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Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.

Radford SJ, Hoang TL, Głuszek AA, Ohkura H, McKim KS - PLoS Genet. (2015)

Bottom Line: We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent.Stabilization of the spindle, however, depends on both central spindle and kinetochore components.We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.

No MeSH data available.


Related in: MedlinePlus

Homologous chromosome bi-orientation depends on kinetochores and CENP-E in oocytes.Confocal images of FISH probes marking the 2nd (red) and 3rd (white) chromosome centromeres. DNA is in blue and tubulin is in green in merged images. Only FISH probes are shown in the panel below each merged image. Scale bars represent 10 μm. (A) Oocytes from wild type and after knockdown of Ndc80 or Spc105R. (B) Dot plot of the distance between pairs of FISH foci in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (C) Oocytes from cana/Df, after knockdown of cmet, and Cenp-E germline clones. The data is summarized in Table 2.
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pgen.1005605.g004: Homologous chromosome bi-orientation depends on kinetochores and CENP-E in oocytes.Confocal images of FISH probes marking the 2nd (red) and 3rd (white) chromosome centromeres. DNA is in blue and tubulin is in green in merged images. Only FISH probes are shown in the panel below each merged image. Scale bars represent 10 μm. (A) Oocytes from wild type and after knockdown of Ndc80 or Spc105R. (B) Dot plot of the distance between pairs of FISH foci in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (C) Oocytes from cana/Df, after knockdown of cmet, and Cenp-E germline clones. The data is summarized in Table 2.

Mentions: Karyosome morphology and immunolocalization of CENP-C in Ndc80- or Spc105R-depleted oocytes suggested kinetochore-microtubule attachments allowed chromosomes to orient towards a spindle pole. To test whether each chromosome associated randomly with a pole, or if homologs oriented towards opposite poles (“bi-orientation”), we used FISH to examine specific chromosomes. Chromosome bi-orientation at meiosis I depends on the establishment of connections between homologous chromosome pairs and opposite spindle poles. When this occurs, tension across the homologous chromosome pair generates an increase in the inter-homolog centromere distance. We used FISH probes to the repetitive sequences present at the centromeres of the second and third chromosomes to determine directly whether the separation of homologous centromeres away from each other depends on kinetochores and their end-on attachment to microtubules in oocytes (Fig 4A). In wild type, we observed an average distance between homologous centromeres of 3.0 μm (Fig 4B). In Ndc80-depleted oocytes, the average distance between homologous centromeres was not significantly reduced (2.7 μm, P = 0.4; Fig 4B), suggesting that end-on kinetochore-microtubule attachments are not required for homologous centromeres to move away from each other. In contrast, in Spc105R-depleted oocytes, the average distance was significantly reduced to 1.8 μm (P = 0.0007; Fig 4B). These results suggest that lateral kinetochore-microtubule attachments are sufficient for homologous centromeres to orient towards a spindle pole and separate from each other in what may be the first step in the bi-orientation process.


Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.

Radford SJ, Hoang TL, Głuszek AA, Ohkura H, McKim KS - PLoS Genet. (2015)

Homologous chromosome bi-orientation depends on kinetochores and CENP-E in oocytes.Confocal images of FISH probes marking the 2nd (red) and 3rd (white) chromosome centromeres. DNA is in blue and tubulin is in green in merged images. Only FISH probes are shown in the panel below each merged image. Scale bars represent 10 μm. (A) Oocytes from wild type and after knockdown of Ndc80 or Spc105R. (B) Dot plot of the distance between pairs of FISH foci in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (C) Oocytes from cana/Df, after knockdown of cmet, and Cenp-E germline clones. The data is summarized in Table 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608789&req=5

pgen.1005605.g004: Homologous chromosome bi-orientation depends on kinetochores and CENP-E in oocytes.Confocal images of FISH probes marking the 2nd (red) and 3rd (white) chromosome centromeres. DNA is in blue and tubulin is in green in merged images. Only FISH probes are shown in the panel below each merged image. Scale bars represent 10 μm. (A) Oocytes from wild type and after knockdown of Ndc80 or Spc105R. (B) Dot plot of the distance between pairs of FISH foci in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (C) Oocytes from cana/Df, after knockdown of cmet, and Cenp-E germline clones. The data is summarized in Table 2.
Mentions: Karyosome morphology and immunolocalization of CENP-C in Ndc80- or Spc105R-depleted oocytes suggested kinetochore-microtubule attachments allowed chromosomes to orient towards a spindle pole. To test whether each chromosome associated randomly with a pole, or if homologs oriented towards opposite poles (“bi-orientation”), we used FISH to examine specific chromosomes. Chromosome bi-orientation at meiosis I depends on the establishment of connections between homologous chromosome pairs and opposite spindle poles. When this occurs, tension across the homologous chromosome pair generates an increase in the inter-homolog centromere distance. We used FISH probes to the repetitive sequences present at the centromeres of the second and third chromosomes to determine directly whether the separation of homologous centromeres away from each other depends on kinetochores and their end-on attachment to microtubules in oocytes (Fig 4A). In wild type, we observed an average distance between homologous centromeres of 3.0 μm (Fig 4B). In Ndc80-depleted oocytes, the average distance between homologous centromeres was not significantly reduced (2.7 μm, P = 0.4; Fig 4B), suggesting that end-on kinetochore-microtubule attachments are not required for homologous centromeres to move away from each other. In contrast, in Spc105R-depleted oocytes, the average distance was significantly reduced to 1.8 μm (P = 0.0007; Fig 4B). These results suggest that lateral kinetochore-microtubule attachments are sufficient for homologous centromeres to orient towards a spindle pole and separate from each other in what may be the first step in the bi-orientation process.

Bottom Line: We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent.Stabilization of the spindle, however, depends on both central spindle and kinetochore components.We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation, but the nature and function of these interactions is not clear. We have examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. We found that the initiation of spindle assembly results from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. We propose that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes.

No MeSH data available.


Related in: MedlinePlus