Abstract
During interphase in Drosophila neuroblasts, the Centrobin (CNB)-positive daughter centriole retains pericentriolar material (PCM) and organizes an aster that is a key determinant of the orientation of cell division. Here we show that daughter centrioles depleted of CNB cannot fulfil this function whereas mother centrioles that carry ectopic CNB can. CNB co-precipitates with a set of centrosomal proteins that include γ-TUB, ANA2, CNN, SAS-4, ASL, DGRIP71, POLO and SAS-6. Following chemical inhibition of POLO or removal of three POLO phosphorylation sites present in CNB, the interphase microtubule aster is lost. These results demonstrate that centriolar CNB localization is both necessary and sufficient to enable centrioles to retain PCM and organize the interphase aster in Drosophila neuroblasts. They also reveal an interphase function for POLO in this process that seems to have co-opted part of the protein network involved in mitotic centrosome maturation.
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Change history
08 February 2013
In the version of this Article that was originally published online, Figure 3 was incorrect. This error has been corrected in the PDF and HTML versions of the Article.
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Acknowledgements
We thank W. Chia (Temasek Life Sciences Laboratory, Singapore), C. Doe (University of Oregon, USA)., T. Kaufman (Indiana University, USA), B. Lange (Max-Planck Institute for Molecular Genetics, Germany), M. Peifer (University of North Carolina at Chapel Hill, USA), the Bloomington stock centre, DGRC, DSHB, Harvard Exelixis and VDRC for providing flies and reagents; A. Olza for transgenesis; and J. Colombelli and L. Bardia from the IRB Advanced Microscopy Facility for technical assistance. Work in our laboratory is supported by grants BFU2009-07975/BMC, BFU2012-32522, CENIT ONCOLOGICA-20091016 and Consolider-Ingenio C3D CSD2006-23, from the Spanish MICINN MIMECO, and CDTI, and SGR Agaur 2009 CG041413, from Generalitat de Catalunya.
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J. Reina, S.L., J.J. and C.G. designed the project; J. Reina, S.L. and J.J. carried out the experiments except for the phosphorylation and allograft assays, which were carried out by T.B. and J. Roig, and by F.R., respectively. C.G. wrote the manuscript, which was discussed and agreed on by all authors.
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Control neuroblasts.
Neuroblasts in culture expressing fluorescent labels for microtubules (GFP–TUB) and centrioles (YFP–ASL). After asymmetric division, the daughter centriole organises an interphase microtubule aster at the apical pole (up), while the mother centriole looses microtubule nucleation activity and becomes motile. The mother centriole resumes microtubule nucleation only at the onset of mitosis when it is located in the basal side of the neuroblast. Z stacks were recorded every 90 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 2182 kb)
CENTROBIN is necessary for microtubule nucleation by daughter centrosomes during interphase.
Neuroblast in culture depleted of CENTROBIN (RNAi-Cnb), expressing GFP–TUB and YFP–ASL. During interphase, both centrosomes are motile and have little or no microtubule organising activity (12:11:47–13:08:12). Microtubule nucleation activity resumes on both centrosomes only at the onset of mitosis (13:14:12). After cytokinesis, both centrioles move to the nearby cortical region and nucleate microtubules (13:48:20), but lose microtubule organising activity and become motile shortly after (14:04:08). Z stacks were recorded every 180 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 1739 kb)
Expression of YFP–PACT does not interfere with asymmetric centrosome behaviour in neuroblasts.
Neuroblast in culture expressing mCherry–TUB (red) and YFP–PACT (green). As wild-type neuroblasts do, this cell displays prominent interphase microtubule asters and a motile centrosome at interphase. The motile centrosome resumes microtubule nucleation at the onset of mitosis. Z stacks were recorded every 90 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 5559 kb)
Ectopically localized CENTROBIN is sufficient to drive microtubule nucleation activity on mother centrosomes in interphase.
