In fission yeast centromeres cluster at the nuclear envelope in a region underlying the spindle pole body during interphase, an arrangement known as a Rabl configuration. We have identified a strain in which one pair of sister kinetochores is unclustered from the others and binds the nuclear envelope at a point distal to the spindle pole body. We show that during mitosis unclustered kinetochores are captured by intranuclear spindle microtubules which then pull the kinetochores back to one of the two spindle poles before they are bi-oriented on the mitotic spindle. We find that kinetochore retrieval occurs at the depolymerising microtubule plus end and is dependent on the non-essential Dam1/DASH complex. In the absence of Dam1 unclustered kinetochores are captured on the lateral surface of spindle microtubule bundles but poleward kinetochore movement does not occur. These data provide the first direct evidence that the Dam1/DASH complex can couple the force generated by microtubule depolymerisation to direct chromosome movement in vivo.

The proper segregation of sister chromatids to daughter cells during mitosis is essential for normal cell proliferation. For this to occur each kinetochore must be captured by a microtubule(s) from one spindle pole and its sister kinetochore bound to a microtubule(s) from the opposite spindle pole, a process known as chromosome bi-orientation (Tanaka et al., 2005, Tanaka et al., 2005). Work in yeast model systems has been instrumental both in the identification of many components of the mitotic spindle and kinetochore, many of which are conserved in humans, and for elucidating the mechanisms governing microtubule interaction with the kinetochore and the processes of chromosome bi-orientation and sister chromatid separation (Nasmyth, 2002; Tanaka, T. U. et al., 2005, Tanaka, T. U. et al., 2005; Westermann et al., 2007). However, our understanding of these events is far from complete.

In budding yeast, Saccharomyces cerevisiae, centromeres cluster near the spindle pole body (SPB). During interphase each kinetochore is bound to a single, short microtubule nucleated from the SPB, which remains embedded in the nuclear envelope (Winey et al., 1995; Jin et al., 2000). This arrangement, known as a Rabl configuration, can be disturbed by microtubule disrupting agents (Rabl, 1885; Jin et al., 2000). As chromosomes are replicated both sister chromatids are initially bound to a spindle microtubule from the same pole, a configuration known as syntelic attachment. In this situation tension cannot be applied across the mitotic spindle. As cells enter mitosis the lack of spindle tension activates the Aurora-like Ipl1 kinase which triggers microtubule detachment so that amphitelic attachment of each sister chromatid to spindle microtubules from opposite poles can be established (Dewar et al., 2004). It has been thought that Ipl1-dependent phosphorylation of Dam1, is particularly important for correcting improper chromosome attachment (Cheeseman et al., 2002; Shang et al., 2003). Dam1 is a subunit of an essential ten component complex (Dam1; also known as DASH) which connects the centromere to the plus end of spindle microtubules throughout the cell cycle (Cheeseman et al., 2001a; Cheeseman et al., 2001b). When reconstituted in vitro the Dam1/DASH complex forms rings around microtubules and can move processively at the plus ends of both polymerising and depolymerising microtubules (Miranda et al., 2005; Westerman et al., 2005; Asbury et al., 2006). The Dam1/DASH ring complex is thought to couple chromosome movement to microtubule dynamics during mitosis, however, direct evidence for this in vivo is lacking. Additional mechanisms are necessary for the capture and retrieval of kinetochores that are not arranged in a Rabl configuration. In particular, Tanaka and colleagues have monitored the behaviour of a chromosome with a regulatable kinetochore which, after prolonged cell cycle arrest in mitosis, is positioned several micrometers from the spindle poles. After reactivation, the kinetochore is captured and transported along the lateral surface of a stabilised microtubule to the spindle pole by the minus end directed kinesin, KAR3 (kinesin-14), before it is bi-oriented on the mitotic spindle (Tanaka, K. et al., 2005, Tanaka, K. et al., 2005). Stabilisation of the captured microtubule occurs by delivery of Stu2 (also known as XMAP215 and Dis1) from the re-activated kinetochore to the microtubule plus end and this is thought to prevent the kinetochore from slipping off the plus end of the microtubule during the retrieval process (Tanaka, K. et al., 2005, Tanaka, K. et al., 2005).

