What do kinetochores attach to

The enrichment of Astrin at kinetochores prior to biorientation points to a novel attachment stabilisation mechanism, independent of already known intra- or inter- kinetochore stretching mediated stabilisation of bioriented attachments reviewed in Conti et al.

To explore this hypothesis, we tracked Astrin recruitment and dynamics at the kinetochores of monopolar spindles that can not biorient kinetochores. To confirm that both Astrin-crescents and sleeves are found at the outer-kinetochore, we analysed YFP-Astrin localisation in cells co-expressing Nuf2-CFP, a bonafide outer-kinetochore marker. Overlap with Nuf2-CFP signal was partial in Astrin-sleeves and complete in Astrin-crescents, confirming the outer-kinetochore localisation of Astrin prior to biorientation Figure 1B.

Each frame is an average-intensity projection of 3 z-planes, nm apart. White arrowhead in main image marks kinetochore magnified in cropped-insets.

YFP inset shows inverted image intensities to highlight Astrin-low to Astrin-high transition. Numbers mark kinetochores magnified in cropped-insets. Each dot represents intensity ratios from one kinetochore. Black bars and whiskers mark average value and standard deviation, respectively, across two experimental repeats. Scale as indicated. E Cartoon shows the orientation of kinetochore-microtubule bridges: the C-term of Astrin resides proximal to the C-term of Ndc To pinpoint precisely where the Astrin-SKAP complex is recruited at the kinetochore, we took clues from previous biochemical studies Wang et al.

Previously, fragments of the two complexes were shown to bind microtubules in vitro Kern et al. In summary, the timing and position of Astrin recruitment to the kinetochore is appropriate for directly stabilising end-on attachments independent of biorientation. First, kinetochore-associated Astrin-sleeve can enrich into an Astrin-crescent prior to biorientation. Second, the C-terminus of Astrin resides proximal to the C-terminus of Ndc80 ideally positioned to strengthen kinetochore-microtubule bridging after the formation of end-on attachments Figure 1E.

To determine how end-on kinetochores are selectively recognised, we searched for the minimal region of Astrin needed for its kinetochore enrichment as crescents. For Astrin recruitment at kinetochores, its C-terminal — a. At the very C-terminus of Astrin, using structure prediction tools Wolf et al.

Removing 70 amino acids — a. A Schematic of Astrin protein domains. Graph of predicted coiled-coil or unstructured region in Astrin C-terminus modelled as a dimer using Multicoil Wolf et al. E Bar graph of percentage of bipolar or multipolar spindles in mitotic cells treated as in D. Bars and whiskers mark average value and standard deviation, respectively, across at least three experimental repeats. Yellow triangle indicates the cell tracked; Yellow asterisks highlight prolonged delay between metaphase and anaphase.

To confirm that the 70 a. While Astrin depleted cells displayed multipolar spindles as reported Thein et al. Using time-lapse microscopy, we tracked the fate of mitotic cells lacking the 70 a. In summary, kinetochore-bound Astrin is required for timely anaphase, but not the congression of chromosomes or the maintenance of bipolar spindles. The C-terminal tail of Astrin enables the selective recognition of end-on attached kinetochores and the timely onset of chromosome segregation.

To understand why the 70 a. Importantly, using the unstructured C-terminal tail, a bioinformatic search could identify Astrin elsewhere in Bilateria Figure 3—figure supplement 1 and Source data 1 , suggesting a previously unrecognised evolutionarily conserved role for the Astrin-Tail. These findings show that the RVxF motif is critical for enriching but not the initial recruitment of Astrin at the kinetochore.

Black bars and whiskers mark average value and standard deviation, respectively, from values across two experimental repeats. Green and magenta circles highlight Astrin-crescents. Yellow asterisk refers to this blinking event.

F Frequency graph of distribution of the number of blinking events as in E in kinetochores tracked for 60 s. KTs and n refers to the kinetochore numbers and blinking events, respectively. G Graph of average distance between Astrin-crescents of kinetochore pairs measured using time-lapse movies as in E. Black bars and whiskers mark average value and standard deviation, respectively, across at least two experimental repeats. We tested whether reducing the kinetochore pool of Astrin using Astrin-4A mutant could delay anaphase onset.

This delay in anaphase onset was despite the normal completion of chromosome congression at the spindle equator Figure 3C. In conclusion, the PP1 docking RVxF motif is dispensable for spindle bipolarity and chromosome congression but is crucial for the enrichment of Astrin-crescents and normal timing of anaphase.

