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. 2015 Oct 8;13(3):460–468. doi: 10.1016/j.celrep.2015.08.008

Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes

Danica Drpic 1,2,3, António J Pereira 1,2, Marin Barisic 1,2, Thomas J Maresca 4, Helder Maiato 1,2,5,∗
PMCID: PMC4623360  PMID: 26456825

Summary

Chromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, we generated Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs). SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.

Keywords: chromokinesins, kinetochore, mitosis, mitosis with unreplicated genomes, spindle assembly checkpoint

Graphical Abstract

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Highlights

  • •

    Spindle assembly checkpoint (SAC) can be satisfied after a delay in cells with mono-oriented chromosomes

  • •

    Mono-oriented chromosomes experience intra-kinetochore stretch

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    Polar ejection forces promote SAC satisfaction independently of bi-orientation

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    Polar ejection forces promote the conversion from lateral to end-on attachments

Tension on bi-oriented chromosomes plays a role in the stabilization of kinetochore-microtubule attachments. However, how kinetochore-microtubule attachments on mono-oriented chromosomes are first stabilized in the absence of tension remained unknown. Drpic et al. now show that polar ejection forces promote the transition from lateral to end-on attachments on mono-oriented chromosomes.

Introduction

During spindle assembly, the initial lateral interactions between chromosomes and microtubules are converted into stable end-on kinetochore-microtubule attachments that lead to chromosome bi-orientation (Magidson et al., 2011). After chromosome bi-orientation, the opposing spindle forces generate tension on centromeres that is important for the stabilization of correct kinetochore–microtubule attachments required for error-free chromosome segregation (Nicklas and Koch, 1969, Nicklas and Ward, 1994). Tension has also been shown to be sufficient to satisfy the spindle-assembly checkpoint (SAC) (Li and Nicklas, 1995), a surveillance mechanism that ensures that all chromosomes are attached to spindle microtubules before anaphase onset (Foley and Kapoor, 2013). Tension from spindle forces affects kinetochore chemistry through changes in phosphorylation of “tension-sensitive” proteins at kinetochores (Gorbsky and Ricketts, 1993, Nicklas et al., 1995). Aurora B, a mitotic kinase present on centromeres, plays a critical role in tension sensing and error correction (Biggins and Murray, 2001, Cheeseman et al., 2002, Lampson et al., 2004) by phosphorylating key substrates at the kinetochore-microtubule interface, such as the KMN network, in response to tension on bi-oriented chromosomes (DeLuca et al., 2006, Liu et al., 2009, Wang et al., 2011, Welburn et al., 2010). Importantly, recent works in human and Drosophila cells have shown that even in the absence of centromeric tension, an intra-kinetochore stretch or structural deformation is sufficient to satisfy the SAC (Maresca and Salmon, 2009, Uchida et al., 2009). However, the underlying mechanism remained unclear.

Chromokinesins are microtubule plus-end-directed motor proteins present on the chromosome arms harboring both chromatin- and microtubule-binding domains. As a consequence of their motor activities, chromokinesins move chromosomes away from the poles by generating random polar ejection forces (PEFs) (Barisic et al., 2014, Brouhard and Hunt, 2005, Levesque and Compton, 2001, Rieder et al., 1986, Wandke et al., 2012, Yajima et al., 2003). Recently, elevated PEFs were shown to stabilize erroneous kinetochore-microtubule attachments (Cane et al., 2013), suggesting a role in the stabilization of kinetochore-microtubule attachments. Here, we found that Chromokinesin-mediated PEFs promote the conversion from lateral to stable end-on kinetochore-microtubule attachments on mono-oriented chromosomes. These findings contribute to explain how initial end-on kinetochore-microtubule attachments are stabilized before bi-orientation.

Results

The SAC Is Satisfied in Cells with Single Chromatids after a Mitotic Delay

To investigate which factors are responsible for kinetochore-microtubule attachment stability before bi-orientation, we established a system in Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs) (Drpic et al., 2013). This was achieved by RNAi-mediated depletion of Double parked (Dup), a conserved protein required for the initiation of DNA replication and post-replication checkpoint response (Whittaker et al., 2000). The main advantage of this system when compared to mammalian cells undergoing MUGs (Brinkley et al., 1988, O’Connell et al., 2009) is that SMUGs preserve their unreplicated genetic material condensed into single chromatids, which never experience bi-orientation due to the absence of sister kinetochores (Drpic et al., 2013). Thus, the function of individual kinetochores in SMUGs can be investigated in their native chromatid context.