Less is known about the mechanisms controlling kinetocore capture and chromosome bi-orientation in fission yeast (Schizosaccharomyces pombe). Although fission yeast centromeres are clustered near the spindle pole body during interphase, the SPB remains outside the nuclear envelope during this period and spindle microtubules are only nucleated when the duplicated SPB enters the nuclear membrane following mitotic initiation (Funabiki et al., 1993; Ding et al., 1997). As such, centromere clustering in fission yeast is independent of microtubules but nevertheless depends on the integrity of the Mis6, Mis12 and Nuf2-Ndc80 kinetochore sub-complexes (Applegren et al., 2003; Asakawa et al., 2005). At present it is not known whether the Rabl configuration in fission yeast aids the speed or efficiency of kinetochore capture or the establishment of chromosome bi-orientation. Recently, McIntosh and colleagues examined the mechanism of kinetochore capture and retrieval after prolonged cell cycle arrest in mitotic nda3-KM311 (β-tubulin) cells, in which kinetochores become dispersed (Grishchuk and McIntosh, 2006). In this case poleward movement of kinetochores was found to be independent of the two kinesin-14 minus end directed motors, Pkl1 and Klp2, and the single member of the dynein family, Dhc1, although a role for Klp2 in controlling the processivity of this process was described. It was concluded that force generated by microtubule depolymerisation alone could drive chromosome retrieval (Grishchuk and McIntosh, 2006; Grishchuk et al., 2005). However, visualisation of microtubule interaction with the kinetochore in nda3-KM311 cells was not possible in this strain for technical reasons, so it remains unclear whether chromosome retrieval occurs along the lateral face of the microtubule, by coupling the kinetochore to the plus end of the depolymerising microtubule or by an alternative mechanism.

We previously showed that the fission yeast Dam1/DASH complex binds to the tips of non-kinetochore-associated spindle microtubules and to the kinetochore-microtubule junction but, unlike in budding yeast, is not essential for viability (Sanchez-Perez et al., 2005; Liu et al., 2005). We have developed a simple live imaging assay to monitor the fate of kinetochores in cells in which the Rabl configuration of one pairs of sister chromatids is disturbed. We demonstrate here that the Dam1/DASH complex is essential for the retrieval of unclustered kinetochores and does so by coupling kinetochore movement to the plus end of depolymerising intranuclear spindle microtubules.

Mto1 is required for proper centromere clustering in fission yeast

In order to examine the mechanisms controlling kinetochore-microtubule interaction in fission yeast we sought a strain in which the normal Rabl configuration of centromeres is disturbed. Association of centromeres to spindle poles is lost during meiosis when telomeres become bound to the spindle pole body (Funabiki et al., 1993; Chikashige et al., 1994). We noted that when cells that arrested before meiotic S phase (mei1-B102) are encouraged to re-enter the mitotic cell cycle by refeeding, de novo formation of the Rabl configuration does not require passage through mitosis, but is nonetheless prevented by microtubule disrupting agents (Goto et al., 2001). From this, we reasoned that interphase microtubules may play a role in Rabl formation. Mto1 (also known as Mod20 or Mbo1) is a centrosomin-like protein that associates to the γ-tubulin complex at interphase microtubule organising centres (iMTOCs) including the outer, but not inner, face of the spindle pole body and is required for nucleation of cytoplasmic microtubules (Sawin et al., 2004; Venkatram et al., 2004). As a consequence, 30% of Δmto1 cells lack cytoplasmic microtubules and the remainder possess a single interphase microtubule bundle. In fission yeast, cytoplasmic microtubules are required for positioning the nucleus in the centre of the cell and for the initial alignment of the mitotic spindle along the longitudinal axis of the cell (Tran et al., 2001; Vogel et al., 2007). To assess the effect of interphase microtubules on Rabl formation, we monitored spindle pole and kinetochore position in Δmto1 ndc80-gfp cdc11-cfp cells. Ndc80 and Cdc11 bind to the kinetochore and spindle pole body, respectively, throughout the cell cycle (Krapp et al., 2001; Wigge and Kilmartin, 2001) We found that 9.1% of Δmto1 cells display an abnormal arrangement of centromeres during interphase. In these cells one pair of kinetochores is not clustered with the other two kinetochore pairs but remains associates with the nuclear periphery, at a point distal to the unseparated spindle pole body (Fig. 1A, supplementary material Fig. S1A). Notably, unclustered kinetochores appeared more frequently in cells with nuclei that were displaced to the cell tip relative to cells with more centrally located nuclei (Fig. 1B). One would presume that, since nuclear positioning is determined by cytoplasmic microtubules, the position of nuclei in Δmto1 cells reflects the efficiency by which the single remaining cytoplasmic microtubule can position the nucleus. By the same reasoning the prevalence of unclustered kinetochores in cells with nuclei at cell tips may also reflect the severity of the defect in interphase microtubule function. Clustering of kinetochore may either require active nucleation of cytoplasmic microtubules from the outer face of the SPB or, alternatively, iMTOCs may provide a docking site for kinetochores on the inner face of the nuclear envelope in the absence of Mto1. Further experiments will be needed to distinguish these and other possibilities.

Unclustered kinetochores are efficiently retrieved during mitosis

Despite the high frequency of unclustered kinetochores during interphase, the frequency of chromosome mis-segregation in Δmto1 cells is only six to eight times higher than that in wild-type cells (Sawin et al., 2004). To monitor the behaviour of unclustered kinetochores we filmed Δmto1 ndc80-gfp cdc11-cfp cells through the cell cycle. Little change in the position of unclustered kinetochores was observed during interphase relative to the unseparated spindle pole (data not shown). However, as the cells entered mitosis (as judged by the appearance of separated spindles poles), unclustered kinetochores moved towards one or other spindle pole before the kinetochores were bi-oriented on the mitotic spindle (Fig. 1C). In some cells (15/25 movies) unclustered kinetochores were retrieved during early pro-metaphase (phase 1), whereas in other cells (9/25) this occurred only after the mitotic spindle had achieved constant length (phase 2) (Fig. 1C,D). The rate of chromosome retrieval varied from cell to cell, having a maximal rate of 0.7 μm/minute. In some cases the retrieval was interrupted by periods where polewards movement paused and then resumed. In most cells (24/25) kinetochores were retrieved before anaphase onset. The average time between spindle pole separation and completion of anaphase A was somewhat extended in Δmto1 cells with unclustered kinetochores (18±2 minutes, n=13) compared to those with normal Rabl configuration (15±2 minutes, n=24), suggesting that the presence of unclustered kinetochores activates the spindle assembly checkpoint. In one movie we failed to observe chromosome retrieval for more than 30 minutes and anaphase onset did not occur (Fig. 1D). The failure to retrieve unclustered kinetochores may account for the elevated level of chromosome mis-segregation in Δmto1 cells.

Kinetochore retrieval occurs at the plus end of intranuclear spindle microtubules

Electron micrographs of the fission yeast pre-anaphase mitotic spindle reveal that it is composed of 12-16 microtubules that emanate from two spindle pole bodies (SPBs) embedded in opposite sides of a persistent nuclear envelope, and that overlap in a central zone (spindle midzone). An additional 10-12 microtubules originate from each SPB and terminate at the three kinetochores (Tanaka and Kanbe, 1986; Ding et al., 1993; Ding et al., 1997). Other highly dynamic intranuclear spindle microtubules (INAs) are nucleated at an angle to the main body of the mitotic spindle and these normally undergo catastrophe when the tips encounter the inside of the nuclear envelope, although the role of these microtubules is unclear (Zimmerman et al., 2004). To determine how chromosome retrieval is achieved we filmed Δmto1 fta2-tdTomato nmt1-gfp-atb2 cells during mitosis. Fta2 is a constitutively bound component of the fission yeast kinetochore (Liu et al., 2005; Kerres et al., 2006). In all the Δmto1 fta2-tdTomato nmt1-gfp-atb2 cells examined we found that unclustered kinetochores were retrieved (20/20 movies), indicating that mild overexpression of atb2 from the nmt1 promoter does not effect the retrieval process. During phase 1 the spindle is formed co-incidentally with depolymerisation of the final SPB-bound interphase microtubule. For this reason it was difficult to distinguish nuclear spindle and cytoplasmic interphase microtubules during early mitosis. To circumvent this problem we examined cells in which unclustered kinetochores were retrieved during phase 2. We found that intranuclear microtubule(s) are nucleated from both spindle pole bodies at an angle to the main body of the mitotic spindle (Fig. 2A). These microtubules make multiple forays into the intranuclear space. Once contact between the plus end of the intranuclear spindle microtubule and the kinetochore is made the kinetochore is rapidly retrieved towards the pole (Fig. 2A). Chromosome bi-orientation and anaphase onset follow some minutes later. In most movies (13/20) we observed chromosome capture close to the plus end of a intranuclear spindle microtubule and retrieval occurring immediately afterwards. In other movies (7/20) we found that the kinetochore is captured on the lateral surface of the intranuclear spindle microtubule but kinetochore movement towards the pole does not occur until the microtubule depolymerises to the point where the kinetochore associates with the depolymerising plus end (Fig. 2B). We observed that some deformations of the nuclear envelope occur during mitosis, although we have been unable, for technical reasons, to tell whether these are due to interactions of intranuclear spindle microtubules with the nuclear envelope (supplementary material Fig. S1B). Importantly, we never observed polewards motion of kinetochores on the lateral surface of a stabilised intranuclear spindle microtubule. These data indicate that, contrary to the situation in budding yeast, unclustered kinetochores are retrieved at the plus end of a depolymerising spindle microtubule (Tanaka, K. et al., 2005, Tanaka, K. et al., 2005).

Fig. 1.

Unclustered kinetochore pairs are efficiently retrieved to the spindle pole before sister chromatid bi-orientation. (A) ndc80-gfp cdc11-cfp and Δmto1 ndc80-gfp cdc11-cfp cells were fixed and stained with DAPI to reveal the position of kinetochores (green), spindle poles (red) and chromatin (blue). (B) The frequency by which unclustered kinetochores appear in Δmto1 ndc80-gfp cdc11-cfp cells when the nucleus is placed in the middle of the cell (region A), displaced to one side of the cell but not at the cell tip (region B) or at the cell tip (region C) was calculated. (C) Images from a movie of Δmto1 ndc80-gfp cdc11-cfp cells in mitosis. Spindle poles are shown in red and kinetochores are shown in green. Note that an unclustered pair of sister chromatids (white arrowhead) are drawn back to the spindle pole and then bi-oriented on the mitotic spindle. Bar, 1 μm. (D) The distance between the unclustered kinetochore pair and the spindle pole was calculated at 30-second intervals in multiple movies of ndc80-gfp cdc11-cfp cells.

Fig. 1.