In Astrin-4A expressing cells, dim kinetochore-crescents gradually declined into Astrin-sleeves referred as, blinking; Figure 3E , explaining reduced Astrin-4A crescent intensities in fixed-cells Figure 3A and Figure 3—figure supplement 3A. In contrast, Astrin-WT crescents were retained brightly at congressed kinetochores Figure 3E , mediating stable kinetochore-microtubule bridging. This confirms that the PP1 docking motif is required for stably retaining Astrin at kinetochores.

We conclude that the putative PP1-docking motif is not essential for the initial arrival of Astrin sleeves at kinetochores; it is essential for the maintenance of Astrin-crescents, revealing a role for the PP1-docking motif in the stable maintenance of kinetochore-microtubule bridges.

If a reduction in Astrin complex weakens kinetochore-microtubule bridging, we are likely to observe reduced pulling of sister kinetochores by microtubule-end mediated forces. To assess whether sister kinetochores experience normal microtubule-end mediated pulling forces in cells lacking the PP1-docking motif, we measured inter-kinetochore distances in live-cells expressing Astrin-4A mutant.

Despite normal kinetochore alignment at the spindle equator, inter-kinetochore distances between Astrin crescents were significantly reduced in cells expressing Astrin-4A compared to Astrin-WT Figure 3G. To determine whether reduced kinetochore life-time of Astrin-4A leads to reduced kinetochore pulling or conversely, an inability to withstand microtubule-mediated pulling leads to a reduction in Astrin-4A at kinetochores, we tested whether a brief treatment with nm Taxol, which pauses microtubule dynamics and reduces microtubule-mediated pulling Draviam et al.

We conclude that reduced microtubule-mediated pulling is a consequence, rather than the cause, of the reduced life-time of Astrin-4A at kinetochores. Astrin enrichment at end-on kinetochores is likely to further strengthen kinetochore-microtubule bridges as it is required to withstand microtubule-mediated kinetochore pulling and the timely onset of chromosome segregation.

We investigated the extent to which Astrin is capable of interacting with PP1 both in vivo and in vitro. We conclude that Astrin and PP1 interact at the outer-kinetochore, and this interaction is dynamic. Cartoons of fusion proteins expressed in each condition are shown on the left. Cyan arrowhead marks FRET-positive kinetochore. Each dot represents a value from a kinetochore KT or a spindle SP area of size 0.

Black bars and whiskers mark average value and standard deviation, respectively, from kinetochores or spindle regions across two experimental repeats. Immunoblot was probed with antibody against Astrin top or gamma-Tubulin bottom, positive control.

Immunoblot is representative of two independent pulldown studies. For methodology details see Figure 4—figure supplement 1A. Areas used for Astrin intensity measurements are shown in Figure 4—figure supplement 1B.

In these pulldown assays, we used recombinantly produced GST immobilised on Glutathione beads as a negative control Figure 4D. To confirm the specificity of the anti-Astrin antibody in immunoblots, we compared lysates of unsynchronised or mitotically enriched cells treated with Control or Astrin siRNA Figure 4—figure supplement 1C and confirmed all three bands to be specific to Astrin in mitotic cell lysates see green arrowheads in Figure 4—figure supplement 1C.

Based on these in vivo and in vitro studies we conclude that Astrin and PP1 interact with each other. We could not assess FRET at all kinetochores of cells expressing the Astrin-4A mutant as the mutant does not present a sustained kinetochore localisation see also, Figure 3.

However, using those timepoints when the Astrin-4A mutant was transiently enriched at the kinetochore, we could quantitatively establish that the Astrin-CFP 4A mutant expressing cells present severely reduced FRET signals compared to Astrin-WT expressing cells Figure 4—figure supplement 2B.

Reduced microtubule-mediated pulling of kinetochores in Astrin-4A expressing cells Figure 3G and Figure 3—figure supplement 4A,B suggests an inability to maintain stable end-on attachments. To directly assess whether the RVxF motif in Astrin is important for the maintenance of end-on attachments, we used a quantitative assay that has previously allowed us to clearly identify the stability of three different attachment steps of the end-on conversion process: kinetochores unattached to microtubules; kinetochores attached laterally to microtubule-walls; and kinetochores attached end-on to microtubule-ends Shrestha and Draviam, ; Shrestha et al.

