Kinetochore spindle attachment

Destruction of cohesin allows the spindle microtubules to pull the separated chromatids to opposite poles of the cell. Failure of spindle attachment to a single kinetochore activates the SAC (spindle assembly checkpoint), which arrests cells at metaphase until corrections are effected and equal distribution of chromosomes has been ensured. A sensory mechanism initiates the wait anaphase signal from an imattached kinetochore and triggers the accu mulation of the checkpoint components that comprise the Bub (budding uninhibited by benomyl)-Mad (mitotic arrest deficient) families of proteins. 

The foregoing description overlooks the extreme complexity of mitosis, each stage of which must occur with precision and in the correct sequence 222-2253 The replication of DNA, which takes place in the S phase of the cell cycle (and is discussed in Chapter 27) must be completed before mitosis begins. This is followed by condensation of the DNA into chromosomes (Chapter 27), breakdown of the nuclear membrane,226 2263 assembly of the kinetochores by which the chromosomes attach to the spindle,222 assembly of the spindle, attachment of chromosomes to the spindle, segregation of the chromosomes to opposite poles in anaphase, and finally the cleavage of the cell. 

Three types of microtubule can readily be defined in the mitotic spindle. Polar microtubules overlap (and probably interact) between the poles and are involved in pushing the poles apart in anaphase. Astral microtubules radiate in all directions and also help separate the poles. Kinetochore microtubules attach themselves to specialized protein structures (kinetochores) located on each side of the centromere of each chromosome. These microtubules are involved in moving the chromosomes to the metaphase plate and in separating sister chromatids at anaphase. The microtubules in the spindle are very dynamic and have a half-life of only a few seconds. This appears to be especially important in the capture of chromosomes by the kinetochore microtubules. Microtubules that miss the target kinetochores are quickly lost because their dynamic instability soon leads to depolymerization. The new microtubules that form may hit the target and be partially stabilized through plus-end capping. 

Fig. 12.13 (a) We distinguish the centrosomes and the kinetochores. There are three classes of microtubules in a mitotic spindle. Attached to the kinetochores are the kinetochore-microtubules and attached to the centrosomes are the astral and the polar microtubules, which become the spindle poles, (b) The centrosomes undergo characteristic changes during the cell cycle. In the S phase, daughter centrioles begin to form. Finally, the centrosome divides to form the mitotic spindle poles. 

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 20-3lb). 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... 

Hauf, S., Cole, R W., LaTerra, S., Zimmer, C., Schnapp, G., Walter, R, et al. (2003). The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. The Journal of CeU Biology, 161, 281-294. 

Amin, M.A., Matsunaga, S., Uchiyama, S., and Fukui, K. (2008) Nucleophosmin is required for chromosome congression, proper mitotic spindle formation, and kinetochore-microtubule attachment in HeLa cells. FEES Lett, 582, 3839-3844. 

Williams B.C., Gatti M., and Goldberg M.L. 1996. Bipolar spindle attachments affect redistribution of ZWIO, a Drosophila centromere/kinetochore component required for accurate chromosome segregation./. Cell Biol. 34 1127-1140... 

Radial arrays are best exemplified by mitotic half-spindles, which have a central MTOC, the centrosome. The centrosome consists of two centrioles (which are homologous with the basal body) surrounded by dense pericentriolar material (Kalt and Schliwa, 1993). In plant cells, the MTOC of the mitotic spindle consists of dense material only without centrioles. The plus ends of microtubules of the mitotic spindle are directed toward the equator of the cell. Some are free, and others attach to kinetochores on chromatids (see Figure 4). 

In terms of evolutionary biology, the complex mitotic process of higher animals and plants has evolved through a progression of steps from simple prokaryotic fission sequences. In prokaryotic cells, the two copies of replicated chromosomes become attached to specialized regions of the cell membrane and are separated by the slow intrusion of the membrane between them. In many primitive eukaryotes, the nuclear membrane participates in a similar process and remains intact the spindle microtubules are extranuclear but may indent the nuclear membrane to form parallel channels. In yeasts and diatoms, the nuclear membrane also remains intact, an intranuclear polar spindle forms and attaches at each pole to the nuclear envelope, and a single kinetochore microtubule moves each chromosome to a pole. In the cells of higher animals and plants, the mitotic spindle starts to form outside of the nucleus, the nuclear envelope breaks down, and the spindle microtubules are captured by chromosomes (Kubai, 1975 Heath, 1980 Alberts et al., 1989). 

Loss of sister chromatid cohesion would therefore be sufficient for the sudden movement of chromatids to opposite poles at the metaphase to anaphase transition. According to this hypothesis, a specific apparatus binds chromatids together during replication, holds them in an orientation that facilitates the attachment of sister kinetochores to spindles extending to opposite poles, and resists the splitting force that results from this bipolar attachment to the spindle. Destruction of this specialized cohesive structure triggers movement of chromatids to opposite poles at the onset of anaphase. 

These proteins form complexes with cdc20, thereby sequestering it from activating the APC/C complex (66) (Fig. 4). The spindle checkpoint is activated in response to various spindle poisons, such as nocodazole, a drug that depolymerizes microtubules and thus prevents the attachment of microtubules to the kinetochores. On the other hand, taxanes inhibit the dynamic instability of the spindle and allow microtubule attachment but prevent the generation of tension across kinetochores. 

Fig. 17.2 Top The normal situation the Bub and Mad proteins dissociate from the kinetochore region when the microtubuies are properly aligned. The released Bub/Mad proteins activate a protein (Cdc20) which regulates entry into mitosis and activates the anaphase-promoting complex (APC) and gives the go-ahead fbr entry into the anaphase. Below. The situation when the kinetochore is not attached properly to the microtubules. In this case, the Bub and Mad proteins remain attached to the kinetochore, Cdc20 remains inactive, and APC is not activated. This arrests the cell in metaphase. Thus, the Bub and Mad and Mps-1 proteins in yeast and mammals, respectively, respond to improper assembly of the spindle by arresting cells. (Reproduced with permission of Professor R. A. Weinberg and Nature from Rg. 2 in ref. 8.)...

Centromere is that site on the chromosome that is attached to the mitotic spindle during divison of the nucleus in mitosis. The centromere is also the point in the condensed chromosome where the two sister chromatids are connected. In the late prophase, the centromere has two kinetochores, one for each sister chromatid. 

Jablonski SA, Chan GK, Cooke CA, Eamshaw WC, Yen TJ. The hBUBl and hBUBRl kinases sequentially assemble onto kinetochores during prophase with hBUBRl concentrating at the kinetochore plates in mitosis. Chromosoma 1998 107 386-396. Zhou J, Yao J, Joshi HC. Attachment and tension in the spindle assembly checkpoint. J. Cell. Sci. 2002 115 3547-3555. 

Whereas the benomyl screens established the existence of the spindle checkpoint and identified some of the key components in checkpoint signaling, a fundamental question that remained unanswered was what exactly is monitored by the checkpoint. Two general models have been proposed. One is that the checkpoint monitors the attachment of spindle microtubules at the kinetochore, a stmcture that forms on each chromosome to mediate microtubule binding. Unattached kinetochores keep the checkpoint active and delay anaphase (27). A second model is that the checkpoint monitors force across the centromere, the region of the chromosome where kinetochores assemble... 

Figure 3 Manipulation of chromosome-microtubule attachments with small molecules, (a) In the absence of microtubule poisons, the attachment of both kinetochores to spindle microtubules creates tension across the centromere, (b) Taxol reduces tension across the centromere by inhibiting microtubule dynamics, (c) Nocodazole creates unattached kinetochores by depolymerizing microtubules.

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