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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

Prometaphase spindle stability depends on both kinetochore and central spindle components in oocytes.In all images, DNA is in blue, tubulin is in green, and the scale bars represent 10 μm. (A,B) Confocal images of wild-type oocytes and after Spc105R knockdown from prometaphase-enriched (A) and metaphase-enriched (B) collections. The CPC component INCENP is in red in merged images, white in single channel images. (C) Confocal images of sub-depleted and sub Spc105R double-depleted oocytes. For sub-depleted oocytes, a tripolar (left) and bipolar (right) spindle are shown. Monopolar spindles were also observed [9, 47]. In all sub-depleted oocytes, the prominent central spindle is missing. For sub Spc105R double-depleted oocytes, the absence of a spindle (left) and a spindle with thin and disorganized microtubules (right) are shown. (D) Graph showing the percentage of weak/absent spindles during prometaphase in wild-type oocytes (n = 171) and after Spc105R (n = 86), sub (n = 23), or sub Spc105R (n = 24) depletion, and metaphase in wild-type oocytes (n = 110) and after Spc105R depletion (n = 42). The frequency of weak/absent spindles at metaphase in sub- and sub Spc105R-depleted oocytes was not determined. Error bars show 95% confidence intervals.
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pgen.1005605.g006: Prometaphase spindle stability depends on both kinetochore and central spindle components in oocytes.In all images, DNA is in blue, tubulin is in green, and the scale bars represent 10 μm. (A,B) Confocal images of wild-type oocytes and after Spc105R knockdown from prometaphase-enriched (A) and metaphase-enriched (B) collections. The CPC component INCENP is in red in merged images, white in single channel images. (C) Confocal images of sub-depleted and sub Spc105R double-depleted oocytes. For sub-depleted oocytes, a tripolar (left) and bipolar (right) spindle are shown. Monopolar spindles were also observed [9, 47]. In all sub-depleted oocytes, the prominent central spindle is missing. For sub Spc105R double-depleted oocytes, the absence of a spindle (left) and a spindle with thin and disorganized microtubules (right) are shown. (D) Graph showing the percentage of weak/absent spindles during prometaphase in wild-type oocytes (n = 171) and after Spc105R (n = 86), sub (n = 23), or sub Spc105R (n = 24) depletion, and metaphase in wild-type oocytes (n = 110) and after Spc105R depletion (n = 42). The frequency of weak/absent spindles at metaphase in sub- and sub Spc105R-depleted oocytes was not determined. Error bars show 95% confidence intervals.

Mentions: Our results thus far show that kinetochores participate in chromosome alignment, bi-orientation, and co-orientation in Drosophila oocytes. Since chromatin-mediated pathways direct spindle assembly in oocytes [45], we investigated the contribution of the kinetochores to spindle assembly and stability at prometaphase I and metaphase I. Metaphase I-arrested spindles tend to be shorter than prometaphase spindles with less prominent central spindles (Fig 6A and 6B). In fact, we observed that spindles were weak, that is very small, faint, or lacking microtubules entirely (indicated below as “weak/absent”), more frequently in metaphase-enriched oocyte samples (33%, P = <0.0001) (see Materials and Methods for details of how metaphase- or prometaphase-enriched samples are collected) than in prometaphase-enriched oocyte samples (11%) (Fig 6D). This difference suggests that in Drosophila oocytes, spindle assembly proceeds via an elongation phase during which spindles are robust (prometaphase), followed by a contraction phase in which microtubule density decreases (metaphase).


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)

Prometaphase spindle stability depends on both kinetochore and central spindle components in oocytes.In all images, DNA is in blue, tubulin is in green, and the scale bars represent 10 μm. (A,B) Confocal images of wild-type oocytes and after Spc105R knockdown from prometaphase-enriched (A) and metaphase-enriched (B) collections. The CPC component INCENP is in red in merged images, white in single channel images. (C) Confocal images of sub-depleted and sub Spc105R double-depleted oocytes. For sub-depleted oocytes, a tripolar (left) and bipolar (right) spindle are shown. Monopolar spindles were also observed [9, 47]. In all sub-depleted oocytes, the prominent central spindle is missing. For sub Spc105R double-depleted oocytes, the absence of a spindle (left) and a spindle with thin and disorganized microtubules (right) are shown. (D) Graph showing the percentage of weak/absent spindles during prometaphase in wild-type oocytes (n = 171) and after Spc105R (n = 86), sub (n = 23), or sub Spc105R (n = 24) depletion, and metaphase in wild-type oocytes (n = 110) and after Spc105R depletion (n = 42). The frequency of weak/absent spindles at metaphase in sub- and sub Spc105R-depleted oocytes was not determined. Error bars show 95% confidence intervals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608789&req=5

pgen.1005605.g006: Prometaphase spindle stability depends on both kinetochore and central spindle components in oocytes.In all images, DNA is in blue, tubulin is in green, and the scale bars represent 10 μm. (A,B) Confocal images of wild-type oocytes and after Spc105R knockdown from prometaphase-enriched (A) and metaphase-enriched (B) collections. The CPC component INCENP is in red in merged images, white in single channel images. (C) Confocal images of sub-depleted and sub Spc105R double-depleted oocytes. For sub-depleted oocytes, a tripolar (left) and bipolar (right) spindle are shown. Monopolar spindles were also observed [9, 47]. In all sub-depleted oocytes, the prominent central spindle is missing. For sub Spc105R double-depleted oocytes, the absence of a spindle (left) and a spindle with thin and disorganized microtubules (right) are shown. (D) Graph showing the percentage of weak/absent spindles during prometaphase in wild-type oocytes (n = 171) and after Spc105R (n = 86), sub (n = 23), or sub Spc105R (n = 24) depletion, and metaphase in wild-type oocytes (n = 110) and after Spc105R depletion (n = 42). The frequency of weak/absent spindles at metaphase in sub- and sub Spc105R-depleted oocytes was not determined. Error bars show 95% confidence intervals.
Mentions: Our results thus far show that kinetochores participate in chromosome alignment, bi-orientation, and co-orientation in Drosophila oocytes. Since chromatin-mediated pathways direct spindle assembly in oocytes [45], we investigated the contribution of the kinetochores to spindle assembly and stability at prometaphase I and metaphase I. Metaphase I-arrested spindles tend to be shorter than prometaphase spindles with less prominent central spindles (Fig 6A and 6B). In fact, we observed that spindles were weak, that is very small, faint, or lacking microtubules entirely (indicated below as “weak/absent”), more frequently in metaphase-enriched oocyte samples (33%, P = <0.0001) (see Materials and Methods for details of how metaphase- or prometaphase-enriched samples are collected) than in prometaphase-enriched oocyte samples (11%) (Fig 6D). This difference suggests that in Drosophila oocytes, spindle assembly proceeds via an elongation phase during which spindles are robust (prometaphase), followed by a contraction phase in which microtubule density decreases (metaphase).

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