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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

Chromosome orientation depends on the kinetochore in oocytes.(A) Confocal images of the centromere protein CENP-C (red in merged images, white in single channel) in wild-type oocytes, after knockdown of Ndc80 or Spc105R, and in ord mutants. Single channel images are zoomed in relative to merged to highlight CENP-C foci. In ord mutants, due to defects in cohesion or crossing over, precocious anaphase is observed [80, 81]. DNA is in blue and tubulin is in green in merged images. Scale bars represent 10 μm in merged, 5 μm in CENP-C single channel images. (B) Bar graph showing the ratio of CENP-C foci closer to (dark gray) and further from (light gray) the spindle than 0.2 μM. Inset shows a centromere in red, a microtubule in green, and the distance measurement denoted by “x”. (C) Dot plot of the number of CENP-C foci per oocyte in wild type, after Ndc80 or Spc105R knockdown, in ord mutants, and in mei-S332 mutants (encoding the Drosophila homolog of Shugoshin). Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (D) Dot plot of the angle of CENP-C foci with respect to the spindle axis in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the median. The red bar in the inset shows a line perpendicular to the spindle axis: centromeres located on this line would result in 90 degree angle measurements, while centromeres in line with the spindle axis measure 0 degrees. Differences in the width of the karyosome, in wild-type oocytes (3.13 μm), and after knockdown of Ndc80 (3.43 μm) or Spc105R (3.32 μm), are not great enough to explain the differences in angles of CENP-C foci.
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pgen.1005605.g003: Chromosome orientation depends on the kinetochore in oocytes.(A) Confocal images of the centromere protein CENP-C (red in merged images, white in single channel) in wild-type oocytes, after knockdown of Ndc80 or Spc105R, and in ord mutants. Single channel images are zoomed in relative to merged to highlight CENP-C foci. In ord mutants, due to defects in cohesion or crossing over, precocious anaphase is observed [80, 81]. DNA is in blue and tubulin is in green in merged images. Scale bars represent 10 μm in merged, 5 μm in CENP-C single channel images. (B) Bar graph showing the ratio of CENP-C foci closer to (dark gray) and further from (light gray) the spindle than 0.2 μM. Inset shows a centromere in red, a microtubule in green, and the distance measurement denoted by “x”. (C) Dot plot of the number of CENP-C foci per oocyte in wild type, after Ndc80 or Spc105R knockdown, in ord mutants, and in mei-S332 mutants (encoding the Drosophila homolog of Shugoshin). Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (D) Dot plot of the angle of CENP-C foci with respect to the spindle axis in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the median. The red bar in the inset shows a line perpendicular to the spindle axis: centromeres located on this line would result in 90 degree angle measurements, while centromeres in line with the spindle axis measure 0 degrees. Differences in the width of the karyosome, in wild-type oocytes (3.13 μm), and after knockdown of Ndc80 (3.43 μm) or Spc105R (3.32 μm), are not great enough to explain the differences in angles of CENP-C foci.

Mentions: To confirm that SPC105R, but not NDC80, is required for kinetochore-microtubule interactions, we measured the distance between each centromere and the nearest microtubules in oocytes not treated with colchicine. In wild type, the majority of centromeres were within 0.2 μm of the microtubules (Fig 3A and 3B). A similar frequency was found with loss of NDC80, suggesting these defective kinetochores still interacted with the microtubules (Fig 3A and 3B, P = 0.07). Oocytes lacking SPC105R, however, had significantly fewer centromeres within 0.2 μm of the microtubules (Fig 3A and 3B, P = <0.0001). Based on the results with colchicine, it is likely that centromeres lacking SPC105R move within 0.2 μm of a microtubule by chance. These results suggest SPC105R, but not NDC80, is required for the kinetochores to attach to the microtubules.


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)

Chromosome orientation depends on the kinetochore in oocytes.(A) Confocal images of the centromere protein CENP-C (red in merged images, white in single channel) in wild-type oocytes, after knockdown of Ndc80 or Spc105R, and in ord mutants. Single channel images are zoomed in relative to merged to highlight CENP-C foci. In ord mutants, due to defects in cohesion or crossing over, precocious anaphase is observed [80, 81]. DNA is in blue and tubulin is in green in merged images. Scale bars represent 10 μm in merged, 5 μm in CENP-C single channel images. (B) Bar graph showing the ratio of CENP-C foci closer to (dark gray) and further from (light gray) the spindle than 0.2 μM. Inset shows a centromere in red, a microtubule in green, and the distance measurement denoted by “x”. (C) Dot plot of the number of CENP-C foci per oocyte in wild type, after Ndc80 or Spc105R knockdown, in ord mutants, and in mei-S332 mutants (encoding the Drosophila homolog of Shugoshin). Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (D) Dot plot of the angle of CENP-C foci with respect to the spindle axis in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the median. The red bar in the inset shows a line perpendicular to the spindle axis: centromeres located on this line would result in 90 degree angle measurements, while centromeres in line with the spindle axis measure 0 degrees. Differences in the width of the karyosome, in wild-type oocytes (3.13 μm), and after knockdown of Ndc80 (3.43 μm) or Spc105R (3.32 μm), are not great enough to explain the differences in angles of CENP-C foci.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005605.g003: Chromosome orientation depends on the kinetochore in oocytes.(A) Confocal images of the centromere protein CENP-C (red in merged images, white in single channel) in wild-type oocytes, after knockdown of Ndc80 or Spc105R, and in ord mutants. Single channel images are zoomed in relative to merged to highlight CENP-C foci. In ord mutants, due to defects in cohesion or crossing over, precocious anaphase is observed [80, 81]. DNA is in blue and tubulin is in green in merged images. Scale bars represent 10 μm in merged, 5 μm in CENP-C single channel images. (B) Bar graph showing the ratio of CENP-C foci closer to (dark gray) and further from (light gray) the spindle than 0.2 μM. Inset shows a centromere in red, a microtubule in green, and the distance measurement denoted by “x”. (C) Dot plot of the number of CENP-C foci per oocyte in wild type, after Ndc80 or Spc105R knockdown, in ord mutants, and in mei-S332 mutants (encoding the Drosophila homolog of Shugoshin). Horizontal dotted lines show the mean, error bars show 95% confidence intervals. (D) Dot plot of the angle of CENP-C foci with respect to the spindle axis in wild-type oocytes and after knockdown of Ndc80 or Spc105R. Horizontal dotted lines show the median. The red bar in the inset shows a line perpendicular to the spindle axis: centromeres located on this line would result in 90 degree angle measurements, while centromeres in line with the spindle axis measure 0 degrees. Differences in the width of the karyosome, in wild-type oocytes (3.13 μm), and after knockdown of Ndc80 (3.43 μm) or Spc105R (3.32 μm), are not great enough to explain the differences in angles of CENP-C foci.
Mentions: To confirm that SPC105R, but not NDC80, is required for kinetochore-microtubule interactions, we measured the distance between each centromere and the nearest microtubules in oocytes not treated with colchicine. In wild type, the majority of centromeres were within 0.2 μm of the microtubules (Fig 3A and 3B). A similar frequency was found with loss of NDC80, suggesting these defective kinetochores still interacted with the microtubules (Fig 3A and 3B, P = 0.07). Oocytes lacking SPC105R, however, had significantly fewer centromeres within 0.2 μm of the microtubules (Fig 3A and 3B, P = <0.0001). Based on the results with colchicine, it is likely that centromeres lacking SPC105R move within 0.2 μm of a microtubule by chance. These results suggest SPC105R, but not NDC80, is required for the kinetochores to attach to the microtubules.

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