Kinetochore structure

Goldstein LS 1981 Kinetochore structure and its role in chromosome orientation during the first meiotic division in male D. melanogaster. Cell 25 591-602... 

Only the major features of spindle organization as now known will be considered here. Spindle chemistry, kinetochore structure, and some details of spindle organization will be discussed in the sections which follow on chromosome movement and distribution. 

MitosiSy and Mitosis Versus Meiosis. Kinetochore structure now becomes a major consideration. Only a brief summary will be given here for a more extensive review see Brinkley and Stubblefield (1970). 

Granted that the proposed explanation is at least reasonable for both meiotic and mitotic chromosome distribution, can the fundamental difference between these divisions also be explained As Ostergren (1951) proposed, a kinetochore structural difference may parallel the kinetochore orientation difference. An opposite polarity of sister kinetochores in mitosis has just been invoked to explain, in part, achievement of bipolar amphiorientation. On the same assumptions, if sister kinetochores lie close together and have a common polarity, their orientation to the same pole (syntely) should at least be facilitated. The required polarity occurs in the one example studied at late prophase (Amphiuma, p. 267). 

Fig. 18 (cont.). B, One of a complete series of twelve serial sections through the manipulated chromosome, this one at the level of the first half-bivalent s kinetochores. Two chromosomal microtubules are indicated by arrows they lie in the direction of bivalent motion, as does a third microtubule seen in an adjacent section. X80,000. C, A section at the level of the second half-bivalent s kinetochores, which had evinced no motion up to the time of fixation. Neither this nor the adjacent sections disclose any microtubules here, but kinetochore structure (the less electron opaque chromosome material indicated by the arrows) is Identical with that of unmanipulated bivalents. X80,000. (From unpublished studies of Brinkley and Nicklas.)... 

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. 

Centromer-specific H3 variants (CenH3 s) are known as CENP-A in mammals, Cid in flies, Cse4 in yeast (Fig. 1 Table 1 reviewed in Smith 2002). CenH3 s are incorporated into nucleosomes independently of centromeric DNA replication. CenH3-containing nucleosomes associate with a number of factors with important roles in centromere structure and kinetochore assembly. Recently, the deposition complex... 

The lower evolutionary constraint on the sequences of CENP-A-type variants in comparison to most other core histone variants could in part reflect the very limited context in which CENP-A functions, i.e., only in centromeric chromatin associated with the kinetochore. Most other variants appear to be rather widely distributed in the chromatin, and therefore, may be involved in a greater variety of interactions and modifications that could constrain their structure during evolution. 

Warburton, P.E., Cooke, C.A., Bourassa, S., Vafa, O., Sullivan, B.A., Stetten, G., Gimelli, G., Warburton, D., Tyler-Smith, C., Sullivan, K.F., Poirier, G.G., and Earnshaw, W.C. (1997) Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Current Biol. 7, 901-904. 

Kinetochore. A structure that attaches laterally to the centromere of a chromosome it is the site of chromosome tubule attachment. 

Garcia-Saez, I., Yen, T., Wade, R. H., and Kozielski, F. (2004). Crystal structure of the motor domain of the human kinetochore protein GENP-E. J. Mol. Biol. 340, 1107-1116. 

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. 

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. 

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.

In higher eukaryotes, a complex protein structure called the kinetochore assembles at centromeres and associates with multiple mitotic spindle fibers during mitosis. Homologs of most of the centromeric proteins found in the yeasts occur in humans and other higher eukaryotes and are thought to be components of kinetochores. The role of the centromere and proteins that bind to it in the segregation of sister chromatids during mitosis is described in Chapters 20 and 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 mitotic 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, trilamlnated 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 7-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 mitotic apparatus In plant cells at the end of this section. 

The taxanes originally were extracted from the bark of the Pacific Yew tree and have found widespread use as anticancer agents. The structurally related taxanes, docetaxel and pacUtaxel, bind to and stabilize polymerized microtubules. Cells that enter mitosis in the presence of paclitaxel attempt to assemble a spindle apparatus, however the inhibition of depolymerization renders this structure unable to properly position chromosomes at the metaphase plate. Therefore, although kinetochores bind to microtubules, there is insufficient tension to inactivate the SAC. Paclitaxel-treated cells become blocked in mitosis, and eventually die by the mechanisms just described. Currently, there... 

Experiments examining the intracellular localization of Mad2 have suggested a model for how the feedback control mechanism might operate [38, 39]. At early stages of mitosis, Mad2 localizes to the kinetochore, a structure that forms on each chromosome and mediates attachment to spindle microtubules. As... 

CENP-A to CENP-G) bind to the DNA sequences of centromeres and direct the formation of fhe kinetochores. Even for the simpler centromere of budding yeasfs, kinetochores have a complex structure.The CENT proteins were first idenhfied as autoantigens in sera of patients with the autoimmune disorder scleroderma (Chapter... 

Paddy, M. R., and Chelsky, D. (1991). Spoke A 120-kD protein associated with a novel fdamentous structure on or near kinetochore microtubules in the mitotic spindle. J. Cell Biol. 113, 161-171. Paddy, M. R., Belmont, A. S., Saumweber, H., Agard, D. A., and Sedat, J. W. (1990). Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell (Cambridge, Mass.) 62, 89-106. 

McEwen, B. F., Arena, J. T.. Frank. J., and Rieder, C. L. (1993). Structure of the colcemid-treated PtKl kinetochore outer plate as determined by high voltage electron microscopic tomography. J. Cell Biol 120,301-312. 

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