To analyse kinetochore-microtubule attachment status, we immunostained cells using an antibody against Tubulin and the CREST antisera centromere marker and imaged using deconvolution microscopy.

Importantly, laterally-attached kinetochores, and not detached kinetochores, were significantly increased in Astrin mutant-expressing cells Figure 5A,B. Thus, Astrin mutant-expressing cells are able to retain kinetochores along microtubule-walls but they cannot maintain kinetochores at microtubule-ends, similar to Astrin depleted cells Shrestha et al.

This reveals that the kinetochore pool of Astrin is important for the selective stabilisation of end-on attachments. These monopolar spindle studies together show that Astrin:PP1 interaction is required specifically to maintain end-on, but not lateral, attachments independent of biorientation status.

Thus, although Astrin-mediated delivery of PP1 is not required for chromosome congression Figure 2D,E and Figure 3—figure supplement 3C,D , it is crucial for the stable maintenance of end-on attachments Figure 5B.

Negative and Crescent refer to the absence and presence of Astrin-crescents, respectively. Each circle represents percentage of kinetochores from one cell. KTs refer to total number of kinetochores assessed. Black bar marks average values from at least three independent experiments. Each dot represents a value from one cell. Yellow arrowheads in uncropped images highlight representative kinetochores staining positive for MAD2.

Number n of cells indicated. Values were obtained from three independent repeats. These data indicate an increased incidence of spindle checkpoint signalling in a small proportion of congressed kinetochores in cells expressing either of the two Astrin mutants lacking the PP1-docking motif.

As expected from low resolution time-lapse studies Figures 2 and 3 , chromosome alignment at the spindle equator i. Thus, the stable maintenance of end-on attachments, but not chromosome congression, requires the evolutionarily conserved PP1-docking motif in Astrin. To assess the potential downstream phospho-targets of Astrin-PP1 at kinetochores, we tested whether Astrin mutant expression induces a change in the levels of phospho-DSN1, expected to be in the vicinity of Astrin C-terminus.

For this study, we used phosphorylation specific antibodies reported previously Welburn et al. These findings show changes in Dsn1 phosphorylation status as a direct or indirect downstream target of Astrin-PP1 at kinetochores.

Cropped images are magnified areas boxed in dashed white lines. Number of cells is indicated. C Cartoon summarises the consequence of abrogating or spatially restricting the delivery of phosphatase by Astrin.

Astrin:PP1 enrichment at kinetochores is required for the maintenance of cold-stable kinetochore-microtubule attachments. Upon a brief exposure to cold, cells depleted of Astrin depolymerise all spindle microtubules due to unstable kinetochore-microtubule attachments Thein et al. In summary, these findings show that the delivery of PP1 near the C-terminus of Ndc80 is sufficient to promote the kinetochore enrichment of Astrin-SKAP complex and stabilisation of end-on attachments Figure 6C.

In conclusion, SKA complex enrichment is not linked to Astrin enrichment at the kinetochore. Aurora-B kinase controls microtubule stability by phosphorylating several kinetochore proteins Hauf et al. Reversing Aurora-B phosphorylation is thought to be sufficient for stabilising kinetochore-microtubule attachments reviewed in Kelly and Funabiki, ; Lampson and Cheeseman, We tested whether the delivery of PP1 by Astrin is required to i simply reverse Aurora-B-kinase mediated phospho-events or ii directly stabilise end-on attachments.

To distinguish between the two scenarios, we first tested whether inhibiting Aurora-B can overcome the need for Astrin-PP1 interaction in stabilising end-on attachments Figure 5A,B. Inhibition of Aurora-B in monopolar spindles will block the destabilisation of attachments leading to stable end-on attachments Lampson et al.

Cropped images are areas boxed in white. Black bars show average across at least two experimental repeats. Absence and presence of Astrin-crescents is highlighted.

Percentage of kinetochores with Astrin-GFP sleeves are in Figure 7—figure supplement 1B and percentage of lateral and detached kinetochores are in Figure 7—figure supplement 1C. We next tested whether Astrin-mediated delivery of PP1 is sufficient to stabilise end-on attachments.

These data show that the stabilisation of end-on attachments following the inhibition of Aurora-B is acutely dependant on the delivery of PP1 by Astrin. We wondered whether a dynamic delivery of PP1 by Astrin is essential to prevent premature stabilisation of incorrect attachments.

To test this hypothesis, we studied the consequence of premature and constitutive delivery of PP1 by Astrin during the conversion of a monopolar spindle into a bipolar one. These observations highlight the need for a dynamic interaction between Astrin and PP1. We next analysed the rate of chromosome congression during monopolar to bipolar spindle conversion a measure of error correction.

For this analysis we excluded spindles that were partly bipolar at the beginning of the time-lapse movie These findings show that the constitutive delivery of PP1 by Astrin disrupts the dynamic regulation of attachment stability leading to a delay in chromosome congression and anaphase onset and also errors in chromosome segregation.

Yellow arrows mark chromosomes not aligned or congressed during a prolonged metaphase arrest. Monopolar to bipolar spindle conversion assay regime outlined in Figure 8—figure supplement 1A. Lysates were harvested at the end of the time-lapse microscopy shown in A.

C Cumulative graph of percentage of cells with monopolar spindles that aligned or congressed all chromosomes along the metaphase plate, assessed from time-lapse movies of cells as shown in A. Only cells that displayed monopolar spindles at the beginning of imaging were considered for analysis. In summary, two important sequential steps drive the rapid and selective stabilisation of end-on attachments: first, the reduction of Aurora-B activity allows the formation of end-on attachments and the initial recruitment of Astrin-sleeves at the kinetochore.

Next, Astrin-mediated delivery of PP1, near the C-terminus of Ndc80, promotes its own enrichment as Astrin-crescents - this positive feedback stabilises end-on attachments to withstand microtubule-mediated pulling Figure 9. Although the inhibition of Aurora-B is essential for the formation of end-on attachment, it is insufficient to stabilise attachments in the absence of Astrin-mediated PP1 delivery. DeLuca, J. Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

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KNL1 facilitates phosphorylation of outer kinetochore proteins by promoting Aurora B kinase activity. Gavet, O. Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Cell 18 , — R Core Team. Wickham, H. Tidy Data. Bates, D. Download references. We thank Keith DeLuca for technical assistance and advice; Geert Kops for helpful discussions and sharing unpublished data; Xiaohu Wan and Ted Salmon for providing the SpeckleTracker program and providing advice; Jonathon Pines and Stephen Taylor for providing reagents; and Steven Markus for critical reading of the manuscript.

You can also search for this author in PubMed Google Scholar. Correspondence to Jennifer G. The cell shown enters anaphase only after all chromosomes are aligned. MOV kb. The cell shown enters anaphase with unaligned chromosomes. Cells were imaged using a 60X oil-immersion objective at 5 min intervals. The cell shown enters mitosis and arrests for min, at which time imaging was terminated.

The cell shown enters mitosis and subsequently exits mitosis after min. The cell shown DIC image enters mitosis and arrests for min, at which time imaging was terminated. These cells were imaged using a 40X objective at 5 min intervals.

The cell shown DIC image enters mitosis and subsequently exits after min. The cell shown DIC image enters mitosis and subsequently exits after 45 min. This work is licensed under a Creative Commons Attribution 4. Reprints and Permissions. Tauchman, E. Nat Commun 6, Download citation. Received : 06 July Accepted : 27 October Published : 01 December Anyone you share the following link with will be able to read this content:.

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Abstract During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. Introduction Accurate segregation of duplicated chromosomes in mitosis is critical for the viability of daughter cells and for the maintenance of genomic integrity.

Full size image. Figure 2: Stable kinetochore—microtubule attachment is sufficient to satisfy the SAC in the absence of chromosome bi-orientation. Figure 3: Stable attachments do not induce large-scale changes in intra-kinetochore distance in STLC-treated cells. Full size table.

Figure 4: Stable kinetochore—microtubule attachment silences the SAC in the absence of spindle pole-mediated pushing or pulling forces. Additional information How to cite this article: Tauchman, E. References 1 Holland, A. Article Google Scholar 9 Maresca, T. Article Google Scholar 14 Sarangapani, K. Article Google Scholar 21 Waters, J.

Article Google Scholar 42 Sacristan, C. Article Google Scholar 55 Bates, D. Article Google Scholar Download references. Acknowledgements We thank Keith DeLuca for technical assistance and advice; Geert Kops for helpful discussions and sharing unpublished data; Xiaohu Wan and Ted Salmon for providing the SpeckleTracker program and providing advice; Jonathon Pines and Stephen Taylor for providing reagents; and Steven Markus for critical reading of the manuscript.

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