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

Radford, Sarah J.; Hoang, Tranchau L.; Głuszek, A. Agata; Ohkura, Hiroyuki; McKim, Kim S.

doi:10.1371/journal.pgen.1005605

Cited by 45 publications

(82 citation statements)

References 79 publications

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“…Second, these oocytes form aberrant spindles that appear to be composed mostly of central spindle. The spindles often appear "hollow," which can reflect loss of kinetochore but not central spindle microtubules (Radford et al 2015). These results are consistent with a role for Polo in stabilizing microtubule-kinetochore attachments (Elowe et al 2007;Lénárt et al 2007;Liu et al 2012;Suijkerbuijk et al 2012) but with no function in the central spindle.…”

Section: Discussionsupporting

confidence: 64%

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins inDrosophilaOocytes

et al. 2015

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Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubulechromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation. KEYWORDS meiosis; synthetic lethal mutation; homolog bi-orientation; spindle; chromosome segregation; Drosophila C HROMOSOMES are segregated during cell division by the spindle, a bipolar array of microtubules. In somatic cells, spindle assembly is guided by the presence of centrosomes at the poles. In this conventional spindle assembly model, the kinetochores attach to microtubules from opposing centrosomes and tension is established. This satisfies the spindle assembly checkpoint, which then allows the cell to proceed to anaphase (Musacchio and Salmon 2007). Cell division is completed by recruiting proteins to a midzone of antiparallel microtubules that forms between the segregated chromosomes, signaling furrow formation (Fededa and Gerlich 2012;D'Avino et al. 2015). However, spindle morphogenesis in oocytes of many animals occurs in the absence of centrosomes. This may contribute to the high rates of segregation errors that are maternal in origin and are a leading cause of miscarriages, birth defects, and infertility (Herbert et al. 2015). How a robust spindle assembles without guidance from the centrosomes is not well understood. While it is clear that the chromosomes can recruit microtubules and drive spindle assembly (Tseng et al. 2010;Dumont and Desai 2012), how a bipolar spindle is organized and chromosomes make the correct attachments to microtubules is not understood.The Drosophila oocyte provides a genetically tractable system for the identification of genes involved in acentrosomal spindle assembly. Substantial evidence in Drosop...

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Section: Discussionsupporting

confidence: 64%

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins inDrosophilaOocytes

et al. 2015

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“…Nnf1b has properties that appear to make it a good target for mutations beneficial for suppression of preferential meiotic segregation of centromere variants into the female pronucleus. Nnf1b predominates in kinetochores during female meiosis where it forms a bridge (Przewloka et al 2011) between Cenp-C, an inner centromere protein with noticeable direct DNA binding activity (Hori et al 2008;Politi et al 2002;Sugimoto et al 1994;Yang et al 1996), and the rest of the KMN network, the main microtubule-binding activity that might have a prominent role in the assembly and positioning of the acentrosomal spindles in oocytes (Radford et al 2015). Clearly, the melanogaster, such Nnf1b specialization would have to be subtle, as we have not detected obvious effects after loss of Nnf1b.…”

Section: Discussionmentioning

confidence: 99%

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function

et al. 2016

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Kinetochores allow attachment of chromosomes to spindle microtubules. Moreover, they host proteins that permit correction of erroneous attachments and prevent premature anaphase onset before bi-orientation of all chromosomes in metaphase has been achieved. Kinetochores are assembled from subcomplexes. Kinetochore proteins as well as the underlying centromere proteins and the centromeric DNA sequences evolve rapidly despite their fundamental importance for faithful chromosome segregation during mitotic and meiotic divisions. During evolution of Drosophila melanogaster, several centromere proteins were lost and a recent gene duplication has resulted in two Nnf1 paralogs, Nnf1a and Nnf1b, which code for alternative forms of a Mis12 kinetochore complex component. The rapid evolutionary divergence of centromere/kinetochore constituents in animals and plants has been proposed to be driven by an intragenome conflict resulting from centromere drive during female meiosis. Thus, a female meiosisspecific paralog might be expected to evolve rapidly under positive selection. While our characterization of the D. melanogaster Nnf1 paralogs hints at some partial functional specialization of Nnf1b for meiosis, we have failed to detect evidence for positive selection in our analysis of Nnf1 sequence evolution in the Drosophilid lineage. Neither paralog is essential, even though we find some clear differences in subcellular localization and expression during development. Loss of both paralogs results in developmental lethality. We therefore conclude that the two paralogs are still in early stages of differentiation. Neither paralog is essential, even though we find some clear differences in subcellular localization and expression during development. Loss of both paralogs results in developmental lethality. We therefore conclude that the two paralogs are still in early stages of differentiation.

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“…Spc105R-depleted oocytes do not assemble any of the core kinetochore complexes (Radford et al 2015). In oocytes double-depleted of Klp61F (GL00441) and Spc105R, the karyosome dispersal was significantly reduced compared to Klp61F-depleted oocytes (45%, n = 29, P = 0.002, Figure 1B,D), but remained elevated compared to wild type (P , 0.0001).…”

Section: Klp61f Promotes Chromosome Organization In Drosophila Oocytesmentioning

confidence: 92%

“…RNAi constructs generated by TRiP were: Klp61F (HMS00552 and GL00441), Spc105R (GL00392) (Radford et al 2015), sub (GL00583) (Radford et al 2015), and asp (GL00108). We designed the ncd shRNA construct using the shRNA predictions from DSIR (http://biodev.extra.cea.fr/DSIR/DSIR.…”

Section: Rnai In Drosophila Oocytesmentioning

confidence: 99%

“…SUB also bundles antiparallel MTs but has less ability to discriminate an even number of MTs. (C) Eventually, two types of MTs are present (Radford et al 2015): antiparallel MTs whose plus-ends overlap (light green) and parallel MTs whose plus-ends interact with kinetochores (dark green). We propose that the antiparallel MTs promote symmetry while parallel MTs promote asymmetry.…”

Section: Mechanisms and Forces That Generate Asymmetric Spindles In Omentioning

confidence: 99%

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Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes

Radford

McKim

2017

Genetics

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The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD, and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster. How multiple microtubulebundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces, resulting in the capacity to accurately segregate chromosomes.

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

Cited by 45 publications

References 79 publications

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins inDrosophilaOocytes

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins inDrosophilaOocytes

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function

Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes

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