We have investigated the mechanisms by which kinetochores are transported toward spindle poles by microtubules in budding yeast. We show that poleward movement can occur in two distinct ways: lateral sliding, in which kinetochores move along the side of a microtubule, and end-on pulling, in which the kinetochore is attached to the end of a microtubule and is pulled poleward as the microtubule shrinks. Kar3 is essential to drive poleward lateral sliding, whereas the Dam1 complex is crucial for end-on pulling ().
It is thought that upon centromere DNA replication in budding yeast, kinetochores are disassembled, causing the release of centromeres from microtubules for a short period (Pearson et al., 2004
; Tanaka, 2005
; our unpublished data). Soon afterward, kinetochores are reassembled and captured by the lateral sides of microtubules extending from spindle poles (K. Tanaka et al., 2005
). Microtubule lateral surfaces can secure initial kinetochore capture by providing a much larger contact surface compared with microtubule tips. The amount of Kar3 loaded at kinetochores probably increases while kinetochores are detached from microtubules (supplemental note 1; Tytell and Sorger, 2006
). Kar3 (and the Dam1 complex) is not required for the initial kinetochore capture by microtubules (supplemental note 22; K. Tanaka et al., 2005
), but, once kinetochores are captured, the motor activity of Kar3 drives kinetochore sliding along microtubule lateral surfaces toward spindle poles. Kar3 is the main and probably the sole factor driving this kinetochore sliding because in the absence of Kar3, kinetochores only show one-dimensional diffusion along microtubules.
Kinetochore sliding, which is promoted by Kar3, occurs toward a spindle pole with frequent pausing. This is perhaps because the Kar3 molecules loaded on kinetochores do not persistently drive their sliding along microtubules. This is a similar situation to that of Ncd, a putative Kar3 orthologue in Drosophila melanogaster
shown to be a nonprocessive motor, whose domain is released from microtubules after each ATPase cycle and must bind microtubules repeatedly to drive motion (Endow, 2003
). Probably because of this frequent pausing, the mean velocity of kinetochore sliding is lower than that of microtubule shrinkage (K. Tanaka et al., 2005
). Therefore, microtubule plus ends often reach kinetochores unless microtubules are rescued and regrow before this happens, a process involving Stu2 transport from kinetochores (K. Tanaka et al., 2005
If microtubule plus ends reach kinetochores, cells must choose one of the following two options: (1) microtubules show regrowth (i.e., are rescued), probably facilitated by Stu2 and other factors loaded at kinetochores (K. Tanaka et al., 2005
), or (2) kinetochores are tethered at microtubule plus ends, probably as a result of association with the Dam1 complex ring structure, which has been pulled poleward as microtubules shrink. Kar3 reduces the frequency of the second choice (i.e., establishment of microtubule end-on pulling), probably by anchoring kinetochores to the microtubule lateral surface. In addition, the establishment of microtubule end-on pulling seems to be partly affected by stochastic elements (supplemental note 9). In any case, in the first option, kinetochores still remain associated with the lateral surface of microtubules and continue to slide poleward along microtubules. In the second, kinetochores at microtubule ends are continuously pulled poleward (end-on pulling) as the attached microtubules shrink without pausing or rescue. It is currently unclear how microtubule rescue is suppressed during end-on pulling, but it is not solely caused by a lack of Stu2 loaded on kinetochores (supplemental note 4).
The microtubule end-on pulling of kinetochores is facilitated by the Dam1 complex. In vitro experiments suggested that several Dam1 complexes could gather together and form a ring structure encircling a microtubule, which could move along the microtubule (Miranda et al., 2005
; Westermann et al., 2005
; Asbury et al., 2006
). We found that the Dam1 complexes along a microtubule were collected at the plus ends of depolymerizing microtubules in vivo (supplemental note 23). The simplest interpretation would be that the Dam1 complexes indeed form a ring encircling a microtubule in vivo, which is pushed poleward by splaying protofilaments as the microtubule depolymerizes. However, it cannot be completely ruled out that the Dam1 complexes do not form a ring in vivo (McIntosh, 2005
) and that unknown mechanisms collect the complexes along a microtubule at its plus end during microtubule shrinkage. In any case, when Dam1 function was impaired, the microtubule end-on pulling of kinetochores became defective. Our in vivo data support the model () in which the Dam1 complex tethers kinetochores and plays a crucial role in converting microtubule depolymerization to kinetochore pulling force as initially proposed from the in vitro experiments.
Kinetochore sliding and microtubule end-on pulling are two distinct modes of microtubule-dependent kinetochore transport and seem to use different energy sources to produce the force necessary for kinetochore transport. When microtubules polymerize, the curvature of GDP-bound tubulin dimers is constrained by microtubule geometry so that the polymer lattice stores energy from GTP hydrolysis (Howard and Hyman, 2003
). During end-on pulling, the free energy is released and converted to kinetochore pulling force by a power-stroke mechanism as microtubule protofilaments change from a straight to curved form (Grishchuk et al., 2005
). The Dam1 complex apparently has an important role in this conversion (). In contrast, kinetochore sliding is driven by the Kar3 motor activity that is dependent on its ATP hydrolysis (i.e., additional energy is consumed; Yun et al., 2001
). In spite of this, kinetochore sliding is less processive and achieves less efficient kinetochore transport.
