubules disassemble at the (+) end just behind the kinetochores, not at the poles. [Adapted from G. J. Gorbsky et al., 1987, J. Cell Biol. 104:9, and 1988, J. Cell Biol. 106:1185.]

microtubules, the reaction mixture was diluted, thus lowering the concentration of free tubulin dimers. Video microscopy analysis then showed that the chromosomes moved toward the (—) end, at a rate similar to that of chromosome movement during anaphase in intact cells. Because no ATP (or any other energy source) was present in these experiments, chromosome movement toward the (— ) end must have been powered, in some way, by microtubule disassembly and must not have been powered by microtubule motor proteins.

The in vivo fluorescence-tagging experiment depicted in Figure 20-39 provides evidence that shortening of kinetochore microtubules at their (+) ends moves chromosomes toward the poles in mammalian cells. A kinetochore-associated kinesin, MCAK, promotes disassembly at the (+) end while CENP-E, also at kinetochores, binds to the progressively shortening end. According to this model, microtubule disassembly drives poleward movement of chromosomes. Although kinetochore kinesins promote this disassembly or keep chromosomes attached to the shortening (+ ) end of kinetochore microtubules, they do not actually move chromosomes along the microtubules.

Spindle Elongation in Anaphase B Findings from several types of experiments have implicated three processes in the separation of the poles in anaphase B: a pushing force gen-

Overlap zone k->1

Overlap zone k->1

ATP; microtubule polymerization

Newly polymerized tubulin

ATP; microtubule polymerization

▲ FIGURE 20-40 Model of spindle elongation and movement of poles during anaphase B. One or more (+) end-directed spindle kinesins (orange) bind to antiparallel polar microtubules in the overlap region and then "walk" along a microtubule in the other half-spindle toward its (+) end. In cells that assemble an aster, cytoplasmic dynein, a (—) end-directed motor protein (green) anchored in the cortex of the plasma membrane, walks along astral microtubules, pulling the poles outward. Tubulin subunits are simultaneously added to the plus ends of all polar microtubules, thereby lengthening the spindle. [Adapted from H. Masuda and W. Z. Cande, 1987, Cell 49:193.]

erated by kinesin-mediated sliding of polar microtubules past one another, a pulling force generated by cortex-associated cytosolic dynein, and lengthening of polar microtubules at their (+) ends. The coordinated effect of these processes is depicted in Figure 20-40.

When metaphase cells are depleted of ATP by mild detergent treatment, poleward chromosome movement (anaphase A) proceeds but the spindle poles do not separate (anaphase B). This finding indicates that microtubule motor proteins take part in separating the spindle poles, as they do in centrosome movement in prometaphase (see Figure 20-34). Experiments with artificial spindles assembled from frog egg extracts were sources of further insight into anaphase B movements. In the presence of calcium, the spindle is activated and elongates, simulating anaphase B, and the zone of overlap between the two halves of the spindle decreases in length. The observation that adjacent antiparallel micro-tubules in this system migrate in the direction of their pole-facing (—) ends suggests that a (+) end-directed kinesin separates spindle poles in anaphase B. In one model, the bipolar kinesin BimC attached to a microtubule in the overlap region walks toward the (+) end of a neighboring but antiparallel micro-tubule, thus pushing the adjacent microtubule in the direction of its (—) end (see Figure 20-40). Involvement of a kinesin motor is supported by experiments in which antibodies raised against a conserved region of the kinesin superfamily inhibit ATP-induced elongation of diatom spindles in vitro.

In the presence of a^-tubulin, reactivated frog spindles and isolated diatom spindles add tubulin subunits to the (+) end of polar microtubules, thus lengthening them. The third process taking place in anaphase B can be demonstrated by cutting the spindle in half with a microneedle at anaphase; the resulting half-spindles move quickly to the poles, at a rate faster than usual during anaphase. This observation suggests that cytosolic dynein, a (—) end-directed motor protein associated with the cortex, pulls on astral microtubules, thereby moving the poles farther apart (see Figure 20-40).

► EXPERIMENTAL FIGURE 20-41 Micromanipulation experiments can determine whether the spindle or the asters control location of the cleavage plane during cytokinesis. A

small glass ball is pressed against a fertilized egg until membranes from opposite sides of the cell touch and fuse, thus changing the spherical egg into a doughnut shape. In the first cell division, a normal spindle develops and the doughnut-shaped cell divides to produce a single C-shaped cell with two normal nuclei. This result is expected whether the asters or the spindle determines the cleavage plane. In the second cell division, the two nuclei each produce a normal spindle. If the spindle determined the cleavage plane, then cleavage of the C-shaped cell would yield three cells, one of them with two nuclei (lower left). If the asters determined the cleavage plane, then a third cleavage plane would form between asters from two different spindles (lower right). Cell division produced four cells, indicating an extra furrow formed between a pair of asters.

Microtubules and Microfilaments Work Cooperatively During Cytokinesis

After the chromosomes have migrated to opposite ends of the cell during anaphase, two closely related events proceed (see Figure 20-29): the nuclear envelope re-forms around each complete set of chromosomes, marking the end of mitosis (telophase), and the cytoplasm divides, a process termed

► FIGURE 20-42 Regulation of myosin light chain by mitosis-promoting factor. The mitosis-promoting factor (MPF) is an activated complex of a cyclin-dependent kinase (CDK1) and a mitotic cyclin protein. Phosphorylation of inhibitory sites on the myosin light chain by the kinase activity of MPF early in mitosis prevents active myosin heavy chains from interacting with actin filaments and sliding along them, a process required for cytokinesis. When MPF activity falls at anaphase, a constitutive phosphatase dephosphorylates the inhibitory sites, permitting cytokinesis to proceed. Another enzyme, myosin light-chain kinase, phosphorylates a different residue, activating myosin to interact with actin filaments. [See L. L. Satterwhite et al., 1992, J. Cell Biol. 118:595; adapted from A. Murray and T Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and Company.]

Myosin light chain Interphase Y//////Z,


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