Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity

Chakraborty, M.; Tarasovetc, Ekaterina V.; Zaytsev, Anatoly V.; Godzi, Maxim; Figueiredo, Ana C.; Ataullakhanov, Fazly I.; Grishchuk, Ekaterina L.

doi:10.1038/s41467-019-09411-7

Cited by 34 publications

(63 citation statements)

References 64 publications

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“…Upon entry into M-phase (mitosis or meiosis) and spindle assembly, chromosomes are often initially transported to the spindle poles, where the microtubule density is highest, and from there to the spindle’s equatorial plane, forming lateral attachments to the microtubule lattice. CENP-E, a kinetochore-localized, microtubule-plus-end-directed motor plays an essential function in this process (Bancroft et al, 2015; Barisic et al, 2014; Cai et al, 2009; Chakraborty et al, 2019; Kapoor et al, 2006; Kim et al, 2008; Kitajima et al, 2011; Magidson et al, 2011; Shrestha et al, 2017; Tanaka et al, 2005). In a poorly understood process of ‘end-conversion’, kinetochores engage the microtubule-binding interface of the KMN network and transition from binding to the lattice to binding to the dynamic plus ends of the microtubules, which become embedded into the kinetochore’s outer plate (Dong et al, 2007; Kuhn and Dumont, 2017; McIntosh et al, 2013; Wan et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Veld

Volkov

Stender

et al. 2019

eLife

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Errorless chromosome segregation requires load-bearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Both the Ndc80-tail, which we reveal to be essential for microtubule end-tracking, and Ndc80-bound Ska stabilize microtubule ends in a stalled conformation. Modulation of force-coupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.

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Section: Introductionmentioning

confidence: 99%

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Veld

Volkov

Stender

et al. 2019

eLife

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show abstract

“…Upon entry into M-phase (mitosis or meiosis) and spindle assembly, chromosomes are initially transported to the spindle poles, where the microtubule density is highest, and from there to the spindle's equatorial plane, forming lateral attachments to the microtubule lattice. CENP-E, a kinetochore-localized, microtubule-plus-end-directed motor plays an essential function in this process (Bancroft et al, 2015;Barisic et al, 2014;Cai et al, 2009;Chakraborty et al, 2019;Kapoor et al, 2006;Kim et al, 2008;Kitajima et al, 2011;Magidson et al, 2011;Shrestha et al, 2017;Tanaka et al, 2005). In a poorly understood process of "endconversion", kinetochores engage the microtubule-binding interface of the KMN network and transition from binding to the lattice to binding to the dynamic plus ends of the microtubules, which become embedded into the kinetochore's outer plate (Dong et al, 2007;Kuhn and Dumont, 2017;McIntosh et al, 2013;Wan et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Veld

Stender

Musacchio

et al. 2019

Preprint

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Errorless chromosome segregation requires loadbearing attachments of the plus ends of spindle microtubules to chromosome structures named kinetochores. How these end-on kinetochore attachments are established following initial lateral contacts with the microtubule lattice is poorly understood. Two microtubule-binding complexes, the Ndc80 and Ska complexes, are important for efficient end-on coupling and may function as a unit in this process, but precise conditions for their interaction are unknown. Here, we report that the Ska-Ndc80 interaction is phosphorylation-dependent and does not require microtubules, applied force, or several previously identified functional determinants including the Ndc80-loop and the Ndc80-tail. Under force, Ska stabilizes end-on microtubule attachments in parallel with the Ndc80-tail, which we reveal to be essential for end-tracking by Ndc80 multimers. Modulation of forcecoupling efficiency demonstrates that the duration of stalled microtubule disassembly predicts whether a microtubule is stabilized and rescued by the kinetochore, likely reflecting a structural transition of the microtubule end.

