<scp>PRC</scp>
            1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

Polak, Bruno; Risteski, Patrik; Lesjak, Sonja; Tolić, Iva Marija

doi:10.15252/embr.201642650

Cited by 94 publications

(134 citation statements)

References 36 publications

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“…It has a reported role specifically in “bridging fibers” [5] whose geometry – connecting sister k-fibers by spanning the centromere [33] – is well-suited to maintaining poleward force on chromosomes [15, 34]. Furthermore, overexpression of PRC1 and concomitant thickening of bridging fibers results in a greater angular deflection of the centromere following ablation in HeLa cells [5].…”

Section: Resultsmentioning

confidence: 99%

“…Thus, while PRC1-mediated bridging microtubules can effectively stiffen the centromere [5], our results suggest they may not significantly bear the load of stretching it. In HeLa cells, PRC1 knockdown leads to a ~10% reduction in interkinetochore distance [33]: there may be species-specific differences, and since this is a steady state measurement, it may or may not report on the same underlying mechanics as those we measure following ablation.…”

Section: Resultsmentioning

confidence: 99%

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Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy

et al. 2017

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Summary Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore-fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how – physically and molecularly – this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet, answering this question is key to understanding how the spindle generates and responds to force, and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time, and dissect local anchorage mechanics and mechanism. To do so, we laser ablate single k-fibers at different spindle locations and in different molecular backgrounds, and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3–4 μm from kinetochores, scaling with k-fiber length, and laterally within ~2 μm of k-fiber sides, without detectable load-sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement, but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, while Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well-suited to support robust chromosome segregation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy

et al. 2017

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“…To gain insight into the spatial regulation of Eg5 by TPX2, the distribution of Eg5-EGFP and TPX2-EGFP in the region of the spindle where overlapping microtubules are present was examined (Mastronarde, McDonald, Ding, & McIntosh, 1993;McIntosh & Landis, 1971;Polak, Risteski, Lesjak, & Tolic, 2017). To do this, we took advantage of cells with spindles oriented perpendicular (90 ) microtubule bundles, which were considerably brighter, consistent with kinetochore fibers, which contain on average 17 microtubules (Simunic & Tolic, 2016;Tolic, 2017;Wendell, Wilson, & Jordan, 1993).…”

Section: Eg5 But Not Tpx2 Localizes To Overlapping Microtubulesmentioning

confidence: 99%

“…To gain insight into the spatial regulation of Eg5 by TPX2, the distribution of Eg5-EGFP and TPX2-EGFP in the region of the spindle where overlapping microtubules are present was examined (Mastronarde, McDonald, Ding, & McIntosh, 1993;McIntosh & Landis, 1971;Polak, Risteski, Lesjak, & Tolic, 2017). To do this, we took advantage of cells with spindles oriented perpendicular (90 ) to the coverslip surface and asked if Eg5-EGFP and/or TPX2-EGFP colocalized with microtubules in the region of the spindle between the two sets of kinetochore fibers (Figure 4a).…”

Section: Eg5 But Not Tpx2 Localizes To Overlapping Microtubulesmentioning

confidence: 99%

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

Mann

Wadsworth

2018

Cytoskeleton

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The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

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“…We used fixed HeLa cells expressing GFP-tagged protein regulator of cytokinesis 1 (PRC1) 9,10 , which binds overlap zones of antiparallel microtubules 11,12 . Almost all overlap bundles in the spindle are associated with a pair of sister kinetochores, acting as a bridge between the respective kinetochore fibers [13][14][15] . Thus, PRC1-GFP shows the position of overlap bundles together with their kinetochore fibres, without interference from other microtubules such as polar and astral ones.…”

mentioning

confidence: 99%

The mitotic spindle is chiral due to torques generated by motor proteins

Novak

Polak

Simunić

et al. 2017

Preprint

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Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins 1,2 . Forces are required for the congression of chromosomes to the metaphase plate and separation of chromatids in anaphase [3][4][5][6] . However, torques may also exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. The average helicity of the bundles with respect to the spindle axis is 1.2 °/µm. Inactivation of kinesin-5 (Eg5/Kif11) abolished the chirality of the spindle, suggesting that this motor generates the helical shape of microtubule bundles. To explain the observed shapes, we introduce a theoretical model for the balance of forces and torques acting in the spindle, and show that torque is required to generate the helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its architecture.We set out to infer torques and forces in the spindle, by using the shape of microtubule bundles. We first used stimulated emission depletion (STED) super-resolution microscopy 7,8 to determine the shapes of microtubule bundles in metaphase spindles. We used HeLa cells with labeled microtubules, kinetochores and centrioles (Fig. 1a) and U2OS cells with labeled microtubules and kinetochores (Extended Data Fig. 1a). Single optical sections of metaphase spindles showed that microtubule bundles are continuous almost from pole to pole and acquire complex curved shapes (Fig. 1a). Typically, the outer bundles are curved and resemble a C-letter shape. Interestingly, some bundles throughout the spindle resemble the letter S. Thus, STED images of the spindle suggest that microtubules are arranged into well-defined bundles exhibiting a variety of shapes, which run continuously almost through the whole spindle.In order to obtain a complete three-dimensional contour of microtubule bundles, we used vertically oriented spindles, which are found occasionally in a population of mitotic cells, and . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/167437 doi: bioRxiv preprint first posted online Jul. 22, 2017; imaged them by confocal microscopy (Fig. 1b). In these spindles, optical sections are roughly perpendicular to the microtubule bundles, allowing for precise determination of the bundle location in each section and thus of the whole three-dimensional contour (Methods). We used fixed HeLa cells expressing GFP-tagged protein regulator of cytokinesis 1 (PRC1) 9,10 , which binds ove...

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PRC 1‐labeled microtubule bundles and kinetochore pairs show one‐to‐one association in metaphase

Cited by 94 publications

References 36 publications

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy

Mapping Load-Bearing in the Mammalian Spindle Reveals Local Kinetochore Fiber Anchorage that Provides Mechanical Isolation and Redundancy

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

The mitotic spindle is chiral due to torques generated by motor proteins

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