For accurate chromosome segregation to occur during mitosis, sister chromatids connect at their kinetochores to bundles of spindle microtubules, known as kinetochore (K) fibres, that originate from opposite spindle poles. Such bi-oriented chromosomes then 'congress' in the metaphase plate in preparation for segregation. Kapoor et al. now show that chromosomes in mammalian cells can congress before becoming bi-oriented — mono-oriented chromosomes can 'hitch a ride' along the K fibres of other, already bi-oriented chromosomes.

...mono-oriented chromosomes can 'hitch a ride' along the K fibres of other, already bi-oriented chromosomes.

Using differential interference contrast (DIC) time-lapse microscopy, the authors frequently observed mono-oriented chromosomes that were moving towards the metaphase plate as if they were trying to congress. To determine whether these chromosomes were indeed mono-oriented, Kapoor and co-workers followed mitotic cells until one of the chromosomes had almost reached the metaphase plate, at which point the cells were fixed. Indeed, electron microscopy (EM) analysis of these fixed cells showed several instances in which the leading kinetochore of several chromosomes was not connected to microtubules from the opposite spindle pole. Instead, the kinetochore laterally interacted with a K fibre that was attached to a kinetochore of another, bi-oriented chromosome in the metaphase plate.

By simultaneously imaging microtubules and kinetochores using live-cell dual-channel fluorescence, Kapoor et al. were able to show that at least one chromosome per cell was mono-oriented (in 12 of 49 cells) and followed the same trajectory of K fibres of other, already bi-oriented cells. This pattern indicates that this mode of transportation of mono-oriented chromosomes towards the metaphase plate is functional and regulated.

To investigate this process further, the authors devised a clever assay for inducing the synchronized congression of mono-oriented chromosomes by treating the cells with several cell-permeable chemical inhibitors. Using this assay, they imaged individual cells by time-lapse DIC and spinning-disk confocal microscopy. Once several mono-oriented chromosomes initiated their movement towards the metaphase plate, the cell was fixed for EM analysis. For as many as six out of seven congressing mono-oriented chromosomes, the leading kinetochore was laterally associated with a K fibre from another, bi-oriented chromosome. As predicted, the lagging kinetochore was attached to a K fibre from the proximal spindle pole.

A HeLa cell that has been depleted of CENP-E and subjected to the authors' chemical inhibitor assay. Immunofluorescence highlights tubulin (green) and kinetochores (red). Image courtesy of Dr Michael Lampson, Wadsworth Center, Albany, New York, USA.

But what is the molecular mechanism that is responsible for the congression of mono-oriented chromosomes? It is likely to be a motor protein that transports cargo towards the microtubule plus ends, and that is present at kinetochores during prometaphase. As the kinesin-7-family protein CENP-E fits the bill, Kapoor and colleagues used small interfering RNA to knock down CENP-E in their chemical-inhibitor assay. The presence of mono-oriented chromosomes in CENP-E-depleted cells indicated that kinetochores remain capable of capturing microtubules. However, these mono-oriented chromosomes largely failed to congress in the metaphase plate (see figure), presumably because they could not be transported in the absence of CENP-E. So, this mechanism for the congression of mono-oriented chromosomes seems to be CENP-E dependent.

The authors propose a cooperative mechanism of congression in which “...the probability that a mono-oriented chromosome will be transported toward the spindle equator increases as more and more chromosomes become bioriented...”.