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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

Analysis of MT trajectories within a K-fiber.(A) Representative ‘side views’ of rendered MTs in single tomograms of individual K-fibers from control (left, red, Video 2) or TACC3 OE (right, blue, Video 3) cells. Tomogram thickness, 66.4 nm (Control) trimmed to 34.2 nm, 34.2 nm (TACC3 OE). For scale, MTs are 25 nm in diameter. (B) Aide memoire of the spherical coordinate system. Trajectories of MTs (green line) in K-fibers were defined and normalized via Euler's rotation (see ‘Materials and methods’) such that the overall trajectory of all fibers pointed to the zenith. Measurements of polar angle (θ) and azimuthal angle (φ) were made from the normalized sets and are presented in C–E. (C) Violin plots of the polar angles for MTs in K-fibers from control and TACC3 overexpressing cells. Box plots show the median, 75th and 25th percentile, and whiskers show the minimum and maximum. Violins show a kernel density estimate of the data and are trimmed to the minima and maxima. Comparison of the median MT angle from each K-fiber, Wilcoxon Rank Sum Test, p = 0.007, Nfiber = 12–15. (D) Illustration of the deviation of MTs from the fiber axis caused by TACC3 overexpression. Cartesian coordinates of the intersection of individual MT vectors that start from a common origin (0, 0, 0), with an x-y plane at z = 100 nm. To account for the difference in number of MTs, the insets show a normalized 2D histogram of these coordinates cropped to a 40 × 40 nm square centered at 0, 0, 100. (E) Plots of azimuthal angle (above) and polar angle (below) as a function of distance from the fiber center (defined as described in the ‘Materials and methods’). Line of best fit is shown with 95% confidence bands (dashed line), r2 = 0.07 (control), and 0.007 (TACC3 overexpression).DOI:http://dx.doi.org/10.7554/eLife.07635.015
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fig6: Analysis of MT trajectories within a K-fiber.(A) Representative ‘side views’ of rendered MTs in single tomograms of individual K-fibers from control (left, red, Video 2) or TACC3 OE (right, blue, Video 3) cells. Tomogram thickness, 66.4 nm (Control) trimmed to 34.2 nm, 34.2 nm (TACC3 OE). For scale, MTs are 25 nm in diameter. (B) Aide memoire of the spherical coordinate system. Trajectories of MTs (green line) in K-fibers were defined and normalized via Euler's rotation (see ‘Materials and methods’) such that the overall trajectory of all fibers pointed to the zenith. Measurements of polar angle (θ) and azimuthal angle (φ) were made from the normalized sets and are presented in C–E. (C) Violin plots of the polar angles for MTs in K-fibers from control and TACC3 overexpressing cells. Box plots show the median, 75th and 25th percentile, and whiskers show the minimum and maximum. Violins show a kernel density estimate of the data and are trimmed to the minima and maxima. Comparison of the median MT angle from each K-fiber, Wilcoxon Rank Sum Test, p = 0.007, Nfiber = 12–15. (D) Illustration of the deviation of MTs from the fiber axis caused by TACC3 overexpression. Cartesian coordinates of the intersection of individual MT vectors that start from a common origin (0, 0, 0), with an x-y plane at z = 100 nm. To account for the difference in number of MTs, the insets show a normalized 2D histogram of these coordinates cropped to a 40 × 40 nm square centered at 0, 0, 100. (E) Plots of azimuthal angle (above) and polar angle (below) as a function of distance from the fiber center (defined as described in the ‘Materials and methods’). Line of best fit is shown with 95% confidence bands (dashed line), r2 = 0.07 (control), and 0.007 (TACC3 overexpression).DOI:http://dx.doi.org/10.7554/eLife.07635.015

Mentions: To look further for any evidence of a role for the mesh in MT positioning, we examined the trajectories of MTs within each fiber. K-fibers are bundles of parallel MTs, and we sought to characterize how parallel the MTs are and to test if this is altered by TACC3 overexpression. Deviations from parallel would suggest that the mesh tethers MTs closer together and interferes with their trajectory. Qualitatively, we could see that MTs in TACC3 overexpressing K-fibers were more disorganized (Figure 6A, Video 2 and Video 3). To measure parallelism more rigorously and to allow comparison of multiple K-fibers, we used several computational approaches (described in ‘Materials and methods’), which allowed us to normalize the 3D trajectory of each fiber and then examine the deviation of MT trajectories from this vector. K-fibers in cells overexpressing TACC3 had a higher proportion of MTs that deviate from parallel. They frequently had significantly larger polar angles compared to MTs in K-fibers from control cells (Figure 6B,C). The deviation of MTs from a completely parallel condition is easier to visualize, by examining the Cartesian intersection coordinates of each MT vector with an x-y plane at a given distance (z) from a common origin (see ‘Materials and methods’). These scatter plots show that MTs in TACC3 overexpressing cells deviate further from parallel than those in control cells (Figure 6D). The fraction of MT vectors intersecting this plane at radial distances of <10 nm from the center is 0.39 and 0.18 in control and TACC3 overexpressing cells, respectively. In other words, there are half as many parallel MTs after TACC3 overexpression.10.7554/eLife.07635.015Figure 6.Analysis of MT trajectories 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)

