Proper mitotic spindle orientation depends on the correct anchorage of astral microtubules to the cortex. It relies on the remodeling of the cell cortex, a process not fully understood. Annexin A2 ...(Anx2; also known as ANXA2) is a protein known to be involved in cortical domain remodeling. Here, we report that in HeLa cell early mitosis, Anx2 recruits the scaffold protein Ahnak at the cell cortex facing spindle poles, and the distribution of both proteins is controlled by cell adhesion. Depletion of either protein or impaired cortical Ahnak localization result in delayed anaphase onset and unstable spindle anchoring, which leads to altered spindle orientation. We find that Ahnak is present in a complex with dynein-dynactin. Furthermore, Ahnak and Anx2 are required for correct dynein and NuMA (also known as NUMA1) cortical localization and dynamics. We propose that the Ahnak-Anx2 complex influences the cortical organization of the astral microtubule-anchoring complex, and thereby mitotic spindle positioning in human cells. This article has an associated First Person interview with the first author of the paper.
The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate ...chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.
Drosophila neural stem cells (NSCs) divide asymmetrically to generate siblings of different sizes. This model system has proved helpful in deciphering the contribution of polarity cues and the ...mitotic spindle in asymmetric cell division. Here, we describe a technique we developed to flatten cultured Drosophila brain explants to accurately image the cytoskeleton in live NCSs. We also describe our approach to efficiently remove centrosomes by laser photo-ablation and to measure daughter cell size after NSC division.
For complete details on the use and execution of this protocol, please refer to Thomas et al. (2021).
Display omitted
•Preparation of incubation chambers for tissue imaging•Preparation of live Drosophila melanogaster larval brain explants•Laser photo-ablation of centrosomes in NSCs•Imaging and analysis of NSCs devoid of their apical or basal centrosome
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Drosophila neural stem cells (NSCs) divide asymmetrically to generate siblings of different sizes. This model system has proved helpful in deciphering the contribution of polarity cues and the mitotic spindle in asymmetric cell division. Here, we describe a technique we developed to flatten cultured Drosophila brain explants to accurately image the cytoskeleton in live NCSs. We also describe our approach to efficiently remove centrosomes by laser photo-ablation and to measure daughter cell size after NSC division.
Accurate chromosome segregation requires the perfect spatiotemporal rearrangement of the cellular cytoskeleton. Isolated more than two decades ago from
, Aurora A is a widespread protein kinase that ...plays key roles during cell division. Numerous studies have described the localisation of Aurora A at centrosomes, the mitotic spindle, and, more recently, at mitotic centromeres. In this review, we will summarise the cytoskeletal rearrangements regulated by Aurora A during cell division. We will also discuss the recent discoveries showing that Aurora A also controls not only the dynamics of the cortical proteins but also regulates the centromeric proteins, revealing new roles for this kinase during cell division.
Sister chromatid cohesion is a multi-step process implemented throughout the cell cycle to ensure the correct transmission of chromosomes to daughter cells. Although cohesion establishment and ...mitotic cohesion dissolution have been extensively explored, the regulation of cohesin loading is still poorly understood. Here, we report that the methyltransferase NSD3 is essential for mitotic sister chromatid cohesion before mitosis entry. NSD3 interacts with the cohesin loader complex kollerin (composed of NIPBL and MAU2) and promotes the chromatin recruitment of MAU2 and cohesin at mitotic exit. We also show that NSD3 associates with chromatin in early anaphase, prior to the recruitment of MAU2 and RAD21, and dissociates from chromatin when prophase begins. Among the two NSD3 isoforms present in somatic cells, the long isoform is responsible for regulating kollerin and cohesin chromatin-loading, and its methyltransferase activity is required for efficient sister chromatid cohesion. Based on these observations, we propose that NSD3-dependent methylation contributes to sister chromatid cohesion by ensuring proper kollerin recruitment and thus cohesin loading.
Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect ...the function aurora, the founding family member in Drosophila, the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants. The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.
Proper assembly of mitotic spindles requires microtubule nucleation not only at the centrosomes but also around chromatin. In this study, we found that the Drosophila tubulin-specific chaperone dTBCE ...is required for the enrichment of tubulin in the nuclear space after nuclear envelope breakdown and for subsequent promotion of spindle microtubule nucleation. These events depend on the CAP-Gly motif found in dTBCE and are regulated by Ran and lamin proteins. Our data suggest that during early mitosis, dTBCE and nuclear pore proteins become enriched in the nucleus, where they interact with the Ran GTPase to promote dynamic tubulin enrichment. We propose that this novel mechanism enhances microtubule nucleation around chromatin, thereby facilitating mitotic spindle assembly.
Display omitted
•During early mitosis, dTBCE promotes tubulin enrichment in the nascent spindle region•dTBCE interacts with nuclear pore proteins and Ran•dTBCE is required for microtubule assembly around chromosomes•dTBCE localization and function require its conserved CAP-Gly motif.
Métivier et al. show that the Drosophila tubulin-binding chaperone E (dTBCE) is required for mitotic spindle assembly in neural stem cells. They propose that dTBCE invades the nuclear space during early mitosis to generate a milieu able to enrich tubulin and for subsequent microtubule nucleation around chromosomes.
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
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.
Neuroblast division is characterized by asymmetric positioning of the cleavage furrow, resulting in a large difference in size between the future daughter cells. In animal cells, furrow placement and ...assembly are governed by centralspindlin that accumulates at the equatorial cell cortex of the future cleavage site and at the spindle midzone. In neuroblasts, these two centralspindlin populations are spatially and temporally separated. A leading pool is located at the basal cleavage site and a second pool accumulates at the midzone before traveling to the cleavage site. The cortical centralspindlin population requires peripheral astral microtubules and the chromosome passenger complex for efficient recruitment. Loss of this pool does not prevent cytokinesis but enhances centralspindlin signaling at the midzone, leading to equatorial furrow repositioning and decreased size asymmetry. These data show that basal furrow positioning in neuroblasts results from a competition between different centralspindlin pools in which the cortical pool is dominant.
Display omitted
•Midzone and cortical centralspindlin pools are spatially separated•The cortical centralspindlin pool is dominant•Peripheral MTs recruit the centralspindlin pool at the cortical cleavage furrow•Midzone centralspindlin becomes dominant when astral peripheral MTs are impaired
Centralspindlin positions the cleavage furrow. Thomas et al. show that astral MTs target a dominant centralspindlin pool to the basal furrow in fly neuroblasts. This pool is separated from spindle-midzone-associated centralspindlin. When centralspindlin cortical recruitment is impaired, the midzone pool takes the lead to reset furrowing apically.
The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila ...melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.
PDF
