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Functional analysis of kinetochore assembly in Caenorhabditis elegans.

Oegema K, Desai A, Rybina S, Kirkham M, Hyman AA - J. Cell Biol. (2001)

Bottom Line: Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore " phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle.Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent.These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. oegema@mpi-cbg.de

ABSTRACT
In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore " phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

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Polarity-cued forces begin moving the spindle towards the embryo posterior before anaphase onset. (A) Stills from a video of a C. elegans embryo expressing GFP-histone to mark the DNA and GFP–γ-tubulin to mark the spindle poles. The embryo anterior (Ant) is on the top left and the embryo posterior (Post) on the lower right. Time in seconds on the upper right of each panel is relative to anaphase onset. For reference, the position of the spindle center (arrow) and the two spindle poles (arrowheads) at the first time point (−64s) are marked on the subsequent three panels. (B) The distance between the spindle center and the embryo posterior (expressed as a fraction of egg length) is plotted. About half of the posterior movement of the spindle occurs before anaphase onset. (C) Chromosome segregation during the first mitotic division of the C. elegans embryo is primarily due to anaphase B. Chromosome-to-pole distance (black triangles), change in chromosome separation (medium gray circles), and change in pole-to-pole distance (light grey squares) are plotted relative to anaphase onset. Chromosome separation is the distance between the polar edges of the separating chromosomes measured along the pole–pole axis. Changes in chromosome separation and pole-to-pole distance were obtained by subtracting the relevant values 4 s before anaphase onset. Online supplemental videos are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. Bar, 5 μm.
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Figure 6: Polarity-cued forces begin moving the spindle towards the embryo posterior before anaphase onset. (A) Stills from a video of a C. elegans embryo expressing GFP-histone to mark the DNA and GFP–γ-tubulin to mark the spindle poles. The embryo anterior (Ant) is on the top left and the embryo posterior (Post) on the lower right. Time in seconds on the upper right of each panel is relative to anaphase onset. For reference, the position of the spindle center (arrow) and the two spindle poles (arrowheads) at the first time point (−64s) are marked on the subsequent three panels. (B) The distance between the spindle center and the embryo posterior (expressed as a fraction of egg length) is plotted. About half of the posterior movement of the spindle occurs before anaphase onset. (C) Chromosome segregation during the first mitotic division of the C. elegans embryo is primarily due to anaphase B. Chromosome-to-pole distance (black triangles), change in chromosome separation (medium gray circles), and change in pole-to-pole distance (light grey squares) are plotted relative to anaphase onset. Chromosome separation is the distance between the polar edges of the separating chromosomes measured along the pole–pole axis. Changes in chromosome separation and pole-to-pole distance were obtained by subtracting the relevant values 4 s before anaphase onset. Online supplemental videos are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. Bar, 5 μm.

Mentions: Quicktime™ videos associated with Fig. 2, Fig. 6, Fig. 7, and Fig. 9A and Fig. B are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. In the videos with Fig. 2 (Videos 1–3), a strain expressing GFP-histone H2B was used to visualize chromosome segregation during the first mitotic cell division. Videos are provided for wild-type, CeCENP-A–depleted and CeCENP-C–depleted embryos. The video with Fig. 6 (Video 4) shows an embryo expressing both GFP–γ-tubulin and GFP-histone H2B during the first cell division. Such videos were used to precisely track spindle position within the embryo relative to anaphase onset. In the videos with Fig. 7 (Videos 5–7), the MT cytoskeleton in embryos expressing GFP–α-tubulin was visualized using spinning disk confocal microscopy. Spindle assembly and dynamics were followed in wild-type, CeCENP-A–depleted, and CeCENP-C–depleted embryos. The videos with Fig. 9 A (Videos 8–12) show chromosome segregation in embryos depleted of one of the three chromosomal passengers: CeINCENP, Air-2, or Bir-1. Three different videos of CeINCENP-depleted embryos are shown, one of which (Video 9) provides a fortuitously clear view of the dynamics of sperm-derived paternal chromosomes. Finally, the video associated with Fig. 9 B (Video 13) shows spindle assembly and dynamics in a CeINCENP-depleted embryo.


Functional analysis of kinetochore assembly in Caenorhabditis elegans.

