Microtubules kinetochoric

Figure 17.7 Kinesins at the kinetochore attach to the lateral surface of microtubules. KInetochores attach to the plus ends of microtubules at which there Is constant exchange for tubulin monomers. This exchange leads to growth and shrinkage. Attachment Is maintained by kinesins that bind to the side of the microtubule, not the unstable end.

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). 

During telophase, a midbody forms between the separating daughter cells (see Figure 4), and disassembly of the remaining spindle microtubules and kinetochores takes place. 

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). 

Gorbsky, G.J. Borisy, G.G. (1989). Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase. J. Cell Biol. 109, 653-662. 

Mitchison, T.J. (1988). Microtubule dynamics and kinetochore function in mitosis. Ann. Rev. Cell Biol. 4, 527-549. 

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. 

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. 3. Schematic illustrations of distinct steps in cell division show the central role of contractile motor action in the process of mitosis. (A) to (C) replication (prophase) (D) formation of the mitotic spindle (metaphase) (E) and (F) chromosome migration (anaphase) and building of the nuclear envelopes, and (G) formation of the contractile ring containing actin and myosin, forming the cleavage furrow and eventually two separate daughter cells. CE, centriole pair A, aster of microtubules N, nucleus M, microtubules C, chromosomes K, kinetochore NR, remnant of nuclear envelope NE, nucelar envelope reforming CR, contractile ring CM, cell membrane]. From Squire (1986).

M. De Brabander, G. Geuens, R. Nuydens, R. Willebrords and J. De Mey, Taxol induces the assembly of free microtubules in living cells and blocks the organizing capacity of the centrosome and kinetochores, Proc. Natl. Acad. Sci. USA 78 (1981) 5608-5612. 

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. 

During anaphase, when the sister chromatids split at the centromere, this balance is lost [Fig. 5-37(/>)J. The dynein motors in the kinetochores drive the sister chromatids to the poles. The speed at which this happens may be regulated by the controlled depolymerization of the kinetochore microtubules. The poles can now move apart and the overlap between polar microtubules shortens. It would be completely lost but for rapid polymerization at the plus ends of these filaments. 

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. 

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.)...

Kinetochore is a complex structure, formed in mitosis from chromatin. The kineto-choie recruits microtubules from the mitotic spindle which move the chromosome to the poles of the cell. 

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... 

When both sister kinetochores are attached correctly, they are pulled in opposite directions by the microtubule fibers and the centromere is under tension (Fig. 3a). In this model, the absence of centromere tension would keep the checkpoint active. Small molecules that target tubulin have provided a way to test these models experimentally. Nocodazole depolymerizes microtubules, which creates unattached kinetochores (Fig. 3c), whereas taxol stabilizes microtubules but inhibits their dynamics, which decreases the centromere tension (Fig. 3b) (29). 

When microtubules are depolymerized with nocodazole (Fig. 3c), Mad2 localizes to all kinetochores, which indicates the activation of the checkpoint. If microtubule dynamics are suppressed with taxol while maintaining kinetochore attachments (Fig. 3b), Mad2 localizes to only a few kinetochores... 

This finding suggests that checkpoint signaling, as determined by Mad2 localization, is sensitive to attachment but does not respond directly to centromere tension. The interpretation of these experiments is complicated, however, because tension is required for kinetochores to bind the full complement of microtubules loss of tension may activate the checkpoint indirectly (32). 

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.

Feedback control of anaphase onset, or mitotic checkpoint signaling, is one mechanism that contributes to ensuring accurate chromosome segregation. Delaying anaphase in response to unattached kinetochores, however, is not sufficient. Chromosomes must attach to spindle microtubules in a particular... 

Figure 4 Correction of improper chromosome attachments by activation of Aurora kinase (45). (a) Assay schematic, (i) Treatment with the Eg5 inhibitor monastrol arrests cells in mitosis with monopolar spindles, in which sister chromosomes often are both attached to the single spindle pole, (ii) Hesperadin, an Aurora kinase inhibitor, is added as monastrol is removed. As the spindle bipolarizes with Aurora kinase inhibited, attachment errors fail to correct so that some sister chromosomes are still attached to the same pole of the bipolar spindle, (iii) Removal of hesperadin activates Aurora kinase. Incorrect attachments are destabilized by disassembling the microtubule fibers, which pulls the chromosomes to the pole, whereas correct attachments are stable, (iv) Chromosomes move from the pole to the center of the spindle as correct attachments form, (b) Structures of the Eg5 inhibitor monastrol and two Aurora kinase inhibitors, hesperadin and AKI-1. (c) Spindles were fixed after bipolarization either in the absence (i) or presence (ii) of an Aurora kinase inhibitor. Arrows indicate sister chromosomes that are both attached to the same spindle pole. Projections of multiple image planes are shown, with optical sections of boxed regions (1 and 2) to highlight attachment errors. Scale bars 5 xm. (d) After the removal of hesperadin, GFP-tubulin (top) and chromosomes (bottom) were imaged live by three-dimensional confocal fluorescence microcopy and DIC, respectively. Arrow and arrowhead show two chromosomes that move to the spindle pole (marked by circle in DIC images) as the associated kinetochore-microtubule fibers shorten and that then move to the center of the spindle. Time (minutes seconds) after the removal of hesperadin. Scale bar 5 (cm.

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