Unclustered kinetochore pairs are efficiently retrieved to the spindle pole before sister chromatid bi-orientation. (A) ndc80-gfp cdc11-cfp and Δmto1 ndc80-gfp cdc11-cfp cells were fixed and stained with DAPI to reveal the position of kinetochores (green), spindle poles (red) and chromatin (blue). (B) The frequency by which unclustered kinetochores appear in Δmto1 ndc80-gfp cdc11-cfp cells when the nucleus is placed in the middle of the cell (region A), displaced to one side of the cell but not at the cell tip (region B) or at the cell tip (region C) was calculated. (C) Images from a movie of Δmto1 ndc80-gfp cdc11-cfp cells in mitosis. Spindle poles are shown in red and kinetochores are shown in green. Note that an unclustered pair of sister chromatids (white arrowhead) are drawn back to the spindle pole and then bi-oriented on the mitotic spindle. Bar, 1 μm. (D) The distance between the unclustered kinetochore pair and the spindle pole was calculated at 30-second intervals in multiple movies of ndc80-gfp cdc11-cfp cells.

Fig. 2.

Chromosome retrieval occurs at the plus end of depolymerising intranuclear spindle microtubules (INAs). Images from two movies (A,B) of Δmto1 fta2-tdtomato nmt1-gfp-atb2 cells with unclustered kinetochores. Microtubules are shown in green and kinetochores are shown in red. The position of the unclustered kinetochores is indicated by a white arrowhead. The plus ends of intranuclear spindle microtubules are indicated by a yellow arrowhead. Bar, 1 μm.

Fig. 2.

Chromosome retrieval occurs at the plus end of depolymerising intranuclear spindle microtubules (INAs). Images from two movies (A,B) of Δmto1 fta2-tdtomato nmt1-gfp-atb2 cells with unclustered kinetochores. Microtubules are shown in green and kinetochores are shown in red. The position of the unclustered kinetochores is indicated by a white arrowhead. The plus ends of intranuclear spindle microtubules are indicated by a yellow arrowhead. Bar, 1 μm.

The Dam1/DASH complex is required for retrieval of unclustered kinetochores

In budding yeast, retrieval of unclustered kinetochores is blocked by deletion of the Kar3 minus end directed motor (kinesin-14 family) but not by mutational inactivation of the Dam1/DASH complex (Tanaka, K. et al., 2005, Tanaka, K. et al., 2005). By contrast, we found that chromosome retrieval in Δmto1 cells occurs in the absence of both members of the kinesin-14 family, Pkl1 and Klp2, consistent with previous observations (supplementary material Fig. S2) (Troxell et al., 2001; Grishchuk and McIntosh, 2006). However, we note that in some Δpkl1 Δklp2 cells it takes longer for the retrieval process to be initiated, particularly when the unclustered kinetochore pair was located further away from the spindle pole (supplementary material Fig. S2). We previously showed that the fission yeast DASH complex binds to both the microtubule-kinetochore junction and the plus tips of non-kinetochore-associated intranuclear spindle microtubules (Sanchez-Perez et al., 2005). To test the role of the Dam1/DASH complex in chromosome retrieval, kinetochore movement was monitored in Δdam1 Δmto1 ndc80-gfp cdc11-cfp cells, 8.8% of which contain unclustered kinetochores. We found that in 25/25 movies of Δdam1 Δmto1 ndc80-gfp cdc11-cfp cells with unclustered kinetochores, polewards kinetochore movement of the unclustered kinetochore pair did not occur and anaphase onset was terminally blocked (Fig. 3A and supplementary material Fig. S3A). The movement of the other two pairs of kinetochores on the spindle was initially normal but then slowed as they became bi-oriented, possibly because dynamic instability of kinetochore microtubules is controlled locally by the application of tension (supplementary material Fig. S3A). By contrast, the kinetochores of Δdam1 Δmto1 ndc80-gfp cdc11-cfp cells with normal Rabl configuration underwent bi-orientation and separation, although this was sometimes accompanied by the appearance of lagging sister chromatids or unequal segregation of sister chromatids, as previously observed (Sanchez-Peres et al., 2005) (data not shown). Notably, one or more pairs of kinetochores become detached from the spindle pole body following protracted cell cycle arrest in nda3-KM311 cells (Grishchuk and McIntosh, 2006). In agreement with our previous observations we found that chromosome retrieval and cell cycle re-entry in nda3-KM311 cells is also completely dependent on the Dam1/DASH complex (A.F. and J.B.A.M., unpublished observations).