Given these disadvantages, why do cells still use kinetochore sliding for their transport? Kinetochore sliding may have the following merits compared with end-on pulling: (1) for the establishment of microtubule end-on pulling, kinetochores must wait until the associated microtubule shrinks and the microtubule plus end finally reaches the kinetochore. Therefore, depending on the situation, kinetochores may reach a spindle pole earlier by sliding than by end-on pulling. (2) A single microtubule plus end is probably able to attach to only a single kinetochore during end-on pulling (Winey and O'Toole, 2001
), but, in contrast, multiple kinetochores could be transported simultaneously by sliding (supplemental note 24). (3) Microtubule rescue, which happens during kinetochore sliding but not during end-on pulling, would increase the chance that kinetochores further afield are also captured by the same microtubule (supplemental note 25).
Because kinetochore sliding is converted into end-on pulling but not vice versa, the population of kinetochores attached to microtubule plus ends increases during poleward kinetochore transport. Both sister kinetochores subsequently interact with microtubules, and the Ipl1 kinase promotes the reorientation of kinetochore–microtubule attachment (T.U. Tanaka et al., 2002
), in which the Dam1 complex is a crucial substrate of the kinase (Cheeseman et al., 2002
). Because this reorientation happens in a tension-dependent manner (Nicklas, 1997
; Dewar et al., 2004
), sister kinetochores eventually attach to microtubules from opposite spindle poles (biorientation). To establish biorientation efficiently, kinetochores must be located within the spindle where microtubules extend from both spindle poles at high density. Because microtubule-dependent transport brings kinetochores close to the spindle, this process should facilitate efficient sister kinetochore biorientation.
The stable maintenance of biorientation crucially requires Dam1 complex function (Janke et al., 2002
). Presumably, in metaphase, the Dam1 complex is necessary to pull sister kinetochores toward opposite spindle poles, generating tension across sister kinetochores and, in turn, stabilizing kinetochore–microtubule attachment (Dewar et al., 2004
), thus simultaneously avoiding breakage of the attachment when this tension is applied (supplemental note 26). Metaphase is followed by anaphase A (Pearson et al., 2001
), in which the kinetochore–spindle pole distance is shortened. We envisage that the Dam1 complex also plays the same role in anaphase A, as we found in prometaphase (i.e., tethering kinetochores at the microtubule plus ends and converting microtubule depolymerization [occurring at kinetochore sides; Maddox et al., 2000
] into kinetochore pulling force). Consistent with this notion, we found that the Dam1 complex colocalizes with kinetochores during anaphase A (supplemental note 27; unpublished data).
Recently, the Dam1 complex orthologue was identified in fission yeast (Liu et al., 2005
; Sanchez-Perez et al., 2005
). In this organism, the Dam1 complex has important roles in sister kinetochore biorientation and kinetochore congression to the spindle midzone, which is consistent with Dam1 complex function in budding yeast. Moreover, kinetochores are still transported poleward in the absence of all of the known microtubule minus end–directed motors (i.e., two kinesin-14s and dynein) in fission yeast (Grishchuk and McIntosh, 2006
); thus, perhaps kinetochores are transported by end-on pulling in this organism, as we have shown directly here in budding yeast.
In vertebrate cells, kinetochores are also captured by the lateral sides of single microtubules and are transported toward spindle poles in prometaphase (Rieder and Alexander, 1990
). How is the kinetochore transport regulated in vertebrate cells? In contrast to mechanisms in budding yeast (supplemental note 8), vertebrate dynein could be involved in fast and processive kinetochore sliding along microtubules (supplemental note 28; Rieder and Alexander, 1990
; King et al., 2000
). If this is the case, the depletion of dynein may reveal the microtubule end-on pulling of kinetochores as a possible redundant mechanism for kinetochore transport in vertebrate cells, just as it was revealed by kar3Δ
in yeast. Although convincing orthologues of the Dam1 complex components have not yet been identified in vertebrate cells (Meraldi et al., 2006
), functional counterparts of the Dam1 complex may have an important role in microtubule end-on pulling. Kinesin-13s (mitotic centromere-associated kinesin, etc.) may be such functional counterparts because they also form rings encircling single microtubules in vitro (Moores et al., 2006
; Tan et al., 2006
), localize at kinetochores in mitosis (Wordeman, 2005
), and act as important substrates of the aurora B kinase in ensuring proper kinetochore–microtubule attachment (Andrews et al., 2004
; Lan et al., 2004
; Ohi et al., 2004
; Sampath et al., 2004
Kinetochore capture and transport by spindle microtubules is the first crucial step for proper chromosome segregation in all eukaryotic cells. Comparison of kinetochore transport between different organisms will uncover the evolution of regulatory mechanisms for this fundamental cellular process.