Publications by year
In Press
Wakefield J, Soeller C, Jeynes J, Tariq A (In Press). In vitro reconstitution of branching microtubule nucleation. eLife, 8
2020
Palumbo V, Tariq A, Borgal L, Metz J, Brancaccio M, Gatti M, Wakefield JG, Bonaccorsi S (2020). <i>Drosophila</i> Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerization.
Journal of Cell ScienceAbstract:
Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerization
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.
Abstract.
Tariq A, Green L, Soeller C, Wakefield JG (2020). Cleavable Affinity Purification (Cl-AP): a One-step Procedure to Affinity Purify Protein Complexes.
Bio Protoc,
10(22).
Abstract:
Cleavable Affinity Purification (Cl-AP): a One-step Procedure to Affinity Purify Protein Complexes.
Cleavable Affinity Purification (Cl-AP) uses a tripartite system of Protein-A-Streptavidin beads and nanobodies, coupled with a biotinylated, thiol-cleavable linker, providing one-step affinity purification from lysates of tissues expressing tagged proteins. This technique allows fluorescent versions of mitotic protein complexes to be isolated intact from cells, for use in biophysical and microscopy-based assays, overcoming the traditional limitations of reductionist approaches. We have used this technique successfully to purify both GFP-tagged and mCherry-tagged proteins, and their interacting partners, expressed in Drosophila melanogaster embryos. Although we demonstrate the efficacy of the GFP-binding protein and RFP-binding protein nanobodies from Chromotek, in theory any antibody could be coupled to the beads and used as a Cl-AP reagent.
Abstract.
Author URL.
Maccallini P, Bavasso F, Scatolini L, Bucciarelli E, Noviello G, Lisi V, Palumbo V, D’Angeli S, Cacchione S, Cenci G, et al (2020). Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development.
Maccallini P, Bavasso F, Scatolini L, Bucciarelli E, Noviello G, Lisi V, Palumbo V, D'Angeli S, Cacchione S, Cenci G, et al (2020). Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development.
PLoS Genet,
16(5).
Abstract:
Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development.
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). 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 versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.
Abstract.
Author URL.
2019
Anderson G, Dupre J, Wakefield JG (2019). Drawing and the dynamic nature of living systems.
ELIFE,
8 Author URL.
Tariq A, Green L, Jeynes JCG, Soeller C, Wakefield JG (2019). In vitro reconstitution of branched microtubule nucleation.
Borgal L, Quiniou M, Wakefield J (2019). Tissue specific vulnerability to mitotic defects caused by mutations in the Drosophila ASPM homologue, Asp.
O'Flaherty L, Shnyder SD, Cooper PA, Cross SJ, Wakefield JG, Pardo OE, Seckl MJ, Tavaré JM (2019). Tumor growth suppression using a combination of taxol-based therapy and GSK3 inhibition in non-small cell lung cancer.
PLoS One,
14(4).
Abstract:
Tumor growth suppression using a combination of taxol-based therapy and GSK3 inhibition in non-small cell lung cancer.
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.
Abstract.
Author URL.
2018
Borgal L, Wakefield JG (2018). Context-dependent spindle pole focusing.
Essays Biochem,
62(6), 803-813.
Abstract:
Context-dependent spindle pole focusing.
The formation of a robust, bi-polar spindle apparatus, capable of accurate chromosome segregation, is a complex process requiring the co-ordinated 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.
Abstract.
Author URL.
Pellacani C, Bucciarelli E, Renda F, Hayward D, Palena A, Chen J, Bonaccorsi S, Wakefield JG, Gatti M, Somma MP, et al (2018). Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation.
Elife,
7Abstract:
Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation.
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.
Abstract.
Author URL.
2017
Chen JWC, Chen ZA, Rogala KB, Metz J, Deane CM, Rappsilber J, Wakefield JG (2017). Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle.
Biol Open,
6(5), 654-663.
Abstract:
Cross-linking mass spectrometry identifies new interfaces of Augmin required to localise the γ-tubulin ring complex to the mitotic spindle.
