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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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CeCENP-C localization to chromosomes requires CeCENP-A but not vice versa. Wild-type, CeCENP-A–, or CeCENP-C–depleted embryos were fixed and stained to visualize DNA (cyan), MTs (red), CeCENP-A, and CeCENP-C. Metaphase embryos (A) and higher magnification views of prophase/prometaphase nuclei (B) are shown. RNAi of either component reduces it to levels undetectable by immunofluorescence. Persistence of the spindle pole staining in CeCENP-A–depleted embryos indicates that this staining is nonspecific (see also Fig. 10 C). In the absence of CeCENP-A (A and B, middle rows), CeCENP-C is visible in nuclei during prophase, but is not observed associated with chromosomes at any point during mitosis. In contrast, in the absence of CeCENP-C, CeCENP-A associates with chromosomes throughout mitosis (A and B, bottom rows). All images are projections of three-dimensional stacks. Bars, 5 μm.
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Figure 3: CeCENP-C localization to chromosomes requires CeCENP-A but not vice versa. Wild-type, CeCENP-A–, or CeCENP-C–depleted embryos were fixed and stained to visualize DNA (cyan), MTs (red), CeCENP-A, and CeCENP-C. Metaphase embryos (A) and higher magnification views of prophase/prometaphase nuclei (B) are shown. RNAi of either component reduces it to levels undetectable by immunofluorescence. Persistence of the spindle pole staining in CeCENP-A–depleted embryos indicates that this staining is nonspecific (see also Fig. 10 C). In the absence of CeCENP-A (A and B, middle rows), CeCENP-C is visible in nuclei during prophase, but is not observed associated with chromosomes at any point during mitosis. In contrast, in the absence of CeCENP-C, CeCENP-A associates with chromosomes throughout mitosis (A and B, bottom rows). All images are projections of three-dimensional stacks. Bars, 5 μm.

Mentions: Next, we filmed embryos depleted of either CeCENP-A or CeCENP-C by RNAi. In C. elegans, dsRNA injected into adult hermaphrodites specifically ablates transcripts with high sequence homology, preventing further production of the protein coded by the targeted gene (Montgomery and Fire 1998). Continued embryo production and protein turnover deplete the maternal cytoplasm of the targeted protein within 20–30 h. We confirmed depletions of CeCENP-A and CeCENP-C to levels undetectable by immunofluorescence (see Fig. 3). Interestingly, depletion of either protein resulted in an essentially identical phenotype (Fig. 2, middle and right). In embryos depleted of either CeCENP-A (n = 15 one-cell embryos) or CeCENP-C (n = 8 one-cell embryos), chromosomes derived from the two pronuclei compact into separate discrete masses and fail to distribute over the spindle equator (Fig. 2/119s). The failure of chromosome distribution is particularly clear in the insets which show end-on views of metaphase spindles. These views are derived from the second division, during which the spindle in the anterior cell often orients perpendicular to the focal plane. At the time when wild-type embryos undergo anaphase, chromosomes in the depleted embryos remain in separate compact masses and no chromosome segregation occurs (Fig. 2, 208 and 210s). Nevertheless, cytokinesis initiates at the same time after NEBD as in wild-type. As a consequence of the cytokinetic furrow and cytoplasmic flows, the two unsegregated DNA masses distribute randomly between the two cells. In most cases, some DNA is trapped in the cleavage furrow (Fig. 2, 600 and 602s). CeCENP-A/C–depleted embryos progress through the cell cycle with wild-type kinetics, consistent with previous work suggesting the absence of a mitotic checkpoint during early divisions of the C. elegans embryo (Gonczy et al. 1999).


Functional analysis of kinetochore assembly in Caenorhabditis elegans.

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

CeCENP-C localization to chromosomes requires CeCENP-A but not vice versa. Wild-type, CeCENP-A–, or CeCENP-C–depleted embryos were fixed and stained to visualize DNA (cyan), MTs (red), CeCENP-A, and CeCENP-C. Metaphase embryos (A) and higher magnification views of prophase/prometaphase nuclei (B) are shown. RNAi of either component reduces it to levels undetectable by immunofluorescence. Persistence of the spindle pole staining in CeCENP-A–depleted embryos indicates that this staining is nonspecific (see also Fig. 10 C). In the absence of CeCENP-A (A and B, middle rows), CeCENP-C is visible in nuclei during prophase, but is not observed associated with chromosomes at any point during mitosis. In contrast, in the absence of CeCENP-C, CeCENP-A associates with chromosomes throughout mitosis (A and B, bottom rows). All images are projections of three-dimensional stacks. Bars, 5 μm.
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Figure 3: CeCENP-C localization to chromosomes requires CeCENP-A but not vice versa. Wild-type, CeCENP-A–, or CeCENP-C–depleted embryos were fixed and stained to visualize DNA (cyan), MTs (red), CeCENP-A, and CeCENP-C. Metaphase embryos (A) and higher magnification views of prophase/prometaphase nuclei (B) are shown. RNAi of either component reduces it to levels undetectable by immunofluorescence. Persistence of the spindle pole staining in CeCENP-A–depleted embryos indicates that this staining is nonspecific (see also Fig. 10 C). In the absence of CeCENP-A (A and B, middle rows), CeCENP-C is visible in nuclei during prophase, but is not observed associated with chromosomes at any point during mitosis. In contrast, in the absence of CeCENP-C, CeCENP-A associates with chromosomes throughout mitosis (A and B, bottom rows). All images are projections of three-dimensional stacks. Bars, 5 μm.
Mentions: Next, we filmed embryos depleted of either CeCENP-A or CeCENP-C by RNAi. In C. elegans, dsRNA injected into adult hermaphrodites specifically ablates transcripts with high sequence homology, preventing further production of the protein coded by the targeted gene (Montgomery and Fire 1998). Continued embryo production and protein turnover deplete the maternal cytoplasm of the targeted protein within 20–30 h. We confirmed depletions of CeCENP-A and CeCENP-C to levels undetectable by immunofluorescence (see Fig. 3). Interestingly, depletion of either protein resulted in an essentially identical phenotype (Fig. 2, middle and right). In embryos depleted of either CeCENP-A (n = 15 one-cell embryos) or CeCENP-C (n = 8 one-cell embryos), chromosomes derived from the two pronuclei compact into separate discrete masses and fail to distribute over the spindle equator (Fig. 2/119s). The failure of chromosome distribution is particularly clear in the insets which show end-on views of metaphase spindles. These views are derived from the second division, during which the spindle in the anterior cell often orients perpendicular to the focal plane. At the time when wild-type embryos undergo anaphase, chromosomes in the depleted embryos remain in separate compact masses and no chromosome segregation occurs (Fig. 2, 208 and 210s). Nevertheless, cytokinesis initiates at the same time after NEBD as in wild-type. As a consequence of the cytokinetic furrow and cytoplasmic flows, the two unsegregated DNA masses distribute randomly between the two cells. In most cases, some DNA is trapped in the cleavage furrow (Fig. 2, 600 and 602s). CeCENP-A/C–depleted embryos progress through the cell cycle with wild-type kinetics, consistent with previous work suggesting the absence of a mitotic checkpoint during early divisions of the C. elegans embryo (Gonczy et al. 1999).

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