Spinning-disk confocal live-cell imaging revealed that single chromatids in SMUGs were scattered along the spindle. Because of their low chromosome number, the status of kinetochore-microtubule attachments could be inferred by careful inspection of the respective z-sections (see Experimental Procedures). This indicated that SMUGs established mainly lateral and only few merotelic kinetochore-microtubule attachments. For instance, 20 min after nuclear envelope breakdown (NEB) we found that, on average, 8.0 ± 1.6 kinetochores per cell were laterally attached and 3.0 ± 0.82 kinetochores established merotelic attachments (mean ± SD, n = 5 cells; Figures 1A and S1A; Movie S1). Consequently, SMUGs significantly delayed mitotic exit (t = 111 ± 43 min, mean ± SD, n = 11 cells, p = < 0.001, t test) when compared to control cells (t = 31 ± 8 min, mean ± SD, n = 11 cells; Figures 1A and 1C; Movie S1). Indeed, while cyclin B1 levels abruptly decreased at the metaphase-anaphase transition in control cells, cyclin B1 levels decreased more slowly over time in SMUGs (Figures S1E and S1F), suggesting a delay in SAC satisfaction (see also Mirkovic et al., 2015 in this issue of Cell Reports). To investigate whether the delayed mitotic exit in SMUGs is SAC dependent, we co-depleted Mad2 and Dup by RNAi (Figures 1C, S1B, and S1C). We found that, similar to control cells, Mad2 co-depletion overcomes the mitotic delay in SMUGs (Mad2/Dup-depleted cells: t = 22.1 ± 6.0 min, mean ± SD, n = 31 cells; Mad2-depleted cells: t = 18.0 ± 5.6 min, mean ± SD, n = 19 cells), indicating that the mitotic delay in SMUGs is SAC dependent.

Figure 1.

Figure 1

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Cells with Single Chromatids Satisfy the SAC after a Mitotic Delay

(A) Live-cell imaging of Drosophila S2 cells (control and Dup-depleted) stably expressing H2B-GFP and mCherry-α-tubulin. Dashed box indicates a single, condensed chromatid.

(B) Similar conditions, but in which cells were treated with 200 μM colchicine immediately after NEB.

(C) Quantification of mitotic duration (control n = 11 cells; Dup-depleted n = 11 cells; control cells treated with colchicine n = 7 cells; Dup-depleted cells treated with colchicine n = 24 cells; Mad2-depleted cells treated with colchicine n = 19 cells; Mad2/dup-depleted cells treated with colchicine, n = 31 cells).

(D) Live-cell imaging of S2 cells stably expressing BubR1-mCherry and GFP-α-tubulin.

(E and F) Quantification of the number of BubR1 positive kinetochores during normal mitosis (n = 10 cells) and SMUGs (n = 10 cells). Zero time point refers to anaphase onset.

∗∗∗p < 0.001. Black lines indicate individual cells and red lines represent the average. Error bars, SD. Time = hr:min. Scale bar, 5 μm. See also Figures S1 and S2 and Movie S1.

Next, we tested SAC response in SMUGs by adding colchicine immediately after NEB to generate unattached kinetochores and monitored mitotic progression by live-cell imaging. Both control cells and SMUGs were arrested in mitosis for more than 10 hr before undergoing slippage (Rieder and Maiato, 2004) (control t = 18.4 ± 1.23 hr, mean ± SD, n = 7 cells; SMUGs t = 10.4 ± 2.6 hr, mean ± SD, n = 24 cells; Figures 1B and 1C). These results indicate that SMUGs have an active SAC, which is, however, less robust than in control cells. Interestingly, the total levels of Mad2 and the recruitment of Mad2 and active Aurora B to unattached kinetochores in SMUGs were unaltered relative to controls; Figures S1D and S2A–S2D). Thus, despite normal SAC signaling at individual kinetochores, the number of cumulative unattached kinetochores that are able to inhibit the Anaphase Promoting Complex/Cyclosome (APC/C) in SMUGs is reduced by half relative to controls cells. This explains the weakened SAC response in SMUGs and is in line with previous reports in human cells (Collin et al., 2013, Dick and Gerlich, 2013). Importantly, these data strongly suggest that SMUGs normally exit mitosis after SAC satisfaction, as they took more than five times longer to slip out of mitosis in the presence of colchicine.