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.
Abstract.
Author URL.
2016
Cicconi A, Micheli E, Vernì F, Jackson A, Gradilla AC, Cipressa F, Raimondo D, Bosso G, Wakefield JG, Ciapponi L, et al (2016). The Drosophila telomere-capping protein verrocchio binds single-stranded DNA and protects telomeres from DNA damage response.
Nucleic Acids Research,
45(6), 3068-3085.
Abstract:
The Drosophila telomere-capping protein verrocchio binds single-stranded DNA and protects telomeres from DNA damage response
© the Author(s) 2016. Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, themulti-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin.
Abstract.
2015
Conduit PT, Hayward D, Wakefield JG (2015). Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos. In (Ed)
, Academic Press Inc.
Abstract:
Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos
Abstract.
Conduit PT, Hayward D, Wakefield JG (2015). Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos. In (Ed)
, 229-249.
Abstract:
Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos
Abstract.
Palumbo V, Pellacani C, Heesom KJ, Rogala KB, Deane CM, Mottier-Pavie V, Gatti M, Bonaccorsi S, Wakefield JG (2015). Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex.
Current Biology,
25(13), 1777-1783.
Abstract:
Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex
Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.
Abstract.
Chen JWC, Barker AR, Wakefield JG (2015). The Ran Pathway in Drosophila melanogaster Mitosis.
Front Cell Dev Biol,
3Abstract:
The Ran Pathway in Drosophila melanogaster Mitosis.
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.
Abstract.
Author URL.
2014
Hayward D, Wakefield JG (2014). Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos.
Communicative and Integrative Biology,
7(4).
Abstract:
Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos
Upon entry into mitosis, many microtubules are nucleated that coordinately integrate into a stable, yet dynamic, mitotic spindle apparatus. In a recent publication, we examined microtubule-generating pathways within a single model system, the Drosophila syncytial embryo. We found that, following depolymerisation of metaphase spindle microtubules by cold treatment, spindles regenerate predominantly from microtubules nucleated within the vicinity of chromatin. We also showed this chromatin-mediated microtubule nucleation is mediated by the Drosophila homolog of a vertebrate spindle assembly factor (SAF), HURP and is dependent on the conserved microtubule amplifying protein complex, Augmin. Here, we expand our investigation into Drosophila SAFs, providing evidence that, in vitro, both D-HURP and D-TPX2 are able to bind to and stabilize microtubules. We show that GFP-D-HURP purified from embryos interacts with Importin-ß and Augmin and, consistent with this, demonstrate that the underlying basis of chromatin-mediated microtubule nucleation in Drosophila syncytial embryos is dependent on Ran-GTP. © 2014 Landes Bioscience.
Abstract.
Palumbo V, Pellacani C, Heesom KJ, Rogala KB, Deane CM, Mottier-Pavie V, Gatti M, Bonaccorsi S, Wakefield JG (2014). Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex.
Current BiologyAbstract:
Misato Controls Mitotic Microtubule Generation by Stabilizing the Tubulin Chaperone Protein-1 Complex
Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated invivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. Palumbo etal. show the conserved protein Misato interacts with the Tubulin Chaperone Protein-1 (TCP-1) complex in Drosophila. In the absence of Misato, TCP-1 subunit levels are reduced, functional Tubulin is unstable, and mitotic microtubule generation is compromised. The predicted structure of Misato suggests it acts as a molecular placeholder in the absence of Tubulin, stabilizing the TCP-1 complex.
Abstract.
Hayward D, Metz J, Pellacani C, Wakefield JG (2014). Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation.
Dev Cell,
28(1), 81-93.
Abstract:
Synergy between multiple microtubule-generating pathways confers robustness to centrosome-driven mitotic spindle formation.
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.
Abstract.
Author URL.
2012
Hayward D, Wakefield JG (2012). Mars and Mei-38: the roles of two spindle assembly factors in <i>Drosophila</i> syncytial embryos.
