Rebecca Heald, "Serving Up a Plate of Chromosomes" (2006)

Serving Up a Plate of Chromosomes
> Rebecca Heald<br> Science 311, 20 January 2006: 343-344.

Chromosomal DNA encodes the blueprint required to maintain eukaryotic cell and organism viability. Chromosomes replicate during each round of the cell division cycle and remain as pairs of sister chromatids until a bipolar apparatus, called the mitotic spindle, precisely segregates them into two sets, each destined for a new daughter cell (1). Accuracy in this process of mitosis is imperative, as transmission of a faulty blueprint can cause cell death or contribute to cancer. On page 388 of this issue, Kapoor et al. (2) address a longstanding question in the field: How do chromosomes efficiently achieve the right kind of spindle attachments so that they can be properly distributed?

To prepare for segregation, sister chromatids connect at their kinetochores to spindle microtubule bundles, called kinetochore fibers (K-fibers), that emanate from opposite spindle poles. The sisters are thus "bi-oriented" and poised to go their separate ways. Chromosomes gradually "congress" to the central region of the spindle, called the metaphase plate. The cell monitors this process and dissolves the glue holding sisters together only when every chromosome is properly attached and aligned at this plate. At anaphase of the cell division cycle, sister chromatids move to opposite poles as their attached K-fibers depolymerize, completing segregation.

A mechanism of chromosome congression that promotes bi-orientation. Microtubules forming a mitotic spindle (red) that contains two chromosomes is shown, each with paired sister chromatids (blue) and kinetochores (yellow). Thicker red lines represent bundled kinetochore fibers. In the scenario depicted, microtubules growing from both spindle poles have been captured by sister kinetochores of one chromosome, and the chromosome is oscillating (congressed) at the metaphase plate. The other chromosome attaches initially to only one pole and becomes mono-oriented in a position where microtubules from the opposite pole are unlikely to make contact. By attaching to and sliding along the kinetochore fiber of the congressed chromosome, the unattached sister kinetochore moves toward the center of the spindle, where it makes microtubule connections to bi-orient.

The question of how chromosomes achieve the prerequisite bi-orientation has intrigued cell biologists for decades. The common view has been that before congressing, each sister chromatid of a pair connects to a K-fiber that is associated with the opposite spindle pole (3). However, spindle attachment is a stochastic process that depends on the interaction of microtubules with a subset of proteins localized at the kinetochore. As soon as one sister kinetochore "captures" a microtubule emanating from one spindle pole, the chromosome (that is, the pair of sister chromatids) is transported toward that pole, becoming "mono-oriented." How then do microtubules from the other spindle pole make contact with the unattached sister? This is a puzzle, because structural analyses of the spindle indicate that microtubules from the distal pole rarely penetrate far enough to be captured by a chromosome that has already moved toward the opposite pole (4). Yet chromosomes still congress to the plate and become bi-oriented.

Using a combination of sophisticated microscopy techniques, Kapoor et al. have documented kinetochore behavior that solves this conundrum. By following congression in living cells through video microscopy and then rapidly preparing the cells for high-resolution electron microscopy, the authors observed mono-oriented chromosomes with the kinetochore of one sister chromatid attached to the ends of microtubules extending from a proximal pole. Interestingly, its sister was laterally associated with the K-fiber of another chromosome that was already bi-oriented and congressed. Visualizing fluorescent kinetochores and micro-tubules revealed "sliding" of mono-oriented chromosomes toward the spindle equator along other K-fibers through this lateral association, bringing the unattached sister kinetochore into range of microtubules from the distal pole (see the figure). Such excursions do not always result in capture, but they increase the probability that bipolar chromosome attachments can form.

