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Article |
Molecular mechanisms of microtubule-dependent kinetochore transport toward spindle poles
Correspondence to Tomoyuki U. Tanaka: t.tanaka{at}lifesci.dundee.ac.uk
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.
Abbreviation used in this paper: MSD, mean squared displacement.
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Introduction |
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In the budding yeast Saccharomyces cerevisiae, centromeres are tethered to spindle poles by microtubules during most of the cell cycle (Winey and O'Toole, 2001; T.U. Tanaka et al., 2005). Nonetheless, centromeres are released from and recaptured by microtubules during a brief period in S phase, probably as a result of kinetochore disassembly and reassembly upon centromere DNA replication (Pearson et al., 2004; Tanaka, 2005; our unpublished data). We have visualized this process in S phase using electron microscopy and live cell fluorescence microscopy (K. Tanaka et al., 2005). Moreover, to analyze individual kinetochore–microtubule interaction with higher resolution, we displaced a selected centromere (CEN3) from the spindle and other centromeres by conditionally inactivating it (K. Tanaka et al., 2005). Then, during metaphase arrest, we reactivated CEN3, which was subsequently captured by the lateral surface of single microtubules (centromere reactivation system; Fig. 1 A).
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The Dam1 complex, which is also called DASH or DDD and is composed of at least 10 proteins, has important roles in ensuring proper kinetochore–microtubule interaction (Cheeseman et al., 2001a; Janke et al., 2002; Li et al., 2002). However, in our CEN3 reactivation system, mutants of the Dam1 complex components did not show substantial defects in CEN3 capture by microtubules or in the subsequent sliding of CEN3 along microtubules (K. Tanaka et al., 2005). It has been recently reported that several Dam1 complexes could gather together and form rings encircling microtubules in vitro (Miranda et al., 2005; Westermann et al., 2005). It is still unclear whether this is the case in vivo or how the complex regulates kinetochore–microtubule interaction.
Here, we studied mechanisms of kinetochore transport by microtubules using our centromere reactivation system (K. Tanaka et al., 2005) as well as in normal cell cycles (i.e., without cell cycle arrest or regulation of centromere activity). We show that poleward movement of kinetochores 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. Our study reveals how Kar3 and the Dam1 complex regulate these processes.
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To address whether Kar3 localizes at kinetochores and directly drives their transport along microtubules, we visualized Kar3 by fusing it with four tandem copies of GFP (Kar3-4GFP). After the CEN3 reactivation, in most cells, Kar3-4GFP was visible at the CFP-labeled CEN3 before its capture by CFP-labeled microtubules and also during its transport along microtubules (Fig. 1 B). Kar3 was also detected at the plus ends of growing microtubules (supplemental note 1; available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1) and at spindle poles as previously reported (Hildebrandt and Hoyt, 2000; Maddox et al., 2003). The amount of Kar3 at kinetochores appeared to decrease after sister kinetochore biorientation (supplemental note 1; Tytell and Sorger, 2006).
Kinetochores attach to microtubule plus ends and are transported poleward as microtubules shrink in the absence of Kar3
Kar3 is involved in kinetochore transport toward spindle poles (K. Tanaka et al., 2005). However, in the majority of kar3
cells, CEN3 still reached spindle poles after being captured by microtubules (K. Tanaka et al., 2005), suggesting the involvement of a redundant mechanism for kinetochore transport. To identify this mechanism, we analyzed poleward kinetochore transport in kar3 cells in greater detail. In 68% of KAR3+ wild-type cells, CEN3 reached the spindle by sliding along the lateral surface of microtubules, whereas in kar3 cells, this occurred in only 11% of cells (Fig. 2 A, supplemental note 2, and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1).
The sliding observed in kar3 cells might depend on other microtubule motors, but, as we discuss below, it probably depends on motor-independent one-dimensional diffusion (supplemental note 3). In many of the kar3 cells, after CEN3 capture by the lateral sides of microtubules, CEN3 was tethered at the distal ends of microtubules extending from spindle poles and was pulled poleward as the microtubules shrank (Fig. 2 B, microtubule end-on pulling of kinetochores; and Video 1). 81% of kar3 cells showed microtubule end-on pulling of CEN3, whereas this occurred in only 13% of KAR3+ wild-type cells (Fig. 2 A). Thus, in KAR3+ cells, kinetochores were transported mainly by sliding along microtubules, but, in the absence of Kar3, microtubule end-on pulling was the main mode for kinetochore transport.
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cells, microtubule end-on pulling transported CEN3 poleward on average more rapidly than sliding and without pausing (Fig. 2, C [right] and D). During end-on pulling, microtubule rescue was rarely observed (supplemental note 4 and Fig. S1 B). In addition, CEN3 occasionally (4.9%) detached from microtubules during sliding, whereas such detachment was never observed during end-on pulling (supplemental note 5). These differences suggest that the two modes of kinetochore transport are distinctly regulated processes. Nonetheless, CEN3 sliding was sometimes converted to end-on pulling (Fig. 2, A [pale green] and E; and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1), whereas the opposite conversion was seldom, if ever, observed.
Kar3 is the main and probably the sole factor driving kinetochore sliding along microtubules
Having established that CEN3 is transported poleward either by sliding or end-on pulling, we were in a position to evaluate the contribution of Kar3 specifically to CEN3 sliding by excluding end-on pulling events from our analysis. To make an unbiased comparison between KAR3+ and kar3 cells, we studied CEN3 movement for a short period, during which CEN3 is associated with the microtubule lateral surface but not at the microtubule distal end after its initial capture by the microtubule lateral surface (supplemental note 6). During such a period, in KAR3+ cells, CEN3 travelled preferentially poleward (Fig. 3 A, top left) except in a small number of cells (supplemental note 7).
In contrast, in kar3 cells, CEN3 moved in both directions apparently equally (Fig. 3 A, top right). Consequently, the mean displacement of CEN3 from its original position (i.e., its position when initially captured by a microtubule) along a microtubule increased with time and was oriented toward a spindle pole in KAR3+ cells at a speed of 0.39 µm/min (Fig. 3 A, bottom left; and supplemental note 6), whereas it remained approximately zero in kar3 cells (Fig. 3 A, bottom right).
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cells, we suspected that CEN3 motility in this mutant might be caused by one-dimensional diffusion along a microtubule. To address this, we plotted the mean squared displacement (MSD) of CEN3 along a microtubule against a change of time (t) in kar3 cells (Fig. 3 B; Saxton and Jacobson, 1997; Helenius et al., 2006). The MSD increased linearly as t increased, which was indeed consistent with the one-dimensional diffusion of CEN3. The diffusion coefficient (D in MSD = 2Dt) of CEN3 motility along microtubules was calculated as 0.11 µm2/min. This result was consistent with our previous observations that single deletion mutants of other microtubule-dependent motor proteins (Cin8, Kip1, Kip2, Kip3, and Dyn1) had no effect on CEN3 transport (K. Tanaka et al., 2005). We also failed to observe a CEN3 association of these other motor proteins during its transport (supplemental note 8 and Fig. S2, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1). These data suggest that Kar3 is the main and probably the sole factor that drives poleward kinetochore sliding along microtubules.
Kar3 partially hinders kinetochores from being tethered at the microtubule plus ends
In most cells that we observed, CEN3 was first captured by a microtubule lateral surface, but, when the microtubule subsequently shrank, its distal end encountered CEN3 (note that the microtubule shrinkage rate exceeds the mean velocity of CEN3 sliding; Fig. 4 A; K. Tanaka et al., 2005) unless either the microtubule was rescued before this or CEN3 reached a spindle pole by sliding.
Immediately after the microtubule end encountered CEN3, we observed either of the following two events: (1) microtubules were rescued, and CEN3 remained on the microtubule lateral surface; or (2) CEN3 became tethered at the microtubule end and subsequently was transported poleward by end-on pulling (Fig. 4 A). In such encounters in wild-type KAR3+ cells, we observed microtubule rescue in 59% of cases and the establishment of end-on pulling in 41% of cases (Fig. 4 B).
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cells (Fig. 4 B). We found that kar3 cells showed a more frequent establishment of end-on pulling than KAR3+ cells, suggesting that Kar3 can suppress the establishment of end-on pulling when microtubule plus ends reached CEN3 (supplemental note 9 and Fig. S2 C).
