Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules - PubMed (original) (raw)

Kinetochores use a novel mechanism for coordinating the dynamics of individual microtubules

Kristin J VandenBeldt et al. Curr Biol. 2006.

Abstract

Chromosome alignment during mitosis is frequently accompanied by a dynamic switching between elongation and shortening of kinetochore fibers (K-fibers) that connect kinetochores and spindle poles . In higher eukaryotes, mature K-fibers consist of 10-30 kinetochore microtubules (kMTs) whose plus ends are embedded in the kinetochore . A critical and long-standing question is how the dynamics of individual kMTs within the K-fiber are coordinated . We have addressed this question by using electron tomography to determine the polymerization/depolymerization status of individual kMTs in the K-fibers of PtK1 and Drosophila S2 cells. Surprisingly, we find that the plus ends of two-thirds of kMTs are in a depolymerizing state, even when the K-fiber exhibits net tubulin incorporation at the plus end . Furthermore, almost all individual K-fibers examined had a mixture of kMTs in the polymerizing and depolymerizing states. Therefore, although K-fibers elongate and shrink as a unit, the dynamics of individual kMTs within a K-fiber are not coordinated at any given moment. Our results suggest a novel control mechanism through which attachment to the kinetochore outer plate prevents shrinkage of kMTs. We discuss the ramifications of this new model on the regulation of chromosome movement and the stability of K-fibers.

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Figures

Figure 1

Figure 1

Illustration of the Use of Electron Tomography to Identify the Plus-End Conformations of kMTs in PtK1 Cells (A) EM image of a 200 nm thick plastic section containing a metaphase chromosome from a PtK1 cell prepared by high-pressure freezing and freeze substitution. Scale bar = 1 μm. (B) Higher magnification image of the boxed area in (A), with the chromosome, kinetochore, and kMTs labeled. Scale bar = 200 nm. (C) A 1.6 nm thick slice from the tomographic reconstruction of the same area represented in (B). The kinetochore outer plate and two kMTs displaying the curved conformation are indicated. Scale bar = 200 nm. (D and E) Examples of the range of straight (D) and curved (E) conformations that were detected for the plus ends of kMTs by electron tomography. Underneath each image is the percentage of kMT plus ends in untreated metaphase PtK1 cells having a similar conformation. Scale bars represent 25 nm.

Figure 2

Figure 2

Bar Graph of kMT Plus-End Conformations The percentage of kMT plus ends classified as straight and curved are plotted for: M, untreated metaphase; T, taxol-treated metaphase; N, nocodazole-treated metaphase; A, untreated anaphase PtK1 cells; and S2, untreated metaphase Drosophila S2 cells. The sample size (number of kMT plus ends classified) is indicated in parentheses on the abscissa.

Figure 3

Figure 3

Plus-End Conformations of the kMTs in Individual K-Fibers from Metaphase PtK1 and S2 Cells (A) A plot of the number of kMT plus ends classified in the straight and curved conformations for ten K-fibers from four different cells. On average, K-fibers in PtK1 cells contain 23 MTs [3], but for technical reasons, we only analyzed 8–16 plus ends per kinetochore as a random sampling of the total. The ordering of kinetochores along the abscissa is by increasing percentage of kMTs found in the curved conformation. (B) Histogram of the percentage of kMTs exhibiting the curved conformation in 27 K-fibers, for which 3–16 kMTs each were analyzed. The broad distribution with a weak secondary peak at a low percentage of curved plus ends indicates that the kMT plus-end conformations within a given K-fiber are not completely random. (C) A plot of the number of kMT plus ends classified in the straight and curved conformations for K-fibers from three pairs of sister kinetochores in PTK metaphase cells. (D) A plot of the number of kMT plus ends classified in the straight and curved conformations for six K-fibers from a S2 cell.

Figure 4

Figure 4

Depth that kMTs Penetrated into the Centromere (A) A single 10 nm thick slice from a tomographic reconstruction, with regional delineations for the corona (CR), outer plate (OP), and heterochromatin (HC). Two kMTs terminating in the outer plate are indicated (kMTs [OP]). Scale bar = 200 nm. (B) Bar graph of kMT penetration. The percentages of kMTs terminating in the three regions delineated in (A) are indicated above the bars for: M, untreated; T, taxol treated; N, nocodazole-treated metaphase PtK1 cells; and A, untreated anaphase PtK1 cells. The sample sizes are indicated in parentheses on the abscissa. Measurements were limited to kinetochores for which a clearly defined outer plate was visible. (C) Curved plus ends showing the kinks (arrows) and tight-radius curvatures (arrowhead) that were frequently observed. Scale bar = 25 nm.

Figure 5

Figure 5

Model for kMT Dynamic Instability on PtK1 and Drosophila S2 Kinetochores A single kMT is shown undergoing a conformational change from straight (left side) to curved (right side). A key feature of the model is that attachment to the outer plate prevents kMT disassembly that is not coupled to chromosome motion. This enables kMTs in the disassembly phase to be rescued, which in turn results in a continual cycling of individual kMTs that are in the growing phase. The cycling of kMTs between the growing and shrinking phases permits net incorporation of tubulin into the plus end of the K-fiber, even though the majority of its kMTs are in the disassembly phase. The catastrophe rate is shown as twice the rescue rate because that generates a 1:2 ratio of straight to curved conformations. Dissociation from the outer plate is low in PtK1 cells but is much higher in cells with a high kMT turnover rate. In the latter case, dynamic cycles of MT growth and shrinkage would occur primarily in the spindle, rather than on the kinetochore outer plate. However, the principle of cycling between which individual kMTs are contributing to K-fiber growth would be the same. The heterochromatin (HC), outer plate (OP), and corona (CR) designations are the same as those given in Figure 4A.

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