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The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle.

Nixon FM, Gutiérrez-Caballero C, Hood FE, Booth DG, Prior IA, Royle SJ - Elife (2015)

Bottom Line: Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs.We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber.Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, Coventry, United Kingdom.

ABSTRACT
Kinetochore fibers (K-fibers) of the mitotic spindle are force-generating units that power chromosome movement during mitosis. K-fibers are composed of many microtubules that are held together throughout their length. Here, we show, using 3D electron microscopy, that K-fiber microtubules (MTs) are connected by a network of MT connectors. We term this network 'the mesh'. The K-fiber mesh is made of linked multipolar connectors. Each connector has up to four struts, so that a single connector can link up to four MTs. Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs. Optimal stabilization of K-fibers by the mesh is required for normal progression through mitosis. We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber. Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.

No MeSH data available.


Related in: MedlinePlus

Inducible expression of GFP-TACC3 in HeLa cells to alter the composition of mesh.(A) Western blot to show the extent of overexpression of TACC3 caused by inducing the expression of GFP-TACC3 in a stable TetOn HeLa cell line using 0.5 μg/ml doxycycline for 24 hr. The blot was probed for TACC3 and tubulin (as a loading control). (B) Widefield fluorescence micrographs of cells inducibly expressing GFP-TACC3. Note the strong fluorescence on K-fibers of the mitotic spindle. (C) Workflow to show the preparation of metaphase cells for analysis by 3D EM. Cells were synchronized and GFP-TACC3 expression was induced before release from RO3306 for 30–40 min. Mitotic cells were shaken off, pelleted, resuspended, and frozen under high pressure. Following freeze substitution and embedding, cells were trimmed and sectioned before imaging by electron microscopy. For details, see ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.07635.009
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fig3s1: Inducible expression of GFP-TACC3 in HeLa cells to alter the composition of mesh.(A) Western blot to show the extent of overexpression of TACC3 caused by inducing the expression of GFP-TACC3 in a stable TetOn HeLa cell line using 0.5 μg/ml doxycycline for 24 hr. The blot was probed for TACC3 and tubulin (as a loading control). (B) Widefield fluorescence micrographs of cells inducibly expressing GFP-TACC3. Note the strong fluorescence on K-fibers of the mitotic spindle. (C) Workflow to show the preparation of metaphase cells for analysis by 3D EM. Cells were synchronized and GFP-TACC3 expression was induced before release from RO3306 for 30–40 min. Mitotic cells were shaken off, pelleted, resuspended, and frozen under high pressure. Following freeze substitution and embedding, cells were trimmed and sectioned before imaging by electron microscopy. For details, see ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.07635.009

Mentions: Previous work indicated that one component of the mesh is a complex of TACC3–ch-TOG–clathrin whose assembly is regulated by Aurora-A kinase (Booth et al., 2011; Hood et al., 2013). Moreover, the amount of this complex on K-fibers can be increased simply by overexpressing TACC3 (Booth et al., 2011). In order to experimentally manipulate the mesh, we therefore made a stable inducible HeLa cell line where GFP-TACC3 could be overexpressed in a controlled manner (Figure 3—figure supplement 1). The most obvious effect of this manipulation was to alter MT organization. K-fibers in cells expressing GFP-TACC3 had more MTs per fiber, and the cross-sectional area that those MTs occupied was larger, compared to fibers in control uninduced HeLa cells (Figure 3A). The MT density was similar between the two groups suggesting that the fiber area scaled with the number of MTs (Figure 3A).10.7554/eLife.07635.008Figure 3.Analysis of MT packing within a K-fiber.


The mesh is a network of microtubule connectors that stabilizes individual kinetochore fibers of the mitotic spindle.

Nixon FM, Gutiérrez-Caballero C, Hood FE, Booth DG, Prior IA, Royle SJ - Elife (2015)

Inducible expression of GFP-TACC3 in HeLa cells to alter the composition of mesh.(A) Western blot to show the extent of overexpression of TACC3 caused by inducing the expression of GFP-TACC3 in a stable TetOn HeLa cell line using 0.5 μg/ml doxycycline for 24 hr. The blot was probed for TACC3 and tubulin (as a loading control). (B) Widefield fluorescence micrographs of cells inducibly expressing GFP-TACC3. Note the strong fluorescence on K-fibers of the mitotic spindle. (C) Workflow to show the preparation of metaphase cells for analysis by 3D EM. Cells were synchronized and GFP-TACC3 expression was induced before release from RO3306 for 30–40 min. Mitotic cells were shaken off, pelleted, resuspended, and frozen under high pressure. Following freeze substitution and embedding, cells were trimmed and sectioned before imaging by electron microscopy. For details, see ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.07635.009
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495718&req=5

fig3s1: Inducible expression of GFP-TACC3 in HeLa cells to alter the composition of mesh.(A) Western blot to show the extent of overexpression of TACC3 caused by inducing the expression of GFP-TACC3 in a stable TetOn HeLa cell line using 0.5 μg/ml doxycycline for 24 hr. The blot was probed for TACC3 and tubulin (as a loading control). (B) Widefield fluorescence micrographs of cells inducibly expressing GFP-TACC3. Note the strong fluorescence on K-fibers of the mitotic spindle. (C) Workflow to show the preparation of metaphase cells for analysis by 3D EM. Cells were synchronized and GFP-TACC3 expression was induced before release from RO3306 for 30–40 min. Mitotic cells were shaken off, pelleted, resuspended, and frozen under high pressure. Following freeze substitution and embedding, cells were trimmed and sectioned before imaging by electron microscopy. For details, see ‘Materials and methods’.DOI:http://dx.doi.org/10.7554/eLife.07635.009
Mentions: Previous work indicated that one component of the mesh is a complex of TACC3–ch-TOG–clathrin whose assembly is regulated by Aurora-A kinase (Booth et al., 2011; Hood et al., 2013). Moreover, the amount of this complex on K-fibers can be increased simply by overexpressing TACC3 (Booth et al., 2011). In order to experimentally manipulate the mesh, we therefore made a stable inducible HeLa cell line where GFP-TACC3 could be overexpressed in a controlled manner (Figure 3—figure supplement 1). The most obvious effect of this manipulation was to alter MT organization. K-fibers in cells expressing GFP-TACC3 had more MTs per fiber, and the cross-sectional area that those MTs occupied was larger, compared to fibers in control uninduced HeLa cells (Figure 3A). The MT density was similar between the two groups suggesting that the fiber area scaled with the number of MTs (Figure 3A).10.7554/eLife.07635.008Figure 3.Analysis of MT packing within a K-fiber.

Bottom Line: Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs.We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber.Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Cell Biology, Warwick Medical School, Coventry, United Kingdom.

ABSTRACT
Kinetochore fibers (K-fibers) of the mitotic spindle are force-generating units that power chromosome movement during mitosis. K-fibers are composed of many microtubules that are held together throughout their length. Here, we show, using 3D electron microscopy, that K-fiber microtubules (MTs) are connected by a network of MT connectors. We term this network 'the mesh'. The K-fiber mesh is made of linked multipolar connectors. Each connector has up to four struts, so that a single connector can link up to four MTs. Molecular manipulation of the mesh by overexpression of TACC3 causes disorganization of the K-fiber MTs. Optimal stabilization of K-fibers by the mesh is required for normal progression through mitosis. We propose that the mesh stabilizes K-fibers by pulling MTs together and thereby maintaining the integrity of the fiber. Our work thus identifies the K-fiber meshwork of linked multipolar connectors as a key integrator and determinant of K-fiber structure and function.

No MeSH data available.


Related in: MedlinePlus