How does Kar3 partially suppress the establishment of the microtubule end-on pulling of CEN3? We speculated that Kar3 might anchor CEN3 to the microtubule lateral surface (close to microtubule plus ends) and that this might hinder CEN3 from forming a specific attachment required for end-on pulling. To test this idea, we used the kar3-1 mutant, which can bind microtubule lateral surfaces but cannot work as a motor because of an ATP hydrolysis defect (Meluh and Rose, 1990; Maddox et al., 2003; K. Tanaka et al., 2005). In contrast to kar3 cells, kar3-1 mutant cells showed a strong reduction in the frequency of establishing end-on pulling (Fig. 4 B, compare kar3-1 with kar3; and supplemental note 10). This result also explains why microtubule-dependent poleward CEN3 transport was more severely delayed in kar3-1 than in kar3 (K. Tanaka et al., 2005). The kar3-1 mutant cannot facilitate CEN3 sliding along microtubules as a result of its defective motor activity yet considerably inhibits the establishment of the microtubule end-on pulling of CEN3 in contrast to kar3, thus causing a considerable delay in CEN3 transport (supplemental notes 10 and 11).
Characterizing microtubule depolymerization during end-on pulling
We next addressed which microtubule end underwent depolymerization during the microtubule end-on pulling of CEN3. We marked a microtubule region midway between CEN3 (bound at the microtubule plus end) and a spindle pole by photobleaching the YFP-Tub1 signal (Fig. 5 A).
The distance between the photobleached region and a spindle pole did not substantially change as CEN3 was pulled poleward by microtubule shrinkage. Thus, depolymerization occurred at the plus end (i.e., bound to CEN3) but not substantially at the minus end (i.e., at a spindle pole). This is consistent with other results indicating that microtubules are dynamic only at the plus ends in budding yeast (Maddox et al., 2000; K. Tanaka et al., 2005).
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In addition, because Kar3 and Kip3 (a kinesin-8 family member) facilitate microtubule depolymerization in vitro (Endow et al., 1994; Chu et al., 2005; Sproul et al., 2005; Gupta et al., 2006; Varga et al., 2006) and foster microtubule disassembly in vivo (Huyett et al., 1998; Hildebrandt and Hoyt, 2000; Severin et al., 2001; West et al., 2001), we addressed whether these motor proteins affect the microtubule shrinkage rate (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1). Deletions of kar3 or kip3 and the kar3 kip3 double mutant did not significantly change the microtubule shrinkage rate either when CEN3 was transported by microtubule end-on pulling or in the absence of CEN3 association. Nonetheless, the mean length of nuclear microtubules was longer in kar3 and kip3 cells than in wild-type cells (unpublished data) as a result of a reduced frequency of microtubule catastrophe (conversion from growth to shrinkage) in these cells (unpublished data).
The Dam1 complexes along a microtubule are collected at the microtubule plus end as the microtubule depolymerizes
We next studied what factors facilitate the microtubule end-on pulling of kinetochores. The Dam1 complex was a candidate for the following reasons: (1) the Dam1 complex is not required for kinetochore sliding (K. Tanaka et al., 2005) but, nonetheless, becomes associated with kinetochores before sister kinetochores biorient in metaphase (Cheeseman et al., 2001a; Janke et al., 2002; Li et al., 2002); (2) the complex forms a ring encircling microtubules in vitro (Miranda et al., 2005; Westermann et al., 2005) and, if this is the case in vivo, it could tether kinetochores at microtubule plus ends while the complex is pushed poleward as microtubule protofilaments splay out during microtubule shrinkage (Fig. 6 A); and (3) dam1 and kar3 mutants show a synergistic effect on chromosome loss (supplemental note 13), which could be explained if both factors are involved in microtubule-dependent kinetochore transport.
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cells, would be defective when the Dam1 complex is impaired. To test this, we attempted to combine kar3 with temperature-sensitive mutants of the Dam1 complex components dam1-1, ask1-3, and spc34-3. However, all of these combinations were lethal even at a permissive temperature for each temperature-sensitive mutant. Therefore, we used the kar3-64 temperature-sensitive mutant instead of kar3 (supplemental note 14). Only the dam1-1 kar3-64 double mutant was viable, whereas the other two combinations were lethal.