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“…These results are consistent with the unique capacity of human CLASPs to bind to curved microtubule protofilaments (Leano et al, 2013; Maki et al, 2015) and recent electron microscopy work showing that both growing and shrinking microtubule plus ends, including those of kinetochore microtubules, are curved (McIntosh et al, 2018). They are also supported by recent in vitro reconstitution experiments that showed that CLASP2-coated spherical beads are able to mediate moderately long-lived attachments with polymerizing microtubule plus-ends (Chakraborty et al, 2019). Unlike other TOG-domain containing proteins, such as members of the XMAP215/ch-TOG family that work as microtubule polymerases that bind to free α,β-tubulin heterodimers (Ayaz et al, 2012; Geyer et al, 2018; Nithianantham et al, 2018), our data is most consistent with a model in which microtubule lattice binding and EB protein-dependent microtubule plus-end tracking of CLASP2 is required at the kinetochore to stabilize, or prevent destabilization, of kinetochore-microtubule attachments as cells progress into metaphase.…”

Section: Resultsmentioning

confidence: 63%

CLASP2 lattice-binding near microtubule plus ends stabilizes kinetochore attachments

Girão

Okada

Figueiredo

et al. 2019

Preprint

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24The fine regulation of kinetochore microtubule dynamics during mitosis ensures 25 proper chromosome segregation by promoting error correction and spindle 26 assembly checkpoint (SAC) satisfaction. CLASPs are widely conserved 27 microtubule plus-end-tracking proteins that regulate microtubule dynamics 28 throughout the cell cycle and independently localize to kinetochores during 29 mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore microtubule 30 dynamics, but the underlying molecular mechanism remains unknown. Here we 31 found that human CLASP2 can dimerize through its C-terminal kinetochore-32 targeting domain, but kinetochore localization was independent of dimerization. 33 CLASP2 kinetochore localization, microtubule plus-end-tracking and microtubule 34 lattice binding through TOG2 and TOG3 (but not TOG1) domains, independently 35 sustained normal spindle length, timely SAC satisfaction, chromosome 36 congression and faithful segregation. Measurements of kinetochore microtubule 37 half-life in living cells expressing RNAi-resistant mutants revealed that CLASP2 38 kinetochore localization, microtubule plus-end-tracking and lattice binding 39 cooperatively modulate kinetochore microtubule stability during mitosis. Thus, 40 CLASP2 regulates kinetochore microtubule dynamics by integrating distinctive 41 microtubule-binding properties at the kinetochore-microtubule interface to ensure 42 chromosome segregation fidelity. 43 44 Keywords 45 Mitosis, kinetochore, microtubule dynamics, CLASP2, TOG, +TIPs 46 47 93 mechanistic view on how the intrinsic properties of CLASPs modulate 94 kinetochore microtubule dynamics. 95 Here we focused on human CLASP2 to investigate how its distinct 96 functional domains affect mitosis, with emphasis on the regulation of kinetochore 97 5 microtubule dynamics. Our findings revealed that CLASP2 integrates multiple 98 independent features, including microtubule plus-end-tracking, microtubule 99 lattice binding through TOG domains and kinetochore localization, to modulate100 kinetochore microtubule dynamics required for faithful chromosome segregation 101 during mitosis in human cells. Previous studies have come to contradictory conclusions regarding the native 121 structure of CLASPs. Based on the hydrodynamic profile of several kinetochore 122 sub-complexes present in Xenopus egg extracts, it was proposed that the single 123 Xenopus CLASP exists as an elongated monomer in solution (Emanuele et al., 124 2005). This was subsequently confirmed by Fluorescence Correlation 125 Spectroscopy and Photon Counting for human GFP-CLASP2 (Drabek et al., 126 2006), as well as single molecule fluorescence intensity quantification of 127 recombinant human GFP-CLASP2 (Aher et al., 2018). However, another set of 128 studies with recombinant human CLASP1 and its counterparts in S. pombe and 129 S. cerevisiae indicated that they can form homodimers in solution through their 130 C-terminal domain (Al-Bassam et al., 2010; Funk et al., 2014; Patel et al., 2012). 131To shed light into...

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Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity

Cited by 34 publications

References 64 publications

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

CLASP2 lattice-binding near microtubule plus ends stabilizes kinetochore attachments

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