Author URL.
Chen JW-C, Wakefield JG (2012). The importance of Augmin in microtubule generation beyond cell division.
Author URL.
Hayward D, Wakefield JG (2012). The inter-relationship between spindle assembly pathways in <i>Drosophila</i> syncytial embryos.
Author URL.
2011
Duncan T, Wakefield JG (2011). 50 ways to build a spindle: the complexity of microtubule generation during mitosis.
Chromosome Res,
19(3), 321-333.
Abstract:
50 ways to build a spindle: the complexity of microtubule generation during mitosis.
The accurate segregation of duplicated chromosomes, essential for the development and viability of a eukaryotic organism, requires the formation of a robust microtubule (MT)-based spindle apparatus. Entry into mitosis or meiosis precipitates a cascade of signalling events which result in the activation of pathways responsible for a dramatic reorganisation of the MT cytoskeleton: through changes in the properties of MT-associated proteins, local concentrations of free tubulin dimer and through enhanced MT nucleation. The latter is generally thought to be driven by localisation and activation of γ-tubulin-containing complexes (γ-TuSC and γ-TuRC) at specific subcellular locations. For example, upon entering mitosis, animal cells concentrate γ-tubulin at centrosomes to tenfold the normal level during interphase, resulting in an aster-driven search and capture of chromosomes and bipolar mitotic spindle formation. Thus, in these cells, centrosomes have traditionally been perceived as the primary microtubule organising centre during spindle formation. However, studies in meiotic cells, plants and cell-free extracts have revealed the existence of complementary mechanisms of spindle formation, mitotic chromatin, kinetochores and nucleation from existing MTs or the cytoplasm can all contribute to a bipolar spindle apparatus. Here, we outline the individual known mechanisms responsible for spindle formation and formulate ideas regarding the relationship between them in assembling a functional spindle apparatus.
Abstract.
Author URL.
Li S, Wakefield J, Noble JA (2011). AUTOMATED SEGMENTATION AND ALIGNMENT OF MITOTIC NUCLEI FOR KYMOGRAPH VISUALISATION. 2011 IEEE International Symposium on Biomedical Imaging: from Nano to Macro.
Wakefield JG (2011). Foreword: chromosomes and microtubules--the dynamic duo of mitosis.
Chromosome Res,
19(3), 269-273.
Author URL.
Antrobus R, Wakefield JG (2011). Isolation, identification, and validation of microtubule-associated proteins from Drosophila embryos. In (Ed)
, 273-291.
Abstract:
Isolation, identification, and validation of microtubule-associated proteins from Drosophila embryos.
Abstract.
Author URL.
2009
Wainman A, Buster DW, Duncan T, Metz J, Ma A, Sharp D, Wakefield JG (2009). A new Augmin subunit, Msd1, demonstrates the importance of mitotic spindle-templated microtubule nucleation in the absence of functioning centrosomes.
Genes Dev,
23(16), 1876-1881.
Abstract:
A new Augmin subunit, Msd1, demonstrates the importance of mitotic spindle-templated microtubule nucleation in the absence of functioning centrosomes.
The Drosophila Augmin complex localizes gamma-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.
Abstract.
Author URL.
Meireles AM, Fisher KH, Colombié N, Wakefield JG, Ohkura H (2009). Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis.
J Cell Biol,
184(6), 777-784.
Abstract:
Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis.
The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex.
Abstract.
Author URL.
2008
Hughes JR, Meireles AM, Fisher KH, Garcia A, Antrobus PR, Wainman A, Zitzmann N, Deane C, Ohkura H, Wakefield JG, et al (2008). A microtubule interactome: complexes with roles in cell cycle and mitosis.
PLoS Biol,
6(4).
Abstract:
A microtubule interactome: complexes with roles in cell cycle and mitosis.
The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.
Abstract.
Author URL.
Buttrick GJ, Beaumont LMA, Leitch J, Yau C, Hughes JR, Wakefield JG (2008). Akt regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo.