We analyzed CEN3 transport in dam1-1 and kar3-64 single mutants and in the dam1-1 kar3-64 double mutant at 35°C, which is a restrictive temperature for these mutant alleles. In the kar3-64 single mutant, CEN3 was transported toward spindle poles by microtubule end-on pulling in the majority (74%) of cells (Fig. 8 A), which is consistent with the behavior of kar3 cells (Fig. 2 A).
In the dam1-1 single mutant, lateral sliding was observed in 66% of the cells, similar to the wild type. However, the amount of successful end-on pulling was dramatically reduced to only 3%. The remainder of the dam1-1 cells ended up in a standstill state, with CEN3 at the end of a microtubule that did not shrink any further (similar to the cell in Fig. 8 B; supplemental note 15). This suggests that Dam1 is required for successful end-on pulling. This conclusion was supported by the behavior of the dam1-1 kar3-64 double mutant, in which virtually all cells ended up in the standstill state (Fig. 8 B and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1). These results suggest that although Kar3 is required for lateral sliding, the Dam1 complex has an essential role in the microtubule end-on pulling of kinetochores.
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Kar3 and the Dam1 complex facilitate kinetochore transport by microtubules in normal S phase
To analyze microtubule-dependent kinetochore transport at high resolution, we have so far studied this process using centromere reactivation in metaphase-arrested cells (Fig. 1 A). Next, we wanted to study whether Kar3 and the Dam1 complex also have important roles in the transport of authentic centromeres (i.e., without regulation by an adjacent GAL1-10 promoter) and in normal cell cycles (i.e., without cell cycle arrest).
Recently, we have been able to visualize the transient detachment of GFP-marked centromeres from microtubules in early S phase (supplemental note 17; our unpublished data). This detachment continued for 0.5–1.5 min, during which centromeres typically moved up to 1.4 ± 0.6 µm (mean ± SD) from a spindle pole. After detachment, centromeres were recaptured by microtubules and swiftly (in 0.5–1.0 min) returned to the vicinity of a spindle pole (<0.6 µm from the spindle pole; our unpublished data).
These results prompted us to compare the behavior of centromeres, which detached from microtubules in early S phase in wild-type, dam1-1, and kar3-64 single mutant and dam1-1 kar3-64 double mutant cells. We labeled CEN5, CEN15, and microtubules by CFP, GFP, and YFP, respectively. Before cells started budding (i.e., before entry into S phase), both CENs localized close to a spindle pole in wild-type and kar3-64 cells but often somewhat more distant (>1.0 µm) from a pole while still attached to microtubules in dam1- 1 and dam1-1 kar3-64 cells. Nonetheless, in all four kinds of cells, both CENs equally showed detachment from microtubules when cells started budding (i.e., during S phase; supplemental note 17) and were recaptured by microtubules extending from a spindle pole after a similar time interval (0.5–1.5 min).
In both wild-type and kar3-64, after CENs were recaptured by microtubules, most centromeres promptly (in 0.5–1.0 min) moved to the vicinity of a spindle pole (Fig. 9 A, pink). Microtubule end-on pulling was more frequently discerned in kar3-64 cells than in wild-type cells (Fig. 9, A [red shaded areas] and B; and supplemental note 18), although in other cases, it was difficult in this experimental condition to distinguish CEN transport by sliding along microtubules and by end-on pulling. In dam1-1 and dam1-1 kar3-64, CENs were captured by microtubules but were not transported to the vicinity of a spindle pole in 53% and 93% of cells, respectively (Fig. 9 A, teal). In such cases, we often discerned that CENs attach at microtubule distal ends without being pulled toward a spindle pole (Fig. 9, A [blue shaded area] and B; and supplemental note 18). We also measured the velocity of CEN transport and found that it was distinctly altered by dam1-1 and kar3-64 mutations (supplemental note 19 and Fig. S4 B).
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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, 2006; 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 (Fig. 10) 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 (Fig. 10). 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, 2005), 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 kar3 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.
