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1.
J Cell Biol ; 217(10): 3431-3445, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30087124

ABSTRACT

The meiotic spindle in oocytes is assembled in the absence of centrosomes, the major microtubule nucleation sites in mitotic and male meiotic cells. A crucial, yet unresolved question in meiosis is how spindle microtubules are generated without centrosomes and only around chromosomes in the exceptionally large volume of oocytes. Here we report a novel oocyte-specific microtubule nucleation pathway that is essential for assembling most spindle microtubules complementarily with the Augmin pathway. This pathway is mediated by the kinesin-6 Subito/MKlp2, which recruits the γ-tubulin complex to the spindle equator to nucleate microtubules in Drosophila oocytes. Away from chromosomes, Subito interaction with the γ-tubulin complex is suppressed by its N-terminal region to prevent ectopic microtubule assembly in oocytes. We further demonstrate in vitro that the Subito complex from ovaries can nucleate microtubules from pure tubulin dimers. Collectively, microtubule nucleation regulated by Subito drives spatially restricted spindle assembly in oocytes.


Subject(s)
Meiosis , Microtubules/metabolism , Oocytes/metabolism , Protein Multimerization , Spindle Apparatus/metabolism , Tubulin/metabolism , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster , Female , Kinesins/genetics , Kinesins/metabolism , Male , Microtubules/genetics , Spindle Apparatus/genetics , Tubulin/genetics
2.
Methods Cell Biol ; 145: 237-248, 2018.
Article in English | MEDLINE | ID: mdl-29957206

ABSTRACT

Studies using Drosophila have played pivotal roles in advancing our understanding of molecular mechanisms of mitosis throughout the past decades, due to the short generation time and advanced genetic research of this organism. Drosophila is also an excellent model to study female meiosis in oocytes. Pathways such as the acentrosomal assembly of the meiotic spindle in oocytes are conserved from fly to humans. Collecting and manipulating large Drosophila oocytes for microscopy and biochemistry are both time and cost efficient, offering advantages over mouse or human oocytes. Therefore, Drosophila oocytes serve as an excellent platform for molecular studies of female meiosis using a combination of genetics, microscopy, and biochemistry. Here we describe key methods to observe the formation of the meiotic spindle either in fixed or in live oocytes. Moreover, biochemical methods are described to identify protein-protein interactions in vivo.


Subject(s)
Drosophila/physiology , Meiosis/physiology , Microscopy/methods , Oocytes/physiology , Spindle Apparatus/physiology , Animals , Drosophila/metabolism , Drosophila Proteins/metabolism , Female , Kinesins/metabolism , Mitosis/physiology , Oocytes/metabolism
3.
J Cell Biol ; 216(10): 3029-3039, 2017 10 02.
Article in English | MEDLINE | ID: mdl-28860275

ABSTRACT

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.


Subject(s)
14-3-3 Proteins/metabolism , Chromosomes, Insect/metabolism , Drosophila Proteins/metabolism , Kinesins/metabolism , Microtubules/metabolism , Oocytes/metabolism , Spindle Apparatus/metabolism , 14-3-3 Proteins/genetics , Animals , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Chromosomes, Insect/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Kinesins/genetics , Microtubules/genetics , Oocytes/cytology , Protein Transport/physiology , Spindle Apparatus/genetics
4.
Nat Commun ; 6: 8879, 2015 Nov 16.
Article in English | MEDLINE | ID: mdl-26568519

ABSTRACT

Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation.


Subject(s)
Aurora Kinase A/genetics , Brain Neoplasms/genetics , Carcinogenesis/genetics , Cyclin B/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/metabolism , Neuroblastoma/genetics , Animals , Blotting, Western , Brain Neoplasms/metabolism , Cell Proliferation , Chromosome Segregation , Drosophila , Fluorescent Antibody Technique , Microtubule-Associated Proteins , Mitosis , Mutation , Neural Stem Cells , Neuroblastoma/metabolism , Neurons
5.
PLoS One ; 6(1): e14600, 2011 Jan 31.
Article in English | MEDLINE | ID: mdl-21297952

ABSTRACT

BACKGROUND: CDK11(p58) is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis. METHODOLOGY/PRINCIPAL FINDINGS: In addition to these previously described roles, our study shows that CDK11(p58) inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11(p58), which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression. CONCLUSION/SIGNIFICANCE: We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11(p58) could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.


Subject(s)
Centrioles/metabolism , Centrosome/metabolism , Cyclin D3/physiology , Mitosis , Protein Serine-Threonine Kinases/metabolism , Cyclin D3/metabolism , Gene Expression , HeLa Cells , Humans , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Transport
6.
J Cell Biol ; 189(4): 651-9, 2010 May 17.
Article in English | MEDLINE | ID: mdl-20479466

ABSTRACT

Aurora A is a spindle pole-associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150(glued) on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150(glued). Although wild-type p150(glued) binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150(glued). Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150(glued) MBD to limit the microtubule binding of the dynein-dynactin complex and thus regulates spindle assembly.


Subject(s)
Drosophila Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Mitosis , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Aurora Kinase A , Aurora Kinases , Binding Sites , Drosophila Proteins/genetics , Drosophila melanogaster/metabolism , Dynactin Complex , Dyneins/metabolism , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Phosphorylation , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Protein Subunits/metabolism , RNA, Small Interfering/metabolism , Spindle Apparatus/metabolism
7.
Med Sci (Paris) ; 26(4): 377-83, 2010 Apr.
Article in French | MEDLINE | ID: mdl-20412742

ABSTRACT

Centrosomes are essential protagonists during cell division through microtubule nucleation and spindle formation which are key to the harmonious distribution of sister chromatids in the two daughter cells. However, during the past decade, a wealth of new observations has extended their role beyond mitosis, particularly in the asymmetrical partition of cell fate determinants. Remarkably, asymmetric centrosome inheritance per se, through the segregation of differently aged mother -centrioles, seems to regulate the differential behaviour of daughter cells, in part through asynchronous expression of primary cilia, governing the response to environmental signals. It is thus understandable why any quantitative or qualitative dysfunction of centrioles contributes to genomic -instability and thus -tumorigenesis.


Subject(s)
Cell Cycle/physiology , Cell Transformation, Neoplastic/genetics , Centrosome/physiology , Spindle Apparatus/physiology , Aneuploidy , Animals , Cell Division/physiology , Centrioles/physiology , Cilia/physiology , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/pathology , Humans , Models, Biological , Neoplastic Stem Cells/cytology , Stem Cells/cytology
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