J Cell Biol,
180(3), 537-548.
Abstract:
Akt regulates centrosome migration and spindle orientation in the early Drosophila melanogaster embryo.
Correct positioning and morphology of the mitotic spindle is achieved through regulating the interaction between microtubules (MTs) and cortical actin. Here we find that, in the Drosophila melanogaster early embryo, reduced levels of the protein kinase Akt result in incomplete centrosome migration around cortical nuclei, bent mitotic spindles, and loss of nuclei into the interior of the embryo. We show that Akt is enriched at the embryonic cortex and is required for phosphorylation of the glycogen synthase kinase-3beta homologue Zeste-white 3 kinase (Zw3) and for the cortical localizations of the adenomatosis polyposis coli (APC)-related protein APC2/E-APC and the MT + Tip protein EB1. We also show that reduced levels of Akt result in mislocalization of APC2 in postcellularized embryonic mitoses and misorientation of epithelial mitotic spindles. Together, our results suggest that Akt regulates a complex containing Zw3, Armadillo, APC2, and EB1 and that this complex has a role in stabilizing MT-cortex interactions, facilitating both centrosome separation and mitotic spindle orientation.
Abstract.
Author URL.
Gao S, Giansanti MG, Buttrick GJ, Ramasubramanyan S, Auton A, Gatti M, Wakefield JG (2008). Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis.
J Cell Biol,
180(3), 521-535.
Abstract:
Australin: a chromosomal passenger protein required specifically for Drosophila melanogaster male meiosis.
The chromosomal passenger complex (CPC), which is composed of conserved proteins aurora B, inner centromere protein (INCENP), survivin, and Borealin/DASRA, localizes to chromatin, kinetochores, microtubules, and the cell cortex in a cell cycle-dependent manner. The CPC is required for multiple aspects of cell division. Here we find that Drosophila melanogaster encodes two Borealin paralogues, Borealin-related (Borr) and Australin (Aust). Although Borr is a passenger in all mitotic tissues studied, it is specifically replaced by Aust for the two male meiotic divisions. We analyzed aust mutant spermatocytes to assess the effects of fully inactivating the Aust-dependent functions of the CPC. Our results indicate that Aust is required for sister chromatid cohesion, recruitment of the CPC to kinetochores, and chromosome alignment and segregation but not for meiotic histone phosphorylation or spindle formation. Furthermore, we show that the CPC is required earlier in cytokinesis than previously thought; cells lacking Aust do not initiate central spindle formation, accumulate anillin or actin at the cell equator, or undergo equatorial constriction.
Abstract.
Author URL.
Buttrick GJ, Wakefield JG (2008). PI3-K and GSK-3: Akt-ing together with microtubules.
Cell Cycle,
7(17), 2621-2625.
Abstract:
PI3-K and GSK-3: Akt-ing together with microtubules.
Phosphoinositide-3-Kinase (PI3-K) and the downstream kinases Akt and Glycogen Synthase Kinase-3 (GSK-3) have recently been implicated in regulating both microtubule (MT) dynamics and organization. Here we review the role of this signalling pathway in controlling MTs, and explore ways in which the kinases and their substrates may co-operate to spatially regulate MTs in different contexts.
Abstract.
Author URL.
Beaumont LMA, Wakefield J, Noble JA (2008). SPATIOTEMPORAL BAYESIAN CELL POPULATION TRACKING AND ANALYSIS WITH LINEAGE CONSTRUCTION. 2008 5th IEEE International Symposium on Biomedical Imaging: from Nano to Macro.
Fisher KH, Deane CM, Wakefield JG (2008). The functional domain grouping of microtubule associated proteins.
Commun Integr Biol,
1(1), 47-50.
Abstract:
The functional domain grouping of microtubule associated proteins.
