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Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen C, Murata M, Chan SW - PLoS Genet. (2011)

Bottom Line: Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced.These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells.Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

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Depletion of GFP-tailswap protein from meiotic kinetochores causes removal of MIS12.GFP-CENH3, GFP-tailswap and MIS12 proteins were immunolocalized in anthers during the pachytene stage of meiosis with anti-GFP and anti-MIS12 antibodies. Somatic cells from the same anther are shown as a control. GFP-CENH3 and MIS12 were visualized at both meiotic and somatic kinetochores of GFP-CENH3 plants. In GFP-tailswap plants, GFP-tailswap and MIS12 were both undetectable in GFP-tailswap meiotic kinetochores but can be seen in somatic kinetochores. Scale bars −5 µm.
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pgen-1002121-g007: Depletion of GFP-tailswap protein from meiotic kinetochores causes removal of MIS12.GFP-CENH3, GFP-tailswap and MIS12 proteins were immunolocalized in anthers during the pachytene stage of meiosis with anti-GFP and anti-MIS12 antibodies. Somatic cells from the same anther are shown as a control. GFP-CENH3 and MIS12 were visualized at both meiotic and somatic kinetochores of GFP-CENH3 plants. In GFP-tailswap plants, GFP-tailswap and MIS12 were both undetectable in GFP-tailswap meiotic kinetochores but can be seen in somatic kinetochores. Scale bars −5 µm.

Mentions: CENH3 is required to recruit a large number of essential kinetochore proteins in other organisms. To further characterize the effects of the GFP-tailswap variant on kinetochore assembly, we performed immunostaining on GFP-tailswap and control GFP-CENH3 anther squashes with antibodies raised against the A. thaliana kinetochore proteins CENP-C and MIS12 [27], [28]. CENP-C antibodies did not yield specific staining of kinetochores in meiocytes from either GFP-CENH3 or GFP-tailswap plants. However, MIS12 staining was observed at kinetochores in GFP-CENH3 meiocytes (n = 44), but not in GFP-tailswap meiocytes (n = 33) (Figure 7C and 7K). Somatic cells from both GFP-CENH3 and GFP-tailswap plants showed kinetochore localization of MIS12 (Figure 7G and 7O). Although MIS12 may be recruited in a CENH3-independent way in human cells [20], our results show that loss of A. thaliana CENH3 in meiosis also depletes MIS12 from the kinetochore. As MIS12 is a component of the KMN network that connects kinetochores to spindle microtubules, we predict that this will compromise kinetochore-microtubule attachment [29]. Furthermore, MIS12 is important for mono-orientation during meiosis in maize [4]. In summary, severe depletion of the GFP-tailswap protein during meiosis and downstream effects on kinetochore assembly can explain the chromosome segregation defects observed in the mutant.


Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Ravi M, Shibata F, Ramahi JS, Nagaki K, Chen C, Murata M, Chan SW - PLoS Genet. (2011)

Depletion of GFP-tailswap protein from meiotic kinetochores causes removal of MIS12.GFP-CENH3, GFP-tailswap and MIS12 proteins were immunolocalized in anthers during the pachytene stage of meiosis with anti-GFP and anti-MIS12 antibodies. Somatic cells from the same anther are shown as a control. GFP-CENH3 and MIS12 were visualized at both meiotic and somatic kinetochores of GFP-CENH3 plants. In GFP-tailswap plants, GFP-tailswap and MIS12 were both undetectable in GFP-tailswap meiotic kinetochores but can be seen in somatic kinetochores. Scale bars −5 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111537&req=5

pgen-1002121-g007: Depletion of GFP-tailswap protein from meiotic kinetochores causes removal of MIS12.GFP-CENH3, GFP-tailswap and MIS12 proteins were immunolocalized in anthers during the pachytene stage of meiosis with anti-GFP and anti-MIS12 antibodies. Somatic cells from the same anther are shown as a control. GFP-CENH3 and MIS12 were visualized at both meiotic and somatic kinetochores of GFP-CENH3 plants. In GFP-tailswap plants, GFP-tailswap and MIS12 were both undetectable in GFP-tailswap meiotic kinetochores but can be seen in somatic kinetochores. Scale bars −5 µm.
Mentions: CENH3 is required to recruit a large number of essential kinetochore proteins in other organisms. To further characterize the effects of the GFP-tailswap variant on kinetochore assembly, we performed immunostaining on GFP-tailswap and control GFP-CENH3 anther squashes with antibodies raised against the A. thaliana kinetochore proteins CENP-C and MIS12 [27], [28]. CENP-C antibodies did not yield specific staining of kinetochores in meiocytes from either GFP-CENH3 or GFP-tailswap plants. However, MIS12 staining was observed at kinetochores in GFP-CENH3 meiocytes (n = 44), but not in GFP-tailswap meiocytes (n = 33) (Figure 7C and 7K). Somatic cells from both GFP-CENH3 and GFP-tailswap plants showed kinetochore localization of MIS12 (Figure 7G and 7O). Although MIS12 may be recruited in a CENH3-independent way in human cells [20], our results show that loss of A. thaliana CENH3 in meiosis also depletes MIS12 from the kinetochore. As MIS12 is a component of the KMN network that connects kinetochores to spindle microtubules, we predict that this will compromise kinetochore-microtubule attachment [29]. Furthermore, MIS12 is important for mono-orientation during meiosis in maize [4]. In summary, severe depletion of the GFP-tailswap protein during meiosis and downstream effects on kinetochore assembly can explain the chromosome segregation defects observed in the mutant.

Bottom Line: Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced.These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells.Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

Show MeSH
Related in: MedlinePlus