To directly test whether SMUGs satisfy the SAC after a mitotic delay, we investigated the behavior of another SAC protein, BubR1, using live-cell imaging of SMUGs stably expressing BubR1-mCherry/α-tubulin-GFP. BubR1 is normally recruited to unattached kinetochores and its levels decrease significantly as chromosomes bi-orient, becoming undetectable on anaphase kinetochores (Howell et al., 2004, Maiato et al., 2002). In contrast, BubR1 remains associated with kinetochores in cells that slip out of mitosis without satisfying the SAC (Brito and Rieder, 2006). We found that, despite of a mitotic delay, SMUGs lost BubR1 from kinetochores just before exiting from mitosis (Figures 1D–1F and Movie S2). This demonstrates that the SAC in SMUGs with single chromatids can be satisfied without bi-orientation.

Single Chromatids in SMUGs Experience Intra-kinetochore Stretch/Structural Deformation after a Mitotic Delay

Intra-kinetochore stretch or structural deformation is sufficient to satisfy the SAC even with reduced centromeric tension (Maresca and Salmon, 2009, Uchida et al., 2009). To investigate whether SMUGs experience intra-kinetochore stretch/structural deformation, we measured the absolute distance between the inner kinetochore protein Cid-mCherry and the outer kinetochore protein Ndc80-GFP (Maresca and Salmon, 2009) at individual kinetochores (see Experimental Procedures) from control cells treated with colchicine (reference for relaxed kinetochores) or MG132 (reference for bi-oriented chromosomes under tension), as well as from Dup-depleted cells treated with MG132 for 2 hr (to normalize the mitotic delay). We found that under these conditions single chromatids in SMUGs experienced a significant intra-kinetochore stretch/structural deformation relative to relaxed kinetochores (Mann-Whitney rank-sum test, p < 0.001) that was almost comparable to bi-oriented chromosomes under tension (Figures 2A and 2C). In line with these measurements, we further observed intermediate levels of Aurora B-mediated phosphorylation of the outer kinetochore protein KNL1 (Welburn et al., 2010) relative to unattached controls and bi-oriented chromosomes (Figures 2B and 2C), suggesting that intra-kinetochore stretch/structural deformation positively correlates with kinetochore-microtubule attachment stability. Taken together, these data indicate that single chromatids in SMUGs experience sufficient intra-kinetochore stretch/structural deformation to satisfy the SAC.

Figure 2.

Figure 2

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Single Chromatids in SMUGs Experience Intra-kinetochore Stretch after a Mitotic Delay

(A) Fixed control cells stably expressing Cid-mCherry/Ndc80-GFP were treated with colchicine or MG132 (2 hr) and compared with Dup-depleted cells treated with MG132 (2 hr).

(B) Immunofluorescence analysis of Aurora-B phosphorylation of the outer kinetochore protein KNL1 in SMUGs and control cells in the same conditions as in (A).

(C) Quantification of pKNL1 and intra-kinetochore stretch (shift) by measuring absolute distance between red (Cid) and green (Ndc80) centroids in control cells versus SMUGs.

PEFs Stabilize Kinetochore-Microtubule Attachments and Promote SAC Satisfaction Independently of Chromosome Bi-orientation

Elevated PEFs on chromosome arms after overexpression of the Chromokinesin Nod lead to the stabilization of syntelic kinetochore-microtubule attachments in Drosophila S2 cells (Cane et al., 2013). To test whether the kinetochore-microtubule stabilizing role of PEFs is involved in SAC satisfaction in SMUGs, we co-depleted Dup and Nod. This resulted in a SAC-dependent increase in mitotic duration when compared to Dup-depleted cells (t = 208 ± 109 min, mean ± SD, n = 25 cells, p = 0.007, t test; Figures 3B and 3D; Movie S3). Co-depletion of both Chromokinesins, Nod and Klp3A, with Dup caused an even longer mitotic delay (t = 304 ± 66 min, mean ± SD, n = 8 cells, p ≤ 0.001, t test; Figures 3D and S3E). Interestingly, Nod depletion in control cells caused chromosome alignment defects and also significantly increased the duration of mitosis (t = 44 ± 12 min, mean ± SD, n = 26, p = 0.005, Mann-Whitney rank-sum test; Figures 3A and 3D; Movie S3), in line with previous findings in human cells (Levesque and Compton, 2001, Magidson et al., 2011). This phenotype was exacerbated when Nod and Klp3A were co-depleted (t = 62 ± 29 min, mean ± SD, n = 20, p = 0.003, t test; Figures 3D and S3E), suggesting that PEFs play an important role in the stabilization of kinetochore-microtubule attachments during a normal mitosis. Thus, in the absence of Chromokinesin-mediated PEFs, SAC satisfaction is delayed and the delay is more pronounced in the absence of chromosome bi-orientation.

