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Proper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule. The consequence of this is unknown. Here, we find that, although two kinetochores (two pairs of sister kinetochores) can interact with the lateral side of one microtubule, only one kinetochore can form a sustained attachment to the microtubule end in budding yeast ( Saccharomyces cerevisiae ). This leads to detachment of the other kinetochore from the microtubule end (or a location in its proximity). Intriguingly, in this context, kinetochore sliding along a microtubule towards a spindle pole delays and diminishes discernible kinetochore detachment. This effect expedites collection of the entire set of kinetochores to a spindle pole. We propose that cells are equipped with the kinetochore-sliding mechanism to mitigate problems associated with multiple kinetochores on one microtubule in early mitosis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT-MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT. KT-derived MTs may facilitate KT capture, but their contribution is elusive. In this study, we find that Stu1 recruits Stu2 to budding yeast KTs, which promotes MT generation there. By removing Stu2 specifically from KTs, we show that KT-derived MTs shorten the half-life of noncaptured KTs from 48-49 s to 28-34 s. Using computational simulation, we found that multiple factors facilitate KT capture redundantly or synergistically. In particular, KT-derived MTs play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial KT encounter with spindle MTs., (© 2017 Vasileva et al.)

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles (bi-orientation). To establish bi-orientation, aberrant kinetochore-microtubule attachments are disrupted (error correction) by aurora B kinase (Ipl1 in budding yeast). Paradoxically, during this disruption, new attachments are still formed efficiently to enable fresh attempts at bi-orientation. How this is possible remains an enigma. Here we show that kinetochore attachment to the microtubule lattice (lateral attachment) is impervious to aurora B regulation, but attachment to the microtubule plus end (end-on attachment) is disrupted by this kinase. Thus, a new lateral attachment is formed without interference, then converted to end-on attachment and released if incorrect. This process continues until bi-orientation is established and stabilized by tension across sister kinetochores. We reveal how aurora B specifically promotes disruption of the end-on attachment through phospho-regulation of kinetochore components Dam1 and Ndc80. Our results reveal fundamental mechanisms for promoting error correction for bi-orientation.

How kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore. This process often fails and, responding to this failure, microtubule rescue (conversion from shrinkage to growth) occurs, preventing kinetochore detachment from the microtubule end. This rescue is promoted by Stu2 transfer (ortholog of vertebrate XMAP215/ch-TOG) from the kinetochore to the microtubule end. Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore. Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules. Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual kinetochore-microtubule interactions because multiple kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual kinetochore-microtubule interactions in Saccharomyces cerevisiae, by isolating one of the kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call 'centromere reactivation system', for dissection of the process of kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of kinetochore-microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.

In early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Segregation of sister chromatids to opposite spindle poles during anaphase is dependent on the prior capture of sister kinetochores by microtubules extending from opposite spindle poles (bi-orientation). If sister kinetochores attach to microtubules from the same pole (syntelic attachment), the kinetochore-spindle pole connections must be re-oriented to be converted to proper bi-orientation. This re-orientation is facilitated by Aurora B kinase (Ipl1 in budding yeast), which eliminates kinetochore-spindle pole connections that do not generate tension. Mps1 is another evolutionarily conserved protein kinase, required for spindle-assembly checkpoint and, in some organisms, for duplication of microtubule-organizing centers. Separately from these functions, however, Mps1 has an important role in chromosome segregation. Here we show that, in budding yeast, Mps1 has a crucial role in establishing sister-kinetochore bi-orientation on the mitotic spindle. Failure in bi-orientation with inactive Mps1 is not due to a lack of kinetochore-spindle pole connections by microtubules, but due to a defect in properly orienting the connections. Mps1 promotes re-orientation of kinetochore-spindle pole connections and eliminates those that do not generate tension between sister kinetochores. We did not find evidence that Ipl1 regulates Mps1 or vice versa; therefore, they play similar, but possibly independent, roles in facilitating bi-orientation.

In the budding yeast Saccharomyces cerevisiae, microtubule-organizing centers called spindle pole bodies (SPBs) are embedded in the nuclear envelope, which remains intact throughout the cell cycle (closed mitosis). Kinetochores are tethered to SPBs by microtubules during most of the cell cycle, including G1 and M phases; however, it has been a topic of debate whether microtubule interaction is constantly maintained or transiently disrupted during chromosome duplication. Here, we show that centromeres are detached from microtubules for 1-2 min and displaced away from a spindle pole in early S phase. These detachment and displacement events are caused by centromere DNA replication, which results in disassembly of kinetochores. Soon afterward, kinetochores are reassembled, leading to their recapture by microtubules. We also show how kinetochores are subsequently transported poleward by microtubules. Our study gives new insights into kinetochore-microtubule interaction and kinetochore duplication during S phase in a closed mitosis.

In mitosis, kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward kinetochore transport in Saccharomyces cerevisiae. First, kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at kinetochores. Second, kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward kinetochore-pulling force. Thus, microtubule-dependent poleward kinetochore transport is ensured by at least two distinct mechanisms.