Visualization of Aberrant Perinuclear Microtubule Aster Organization by Microtubule-Destabilizing Agents

Sakaushi, Shinji; Senda-Murata, Kaori; Oka, Saori; Sugimoto, Kenji

doi:10.1271/bbb.80754

Cited by 10 publications

(9 citation statements)

References 9 publications

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“…6d ). The observation that nocodazole promotes a basal level of aster formation is consistent with published results 34 , as endogenous aster-promoting factors are present in these cells. Finally, we used a live-imaging approach to observe EGFP-HSET during aster formation over time.…”

Section: Resultssupporting

confidence: 92%

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Norris

Jung

Singh

et al. 2018

Preprint

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16Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two 17 architectures: bundles and asters. Although both architectures are critical for cellular function, 18 the molecular pathways that drive aster formation are poorly understood. Here, we study aster 19 formation by human minus-end directed kinesin-14 (HSET/KIFC1). We show that HSET is 20 incapable of forming asters from pre-formed, non-growing MTs, but rapidly forms MT asters in 21 the presence of soluble tubulin. HSET binds soluble (non-polymer) tubulin via its N-terminal tail 22 domain to form heterogeneous HSET-tubulin "clusters" containing multiple motors. Cluster 23 formation induces motor processivity and rescues the formation of asters from non-growing 24MTs. We then show that excess soluble tubulin stimulates aster formation in HeLa cells 25 overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT 26 bundle and aster formation in a manner governed by the availability of soluble tubulin. 27 28 65 processivity is essential for promoting aster formation. Finally, we present data suggesting that 66HSET's aster-forming behavior can be activated by excess soluble tubulin in mitotic HeLa cells. 67Our work establishes a general principle of aster formation by MT-crosslinking motor proteins, 68and demonstrates how motor regulation on a molecular level dictates the formation of higher-69 order MT structures. 70 71Results 72 Aster and bundle formation by HSET is context-dependent 73To determine if HSET, like its Xenopus homologue XCTK2, could form MT asters, we 74 expressed and purified four HSET truncations tagged with an N-terminal EGFP (Fig. 1a, 75 Supplementary Fig. S1a) for use in self-organization assays 17 . We verified that each construct 76 is a dimer by comparing their stepwise photobleaching traces 28 to EGFP-XMCAK, a known 77 dimer 29 (Supplementary Fig. S1b-c). When we combined 100 nM full-length EGFP-HSET, 20 78 µ M tubulin, and saturating ATP/GTP at 37°C, we observed MT aster formation which reached a 79 steady state within ~10 minutes (Fig. 1b, Supplementary Fig. S1d, and Supplementary Movie 80 1), consistent with published studies using Ncd multimers 7 or XCTK2 17 . Deletion of either the 81 N-terminal tail (EGFP-HSETΔTail), or the conserved C-terminal motor domain (EGFP-82HSETΔMotor) eliminated aster formation (Fig. 1b, middle). When the motor domain was 83 replaced by another copy of the N-terminal tail domain (EGFP-HSET-DoubleTail, see 17 ), we 84 observed bundle formation, but no MT asters (Fig. 1b, bottom). The entire HSET molecule is 85 thus required for aster formation of growing MTs. 86We next asked whether HSET was capable of forming asters from preformed, non-87 growing MTs stabilized by GMPCPP 30 . When we combined 100 nM full-length EGFP-HSET 88 with preformed MTs (1

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Section: Resultssupporting

confidence: 92%

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Norris

Jung

Singh

et al. 2018

Preprint

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“…19,33 The outcome of premature aster formation ranges from mild effects on the spindle as seen with epothilone A, 37 where the bipolar spindle is retained, to severe as seen with paclitaxel, 12 epothilone B 12,37 and docetaxel, 19 where multipolar spindles are formed with inhibition of pole separation at higher doses. When compared with these other MT-stabilizing agents, laulimalide favors an intermediate spindle phenotype, as long as the drug is present before nuclear envelope breakdown.…”

Section: Discussionmentioning

confidence: 99%

“…The formation of multiple asters at this stage appears to be a common property of all types of microtubule targeting agents, including stabilizers, destabilizers and microtubule-modulating drugs like EM011. [32][33][34] Precisely how MT asters are formed is not well understood. Several studies suggest these asters arise from increased polymerization of MT states in the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

et al. 2012

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“…Live cell imaging has advanced rapidly in recent years because of the improvement of microscopy techniques [1]- [3]. In particular, elucidation of cellular transportation paths is important for understanding clinical state.…”

Section: Introductionmentioning

confidence: 99%

Melanosome Tracking Using Automatic Error Correction

Okabe

2014

IEICE Trans. Inf. & Syst.

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This paper proposes an automatic error correction method for melanosome tracking. Melanosomes in intracellular images are currently tracked manually when investigating diseases, and an automatic tracking method is desirable. We detect all melanosome candidates by SIFT with 2 different parameters. Of course, the SIFT also detects non-melanosomes. Therefore, we use the 4-valued difference image (4-VDimage) to eliminate non-melanosome candidates. After tracking melanosome, we re-track the melanosome with low confidence again from t + 1 to t. If the results from t to t + 1 and from t + 1 to t are different, we judge that initial tracking result is a failure, the melanosome is eliminated as a candidate and re-tracking is carried out. Experiments demonstrate that our method can correct the error and improves the accuracy.

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Visualization of Aberrant Perinuclear Microtubule Aster Organization by Microtubule-Destabilizing Agents

Cited by 10 publications

References 9 publications

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

Melanosome Tracking Using Automatic Error Correction

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