Figure 3.

Figure 3

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PEFs Are Involved in SAC Satisfaction Independently of Chromosome Bi-orientation

(A and B) Live-cell imaging of Drosophila S2 cells stably expressing H2B-GFP and mCherry-α-tubulin. The panels illustrate control, Nod-depleted, Dup-depleted, as well as Nod and Dup co-depleted situations, as indicated.

(C) Live-cell imaging of Nod-mCherry-overexpressing cells with and without Dup depletion.

(D) Mitotic duration of control (n = 11 cells), Nod-depleted (n = 26 cells), Nod/Klp3A-depleted (n = 20 cells), Nod-overexpressing (OX) (n = 22 cells), Dup-depleted (n = 10), Nod/Dup-depleted (n = 25), Nod/Klp3A/Dup-depleted (n = 8), and Nod OX SMUGs (n = 12). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Time = hr:min. Scale bar, 5 μm. Error bars, SD. See also Figure S3.

One prediction from these data is that elevated PEFs should promote the stabilization of kinetochore-microtubule attachments and consequently accelerate SAC satisfaction in SMUGs. To test this, we overexpressed Nod-mCherry in Dup-depleted cells stably expressing GFP-α-tubulin (Cane et al., 2013). In agreement with our prediction, Nod overexpression significantly shortened the mitotic duration in Dup-depleted cells (t = 46.5 ± 22 min, mean ± SD, n = 12 cells, p ≤ 0.001, t test; Figures 3C and 3D; Movie S4). In contrast, elevated PEFs caused by Nod overexpression in control cells increased mitotic duration (t = 67 ± 27 min, mean ± SD, n = 22 cells p = 0.003, Mann-Whitney rank-sum test; Figures 3C and 3D; Movie S4), which might be due to random ejection of chromosomes after stabilization of monotelic attachments, thereby preventing bi-orientation and timely SAC satisfaction (Barisic et al., 2014). Overall, these data suggest that Chromokinesin-mediated PEFs promote SAC satisfaction in SMUGs by stabilizing kinetochore-microtubule attachments independently of chromosome bi-orientation.

PEFs Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes

HeLa cells undergoing MUGs satisfy the SAC independently of bi-orientation mainly by establishing merotelic attachments (O’Connell et al., 2008). Due to opposite spindle forces, merotelic attachments might cause kinetochore deformation that generates sufficient intra-kinetochore stretch that would satisfy the SAC (Maresca and Salmon, 2009, Uchida et al., 2009). Importantly, the contribution of PEFs for SAC satisfaction could not be investigated in this system because kinetochores detach from chromatin, which remains decondensed during MUGs (Brinkley et al., 1988, O’Connell et al., 2009). Although we cannot exclude that, in addition to PEFs, some merotelic attachments contribute to SAC silencing in SMUGs, these attachments were rare, as indicated by our live-cell recordings and careful inspection of the respective z stacks (Figures 1A and S1A; Movie S1) (see also Mirkovic et al., this issue).

To test whether PEFs are required to satisfy the SAC in SMUGs, independently of chromosome bi-orientation and the establishment of merotelic attachments, we investigated the duration of mitosis in Nod-depleted cells with a monopolar spindle configuration (in which only monotelic attachments can be established), after co-depletion of the Kinesin-5 protein Klp61F by RNAi (Cane et al., 2013) (Figure 4A; Movie S5). We found that SMUGs with monopolar spindles were also able to exit mitosis after a delay (t = 178 ± 59 min, mean ± SD, n = 9; Figure 4A; Movie S5), which was exacerbated after Nod co-depletion (t = 379 min ± 132 min, mean ± SD, n = 4, p = 0.011, Mann-Whitney rank-sum test; Figure 4A; Movie S5). Closer inspection of z stacks from live-cell images of monopolar spindles in SMUGs revealed a clear transition from lateral to end-on kinetochore-microtubule attachments prior to mitotic exit, and the presence of Nod-mediated PEFs promoted this transition (Figures 4B and 4C; Movie S5). Immunofluorescence analysis with a Mad1 antibody confirmed that the percentage of unattached kinetochores in SMUGs with monopolar spindles (35%) increased after Nod depletion (62%, p = 0.028, t test; Figure 4D). Overall, these data demonstrate that Chromokinesin-mediated PEFs promote the conversion from lateral to stable end-on kinetochore-microtubule attachments, independently of bi-orientation and merotely.

