The far C-terminus of MCAK regulates its conformation and spindle pole focusing

Zong, Hailing; Carnes, Stephanie K.; Moe, Christina; Walczak, Claire; Ems-McClung, Stephanie C.

doi:10.1091/mbc.e15-10-0699

Cited by 14 publications

(25 citation statements)

References 46 publications

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“…Specifically, it is possible that the addition of a second MT-binding domain might alter the quaternary structure of the probe (as compared with SH-TSmod), locking it in a conformation that adversely affects binding to MTs within the spindle midzone. The change in localization resulting from the addition of a second mTMBD is conceptually similar to the different localization patterns observed for different conformations of kinesin-13 in Xenopus egg extract spindles (Zong et al 2016). …”

Section: Discussionsupporting

confidence: 61%

Tau‐based fluorescent protein fusions to visualize microtubules

et al. 2017

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The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule-dependent phenomena such as cell motility, cell division, and mitosis. Here we describe a novel set of fluorescent protein fusions designed specifically to visualize microtubules in living systems using fluorescence microscopy. Each fusion contains a fluorescent protein module linked in frame to a modified phospho-deficient version of the microtubule-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the microtubule-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, microtubule dynamics were not grossly perturbed by the presence of Tau-based fluorescent protein fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based fluorescent protein fusions as minimally perturbing tools to accurately visualize microtubules in living systems.

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Section: Discussionsupporting

confidence: 61%

Tau‐based fluorescent protein fusions to visualize microtubules

et al. 2017

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“…Two recent studies showed that GTSE1 localizes to the spindle during mitosis, and like TACC3, is necessary for astral microtubule stabilization and efficient chromosome alignment (Bendre et al, 2016;Tipton et al, 2017). We showed that GTSE1 stabilizes MTs in mitosis by inhibiting MCAK activity (Bendre et al, 2016). In that study, cancer cells either depleted or knocked-out of GTSE1 showed destabilization of both astral MTs and kinetochore-MT attachments.Another study reported that a specific "slow turnover" population of MTs become stabilized upon depletion of GTSE1(Tipton et al, 2017).…”

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confidence: 58%

“…However, whether clathrin (or the CHC/TACC3 complex) is required for astral microtubule stabilization has not been addressed.Another potential functional member of the CHC/TACC3 complex is GTSE1 (pronounced "jitsee-one" (Monte et al, 2000)), an intrinsically disordered protein that has been shown by proteomic analysis to interact with the CHC/TACC3 complex in cells (Hubner et al, 2010). Two recent studies showed that GTSE1 localizes to the spindle during mitosis, and like TACC3, is necessary for astral microtubule stabilization and efficient chromosome alignment (Bendre et al, 2016;Tipton et al, 2017). We showed that GTSE1 stabilizes MTs in mitosis by inhibiting MCAK activity (Bendre et al, 2016).…”

mentioning

confidence: 77%

“…The reason for these seemingly contradictory results regarding MT stability after GTSE1 depletion is unclear. GTSE1 requires both CHC and TACC3 to localize to spindles, but does not appear to influence their localization (Hubner et al, 2010;Bendre et al, 2016;Cheeseman et al, 2013). The interactions and mechanism by which the CHC/TACC3 complex recruits GTSE1 to the spindle is unknown.The ability of clathrin to function in endocytosis and vesicle trafficking is dependent on interactions between the CHC N-terminal domain (NTD) and clathrin "adaptor" proteins, named for their ability to provide a physical link between specific cargo receptors and clathrin to initiate recruitment of clathrin to membranes and ultimately form a lattice and coated vesicle (Lemmon and Traub, 2012;Robinson, 2015).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Rondelet

Lin

Singh

et al. 2019

Preprint

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Clathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle.Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes 2 microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis. Rizk et al., 2009; Walczak et al., 1996;Wordeman and Mitchison, 1995) (Moore et al., 2005). It was proposed that a function of TACC3 is to antagonize MCAK's potent activity at centrosomes to stabilize astral MTs, presumably by promoting activity of the microtubule depolymerase XMAP215/ch-Tog (Kinoshita et al., 2005). However, whether clathrin (or the CHC/TACC3 complex) is required for astral microtubule stabilization has not been addressed.Another potential functional member of the CHC/TACC3 complex is GTSE1 (pronounced "jitsee-one" (Monte et al., 2000)), an intrinsically disordered protein that has been shown by proteomic analysis to interact with the CHC/TACC3 complex in cells (Hubner et al., 2010). Two recent studies showed that GTSE1 localizes to the spindle during mitosis, and like TACC3, is necessary for astral microtubule stabilization and efficient chromosome alignment (Bendre et al., 2016;Tipton et al., 2017). We showed that GTSE1 stabilizes MTs in mitosis by inhibiting MCAK activity (Bendre et al., 2016). In that study, cancer cells either depleted or knocked-out of GTSE1 showed destabilization of both astral MTs and kinetochore-MT attachments.Another study reported that a specific "slow turnover" population of MTs become stabilized upon depletion of GTSE1(Tipton et al., 2017). The reason for these seemingly contr...

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Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

Goldstein

Siegler

Goldman

et al. 2017

Cell. Mol. Life Sci.

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The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.

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The far C-terminus of MCAK regulates its conformation and spindle pole focusing

Abstract: Spatial regulation of microtubule dynamics is critical for proper spindle assembly. The far C-terminus of the microtubule-depolymerizing kinesin-13 MCAK regulates MCAK localization at spindle poles, which is needed for proper pole focusing.

Cited by 14 publications

References 46 publications

Tau‐based fluorescent protein fusions to visualize microtubules

Tau‐based fluorescent protein fusions to visualize microtubules

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

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