The observed frequency of these chromosome movements suggested that sliding of unattached kinetochores along other K-fibers occurs commonly. In fact, under conditions (chemical inhibitors that perturb mitotic progression) that allowed congression and bi-orientation to be followed in a large population of chromosomes synchronously, Kapoor et al. observed that ~85% of kinetochores were paired such that one sister chromatid attached to a pole and the other laterally associated with a mature K-fiber from a different, bi-oriented chromosome that stretched toward the metaphase plate. This indicates the predominance of this congression mechanism to promote chromosome bi-orientation. By combining their chemical inhibitor-based assay with RNA interference, a technique capable of depleting a specific protein from cells, Kapoor et al. could investigate the factors behind kinetochore sliding along a lateral K-fiber. Their hunch was that the microtubule-based motor CENP-E, a member of the kinesin-7 family, was involved, because this protein localizes to the kinetochore during congression and moves with the correct polarity--toward microtubule "plus" ends that are uniformly oriented toward the metaphase plate in kinetochore fibers (5). Although kinetochores could still capture microtubules after CENP-E depletion, mono-oriented chromosomes that were not transported toward the metaphase plate accumulated at spindle poles. Thus, CENP-E is likely the motor responsible for gliding unattached sister kinetochores along neighboring K-fibers, helping mono-oriented chromosomes achieve congression before bi-orientation. These findings are the first to indicate bona fide kinetochore motility depending on CENP-E. Chromosome congression defects observed upon CENP-E inhibition were previously attributed to a role in microtubule capture and/or in maintaining kinetochore attachment to dynamic microtubules (6). CENP-E also contributes to a checkpoint signaling pathway that monitors kinetochore status (7), making it a central player in both the process and the fidelity of spindle function.

The spindle is a remarkable cellular machine, and the work by Kapoor et al. demonstrates that we are still uncovering its fundamental mechanisms. The findings explain why chromosome congression is a cooperative process, accelerating as more and more chromosomes gain bipolar attachments that can serve as tracks for mono-oriented chromosomes to congress. Once chromosomes are bi-oriented, they oscillate along the spindle axis as K-fiber microtubules coordinately polymerize and depolymerize at their plus ends. Although a large constellation of kinetochore proteins has been identified, it remains unclear which factors are operating at the dynamic kinetochoremicrotubule interface, and how they are functioning. Combining state-of-the-art imaging, chemical biology, and molecular dissection is a great paradigm for elucidating the mechanistic principles of mitosis.

References
> (1) S. Gadde, R. Heald, Curr. Biol. 14, R797 (2004). <br> (2) T. M. Kapoor et al., Science 311, 388 (2006).
> (3) A. W. Murray, T. J. Mitchison, Curr. Biol. 4, 38 (1994). <br> (4) D. N. Mastronarde, K. L. McDonald, R. Ding, J. R. McIntosh, J. Cell Biol. 123, 1475 (1993).
> (5) K. W. Wood, R. Sakowicz, L. S. B. Goldstein, D. W. Cleveland, Cell 91, 357 (1997). <br> (6) B. T. Schaar, G. K. T. Chan, P. Maddox, E. D. Salmon, T. J. Yen, J. Cell Biol. 139, 1373 (1997).
> (7) Y. Mao, A. Desai, D. W. Cleveland, J. Cell Biol. 170, 873 (2005). <p>

> <strong>Chromosomes Can Congress to the Metaphase Plate Before Biorientation <br> Tarun M. Kapoor, Michael A. Lampson, Polla Hergert, Lisa Cameron, Daniela Cimini, E. D. Salmon, Bruce F. McEwen, and Alexey Khodjakov
> <em>Science <em>20 January 2006: 388-391.
> &nbsp;<br> Abstract: The stable propagation of genetic material during cell division depends on the congression of chromosomes to the spindle equator before the cell initiates anaphase. It is generally assumed that congression requires that chromosomes are connected to the opposite poles of the bipolar spindle ("bioriented"). In mammalian cells, we found that chromosomes can congress before becoming bioriented. By combining the use of reversible chemical inhibitors, live-cell light microscopy, and correlative electron microscopy, we found that monooriented chromosomes could glide toward the spindle equator alongside kinetochore fibers attached to other already bioriented chromosomes. This congression mechanism depended on the kinetochore-associated, plus end*directed microtubule motor CENP-E (kinesin-7).