Figure 4.

Figure 4

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PEFs Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes

(A) Live-cell imaging of Klp61F/Dup and Klp61F/Dup/Nod-depleted S2 cells stably expressing GFP-α-tubulin and Cid-mCherry.

(B) Respective higher magnifications of lateral and end-on attachments from (A).

(C) Quantification of the different kinetochore-microtubule attachments (through z stacks) in Klp61F/Dup/Nod RNAi and Klp61F/Dup RNAi cells. The difference in the percentage of end-on attachments between Klp61F/Dup RNAi (n = 7 cells) and Klp61F/Dup/Nod RNAi cells (n = 5 cells) at 80 min and 120 min after NEB are statistically significant (Z-test compare proportions, p < 0.05).

(D) Immunofluorescence of Klp61F/Dup and Klp61F/Dup/Nod-depleted S2 cells. Nod depletion in monopolar SMUGs lead to increased number of Mad1 positive kinetochores. Time = hr:min. Scale bar, 5 μm.

Scale bar in higher magnification panels, 2 μm. ∗p < 0.05 relative to the previous time point, t test. Error bars, SD.

Discussion

Chromosome bi-orientation is a critical requirement for accurate chromosome segregation during mitosis and requires that both kinetochores are stably attached to spindle microtubules. Tension from spindle forces has long been known to stabilize correct kinetochore-microtubule attachments (King and Nicklas, 2000), but how the first end-on attachments are stabilized before the development of tension has remained unknown. Here, we found that PEFs promote the conversion from lateral to stable end-on kinetochore-microtubule attachments on mono-oriented chromosomes. Lateral attachments to spindle microtubules are insensitive to Aurora B activity (Kalantzaki et al., 2015) and are initially mediated by kinetochore Dynein, which is dominant over PEFs at the spindle poles (Barisic et al., 2014) and inhibits the action of the Ndc80 complex required for stable end-on attachments (Cheerambathur et al., 2013). Despite not being dominant at this stage, PEFs promote the exclusion of chromosomes from the central area of the mitotic spindle (Magidson et al., 2011), but chromosomes remain tethered to the microtubule walls by CENP-E/Kinesin-7 (Shrestha and Draviam, 2013), which slides chromosomes preferentially along detyrosinated microtubules toward the spindle equator (Barisic et al., 2015). At the equator PEFs become critical to stabilize end-on kinetochore-microtubule attachments required for chromosome bi-orientation (Barisic et al., 2014, Magidson et al., 2011, Wandke et al., 2012). In this context, our data can be best explained by a model in which the lateral to end-on conversion of kinetochore-microtubule attachments near the equator requires the contribution of Chromokinesin-mediated PEFs acting on the arms of mono-oriented chromosomes to counteract microtubule depolymerization-driven poleward motion. This might generate sufficient intra-kinetochore stretch or structural deformation (Maresca and Salmon, 2009, Uchida et al., 2009) that leads to the stabilization of end-on kinetochore-microtubule attachments. Cdk1 downregulation due to cyclin A and B1 degradation might generate positive feedback loops that, in coordination with PEFs, further stabilize kinetochore-microtubule attachments (Collin et al., 2013, Kabeche and Compton, 2013, Mirkovic et al., 2015). While this eventually leads to SAC satisfaction after a significant mitotic delay in SMUGs, we propose that during normal mitosis this mechanism contributes to the stabilization of initial end-on kinetochore-microtubule attachments, before tension from opposing spindle forces is established during bi-orientation.

Experimental Procedures

Quantification of Kinetochore-Microtubule Attachments

In order to distinguish the different types of kinetochore-microtubule attachments in SMUGs, we performed live-cell imaging in Drosophila S2 cells stably expressing GFP-α-tubulin/Cid-mCherry. Images were analyzed using FIJI (ImageJ) software through z stacks (0.5 μm). Kinetochore-microtubule attachments were quantified after tracing microtubule positioning in relation to the Cid signal (kinetochores). When microtubules passed by the Cid signal the attachment was considered as lateral. When microtubules ended at the kinetochore they were considered as end-on attachments. Since in SMUGs chromosomes do not align in the spindle equator, merotelic attachments were rarely observed and were distinguished as having long K-fibers coming from opposite poles that ended on the same kinetochore.

