Papers by James Wakefield
Chromosome Research, Jan 7, 2011
eLife, 2020
Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which a... more Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a po...
PLOS ONE, 2019
Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung car... more Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer.
eLife, 2019
Representing the dynamic nature of biological processes is a challenge. This article describes a ... more Representing the dynamic nature of biological processes is a challenge. This article describes a collaborative project in which the authors – a philosopher of biology, an artist and a cell biologist – explore how best to represent the entire process of cell division in one connected image. This involved a series of group Drawing Labs, one-to-one sessions, and discussions between the authors. The drawings generated during the collaboration were then reviewed by four experts in cell division. We propose that such an approach has value, both in communicating the dynamic nature of biological processes and in generating new insights and hypotheses that can be tested by artists and scientists.
Journal of Cell Biology, May 7, 2001
Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been s... more Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and ␥-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in aster-less , a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.
Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimeth... more Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice...
Journal of Cell Science, 2020
Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing ... more Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of morgana (mora) causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Morgana localizes to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT super-complex, and with additional well-known Hsp90 co-chaperones. Acute inhibition of Morgana function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds microtubules directly and promotes microtubule polymerization in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role.
Current Biology, Aug 1, 2015
Our original article mistakenly used ''Tubulin Chaperone Protein-1'' as the basis for the abbrevi... more Our original article mistakenly used ''Tubulin Chaperone Protein-1'' as the basis for the abbreviation of the molecular chaperone complex TCP-1, including in the article title. We would like to clarify that the acronym TCP-1 is in fact derived from T-complex protein-1 (http://www.ncbi.nlm.nih.gov/gene/6950). As an alternative acronym for this same protein complex is the CCT (Chaperonin Containing TCP-1) complex, we have updated the nomenclature in the article to refer interchangeably to the protein complex that associated with Misato as ''TCP-1/CCT.'' The article title has also been updated (as shown here) to correct the erroneous ''Tubulin Chaperone Protein-1'' nomenclature. The authors sincerely apologize for the confusion, and for any inconvenience caused.
This paper presents a real-time control architecture for SBR systems with no holding tank. The co... more This paper presents a real-time control architecture for SBR systems with no holding tank. The control approach has been divided into three separated layers in order to accommodate different full-scale scenarios. The lower control layer processes the ORP and DO trajectories to detect the depletion of the denitrification and nitrification reactions in order to automatically interrupt the reaction phases. The intermediate layer has been designed to maintain the total solid mass constant in reactors. Finally, an ammonia controller has been implemented in the supervisory layer that optimises the aeration costs to fulfil the effluent ammonia requirements. The overall control system has been verified experimentally in a 150L pilot plant. In this study, the lower control layer reduced the length of mixing and aeration by approximately 20%. In addition, the experimental validation of the ammonia controller led to reductions of about 30% in the overall aerated fraction.
Chromosome Research, Apr 1, 2011
Communicative & Integrative Biology, Mar 1, 2014
between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic ... more between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation.
Frontiers in Cell and Developmental Biology, Nov 26, 2015
Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosi... more Over the last two decades, the small GTPase Ran has emerged as a central regulator of both mitosis and meiosis, particularly in the generation, maintenance, and regulation of the microtubule (MT)-based bipolar spindle. Ran-regulated pathways in mitosis bear many similarities to the well-characterized functions of Ran in nuclear transport and, as with transport, the majority of these mitotic effects are mediated through affecting the physical interaction between karyopherins and Spindle Assembly Factors (SAFs)-a loose term describing proteins or protein complexes involved in spindle assembly through promoting nucleation, stabilization, and/or depolymerization of MTs, through anchoring MTs to specific structures such as centrosomes, chromatin or kinetochores, or through sliding MTs along each other to generate the force required to achieve bipolarity. As such, the Ran-mediated pathway represents a crucial functional module within the wider spindle assembly landscape. Research into mitosis using the model organism Drosophila melanogaster has contributed substantially to our understanding of centrosome and spindle function. However, in comparison to mammalian systems, very little is known about the contribution of Ran-mediated pathways in Drosophila mitosis. This article sets out to summarize our understanding of the roles of the Ran pathway components in Drosophila mitosis, focusing on the syncytial blastoderm embryo, arguing that it can provide important insights into the conserved functions on Ran during spindle formation.