Microtubules (MTs), which play crucial roles in normal cell function, are regulated by MT associated proteins (MAPs). Using a combinatorial approach that includes biochemistry, proteomics and bioinformatics, we have recently identified 270 putative MAPs from Drosophila embryos and characterized some of those required for correct progression through mitosis. Here we identify functional groups of these MAPs using a reciprocal hits sequence alignment technique and assign InterPro functional domains to 28 previously uncharacterized proteins. This approach gives insight into the potential functions of MAPs and how their roles may affect MTs.
Abstract.
Author URL.
2007
Yau C, Wakefield J (2007). QUANTITATIVE IMAGE ANALYSIS OF CHROMOSOME DYNAMICS IN EARLY DROSOPHILA EMBRYOS. 2007 4th IEEE International Symposium on Biomedical Imaging: from Nano to Macro.
2004
Butcher RDJ, Chodagam S, Basto R, Wakefield JG, Henderson DS, Raff JW, Whitfield WGF (2004). The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division.
J Cell Sci,
117(Pt 7), 1191-1199.
Abstract:
The Drosophila centrosome-associated protein CP190 is essential for viability but not for cell division.
The Drosophila CP190 and CP60 proteins interact with each other and shuttle between the nucleus in interphase and the centrosome in mitosis. Both proteins can bind directly to microtubules in vitro, and have been shown to associate with a specific pattern of loci on salivary gland polytene chromosomes, but their functions are unknown. Here we show that reducing the level of CP190 or CP60 by >90% in tissue culture cells does not significantly interfere with centrosome or microtubule organisation, with cell division, or with cell viability. However, CP190 is an essential protein, as flies homozygous for mutations in the Cp190 gene die at late pupal stages of development. In larval brains of Cp190 mutants, mitosis is not radically perturbed, and a mutated form of CP190 (CP190DeltaM), that cannot bind to microtubules or associate with centrosomes, can rescue the lethality associated with mutations in the Cp190 gene. Thus, CP190 plays an essential role in flies that is independent of its association with centrosomes or microtubules.
Abstract.
Author URL.
2003
Wakefield JG, Stephens DJ, Tavaré JM (2003). A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment.
J Cell Sci,
116(Pt 4), 637-646.
Abstract:
A role for glycogen synthase kinase-3 in mitotic spindle dynamics and chromosome alignment.
Glycogen synthase kinase-3 (GSK-3) is a conserved, multifunctional kinase that is constitutively active in resting cells, and inactivated through phosphorylation by protein kinase B (PKB). We have investigated the temporal and spatial control of GSK-3 phosphorylation during the cell cycle in mammalian cells. We show that GSK-3 is present along the length of spindle microtubules and that a fraction of GSK-3 is phosphorylated during mitosis. Phospho-GSK-3 is abundant at the centrosomes and spindle poles but absent from other areas of the spindle. GSK-3 phosphorylation occurs concomitantly with the appearance of phosphorylated and active PKB at the centrosome, which suggests that PKB is the kinase responsible for phosphorylating and inactivating GSK-3 at the centrosome during mitosis. We demonstrate that lithium and two structurally distinct inhibitors of GSK-3 promote defects in microtubule length and chromosomal alignment during prometaphase. Treated cells contain mono-oriented chromosomes concentrated at the plus ends of astral microtubules, which are longer than in untreated cells. Live microscopy of cells expressing Histone-2B-GFP confirms that the inhibition of GSK-3 suppresses mitotic chromosome movement and leads to a prometaphase-like arrest. We propose that GSK-3 is regulated in a temporal and spatial manner during mitosis and, through controlling microtubule dynamics, plays an important role in chromosomal alignment on the metaphase plate.
Abstract.
Author URL.
2001
Tavaré JM, Fletcher LM, Oatey PB, Tyas L, Wakefield JG, Welsh GI (2001). Lighting up insulin action.
Diabet Med,
18(4), 253-260.
Abstract:
Lighting up insulin action.
