M Experimental Figure 2032

Kinetochore proteins mediate attachment of chromosomes to microtubules. (a)

Electron micrograph section through a kinetochore reveals the microtubules (MT), inner and outer kinetochore layers (IL and OL), and chromosome. (b) In animal cells, the kinetochore consists of an inner layer containing proteins that bind centromeric DNA and an outer layer connected to the (+) ends of kinetochore microtubules. The microtubules embedded in the outer layer extend toward one of the two poles of the cell. The outer layer and fibrous corona around the microtubule ends contain microtubule-binding proteins and motor proteins, including CLIP170, cytosolic dynein, and the kinesins CENP-E and MCAK. [Part (a) from B. McEwen et al., 1998, Chromosoma 107:366; courtesy of B. McEwen.]

Centromere

- Kinetochore

Centromere

► FIGURE 20-33 Relation of centrosome duplication to the cell cycle. After the pair of parent centrioles (red) separates slightly, a daughter centriole (blue) buds from each and elongates. By G2, growth of the daughter centrioles is complete, but the two pairs remain within a single centrosomal complex. Early in mitosis, the centrosome splits, and each centriole pair migrates to opposite ends of the cell.

anaphase continue their movement to new locations in the daughter cells, where they release the chromosomes and organize the cytosolic microtubules.

Microtubule dynamics change drastically at the onset of mitosis, when long interphase microtubules disappear and are replaced by astral and spindle microtubules. These mitotic microtubules, which are nucleated from the newly duplicated centrosomes, are more numerous, shorter, and less stable than interphase microtubules. The average lifetime of a microtubule decreases from 10 minutes in interphase cells to 60-90 seconds in the mitotic apparatus. This increase in dynamic instability enables microtubules to assemble and disassemble quickly in mitosis.

The results of genetic and cell biological studies, primarily in yeast and flies, have implicated several kinesins in organ-

▲ FIGURE 20-34 Model for participation of microtubule motor proteins in centrosome movements at prophase. (a) At prophase, polar microtubules growing randomly from opposite poles are aligned with the aid of (—) end-directed motors (orange). (b) After alignment, (+) end-directed mitotic kinesins (yellow), including the bipolar kinesin BimC, generate pushing forces that separate the poles. In addition, a (—) end-directed force exerted by cytosolic dynein (green) located at the cortex may pull asters toward the poles. Similar forces act later at anaphase.

izing polar microtubules into a bipolar array, thereby orienting assembly of the spindle and spindle asters. For instance, antibodies against either a (+) or a (—) end-directed kinesin inhibit the formation of a bipolar spindle when they are mi-croinjected into a cell before but not after prophase. A (—) end-directed kinesin protein, such as Kin-C, is thought to help align the oppositely oriented polar microtubules extending from each centrosome. Then a (+ ) end-directed kinesin, most likely the bipolar BimC, cross-links antiparallel microtubules and pushes them apart. In addition, findings from localization experiments with anti-dynein antibodies have demonstrated the presence of cytosolic dynein in the centrosomes and cortex of dividing animal cells. The results of other studies with yeast mutants lacking cytosolic dynein suggest that dynein at the cortex simultaneously helps tether the astral microtubules and orient the poles of the spindle. Thus the alignment and initial separation of centrosomes at prophase depend on the growth of polar and astral microtubules and on the action of several motor proteins, as depicted in Figure 20-34.

Although centrosomes facilitate the formation of the spindle poles, recent findings show that polar microtubules can be assembled and organized into antiparallel bundles in the absence of centrosomes. For instance, the Ran GTPase that functions in nuclear import and export (Chapter 12) appears to promote the polymerization of tubulin subunits. Ran acts with other proteins, possibly motor proteins, to stabilize microtubules by increasing their frequency of rescue.

Formation of the Metaphase Mitotic Spindle Requires Motor Proteins and Dynamic Microtubules

When the duplicated centrosomes have become aligned, formation of the spindle proceeds, driven by simultaneous events at centrosomes and chromosomes. As just discussed, the cen-trosome facilitates spindle formation by nucleating the assembly of the spindle microtubules. In addition, the (—) ends of microtubules are gathered and stabilized at the pole by dynein-dynactin working with the nuclear/mitotic apparatus protein. The role of dynein in spindle pole formation has been demonstrated by reconstitution studies in which bipolar spindles form in Xenopus egg extracts in the presence of centrosomes, microtubules, and sperm nuclei. The addition of antibodies against cytosolic dynein to this in vitro system releases and splays the spindle microtubules but leaves the cen-trosomal astral microtubules in position (Figure 20-35).

The dynamic instability of spindle microtubules at the other end, the (+ ) end, is critical to their capture of chromosomes during late prophase as the nuclear membrane begins to break down. By quickly lengthening and shortening at its (+) end, a dynamic microtubule probes into the chromosome-rich environment of the cell. Sometimes the (+) end of a microtubule directly contacts a kinetochore, scoring a "bull's-eye. " More commonly, a kinetochore contacts the side of a micro-tubule and then slides along the microtubule to the (+ ) end in a process that includes cytosolic dynein and mitotic kinesins

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