Biology Open, 2017
The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-exi... more The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation.
Genes & Development, Aug 14, 2009
The Drosophila Augmin complex localizes g-tubulin to the microtubules of the mitotic spindle, reg... more The Drosophila Augmin complex localizes g-tubulin to the microtubules of the mitotic spindle, regulating the density of spindle microtubules in tissue culture cells. Here, we identify the microtubule-associated protein Msd1 as a new component of the Augmin complex and demonstrate directly that it is required for nucleation of microtubules from within the mitotic spindle. Although Msd1 is necessary for embryonic syncytial mitoses, flies possessing a mutation in msd1 are viable. Importantly, however, in the absence of centrosomes, microtubule nucleation from within the spindle becomes essential. Thus, the Augmin complex has a crucial role in the development of the fly.
Journal of Cell Biology, Mar 16, 2009
Misregulation of candidate stem cell marker ASPM, and its Drosophila homologue Asp, leads to eith... more Misregulation of candidate stem cell marker ASPM, and its Drosophila homologue Asp, leads to either tumour formation or microcephaly, but the functional roles contributing to each are not understood. We reverse-engineered flies to express a version of Asp (Asp LIE), predicted to have lost its ability to bind the phosphatase PP2A-B'. Although Asp LIE flies were viable, they exhibited splayed neural stem cell spindle poles under stress, and development was substantially delayed. A tissue-level analysis of microcephaly and midgut abnormalities in Asp mutants with a compromised spindle assembly checkpoint (SAC) demonstrates tissue-specific vulnerability to mitotic defects. .
Essays in Biochemistry, Nov 14, 2018
The formation of a robust, bi-polar spindle apparatus, capable of accurate chromosome segregation... more The formation of a robust, bi-polar spindle apparatus, capable of accurate chromosome segregation, is a complex process requiring the coordinated nucleation, sorting, stabilization and organization of microtubules (MTs). Work over the last 25 years has identified protein complexes that act as functional modules to nucleate spindle MTs at distinct cellular sites such as centrosomes, kinetochores, chromatin and pre-existing MTs themselves. There is clear evidence that the extent to which these different MT nucleating pathways contribute to spindle mass both during mitosis and meiosis differs not only between organisms, but also in different cell types within an organism. This plasticity contributes the robustness of spindle formation; however, whether such plasticity is present in other aspects of spindle formation is less well understood. Here, we review the known roles of the protein complexes responsible for spindle pole focusing, investigating the evidence that these, too, act co-ordinately and differentially, depending on cellular context. We describe relationships between MT minus-end directed motors dynein and HSET/Ncd, depolymerases including katanin and MCAK, and direct minus-end binding proteins such as nuclear-mitotic apparatus protein, ASPM and Patronin/CAMSAP. We further explore the idea that the focused spindle pole acts as a non-membrane bound condensate and suggest that the metaphase spindle pole be treated as a transient organelle with context-dependent requirements for function.
eLife, Nov 26, 2018
Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mito... more Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2-and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells.
Developmental Cell, 2014
The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromo... more The mitotic spindle is defined by its organized, bipolar mass of microtubules, which drive chromosome alignment and segregation. Although different cells have been shown to use different molecular pathways to generate the microtubules required for spindle formation, how these pathways are coordinated within a single cell is poorly understood. We have tested the limits within which the Drosophila embryonic spindle forms, disrupting the inherent temporal control that overlays mitotic microtubule generation, interfering with the molecular mechanism that generates new microtubules from preexisting ones, and disrupting the spatial relationship between microtubule nucleation and the usually dominant centrosome. Our work uncovers the possible routes to spindle formation in embryos and establishes the central role of Augmin in all microtubule-generating pathways. It also demonstrates that the contributions of each pathway to spindle formation are integrated, highlighting the remarkable flexibility with which cells can respond to perturbations that limit their capacity to generate microtubules.
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Papers by James Wakefield