A tog protein confers tension sensitivity to kinetochore microtubule attachments

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A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments Graphical Abstract Highlights dch-TOG and Stu2 exhibit a conserved interaction with the Ndc80 kinetochore complex dKinetochore-bound Stu2 directly contributes to microtubule attachment stability

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Why does tension stabilize kinetochore microtubule attachments?

Second, tension promotes microtubule assembly, which therefore reinforces kinetochore-microtubule attachments at higher forces. Although these properties are sufficient to explain the stabilization of kinetochore-microtubule attachments by tension, specific factors that mediate this activity have not yet been identified.

How does tension affect microtubular growth and rescue rates?

For microtubules attached to wild-type kinetochores, the growth speeds and rescue rates increased with tension, while the shortening speeds and catastrophe rates decreased with tension, consistent with our previous observations using recombinant components or native kinetochores ( Akiyoshi et al., 2010, Franck et al., 2007 ).

Does kinetochore bound stu2 contribute to the attachment strength of purified kinetochores?

Together, these results show that kinetochore-bound Stu2 significantly contributes to the overall attachment strength of purified kinetochore particles. Figure S4.

Why do kinetochores become unstable?

Once kinetochores biorient, they come under tension from opposing microtubule pulling forces. Pioneering work showed that incorrect kinetochore attachments are unstable due to the absence of tension ( Dietz, 1958, Nicklas and Koch, 1969 ).

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Which protein anchors microtubule to kinetochore?

The kinetochore interacts with the microtubule and with SAC signaling proteins through a network of three protein complexes: KNL1Spc105, Mis12Mtw1, and Ndc80, collectively referred to as the KMN network 13, 14, 15.


How tension increases attachment of microtubules to the chromosome kinetochore?

Tension probably increases the number of kinetochore microtubules by slowing their turnover rate. The limited effect of tension at prometaphase kinetochores suggests that they have fewer microtubule binding sites than at late metaphase.


What is microtubule kinetochore attachment?

The kinetochore attaches chromosomes to spindle microtubules, modulates the stability of these attachments, and relays microtubule-binding status to the spindle assembly checkpoint, a cell cycle surveillance pathway that delays chromosome segregation in response to unattached kinetochores.


What is the attachment site for microtubules?

At the spindle poles, centrosomes are the major site of microtubule nucleation. Pairs of replicated chromosomes attach to the spindle via kinetochores (middle).


What is the role of the kinetochores and the microtubules?

The kinetochore plays key roles throughout mitosis, both to mediate direct attachments between microtubules and centromeric DNA (Fig. 1) and as a hub for the signaling molecules required to monitor and control faithful chromosome segregation and cell cycle progression.


What is the function of kinetochore microtubules?

Microtubules that bind a chromosome are called kinetochore microtubules. Kinetochore fibers extend from the kinetochore region and attach chromosomes to microtubule spindle polar fibers. These fibers work together to separate chromosomes during cell division.


In what phase do microtubules attach to kinetochores?

In metaphase I, the spindle microtubules attach to the kinetochore of .


What is the name of the multiprotein complex attached to each centromere to which microtubules can bind?

Chromosome attachment, alignment and movement is dependent on a single multi-protein complex, called the kinetochore, that assembles on each chromosome as cells enter mitosis [1]. The kinetochore promotes and monitors stable attachments between the microtubules of the mitotic spindle and the chromosome.


Which molecule is inhibited when chromosomes are attached to microtubules?

SACMicrotubule attachment at all of the kinetochore binding sites is not necessary for deactivation of the SAC and progression to anaphase. Therefore, microtubule-attached and microtubule-unattached states coexist in the animal kinetochore while the SAC is inhibited.


Who provided purified ch-TOG?

We are grateful to Dan Gestaut and Trisha Davis for kindly providing purified Hec1/Ndc80 complex, Shih-Chieh Ti and Tarun Kapoor for generously providing purified ch-TOG, Arshad Desai for providing antibodies, Angelika Amon, Leon Chan, Eris Duro, and Adèle Marston for reagents, Geert Kops for generating an spc105-AID allele, and Neil Umbreit and Krishna Sarangapani for technical assistance. We thank Bungo Akiyoshi, Trisha Davis, Geert Kops, Luke Rice, and members of the S.B. lab for their critical reading of this manuscript. M.P.M. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation. This work was supported by a Packard Fellowship 2006-30521 (to C.L.A.) and NIH grants R01GM079373 (to C.L.A) and R01GM064386 (to S.B.). S.B. is also an investigator of the Howard Hughes Medical Institute.


What is the function of Stu2?

