C. Fiona Cullen

Msps protein is localized to acentrosomal poles to ensure bipolarity of Drosophila meiotic spindles.

The female meiotic spindle is commonly formed in a centrosome-independent manner. Here we report the identification of proteins at acentrosomal poles in the female meiotic spindle of Drosophila. The acentrosomal poles contain at least two proteins, Mini-spindles (Msps) and D-TACC, which are also associated with mitotic centrosomes. These proteins interact with one another and are both required for maintaining the bipolarity of acentrosomal spindles. The polar localization of Msps is dependent on D-TACC and Ncd, a kinesin-like microtubule motor. We propose that the polar localization of Msps mediated by D-TACC and Ncd may be crucial for the stabilization of meiotic spindle bipolarity.

A PAR-1–dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

A PAR-1–mediated bias in microtubule organization in the Drosophila oocyte underlies posterior-directed mRNA transport.

NHK-1 phosphorylates BAF to allow karyosome formation in the Drosophila oocyte nucleus

Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. In this study, we report that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. We find that the noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. We propose that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms.

The conserved kinase NHK-1 is essential for mitotic progression and unifying acentrosomal meiotic spindles in Drosophila melanogaster

Conventional centrosomes are absent from the spindle in female meiosis in many species, but it is not clear how multiple chromosomes form one shared bipolar spindle without centrosomes. We identified a female sterile mutant in which each bivalent chromosome often forms a separate bipolar metaphase I spindle. Unlike wild type, prophase I chromosomes fail to form a single compact structure within the oocyte nucleus, although the integrity of metaphase I chromosomes appears to be normal. Molecular analysis indicates that the mutant is defective in the conserved kinase nucleosomal histone kinase-1 (NHK-1). Isolation of further alleles and RNA interference in S2 cells demonstrated that NHK-1 is also required for mitotic progression. NHK-1 itself is phosphorylated in mitosis and female meiosis, suggesting that this kinase is part of the regulatory system coordinating progression of mitosis and meiosis.

Polo boxes and Cut23 (Apc8) mediate an interaction between polo kinase and the anaphase-promoting complex for fission yeast mitosis

The fission yeast plo1 + gene encodes a polo-like kinase, a member of a conserved family of kinases which play multiple roles during the cell cycle. We show that Plo1 kinase physically interacts with the anaphase-promoting complex (APC)/cyclosome through the noncatalytic domain of Plo1 and the tetratricopeptide repeat domain of the subunit, Cut23. A new cut23 mutation, which specifically disrupts the interaction with Plo1, results in a metaphase arrest. This arrest can be rescued by high expression of Plo1 kinase. We suggest that this physical interaction is crucial for mitotic progression by targeting polo kinase activity toward the APC.

The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis

Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. We have screened female sterile mutations for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupted bipolar spindle morphology and chromosome alignment in non‐activated oocytes. We found that rem encodes a conserved subunit of Cdc2 (Cks30A). As Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase–anaphase transition. In addition, we found that the essential pole components, Msps and D‐TACC, were often mislocalized to the equator, which may explain part of the spindle defect. We showed that the second cks gene cks85A, in contrast, has an important role in mitosis. In conclusion, this study describes a new pre‐anaphase role for a Cks in acentrosomal meiotic spindle formation.

The conserved kinase SRPK regulates karyosome formation and spindle microtubule assembly in Drosophila oocytes

Summary In Drosophila oocytes, after the completion of recombination, meiotic chromosomes form a compact cluster called the karyosome within the nucleus, and later assemble spindle microtubules without centrosomes. Although these oocyte-specific phenomena are also observed in humans, their molecular basis is not well understood. Here, we report essential roles for the conserved kinase SRPK in both karyosome formation and spindle microtubule assembly in oocytes. We have identified a female-sterile srpk mutant through a cytological screen for karyosome defects. Unlike most karyosome mutants, the karyosome defect is independent of the meiotic recombination checkpoint. Heterochromatin clustering found within the wild-type karyosome is disrupted in the mutant. Strikingly, a loss of SRPK severely prevents microtubule assembly for acentrosomal spindles in mature oocytes. Subsequently, bi-orientation and segregation of meiotic chromosomes are also defective. Therefore, this study demonstrates new roles of this conserved kinase in two independent meiotic steps specific to oocytes.

The microtubule catastrophe promoter Sentin delays stable kinetochore–microtubule attachment in oocytes

The microtubule catastrophe-promoting complex Sentin-EB1 delays stable kinetochore–microtubule attachment and facilitates bipolar attachment of homologous chromosomes in Drosophila oocytes.

14-3-3 regulation of Ncd reveals a new mechanism for targeting proteins to the spindle in oocytes

The meiotic spindle is formed without centrosomes in a large volume of oocytes. Local activation of crucial spindle proteins around chromosomes is important for formation and maintenance of a bipolar spindle in oocytes. We found that phosphodocking 14-3-3 proteins stabilize spindle bipolarity in Drosophila melanogaster oocytes. A critical 14-3-3 target is the minus end–directed motor Ncd (human HSET; kinesin-14), which has well-documented roles in stabilizing a bipolar spindle in oocytes. Phospho docking by 14-3-3 inhibits the microtubule binding activity of the nonmotor Ncd tail. Further phosphorylation by Aurora B kinase can release Ncd from this inhibitory effect of 14-3-3. As Aurora B localizes to chromosomes and spindles, 14-3-3 facilitates specific association of Ncd with spindle microtubules by preventing Ncd from binding to nonspindle microtubules in oocytes. Therefore, 14-3-3 translates a spatial cue provided by Aurora B to target Ncd selectively to the spindle within the large volume of oocytes.