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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

MT bundling using mesh complex immunoisolated from mitotic HeLa cells.(A) Schematic diagram of the procedure to release mitotic spindle proteins from mitotic spindles at metaphase (described in Booth et al., 2011). (B) Western blot to show specific co-immunoprecipitation of TACC3/clathrin from spindle fractions (F5-7) but not from mitotic cytosol (F1) using an anti-clathrin light chain (CON.1). No immunoprecipitation of clathrin or TACC3 was seen with a control antibody (anti-myc). Western blots were probed with anti-TACC3 (rabbit polyclonal) or anti-clathrin heavy chain (CHC, TD.1). (C) Representative fluorescence micrographs of rhodamine-labeled MTs incubated with beads from the immunoprecipitation shown in B. Significant bundling was seen for clathrin IPs from F5-7 only. No bundling activity was seen with control IPs or clathrin IPs from F1. Beads are out of frame for clarity. Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.07635.014
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fig5s2: MT bundling using mesh complex immunoisolated from mitotic HeLa cells.(A) Schematic diagram of the procedure to release mitotic spindle proteins from mitotic spindles at metaphase (described in Booth et al., 2011). (B) Western blot to show specific co-immunoprecipitation of TACC3/clathrin from spindle fractions (F5-7) but not from mitotic cytosol (F1) using an anti-clathrin light chain (CON.1). No immunoprecipitation of clathrin or TACC3 was seen with a control antibody (anti-myc). Western blots were probed with anti-TACC3 (rabbit polyclonal) or anti-clathrin heavy chain (CHC, TD.1). (C) Representative fluorescence micrographs of rhodamine-labeled MTs incubated with beads from the immunoprecipitation shown in B. Significant bundling was seen for clathrin IPs from F5-7 only. No bundling activity was seen with control IPs or clathrin IPs from F1. Beads are out of frame for clarity. Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.07635.014

Mentions: These MT-bundling experiments were complicated by the purification of several large proteins (Figure 5—figure supplement 1). As an alternative source, we immunoprecipitated protein complexes, which contained clathrin and TACC3 directly from mitotic spindle of HeLa cells at metaphase (Figure 5—figure supplement 2A,B). We observed bundling of Taxol-stabilized MTs in vitro, using this complex specifically (Figure 5—figure supplement 2C).


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)

MT bundling using mesh complex immunoisolated from mitotic HeLa cells.(A) Schematic diagram of the procedure to release mitotic spindle proteins from mitotic spindles at metaphase (described in Booth et al., 2011). (B) Western blot to show specific co-immunoprecipitation of TACC3/clathrin from spindle fractions (F5-7) but not from mitotic cytosol (F1) using an anti-clathrin light chain (CON.1). No immunoprecipitation of clathrin or TACC3 was seen with a control antibody (anti-myc). Western blots were probed with anti-TACC3 (rabbit polyclonal) or anti-clathrin heavy chain (CHC, TD.1). (C) Representative fluorescence micrographs of rhodamine-labeled MTs incubated with beads from the immunoprecipitation shown in B. Significant bundling was seen for clathrin IPs from F5-7 only. No bundling activity was seen with control IPs or clathrin IPs from F1. Beads are out of frame for clarity. Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.07635.014
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4495718&req=5

fig5s2: MT bundling using mesh complex immunoisolated from mitotic HeLa cells.(A) Schematic diagram of the procedure to release mitotic spindle proteins from mitotic spindles at metaphase (described in Booth et al., 2011). (B) Western blot to show specific co-immunoprecipitation of TACC3/clathrin from spindle fractions (F5-7) but not from mitotic cytosol (F1) using an anti-clathrin light chain (CON.1). No immunoprecipitation of clathrin or TACC3 was seen with a control antibody (anti-myc). Western blots were probed with anti-TACC3 (rabbit polyclonal) or anti-clathrin heavy chain (CHC, TD.1). (C) Representative fluorescence micrographs of rhodamine-labeled MTs incubated with beads from the immunoprecipitation shown in B. Significant bundling was seen for clathrin IPs from F5-7 only. No bundling activity was seen with control IPs or clathrin IPs from F1. Beads are out of frame for clarity. Scale bar, 10 µm.DOI:http://dx.doi.org/10.7554/eLife.07635.014
Mentions: These MT-bundling experiments were complicated by the purification of several large proteins (Figure 5—figure supplement 1). As an alternative source, we immunoprecipitated protein complexes, which contained clathrin and TACC3 directly from mitotic spindle of HeLa cells at metaphase (Figure 5—figure supplement 2A,B). We observed bundling of Taxol-stabilized MTs in vitro, using this complex specifically (Figure 5—figure supplement 2C).

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