Our findings reveal an uncharacterized function of Stu2 that is regulated mechanically, such that kinetochore-microtubule attachments are intrinsically stabilized by tension, implicating it in the correction of erroneous kinetochore-microtubule attachments. Stu2’s association with kinetochores and its ability to strengthen kinetochore-tip attachments are properties shared by its human ortholog, ch-TOG. Chromosome segregation errors are the most prevalent genetic alteration in tumor cells and have been proposed to be a major factor in the evolution of cancer (reviewed in Gordon et al., 2012 ). Because ch-TOG is overexpressed in various tumor types (named for colonic and hepatic tumor overexpressed gene; Charrasse et al., 1995, Charrasse et al., 1998 ), it will be important to determine whether its function at kinetochores contributes to tumorigenesis and, ultimately, whether kinetochore-associated ch-TOG might be a useful therapeutic target.


What is the most prevalent genetic alteration in tumor cells?

Cellular and organismal fitness requires proper partitioning of genetic material during cell division. Failure to accurately segregate chromosomes causes aneuploidy, the most prevalent genetic alteration in tumor cells and a potential factor in the evolution of cancer (reviewed in Gordon et al., 2012 ). Chromosome segregation is driven by microtubule-based forces, which are generated at kinetochores. The kinetochores must stay bound to microtubule “plus ends,” where tubulin subunits are added and lost at a high rate and where the microtubule filaments switch stochastically between phases of assembly and disassembly ( Mitchison and Kirschner, 1984 ).


Does Stu2 strengthen kinetochores?

If Stu2 strengthens kinetochores via its association with Ndc80c, we reasoned that it might also strengthen tip attachments formed by Nd c80c alone. When Ndc80c (described in Figure 1 C) was bound to polystyrene beads at sufficiently high density, such that multiple complexes could engage simultaneously with the microtubule tip, it maintained attachments to growing microtubule tips with an average rupture strength of 3.7 ± 0.3 pN as previously seen ( Figure 3 F; Powers et al., 2009 ). The addition of purified Stu2 increased the rupture strength of these Ndc80c-coated beads dramatically, to an average of 10.6 ± 0.6 pN ( Figure 3 F). We observed similar results using recombinant Ndc80c instead of native Ndc80c purified from yeast (data not shown). The Xenopus Stu2 family member XMAP215 alone forms load-bearing attachments to dynamic microtubule tips ( Trushko et al., 2013 ), suggesting that Stu2 by itself might also possess an inherent tip-coupling activity. To test this, we measured rupture force distributions for Stu2-decorated beads and found an average strength of 3.8 ± 0.6 pN ( Figures S4 C and S4D). Together, these results demonstrate that Stu2 binding to Ndc80c enhances tip coupling, possibly through the addition of its own inherent microtubule binding activity.


Does Stu2 interact with NDC80?

Although the fission yeast Stu2 homologs interact with the Ndc80 complex ( Hsu and Toda, 2011, Tang et al., 2013 ), the budding yeast Stu2 and Ndc80 proteins were reported to not interact in a yeast two-hybrid assay ( Maure et al., 2011 ). We therefore directly tested whether Stu2 and Ndc80 complex (Ndc80c) associate. We independently isolated them via single-step immunoprecipitation of Stu2-V5 or an Ndc80c component, Spc24-His-Flag, followed by high-salt washes to remove co-purifying proteins ( Figures 1 C and S1 B). These conditions result in the isolation of the heterotetrameric Ndc80c or Stu2 to high purity ( Figures 1 C). We then incubated immobilized Ndc80c with purified Stu2-V5 and detected a specific interaction ( Figures 1 C and S1 D), suggesting that Stu2 associates with kinetochores via Ndc80c.


Is Stu2 a microtubule polymerase?

Because Stu2 is a microtubule polymerase ( Podolski et al., 2014 ), its role at the kinetochore might be mediated through changes in the dynamics of kinetochore-attached microtubules, as previously proposed ( Hsu and Toda, 2011, Tang et al., 2013 ). To address this, we used an optical trapping-based “force-clamp” assay. As before, bead-bound kinetochores were attached to microtubule tips using an optical trap. However, rather than gradually increasing the force, we instead applied fixed levels of tension in the direction of microtubule growth. In this assay, kinetochores track continuously with tip growth and shortening, allowing us to monitor the dynamic instability of kinetochore-attached microtubules with high spatiotemporal resolution ( Figures 5 A and 5B; Akiyoshi et al., 2010 ). We examined a range of forces (from 1 to 5 pN) and compared growth and shortening speeds as well as switch rates (catastrophe and rescue frequencies) for microtubule tips attached to kinetochores that either contained or lacked Stu2 (wild-type or Stu2-AID kinetochores, respectively). For microtubules attached to wild-type kinetochores, the growth speeds and rescue rates increased with tension, while the shortening speeds and catastrophe rates decreased with tension, consistent with our previous observations using recombinant components or native kinetochores ( Akiyoshi et al., 2010, Franck et al., 2007 ). Surprisingly, however, when attached to kinetochores lacking Stu2, the microtubules behaved indistinguishably from those attached to kinetochores that retained Stu2. We found no clear differences in any of the four dynamic rate parameters for microtubules attached to Stu2-AID kinetochores versus wild-type kinetochores ( Figures 5 C and 5D), measured over the full range of experimentally accessible forces. Thus, the kinetochore-bound pool of Stu2 does not contribute to the tension-induced changes in microtubule dynamics that we observe in vitro.