Oegema K, Desai A, Rybina S, Kirkham M, Hyman AA - J. Cell Biol. (2001)

Polarity-cued forces begin moving the spindle towards the embryo posterior before anaphase onset. (A) Stills from a video of a C. elegans embryo expressing GFP-histone to mark the DNA and GFP–γ-tubulin to mark the spindle poles. The embryo anterior (Ant) is on the top left and the embryo posterior (Post) on the lower right. Time in seconds on the upper right of each panel is relative to anaphase onset. For reference, the position of the spindle center (arrow) and the two spindle poles (arrowheads) at the first time point (−64s) are marked on the subsequent three panels. (B) The distance between the spindle center and the embryo posterior (expressed as a fraction of egg length) is plotted. About half of the posterior movement of the spindle occurs before anaphase onset. (C) Chromosome segregation during the first mitotic division of the C. elegans embryo is primarily due to anaphase B. Chromosome-to-pole distance (black triangles), change in chromosome separation (medium gray circles), and change in pole-to-pole distance (light grey squares) are plotted relative to anaphase onset. Chromosome separation is the distance between the polar edges of the separating chromosomes measured along the pole–pole axis. Changes in chromosome separation and pole-to-pole distance were obtained by subtracting the relevant values 4 s before anaphase onset. Online supplemental videos are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192036&req=5

Figure 6: Polarity-cued forces begin moving the spindle towards the embryo posterior before anaphase onset. (A) Stills from a video of a C. elegans embryo expressing GFP-histone to mark the DNA and GFP–γ-tubulin to mark the spindle poles. The embryo anterior (Ant) is on the top left and the embryo posterior (Post) on the lower right. Time in seconds on the upper right of each panel is relative to anaphase onset. For reference, the position of the spindle center (arrow) and the two spindle poles (arrowheads) at the first time point (−64s) are marked on the subsequent three panels. (B) The distance between the spindle center and the embryo posterior (expressed as a fraction of egg length) is plotted. About half of the posterior movement of the spindle occurs before anaphase onset. (C) Chromosome segregation during the first mitotic division of the C. elegans embryo is primarily due to anaphase B. Chromosome-to-pole distance (black triangles), change in chromosome separation (medium gray circles), and change in pole-to-pole distance (light grey squares) are plotted relative to anaphase onset. Chromosome separation is the distance between the polar edges of the separating chromosomes measured along the pole–pole axis. Changes in chromosome separation and pole-to-pole distance were obtained by subtracting the relevant values 4 s before anaphase onset. Online supplemental videos are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. Bar, 5 μm.
Mentions: Quicktime™ videos associated with Fig. 2, Fig. 6, Fig. 7, and Fig. 9A and Fig. B are available at http://www.jcb.org/cgi/content/full/153/6/1209/DC1. In the videos with Fig. 2 (Videos 1–3), a strain expressing GFP-histone H2B was used to visualize chromosome segregation during the first mitotic cell division. Videos are provided for wild-type, CeCENP-A–depleted and CeCENP-C–depleted embryos. The video with Fig. 6 (Video 4) shows an embryo expressing both GFP–γ-tubulin and GFP-histone H2B during the first cell division. Such videos were used to precisely track spindle position within the embryo relative to anaphase onset. In the videos with Fig. 7 (Videos 5–7), the MT cytoskeleton in embryos expressing GFP–α-tubulin was visualized using spinning disk confocal microscopy. Spindle assembly and dynamics were followed in wild-type, CeCENP-A–depleted, and CeCENP-C–depleted embryos. The videos with Fig. 9 A (Videos 8–12) show chromosome segregation in embryos depleted of one of the three chromosomal passengers: CeINCENP, Air-2, or Bir-1. Three different videos of CeINCENP-depleted embryos are shown, one of which (Video 9) provides a fortuitously clear view of the dynamics of sperm-derived paternal chromosomes. Finally, the video associated with Fig. 9 B (Video 13) shows spindle assembly and dynamics in a CeINCENP-depleted embryo.

Bottom Line: Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore " phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle.Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent.These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany. oegema@mpi-cbg.de

ABSTRACT
In all eukaryotes, segregation of mitotic chromosomes requires their interaction with spindle microtubules. To dissect this interaction, we use live and fixed assays in the one-cell stage Caenorhabditis elegans embryo. We compare the consequences of depleting homologues of the centromeric histone CENP-A, the kinetochore structural component CENP-C, and the chromosomal passenger protein INCENP. Depletion of either CeCENP-A or CeCENP-C results in an identical "kinetochore " phenotype, characterized by complete failure of mitotic chromosome segregation as well as failure to recruit other kinetochore components and to assemble a mechanically stable spindle. The similarity of their depletion phenotypes, combined with a requirement for CeCENP-A to localize CeCENP-C but not vice versa, suggest that a key step in kinetochore assembly is the recruitment of CENP-C by CENP-A-containing chromatin. Parallel analysis of CeINCENP-depleted embryos revealed mitotic chromosome segregation defects different from those observed in the absence of CeCENP-A/C. Defects are observed before and during anaphase, but the chromatin separates into two equivalently sized masses. Mechanically stable spindles assemble that show defects later in anaphase and telophase. Furthermore, kinetochore assembly and the recruitment of CeINCENP to chromosomes are independent. These results suggest distinct roles for the kinetochore and the chromosomal passengers in mitotic chromosome segregation.

Show MeSH
Related in: MedlinePlus