Understanding the mechanism of insulin action remains one of the most important challenges in modern medical biology. Recent advances in cell imaging techniques, increased processing power of computers and the internet, and the introduction of novel fluorescent reagents such as green fluorescent proteins (GFPs) have revolutionized our ability to scrutinize insulin action by time-lapse microscopy at the single-cell level. This article outlines some of the advances made in the authors' laboratory, with particular reference to imaging the movements of the insulin-sensitive glucose transporter, GLUT4, and the generation of phosphoinositide lipids.
Abstract.
Author URL.
Wakefield JG, Bonaccorsi S, Gatti M (2001). The Drosophila protein Asp is involved in microtubule organization during spindle formation and cytokinesis.
Journal of Cell Biology,
153(4), 637-647.
Abstract:
The Drosophila protein Asp is involved in microtubule organization during spindle formation and cytokinesis
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 γ-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, 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.
Abstract.
2000
Raff JW, Huang JY, Wakefield JG (2000). A role for centrosomes in initiating the destruction of cyclin B in early Drosophila embryos.
MOLECULAR BIOLOGY OF THE CELL,
11, 91A-91A.
Author URL.
Wakefield JG, Huang JY, Raff JW (2000). Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos.
Curr Biol,
10(21), 1367-1370.
Abstract:
Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos.
We reported previously that the disappearance of cyclin B at the end of mitosis in early Drosophila embryos starts at centrosomes and spreads into the spindle [1]. Here, we used a novel mutation, centrosome fall off (cfo), to investigate whether centrosomes are required to initiate the disappearance of cyclin B from the spindle. In embryos laid by homozygous cfo mutant mothers, the centrosomes co-ordinately detached from the mitotic spindle during mitosis, and the centrosomeless spindles arrested at anaphase. Cyclin B levels decreased on the detached centrosomes, but not on the arrested centrosomeless spindles, presumably explaining why the spindles arrest in anaphase in these embryos. We found that the expression of a non-degradable cyclin B in embryos also caused an anaphase arrest, but most centrosomes remained attached to the arrested spindles, and non-degradable cyclin B levels remained high on both the centrosomes and spindles. These findings suggest that the disappearance of cyclin B from centrosomes and spindles is closely linked to its destruction, and that a connection between centrosomes and spindles is required for the proper destruction of the spindle-associated cyclin B in early Drosophila embryos. These results may have important implications for the mechanism of the spindle-assembly checkpoint, as they suggest that unattached kinetochores may arrest cells in mitosis, at least in part, by signalling to centrosomes to block the initiation of cyclin B destruction.
Abstract.
Author URL.
Gergely F, Kidd D, Jeffers K, Wakefield JG, Raff JW (2000). D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo.
EMBO J,
19(2), 241-252.
Abstract:
D-TACC: a novel centrosomal protein required for normal spindle function in the early Drosophila embryo.
We identify Drosophila TACC (D-TACC) as a novel protein that is concentrated at centrosomes and interacts with microtubules. We show that D-TACC is essential for normal spindle function in the early embryo; if D-TACC function is perturbed by mutation or antibody injection, the microtubules emanating from centrosomes in embryos are short and chromosomes often fail to segregate properly. The C-terminal region of D-TACC interacts, possibly indirectly, with microtubules, and can target a heterologous fusion protein to centrosomes and microtubules in embryos. This C-terminal region is related to the mammalian transforming, acidic, coiled-coil-containing (TACC) family of proteins. The function of the TACC proteins is unknown, but the genes encoding the known TACC proteins are all associated with genomic regions that are rearranged in certain cancers. We show that at least one of the mammalian TACC proteins appears to be associated with centrosomes and microtubules in human cells. We propose that this conserved C-terminal 'TACC domain' defines a new family of microtubule-interacting proteins.
Abstract.
Author URL.
1998
Wakefield JG, Oegema K, Raff JW (1998). The centrosomal protein CP60 is required for the interaction between centrosomes and microtubules during anaphase.
Author URL.
1996
Wakefield JG, Raff JW (1996). Characterisation of CP60, a Drosophila centrosomal map.
Author URL.