Who provided purified ch-TOG?

We are grateful to Dan Gestaut and Trisha Davis for kindly providing purified Hec1/Ndc80 complex, Shih-Chieh Ti and Tarun Kapoor for generously providing purified ch-TOG, Arshad Desai for providing antibodies, Angelika Amon, Leon Chan, Eris Duro, and Adèle Marston for reagents, Geert Kops for generating an spc105-AID allele, and Neil Umbreit and Krishna Sarangapani for technical assistance. We thank Bungo Akiyoshi, Trisha Davis, Geert Kops, Luke Rice, and members of the S.B. lab for their critical reading of this manuscript. M.P.M. is an HHMI Fellow of the Damon Runyon Cancer Research Foundation. This work was supported by a Packard Fellowship 2006-30521 (to C.L.A.) and NIH grants R01GM079373 (to C.L.A) and R01GM064386 (to S.B.). S.B. is also an investigator of the Howard Hughes Medical Institute.


What is the most prevalent genetic alteration in tumor cells?

Cellular and organismal fitness requires proper partitioning of genetic material during cell division. Failure to accurately segregate chromosomes causes aneuploidy, the most prevalent genetic alteration in tumor cells and a potential factor in the evolution of cancer (reviewed in


Does Aurora B stabilize microtubules?

Although tension-dependent stabilization is widely accepted as the basis for mitotic accuracy, how tension stabilizes kinetochore-microtubule attachments remains unclear. The Aurora B kinase promotes the release of erroneous attachments through phosphorylation of various kinetochore components (reviewed in


Does Stu2 affect microtubule attachment?

Stu2 confers opposite effects on kinetochore-microtubule attachment stability depending on the level of tension and on the state of the microtubule tip. It is not yet possible to assign these Stu2-dependent effects to established structural features of the kinetochore-microtubule interface, because kinetochore-microtubule coupling remains poorly understood in mechanistic detail. However, some candidate mechanisms are suggested by the selective binding of Stu2 and Ndc80c to curved and straight conformations of tubulin, respectively (


Is Stu2 regulated mechanically?

Our findings reveal an uncharacterized function of Stu2 that is regulated mechanically, such that kinetochore-microtubule attachments are intrinsically stabilized by tension, implicating it in the correction of erroneous kinetochore-microtubule attachments. Stu2’s association with kinetochores and its ability to strengthen kinetochore-tip attachments are properties shared by its human ortholog, ch-TOG. Chromosome segregation errors are the most prevalent genetic alteration in tumor cells and have been proposed to be a major factor in the evolution of cancer (reviewed in


Is Stu2 a microtubule?

Microtubule Dynamics Because Stu2 is a microtubule poly merase (Podolski et al.,2014), its role at the kinetochore might be mediated throughchanges in the dynamics of kinetochore-attached microtu-bules, as previously proposed (Hsu and Toda, 2011; Tanget al., 2013). To address this, we used an optical trapping-


Does Stu2 affect microtubules?

Stu2 confers opposite effects on kinetochore-microtubule attach-ment stability depending on the level of tension and on the stateof the microtubule tip. It is not yet possible to assign theseStu2-dependent effects to established structural features of thekinetochore-microtubule interface, because kinetochore-micro-tubule coupling remains poorly understood in mechanistic detail.However, some candidate mechanisms are suggested by theselective binding of Stu2 and Ndc80c to curved and straightconformations of tubulin, respectively (Alushin et al., 2010; Ayazet al., 2012, 2014), and by the presumed arrangements of thesetubulin conformations at the assembling and disassembling tipsof kinetochore-attached microtubules. We speculate that byselectively binding curved tubulins at the tip, kinetochore-associ-ated Stu2 might form microtubule links that do not interfere withNdc80c, which binds straight tubulins that are presumablylocated within the microtubule lattice (Figure 7A). Faster tipgrowth at higher tension could increase the number of curved tu-bulins at the tip, thereby enhancing the contribution of Stu2 tokinetochore attachment stability. During tip disassembly at lowtension, the kinetochore-associated Stu2 has a destabilizingeffect on attachment. Under these conditions it may directlycompete with or occlude the microtubule-binding activities ofother kinetochore components (such as Ndc80c;Figure 7B), orit may alter the structure of the disassembling tip in a mannerthat inhibits their binding. In any case, the interference by Stu2is relieved as tension is increased. A speculative explanation isthat tension-dependent stretching of the kinetochore structure it-self might relieve this inhibition by spatially separating Stu2 fromthe other microtubule-binding kinetochore elements (Figure 7B).Regardless of the mechanism, our observation that Stu2 affectsattachment stability in a direct and tension-dependent manner

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