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Microscopy
The procedures for time-lapse fluorescence microscopy were described previously (Dewar et al., 2004; K. Tanaka et al., 2005). Time-lapse images were collected every 15 s for 30 min at 23°C (ambient temperature) unless otherwise stated. For image acquisition, we used a microscope (DeltaVision RT; Applied Precision), a UPlanSApo 100x NA 1.40 objective lens (Olympus), a CCD camera (CoolSnap HQ; Photometrics), and SoftWoRx software (Applied Precision). We acquired three to seven (0.7 µm apart) z sections, which were subsequently deconvoluted, projected to two-dimensional images, and analyzed with SoftWoRx and Volocity (Improvision) software. CFP signals were discriminated from either YFP or GFP signals using the JP4 filter set (Chroma Technology Corp.). YFP signals were discriminated from CFP and GFP signals using the JP3 filter set (Chroma Technology Corp.). To collect GFP and YFP signals together without distinguishing the two, the YFP channel of the JP4 filter set was used.
Analyzing the dynamics of kinetochores and microtubules
To evaluate the length of microtubules and position of centromeres, we took account of the distance along the z axis as well as the distance on a projected image. To score modes of kinetochore transport in the CEN3 reactivation system (Figs. 2 A and 8 A), we selected cells in which CEN3 was captured by microtubules (CEN3–spindle pole distance was 2 µm or longer at the initial capture) and subsequently reached spindle poles during a time-lapse observation of 30-min duration. Cells were scored for sliding when CEN3 was transported along microtubules for 1 µm or longer (in most cases toward a spindle pole) and tubulin signal intensity was similar distal and proximal to CEN3 (i.e., CEN3 was not transported with end-on pulling by shorter overlapping microtubules; supplemental note 2). Cells were scored for microtubule end-on pulling when CEN3 was transported poleward for 1 µm or longer with CEN3 attached to the microtubule distal end. Standstill was scored when CEN3 was almost at the same position on microtubules for considerable time (see supplemental note 30 for details). The rate of microtubule growth and shrinkage was evaluated only for approximate linear changes (R2 > 0.85 in linear regression analyses) of microtubule lengths >3 µm. MSD was calculated as described in Helenius et al. (2006). Statistical analyses were performed with the Fisher's exact test (Figs. 4 B, 9 A, and S4 A) or with an unpaired t test (all others) using Prism software (GraphPad) unless otherwise stated. All p-values are two tailed. All error bars in figures represent SEM. For more information, see supplemental note 30.
Online supplemental material
Supplemental notes 1–32 describe more results, discussions, and methods. Fig. S1 A shows kinetochore transport in cells in which microtubule plus ends are labeled, and B shows Stu2 localization at CEN3 during microtubule end-on pulling. Fig. S2 A shows the localization of motor proteins Cin8, Kip1, Kip2, Kip3, and Dyn1, B shows kinetochore transport in kar3 dyn1 and kar3 kip3 double mutants, and C shows Kar3 localization during microtubule end-on pulling. Fig. S3 A shows that Kar3 and Kip3 do not significantly affect the microtubule shrinkage rate, and B shows defects in kinetochore capture and transport in dam1, ask1, and spc34 mutants. Fig. S4 A shows that kinetochores sometimes detach from microtubules in dam1-1-td, B shows CEN transport velocity in normal S phase, and C shows Kar3 localization at kinetochores in normal S phase. Video 1 shows a kar3 cell showing the microtubule end-on pulling of CEN3 (video of the cell shown in Fig. 2 B). Video 2 shows the conversion of CEN3 sliding along a microtubule into microtubule end-on pulling (video of the cell shown in Fig. 2 E). Video 3 shows the Dam1 complexes accumulate at a microtubule plus end as it depolymerizes (video of the cell shown in Fig. 6 B). Video 4 shows that the Dam1 complexes continuously colocalize with CEN3 during microtubule end-on pulling (video of the cell shown in Fig. 7 A). Video 5 shows that the Dam1 complexes do not continuously colocalize with CEN3 during sliding along a microtubule (video of the cell shown in Fig. 7 B). Video 6 presents a dam1-1 kar3-64 cell showing the standstill of CEN3 attached at the plus end of a microtubule (video of the cell shown in Fig. 8 B). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200702141/DC1.
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Acknowledgments |
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This work was supported by Cancer Research UK, Wellcome Trust, the Human Frontier Science Program, the Lister Research Institute Prize, and the Association for International Cancer Research. T.U. Tanaka is a Senior Research Fellow of Cancer Research UK.
Submitted: 20 February 2007
Accepted: 15 June 2007
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