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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

Loss of NDC80 or SPC105R disrupts interactions between kinetochores and microtubules in oocytes.Confocal images of wild-type oocytes (A,B) and after knockdown of Ndc80 (C,D) or Spc105R (E,F). Oocytes were treated with either ethanol (EtOH) (A,C,E) or colchicine (B,D,F). DNA is shown in blue, tubulin is shown in green, and CENP-C is in red. Scale bars represent 10 μm
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pgen.1005605.g002: Loss of NDC80 or SPC105R disrupts interactions between kinetochores and microtubules in oocytes.Confocal images of wild-type oocytes (A,B) and after knockdown of Ndc80 (C,D) or Spc105R (E,F). Oocytes were treated with either ethanol (EtOH) (A,C,E) or colchicine (B,D,F). DNA is shown in blue, tubulin is shown in green, and CENP-C is in red. Scale bars represent 10 μm

Mentions: To determine if kinetochore-microtubule attachments are affected in oocytes depleted of kinetochore components, we examined microtubule localization relative to the centromere protein, CENP-C. The robust central spindle makes it difficult to directly observe kinetochore microtubules; therefore, we used conditions that depolymerize central spindle microtubules to directly observe kinetochore microtubules. Wild-type oocytes exposed to colchicine (see Materials and Methods for details) resulted in the loss of most spindle microtubules except for those that ended at the centromeres (Fig 2B, 17/18 oocytes). In contrast, in colchicine-treated oocytes lacking NDC80, the microtubules were weaker, and those remaining often appeared to be interacting laterally with the centromeres (Fig 2D, 7/12 oocytes). In colchicine-treated oocytes lacking SPC105R, no end-on kinetochore-microtubule attachments were observed (0/12 oocytes), and we observed some oocytes in which all of the microtubules were eliminated (Fig 2F, 3/12 oocytes). These results suggest that NCD80 is required for end-on kinetochore-microtubule attachments, while all kinetochore-microtubule interactions depend on SCP105R.


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)

Loss of NDC80 or SPC105R disrupts interactions between kinetochores and microtubules in oocytes.Confocal images of wild-type oocytes (A,B) and after knockdown of Ndc80 (C,D) or Spc105R (E,F). Oocytes were treated with either ethanol (EtOH) (A,C,E) or colchicine (B,D,F). DNA is shown in blue, tubulin is shown in green, and CENP-C is in red. Scale bars represent 10 μm
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005605.g002: Loss of NDC80 or SPC105R disrupts interactions between kinetochores and microtubules in oocytes.Confocal images of wild-type oocytes (A,B) and after knockdown of Ndc80 (C,D) or Spc105R (E,F). Oocytes were treated with either ethanol (EtOH) (A,C,E) or colchicine (B,D,F). DNA is shown in blue, tubulin is shown in green, and CENP-C is in red. Scale bars represent 10 μm
Mentions: To determine if kinetochore-microtubule attachments are affected in oocytes depleted of kinetochore components, we examined microtubule localization relative to the centromere protein, CENP-C. The robust central spindle makes it difficult to directly observe kinetochore microtubules; therefore, we used conditions that depolymerize central spindle microtubules to directly observe kinetochore microtubules. Wild-type oocytes exposed to colchicine (see Materials and Methods for details) resulted in the loss of most spindle microtubules except for those that ended at the centromeres (Fig 2B, 17/18 oocytes). In contrast, in colchicine-treated oocytes lacking NDC80, the microtubules were weaker, and those remaining often appeared to be interacting laterally with the centromeres (Fig 2D, 7/12 oocytes). In colchicine-treated oocytes lacking SPC105R, no end-on kinetochore-microtubule attachments were observed (0/12 oocytes), and we observed some oocytes in which all of the microtubules were eliminated (Fig 2F, 3/12 oocytes). These results suggest that NCD80 is required for end-on kinetochore-microtubule attachments, while all kinetochore-microtubule interactions depend on SCP105R.

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