Neuroblast in culture expressing mCherry–TUB (red) and YFP–CNB–PACT (green). During interphase (15:41:21), two YFP–CNB–PACT positive dots are associated with strong microtubule signal near the apical cortex. Microtubule nucleation is maintained on both centrosomes upon splitting. The two resulting asters migrate away from each other (15:50:21–16:01:49). After nuclear envelope breakdown (16:04:44), one of the spindle asters attaches to the apical cortex, resulting in spindle rotation (16:06:14–16:10:44). At cytokinesis, the daughter cell is delivered into the cluster of daughter cells from previous divisions (16:21:17). Z stacks were recorded every 90 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 1012 kb)
CENTROBIN-driven acquisition of daughter centriole traits by the mother centriole does not interfere with spindle alignment along the apico-basal axis.
Neuroblast in culture expressing mKATE–ASL (red), mCherry-MIRA (red) and YFP–CNB–PACT (green). The corresponding DIC channel is also shown (grey). Two ASL-labelled dots are always associated with YFP–CNB–PACT signal, which also forms ASL-free aggregates. Centrioles remain apical during most of interphase and move laterally, away from each other, after centrosome splitting (first cell cycle: 12:47:21–12:56:18; second cell cycle: 13:50:00–14:14:11). Hereafter, one centrosome stays at the apical pole, while the other centrosome ends up at the basal site of the cell (first cell cycle: 12:57:48 (NEB)-13:03:49; second cell cycle: 14:15:41–14:24:41). The basal centrosome is always close to the centre of the mCherry-MIRA crescent, which becomes visible shortly after (first cell cycle: 13:08:18; second cell cycle: 14:26:12). Z stacks were recorded every 90 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 1606 kb)
Ectopic microtubule nucleation on both centrosomes caused by YFP–CNB–PACT requires pins.
A pinsP62/pinsP98 neuroblast in culture expressing mCherry–TUB (red) and YFP–CNB–PACT (green). After mitosis, centrioles labelled with YFP–CNB–PACT move to the nearest cortex (18:02:57). Microtubule nucleation is reduced significantly soon after, and both centrosomes become motile (18:10:57). At the onset of the next mitosis, both YFP–CNB–PACT dots resume microtubule nucleation (18:30:57) and asymmetric mitosis occurs. The small daughter cell is delivered to almost the opposite site to the previous (19:20:57). In the resulting neuroblast, once more, YFP–CNB–PACT signal is initially cortical and associated with microtubule nucleation (19:30:57), which is lost shortly after when centrioles become motile again (19:40:57). Z stacks were recorded every 120 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 1469 kb)
Ectopic microtubule nucleation on both centrosomes induced by YFP–CNB–PACT is lost upon addition of BI2536 and recovered after drug removal.
Neuroblast in culture expressing YFP–CNB–PACT (green) and mCherry–TUB (red) undergoes normal mitosis. Upon addition of 20 nM BI2536 (13:41:05), both YFP–CNB–PACT-positive dots fall off the apical cortex (14:12:12) and become migratory. Shortly after removal of the drug from the culture medium (14:45:05), both YFP–CNB–PACT dots resume microtubule nucleation (14:48:05) and the cell cycle continues with NEB (15:27:22) and the formation of the mitotic spindle. Z stacks were recorded every 90 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 2774 kb)
The fusion protein YFP–CNBT4AT9AS82A, a mutant form of YFP–CNB that lacks the three conserved consensus phosphorylation sites for POLO does not rescue the loss of centrobin mutant phenotypes.
A Cnb mutant NB (CnbPe00267/DfED4284) in culture expressing YFP–CNBT4AT9AS82A (green) and mCherry–TUB (red). No stable asters are observed during interphase (12:20:43–12:44:43). At the onset of mitosis (12:49:24), the YFP-labelled daughter centriole upregulates microtubule nucleation and the second microtubule aster appears shortly after (12:53:23). The YFP-containing aster then rotates towards the basal pole at NEB (13:05:23–13:17:56) and is inherited by the GMC. Early in the following interphase, microtubule nucleation ceases again and the YFP signal migrates away from the apical side (13:53:28). Microtubule nucleation resumes once more at the onset of mitosis (15:08:15). Z stacks were recorded every 120 s and their z-projections are displayed at 5 frames per second. Time is shown in hours:minutes:seconds. (MOV 5661 kb)
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Januschke, J., Reina, J., Llamazares, S. et al. Centrobin controls mother–daughter centriole asymmetry in Drosophila neuroblasts. Nat Cell Biol 15, 241–248 (2013). https://doi.org/10.1038/ncb2671
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DOI: https://doi.org/10.1038/ncb2671
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