Analysis of MT trajectories within a K-fiber.(A) Representative ‘side views’ of rendered MTs in single tomograms of individual K-fibers from control (left, red, Video 2) or TACC3 OE (right, blue, Video 3) cells. Tomogram thickness, 66.4 nm (Control) trimmed to 34.2 nm, 34.2 nm (TACC3 OE). For scale, MTs are 25 nm in diameter. (B) Aide memoire of the spherical coordinate system. Trajectories of MTs (green line) in K-fibers were defined and normalized via Euler's rotation (see ‘Materials and methods’) such that the overall trajectory of all fibers pointed to the zenith. Measurements of polar angle (θ) and azimuthal angle (φ) were made from the normalized sets and are presented in C–E. (C) Violin plots of the polar angles for MTs in K-fibers from control and TACC3 overexpressing cells. Box plots show the median, 75th and 25th percentile, and whiskers show the minimum and maximum. Violins show a kernel density estimate of the data and are trimmed to the minima and maxima. Comparison of the median MT angle from each K-fiber, Wilcoxon Rank Sum Test, p = 0.007, Nfiber = 12–15. (D) Illustration of the deviation of MTs from the fiber axis caused by TACC3 overexpression. Cartesian coordinates of the intersection of individual MT vectors that start from a common origin (0, 0, 0), with an x-y plane at z = 100 nm. To account for the difference in number of MTs, the insets show a normalized 2D histogram of these coordinates cropped to a 40 × 40 nm square centered at 0, 0, 100. (E) Plots of azimuthal angle (above) and polar angle (below) as a function of distance from the fiber center (defined as described in the ‘Materials and methods’). Line of best fit is shown with 95% confidence bands (dashed line), r2 = 0.07 (control), and 0.007 (TACC3 overexpression).DOI:http://dx.doi.org/10.7554/eLife.07635.015
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fig6: Analysis of MT trajectories within a K-fiber.(A) Representative ‘side views’ of rendered MTs in single tomograms of individual K-fibers from control (left, red, Video 2) or TACC3 OE (right, blue, Video 3) cells. Tomogram thickness, 66.4 nm (Control) trimmed to 34.2 nm, 34.2 nm (TACC3 OE). For scale, MTs are 25 nm in diameter. (B) Aide memoire of the spherical coordinate system. Trajectories of MTs (green line) in K-fibers were defined and normalized via Euler's rotation (see ‘Materials and methods’) such that the overall trajectory of all fibers pointed to the zenith. Measurements of polar angle (θ) and azimuthal angle (φ) were made from the normalized sets and are presented in C–E. (C) Violin plots of the polar angles for MTs in K-fibers from control and TACC3 overexpressing cells. Box plots show the median, 75th and 25th percentile, and whiskers show the minimum and maximum. Violins show a kernel density estimate of the data and are trimmed to the minima and maxima. Comparison of the median MT angle from each K-fiber, Wilcoxon Rank Sum Test, p = 0.007, Nfiber = 12–15. (D) Illustration of the deviation of MTs from the fiber axis caused by TACC3 overexpression. Cartesian coordinates of the intersection of individual MT vectors that start from a common origin (0, 0, 0), with an x-y plane at z = 100 nm. To account for the difference in number of MTs, the insets show a normalized 2D histogram of these coordinates cropped to a 40 × 40 nm square centered at 0, 0, 100. (E) Plots of azimuthal angle (above) and polar angle (below) as a function of distance from the fiber center (defined as described in the ‘Materials and methods’). Line of best fit is shown with 95% confidence bands (dashed line), r2 = 0.07 (control), and 0.007 (TACC3 overexpression).DOI:http://dx.doi.org/10.7554/eLife.07635.015
Mentions: To look further for any evidence of a role for the mesh in MT positioning, we examined the trajectories of MTs within each fiber. K-fibers are bundles of parallel MTs, and we sought to characterize how parallel the MTs are and to test if this is altered by TACC3 overexpression. Deviations from parallel would suggest that the mesh tethers MTs closer together and interferes with their trajectory. Qualitatively, we could see that MTs in TACC3 overexpressing K-fibers were more disorganized (Figure 6A, Video 2 and Video 3). To measure parallelism more rigorously and to allow comparison of multiple K-fibers, we used several computational approaches (described in ‘Materials and methods’), which allowed us to normalize the 3D trajectory of each fiber and then examine the deviation of MT trajectories from this vector. K-fibers in cells overexpressing TACC3 had a higher proportion of MTs that deviate from parallel. They frequently had significantly larger polar angles compared to MTs in K-fibers from control cells (Figure 6B,C). The deviation of MTs from a completely parallel condition is easier to visualize, by examining the Cartesian intersection coordinates of each MT vector with an x-y plane at a given distance (z) from a common origin (see ‘Materials and methods’). These scatter plots show that MTs in TACC3 overexpressing cells deviate further from parallel than those in control cells (Figure 6D). The fraction of MT vectors intersecting this plane at radial distances of <10 nm from the center is 0.39 and 0.18 in control and TACC3 overexpressing cells, respectively. In other words, there are half as many parallel MTs after TACC3 overexpression.10.7554/eLife.07635.015Figure 6.Analysis of MT trajectories 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