Measurement of Intra-kinetochore Stretch/Deformation

Drosophila S2 cells stably expressing Cid-mCherry/Ndc80-GFP (Maresca and Salmon, 2009) were used for intra-kinetochore stretch measurements in fixed (4% paraformaldehyde) material and for live-cell imaging (intra-kinetochore stretch measurements over time). Sub-pixel determination of fluorescent spot localization was performed using a home-written MATLAB script (MathWorks). A sequential refinement of the spot position starts with manual (mouse) selection of the kinetochore ensemble to be measured. A neighborhood region of interest (ROI) (11 × 11 pixels) is defined around each selected point, the boundary of which is used to estimate average background signal per pixel. This background value is subtracted, and the centroid is then calculated to allow recentering of the ROI. This first part of the script is meant as a coarse correction of the mouse-defined points. Before fitting a circular two-dimensional Gaussian function to each ROI intensity map, an empirical parameter of 1/2 was chosen as the fraction of (highest gray value) ROI pixels to be fed into the fitting procedure thus avoiding the bias induced by residual fluorescence of adjacent structures (e.g., defocused adjacent kinetochores). Fitting is performed using the least-squares fitting routine lsqcurvefit.

Statistical Analysis

Statistical analyses were performed using SigmaStat. Additional procedures are available in Supplemental Experimental Procedures.

Author Contributions

D.D. performed and analyzed all the experiments; A.J.P. developed the algorithm to measure intra-kinetochore stretch on individual kinetochores; M.B. performed data analysis and designed experiments; T.J.M. provided reagents; H.M. performed data analysis, designed experiments, and supervised the work; D.D. and H.M. wrote the paper.

Acknowledgments

We thank Raquel Oliveira for communicating results prior to publication. D.D. is supported by a fellowship from the GABBA PhD program from the University of Porto. T.J.M. is supported by an NIH grant (5 R01 GM107026) and by Research Grant No. 5-FY13-205 from the March of Dimes Foundation and support from the Charles H. Hood Foundation, Boston, MA. H.M. is funded by FLAD Life Science 2020 and PRECISE grant from the European Research Council.

Published: October 8, 2015

Footnotes

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Supplemental Information includes Supplemental Experimental Procedures, three figures, and five movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.08.008.

Supplemental Information

Document S1. Supplemental Experimental Procedures and Figures S1–S3
mmc1.pdf (739.3KB, pdf)
Movie S1. Cells with Single Chromatids Satisfy the SAC after a Mitotic Delay
mmc2.jpg (696.3KB, jpg)
Movie S2. SMUGs Lose BubR1 from Kinetochores Just before Exiting from Mitosis
mmc3.jpg (517.5KB, jpg)
Movie S3. PEFs Are Involved in SAC Satisfaction Independently of Chromosome Bi-orientation
mmc4.jpg (238.7KB, jpg)
Movie S4. Nod Overexpression Promotes SAC Satisfaction Independently of Chromosome Bi-orientation
mmc5.jpg (327.7KB, jpg)
Movie S5. PEFs Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes
mmc6.jpg (291.5KB, jpg)
Document S2. Article plus Supplemental Information
mmc7.pdf (4.8MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Supplemental Experimental Procedures and Figures S1–S3
mmc1.pdf (739.3KB, pdf)
Movie S1. Cells with Single Chromatids Satisfy the SAC after a Mitotic Delay
mmc2.jpg (696.3KB, jpg)
Movie S2. SMUGs Lose BubR1 from Kinetochores Just before Exiting from Mitosis
mmc3.jpg (517.5KB, jpg)
Movie S3. PEFs Are Involved in SAC Satisfaction Independently of Chromosome Bi-orientation
mmc4.jpg (238.7KB, jpg)
Movie S4. Nod Overexpression Promotes SAC Satisfaction Independently of Chromosome Bi-orientation
mmc5.jpg (327.7KB, jpg)
Movie S5. PEFs Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes
mmc6.jpg (291.5KB, jpg)
Document S2. Article plus Supplemental Information
mmc7.pdf (4.8MB, pdf)

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