Papers by Paul T. Conduit
Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of... more Centrosomes 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 <i>Drosophila</i> syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the c...
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Centrosomes are important organisers of microtubules within animal cells. They comprise a pair of... more Centrosomes are important organisers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material (PCM), which nucleates and organises 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 attach...
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Microtubules are polarised polymers nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs)... more Microtubules are polarised polymers nucleated by multi-protein γ-tubulin ring complexes (γ-TuRCs). Within neurons, microtubule polarity is plus-end-out in axons and mixed or minus-end-out in dendrites. Previously we showed that within the soma of Drosophila sensory neurons γ-tubulin localises asymmetrically to Golgi stacks, Golgi-derived microtubules grow asymmetrically towards the axon, and growing microtubule plus-ends are guided towards the axon and restricted from entering dendrite in a Kinesin-2-dependent manner (Mukerjee et al., 2020). Here we show that depleting γ-TuRCs perturbs the direction of microtubule growth from the Golgi stacks, consistent with a model for asymmetric microtubule nucleation involving γ-TuRCs and other nucleation-promoting factors. We also directly observe microtubule turning along microtubule bundles and show that depleting APC, proposed to link Kinesin-2 to plus ends, reduces microtubule turning and increases plus end growth into dendrites. These resu...
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Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nuc... more Neurons contain polarised microtubule arrays essential for neuronal function. How microtubule nucleation and polarity are regulated within neurons remains unclear. We show that γ-tubulin localises asymmetrically to the somatic Golgi withinDrosophilaneurons. Microtubules originate from the Golgi with an initial growth preference towards the axon. Their growing plus ends also turn towards and into the axon, adding to the plus-end-out microtubule pool. Any plus ends that reach a dendrite, however, do not readily enter, maintaining minus-end-out polarity. Both turning towards the axon and exclusion from dendrites depend on Kinesin-2, a plus-end-associated motor that guides growing plus ends along adjacent microtubules. We propose that Kinesin-2 engages with a polarised microtubule network within the soma to guide growing microtubules towards the axon; while at dendrite entry sites engagement with microtubules of opposite polarity generates a backward stalling force that prevents entry i...
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eLife, 2019
Centrosomes are formed when mother centrioles recruit pericentriolar material (PCM) around themse... more 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 d...
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Current Biology, 2018
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Current Biology, 2019
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Essays in Biochemistry, 2018
In this short review, we give an overview of microtubule nucleation within cells. It is nearly 30... more 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 pr...
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Cell, 2017
In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to ... more 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 ...
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The Journal of cell biology, Jan 20, 2016
Microtubule nucleation within cells is catalyzed by γ-tubulin ring complexes localized at specifi... more 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.
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The Journal of Cell Biology, 2006
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eLife, 2015
Centrosomes have many important functions and comprise a ‘mother’ and ‘daughter’ centriole surrou... more 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 (<xref ref-type="bibr" rid="bib5">Conduit et al., 2014b</xref>) 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 rec...
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Nature reviews. Molecular cell biology, Jan 16, 2015
It has become clear that the role of centrosomes extends well beyond that of important microtubul... more It has become clear that the role of centrosomes extends well beyond that of important microtubule organizers. There is increasing evidence that they also function as coordination centres in eukaryotic cells, at which specific cytoplasmic proteins interact at high concentrations and important cell decisions are made. Accordingly, hundreds of proteins are concentrated at centrosomes, including cell cycle regulators, checkpoint proteins and signalling molecules. Nevertheless, several observations have raised the question of whether centrosomes are essential for many cell processes. Recent findings have shed light on the functions of centrosomes in animal cells and on the molecular mechanisms of centrosome assembly, in particular during mitosis. These advances should ultimately allow the in vitro reconstitution of functional centrosomes from their component proteins to unlock the secrets of these enigmatic organelles.
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Current Biology, 2015
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Centrosome & Centriole, 2015
Microinjection is a powerful technique that can be used to study protein function. Early Drosophi... more Microinjection is a powerful technique that can be used to study protein function. Early Drosophila embryos are particularly amenable to microinjection due to their large size and their single cell status. Here, we report methods to microinject these embryos with various reagents to study the function of proteins at centrosomes and centrosome function more generally. Although precise details vary between laboratories, many aspects of the process are conserved. We describe the process from setting up a fly cage to imaging the injected embryos on a spinning disk confocal microscope and use specific examples to highlight the potency of this technique.
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Current Biology, 2014
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Nature Cell Biology, 2013
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Developmental Cell, 2014
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Papers by Paul T. Conduit