Fig. 3.

Dam1 but not Klp5 is required for the retrieval of unclustered kinetochores. The distance between the unclustered kinetochore pair and the spindle pole was calculated at 30 second intervals in multiple movies of (A) Δdam1 Δmto1 ndc80-gfp cdc11-cfp cells and Δklp5 Δmto1 ndc80-gfp cdc11-cfp cells.

Fig. 3.

Dam1 but not Klp5 is required for the retrieval of unclustered kinetochores. The distance between the unclustered kinetochore pair and the spindle pole was calculated at 30 second intervals in multiple movies of (A) Δdam1 Δmto1 ndc80-gfp cdc11-cfp cells and Δklp5 Δmto1 ndc80-gfp cdc11-cfp cells.

Fig. 4.

Unclustered kinetochores associate with the lateral surface of spindle microtubule bundles in the absence of Dam1. Images from three movies (A, B and C) of Δmto1 Δdam1 fta2-tdtomato nmt1-gfp-atb2 cells with unclustered kinetochores. Microtubules are shown in green and kinetochores are shown in red. The position of the unclustered kinetochores is indicated by a white arrowhead. The plus ends of intranuclear spindle microtubules are indicated by a yellow arrowhead. Bar, 1 μm (2 μm in C).

Fig. 4.

Unclustered kinetochores associate with the lateral surface of spindle microtubule bundles in the absence of Dam1. Images from three movies (A, B and C) of Δmto1 Δdam1 fta2-tdtomato nmt1-gfp-atb2 cells with unclustered kinetochores. Microtubules are shown in green and kinetochores are shown in red. The position of the unclustered kinetochores is indicated by a white arrowhead. The plus ends of intranuclear spindle microtubules are indicated by a yellow arrowhead. Bar, 1 μm (2 μm in C).

We previously showed that the DASH complex shares an essential overlapping role with Klp5 and Klp6, two members of the kinesin-8 family (Garcia et al., 2002; West et al., 2002; Sanchez-Perez et al., 2005). Proteins in the KIP3 family of microtubule depolymerising kinesins bind to the plus end of kinetochore associated microtubules and co-ordinate poleward movement of sister chromatids during anaphase A (Gupta, Jr et al., 2006; Varga et al., 2006; Tytell and Sorger, 2006). However, we found that chromosome retrieval of unclustered kinetochores in Δklp5 Δmto1 ndc80-gfp cdc11-cfp cells occurred with the same speed and efficiency as in wild-type cells (Fig. 3B and supplementary material Fig. S3B). These results suggest that the Dam1/DASH complex and Klp5 and Klp6 kinesin have distinct functions in the retrieval of unclustered kinetochore pairs but perhaps overlapping roles in the polewards movement of individual sister chromatids during prometaphase, metaphase and anaphase A.

Kinetochores associate to the lateral surface of spindle microtubule bundles in the absence of Dam1/DASH complex

To distinguish whether the Dam1/DASH complex is required for kinetochore capture by spindle microtubules or for coupling kinetochore movement to microtubule disassembly we monitored microtubule dynamics and kinetochore behaviour in Δdam1 Δmto1 nmt1-gfp-atb2 fta2-tdtomato cells. In eight out of 21 movies no association of intranuclear spindle microtubules to unclustered kinetochores was observed (Fig. 4A). In these cells kinetochores were not retrieved and anaphase did not take place. We were unable to visualise interaction of individual intranuclear spindle microtubules with unclustered kinetochores in the absence of the Dam1/DASH complex, possibly because individual intranuclear spindle microtubules are more unstable or because they rapidly slip off the unclustered kinetochore following microtubule depolymerisation. Despite this, we found that in 12 out of 21 cells, unclustered kinetochores bound to the lateral face of an intranuclear spindle microtubule, but these were always microtubule bundles, as judged by fluorescence intensity and more stable relative to individual intranuclear spindle microtubules (Fig. 4B). In addition, interaction of unclustered kinetochore with spindle microtubules was only observed after a protracted delay in anaphase onset. In most cells the bound kinetochore pairs were split, possibly because they are attached to distinct microtubules within the bundle, but they did not move polewards (Fig. 4B). However, in one movie we found that after a protracted delay in anaphase onset an unclustered kinetochore remained distal to the main body of the mitotic spindle but was not retrieved by intranuclear spindle microtubules from the spindle pole closest to it (Fig. 4C). The spindle continued to elongate and eventually bent and broke in the middle. One half of the broken spindle microtubule bundle from the opposite spindle pole then extended and interacted with the unclustered kinetochore. This resulted in some polewards movement but the retrieval was not completed and the kinetochore pair never became bi-oriented (Fig. 4C). These data provide the first direct evidence that the Dam1/DASH complex can couple movement of kinetochores to the plus end of depolymerising intranuclear spindle microtubules in vivo. The development of a robust live imaging assay for chromosome retrieval will help us to not only understand more precisely how the Dam1/DASH complex is regulated by post-translational modifications but also to define more precisely the mechanisms governing kinetochore interaction with microtubules in fission yeast.

During revision of this manuscript Tanaka and colleagues demonstrated, in budding yeast, that when the Kar3 minus-end directed kinesin is absent the Dam1/DASH complex is required for end-on polewards kinetochore transport (Tanaka et al., 2007). In this system the `inactivated' or `lost' kinetochore is located at greater distances from the spindle pole (>4 μm) than we observe for unclustered kinetochores in fission yeast cells lacking Mto1. It is possible the requirement for polewards transport by minus-end directed kinesin motors of the Kar3 family and the Dam1/DASH complex may reflect the initial distance between the unclustered kinetochore and the spindle pole rather than, or in addition to, a species-specific difference between the two yeasts.

Cell culture

Media, growth and maintenance of strains were as described previously (Moreno et al., 1991). Strains used in this study are listed in supplementary material Table S1. All experiments were performed at 30°C unless otherwise stated. Cultures of nmt1-gfp-atb2 cells were grown at 30°C in YES medium and grew at a rate indistinguishable from that of wild type.

Strain construction

Gene deletions of mto1, dam1, pkl1 and klp2 and carboxyl-terminal epitope tagging of cut11 with GFP and fta2 with tdTomato were performed by two-step PCR-based gene targeting, as previously described (Bähler et al., 1998; Krawchuk and Wahls, 1999). In all deletions the entire ORF was removed. The genotypes of the strains used in this study are detailed in supplementary material Table S1. A list of oligonucleotides used is provided in supplementary material Table S2. Compound tagged and mutant strains were constructed by standard genetic methods (Moreno et al., 1991).

Microscopy

Analysis of live cells was performed in an imaging chamber (CoverWell PCI-2.5, Grace Bio-labs) filled with 1 ml of 1% agarose in minimal medium and sealed with a 22×22 mm glass coverslip. Fluorescence microscopy was performed on a Deltavision Spectris system containing a Photometrics CH350L liquid-cooled CCD camera and Olympus IX70 inverted microscope with a 100× 1.4 NA objective equipped with Deltavision data collection system (Applied Precision, Issaquah, WA). Stacks of 6 z-sections (0.3 μm apart) were taken at 30-second intervals with exposure times of 1 second for GFP, tdTomato and CFP. All imaging was performed at 30°C. Projected images were made for each time point followed by intensity adjustments and conversion to 24 bit TIFF images. The position of the spindle poles and kinetochores were determined using Softworx software and downloaded to Microsoft Excel for analysis.

We thank Takashi Toda, Kathy Gould and Ken Sawin for strains and reagents and Kate Sullivan (Imaging Facility, NIMR) and Vicky Buck for technical support. J.B.A.M. is supported by grants from the Association of International Cancer Research and the Medical Research Council, UK.

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