Different Drosophila cell types exhibit differences in mitotic centrosome assembly dynamics.

Centrosomes are major microtubule organising centres comprising a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, and we previously showed that two key PCM components, Centrosomin (Cnn) and Spd-2, cooperate to form a scaffold structure around the centrioles that recruits the mitotic PCM in Drosophila; the SPD-5 and SPD-2 proteins appear to play a similar function in C. elegans[1–3]. In fly syncytial embryos, Cnn and Spd-2 are initially recruited into a central region of the PCM and then flux outwards [4–6]. This centrosomal flux is potentially important, but it has so far not been reported in any other cell type. Here we examine the dynamic behaviour of Cnn and Spd-2 in Drosophila larval brain cells. Spd-2 fluxes outwards from the centrioles in both brains and embryos in a microtubule-independent manner. In contrast, although Cnn is initially incorporated into the region of the PCM occupied by Spd-2 in both brains and embryos, Cnn fluxes outwards along microtubules in embryos, but not in brain cells, where it remains concentrated around the centrosomal Spd-2. Thus, the microtubule-independent centrosomal-flux of Spd-2 occurs in multiple fly cell types, while the microtubule-dependent outward flux of Cnn appears to be restricted to the syncytial embryo.P.T.C. is supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (105653/Z/14/Z). J.R. is supported by a Senior Investigator award funded by the Wellcome Trust (104575/Z/14/Z).This is the published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0960982215006648

Re-examining the role of Drosophila Sas-4 in centrosome assembly using two-colour-3D-SIM FRAP.

Centrosomes have many important functions and comprise a 'mother' and 'daughter' centriole surrounded by pericentriolar material (PCM). The mother centriole recruits and organises the PCM and templates the formation of the daughter centriole. It has been reported that several important Drosophila PCM-organising proteins are recruited to centrioles from the cytosol as part of large cytoplasmic 'S-CAP' complexes that contain the centriole protein Sas-4. In a previous paper (Conduit et al., 2014b) we showed that one of these proteins, Cnn, and another key PCM-organising protein, Spd-2, are recruited around the mother centriole before spreading outwards to form a scaffold that supports mitotic PCM assembly; the recruitment of Cnn and Spd-2 is dependent on another S-CAP protein, Asl. We show here, however, that Cnn, Spd-2 and Asl are not recruited to the mother centriole as part of a complex with Sas-4. Thus, PCM recruitment in fly embryos does not appear to require cytosolic S-CAP complexes.PTC was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (105653/Z/14/Z) and by an Issac Newton Trust Research Grant from the University of Cambridge awarded to TTW (RG78799). AW, ZN and JWR were supported by a Senior Investigator Award awarded to JWR and funded by the Wellcome Trust (104575/Z/14/Z). The OMX microscope used in this study is part of the Oxford Micron Advanced Bioimaging Unit supported by a Wellcome Trust Strategic Award (091911).This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.7554/eLife.0848

Structural Basis for Mitotic Centrosome Assembly in Flies.

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.Z.F. and A.F.M.H. were supported by Sir William Dunn School EPA PhD studentships and also a Clarendon Scholarship and a Santander Graduate Award to A.F.M.H; A.C., A.W., M.A.C., P.T.C., and J.W.R. were supported by a Wellcome Trust Senior Investigator Award (104575); S.J. and S.M.L. were supported by a Wellcome Trust Senior Investigator Award (100298); A.W. was also partially supported by a Wellcome Trust Strategic Award to the Micron Oxford Advanced Bioimaging Unit (107457)

Evidence that a positive feedback loop drives centrosome maturation in fly embryos.

Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themselves. The PCM expands dramatically as cells prepare to enter mitosis (a process termed centrosome maturation), but it is unclear how this expansion is achieved. In flies, Spd-2 and Cnn are thought to form a scaffold around the mother centriole that recruits other components of the mitotic PCM, and the Polo-dependent phosphorylation of Cnn at the centrosome is crucial for scaffold assembly. Here, we show that, like Cnn, Spd-2 is specifically phosphorylated at centrosomes. This phosphorylation appears to create multiple phosphorylated S-S/T(p) motifs that allow Spd-2 to recruit Polo to the expanding scaffold. If the ability of Spd-2 to recruit Polo is impaired, the scaffold is initially assembled around the mother centriole, but it cannot expand outwards, and centrosome maturation fails. Our findings suggest that interactions between Spd-2, Polo and Cnn form a positive feedback loop that drives the dramatic expansion of the mitotic PCM in fly embryos

Microtubule organization: A complex solution

Microtubule nucleation within cells is catalyzed by γ-tubulin ring complexes localized at specific microtubule-organizing centers. In this issue, Muroyama et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601099) reveal heterogeneity in the composition and function of these complexes, with wide implications for how cells organize their microtubule arrays.P.T. Conduit was funded by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (105653/Z/14/Z)

Microtubule nucleation by γ-tubulin complexes and beyond.

In this short review, we give an overview of microtubule nucleation within cells. It is nearly 30 years since the discovery of γ-tubulin, a member of the tubulin superfamily essential for proper microtubule nucleation in all eukaryotes. γ-tubulin associates with other proteins to form multiprotein γ-tubulin ring complexes (γ-TuRCs) that template and catalyse the otherwise kinetically unfavourable assembly of microtubule filaments. These filaments can be dynamic or stable and they perform diverse functions, such as chromosome separation during mitosis and intracellular transport in neurons. The field has come a long way in understanding γ-TuRC biology but several important and unanswered questions remain, and we are still far from understanding the regulation of microtubule nucleation in a multicellular context. Here, we review the current literature on γ-TuRC assembly, recruitment, and activation and discuss the potential importance of γ-TuRC heterogeneity, the role of non-γ-TuRC proteins in microtubule nucleation, and whether γ-TuRCs could serve as good drug targets for cancer therapy.Wellcome Trust and Royal Society (105653/Z/14/Z) BBSRC BB/M011194/

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Peer reviewed: TrueCentrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site

γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila

Peer reviewed: TrueAcknowledgements: We thank Melissa Rolls, Bing Ye and Yuh-Nung Jan for sharing fly lines. The work benefited from the Imaging Facility, Department of Zoology, University of Cambridge, supported by Matt Wayland and a Sir Isaac Newton Trust Research Grant (18.07ii(c)). We also acknowledge the ImagoSeine core facility of Institut Jacques Monod, member of France-BioImaging (ANR-10-INBS-04) and IBiSA, with the support of Labex “Who Am I”, Inserm Plan Cancer, Region Ile-de-France and Fondation Bettencourt Schueller.Publication status: PublishedFunder: Centre National de la Recherche Scientifique; Id: http://dx.do10.13039/10.13039/501100004794Funder: Université Paris Cité; Id: http://dx.do10.13039/10.13039/501100009516Funder: Royal Society; Id: http://dx.do10.13039/10.13039/501100000288Funder: Isaac Newton Trust; Id: http://dx.do10.13039/10.13039/501100004815Funder: University of Cambridge; Id: http://dx.do10.13039/10.13039/501100000735Funder: Fondation Bettencourt Schueller; Id: http://dx.do10.13039/10.13039/501100007492Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology

γ-TuRCs and Augmin are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborisation (da) neurons. This relatively mild phenotype was attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analysing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the Augmin complex, which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching, likely by increasing the frequency of microtubule growth in terminal dendrites. Consistently, we find that Augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the Augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology