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Professor James Wakefield
Professor of Integrative Cellular Biology
Geoffrey Pope
My research interest has always been that of mitosis and cell division, stimulated by the fundamental beauty of the process as viewed using a fluorescence microscope, and its key role in diseases such as cancer. Cell division is orchestrated by two main microtubule (MT) based structures called the 'spindle apparatus' and the 'central spindle', which function at different times to co-ordinate this dynamic process. We want to understand how these systems organise themselves, and what governs the similarities and differences between them in diverse types of cell, at different stages of development, and during disease. In order to learn as much as possible, we take a multi-disciplinary approach, combining proteomics, bioinformatics and quantitative image analysis with qualitative, descriptive cell and developmental biology, and genetics. I am a member of the Cell Biology research group.
1999 PhD Wellcome/CRUK Institute for Cancer Research and Developmental Biology, University of Cambridge, UK
1995 BSc (Hons) in Biochemistry, University of Bristol
2018-current Professor of Integrative Cellular Biology and Head of Department of Biosciences, University of Exeter
2013-2018 Associate Professor if Integrative Cellular Biology, University of Exeter
2010-2013 Senior Lecturer in Cell Biology, School of Biosciences, University of Exeter
2007-2009 Director, Life Sciences Interface Doctoral Training Centre, University of Oxford
2004-2007 Lecturer in Biology (Deputy Director) Life Sciences Interface Doctoral Training Centre, University of Oxford
2003–2004 Departmental Lecturer in Biology / Co-ordinator, MSc in Integrative Biosciences, University of Oxford
2000-2002 MRC Postdoctoral Research Associate, University of Bristol
1999-2000 EU TMR Postdoctoral Research Associate, Dipartmento di Genetica e Biologie Molecolare, University of Rome, Italy
The Wakefield Lab website
Background (General)
Every living thing renews itself by a process of cell division. In order to produce two new identical cells from one existing cell, the genetic material, the chromosomes, must first be replicated and the volume of the cell must increase. During the process defined as cell division itself, these duplicated chromosomes align in the centre of the cell before one copy of each chromosome is moved to opposite sides of the cell. Finally, the cell physically cleaves in two, usually at the centre of the cell, producing two daughter cells, each identical to themselves and to the original cell.
All cells use protein fibres termed microtubules (MTs) order to faithfully divide. These fibres are made up of repeating units of a protein called tubulin that can be added or taken away from the ends of existing MTs, allowing them to grow and shrink. Other proteins in the cell, called MT associated proteins (MAPs) are able to alter the properties of the MTs, causing them to grow or shrink more quickly, to link existing MTs to each other, or to link the ends of the MTs to chromosomes or other structures in the cell. In this way, MTs can be organised into dynamic structures, capable of doing different things. During cell division, two main MT structures are important - the spindle apparatus, which makes sure the chromosomes line up and are moved apart correctly, and the central spindle, that allows the cell to cleave precisely in two.
Background (Technical)
Cell division is orchestrated by a microtubule (MT) based structure called the spindle apparatus. In most animal cells, the formation of the spindle apparatus is mediated by complex interactions between MTs nucleated by the centrosome, those organised by mitotic DNA, and those seeded by the growing spindle itself. These populations of MTs are organised by a multitude of MT associated proteins (MAPs), including protein motor complexes, to form a polarized and focused spindle capable of chromosome segregation. Following movement of sister chromatids to the spindle poles in anaphase, a bundle of MTs form between the separating DNA. This central spindle assists in the formation of the contractile ring, a transient actin and myosin-II based structure, which is then able to direct cytokinesis.
Aim of the lab
We want to understand how the spindle apparatus and central spindle are organised, and what governs the similarities and differences between these structures in different types of cell. In order to learn as much as possible about this process, we use a combination of quantitative analytical methods and qualitative, descriptive biology.
Why fruit flies?
Drosophila melanogaster has proved to be an excellent system both for studying cell division, and for the isolation of proteins that function during mitosis. There are a number of other labs throughout the world working on distinct aspects of cell division in this model organism. It is a close-knit community of scholars who are open and enthusiastic about advancing the field as quickly as possible.
Other research interests
Obviously, I look to oversee the research being undertaken in the lab in general. In addition, I am interested in the relationship between different scientific approaches, and the way in which they can be combined to provide advances in our understanding of biological processes. The move towards quantitative techniques, driven by technological advances in physical science methods, coupled to the wealth of information accumulated within biology using high-throughput approaches, is now allowing us to measure and analyse sub-cellular processes with a degree of accuracy not previously possible. This provides the researcher with "real" data, which can be combined with statistical methods and powerful computational tools to build models of biological processes that can be tested. However, I believe it is important to view such analyses within the context of the biological process being studied. Humans are able to process visual information and relate it to their previous experiences and accumulated knowledge in a manner impossible for computers. Immersing oneself in the viewing experience can lead to a greater understanding of the way in which a biological process 'works'. Such experiential, intuitive biology has been practiced for centuries and has led to many of the great advances in biology, medicine and related disciplines. We currently have a research grant, funded by the Arts and Humanities Research Council (AHRC), in collaboration with a Philosopher of Biology, Prof John Dupre, and artist Gemma Anderson, exploring the value of drawing in the hypothesis-forming process. By combining the recently advanced quantitative techniques and historically valuable qualitative approaches, I believe science will be able to move towards its goal of understanding the world in which we live more quickly than using one approach alone.
1. Identification and characterisation of novel MAPs with roles in cell divis
2. Understanding the way in which the Augmin complex works to supplement microtubule nucleation from within the mitotic spindle
3. Understanding the role of the chromosomal passenger complex (CPC) in central spindle assembly and cytokine
4. Automated quantitative analysis of microtubule and chromosome dynamics
Hiro Ohkura, University of Edinburgh, UK
David Sharp, Einstein College of Medicine, NY, USA
Maria Grazia Giansanti, Universita di Roma 'La Sapienza', Italy
Charlotte Deane, Department of Statistics, University of Oxford, UK
Alison Noble, Department of Engineering, University of Oxford, UK
Wakefield J, Soeller C, Jeynes J, Tariq A (In Press). In vitro reconstitution of branching microtubule nucleation. eLife, 8 Full text.
Palumbo V, Tariq A, Borgal L, Metz J, Brancaccio M, Gatti M, Wakefield JG, Bonaccorsi S (2020). Drosophila Morgana is an Hsp90-interacting protein with a direct role in microtubule polymerization. Journal of Cell Science
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.
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).
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.
Anderson G, Dupre J, Wakefield JG (2019). Drawing and the dynamic nature of living systems. ELIFE, 8 Author URL. Full text.
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, 7
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.
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.
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.
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).
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.
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).
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.
Borgal L, Wakefield JG (2018). Context-dependent spindle pole focusing. Essays Biochem, 62(6), 803-813.
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.
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.
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.
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.
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.
Chen JWC, Barker AR, Wakefield JG (2015). The Ran Pathway in Drosophila melanogaster Mitosis. Front Cell Dev Biol, 3
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.
Hayward D, Wakefield JG (2014). Chromatin-mediated microtubule nucleation in Drosophila syncytial embryos. Communicative and Integrative Biology, 7(4).
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.
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 Biology
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.
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.
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.
Duncan T, Wakefield JG (2011). 50 ways to build a spindle: the complexity of microtubule generation during mitosis. Chromosome Res, 19(3), 321-333.
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.
Wakefield JG (2011). Foreword: chromosomes and microtubules--the dynamic duo of mitosis. Chromosome Res, 19(3), 269-273. Author URL.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
Buttrick GJ, Wakefield JG (2008). PI3-K and GSK-3: Akt-ing together with microtubules. Cell Cycle, 7(17), 2621-2625.
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.
Fisher KH, Deane CM, Wakefield JG (2008). The functional domain grouping of microtubule associated proteins. Commun Integr Biol, 1(1), 47-50.
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.
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.
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.
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.
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.
Tavaré JM, Fletcher LM, Oatey PB, Tyas L, Wakefield JG, Welsh GI (2001). Lighting up insulin action. Diabet Med, 18(4), 253-260.
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.
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.
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.
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.
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.
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.
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.
Conduit PT, Hayward D, Wakefield JG (2015). Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos. In (Ed) , Academic Press Inc.
Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos
Conduit PT, Hayward D, Wakefield JG (2015). Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos. In (Ed) , 229-249.
Antrobus R, Wakefield JG (2011). Isolation, identification, and validation of microtubule-associated proteins from Drosophila embryos. In (Ed) , 273-291.
Isolation, identification, and validation of microtubule-associated proteins from Drosophila embryos.
Hayward D, Wakefield JG (2012). Mars and Mei-38: the roles of two spindle assembly factors in Drosophila 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 Drosophila syncytial embryos. 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.
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.
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.
Wakefield JG, Oegema K, Raff JW (1998). The centrosomal protein CP60 is required for the interaction between centrosomes and microtubules during anaphase. Author URL.
Wakefield JG, Raff JW (1996). Characterisation of CP60, a Drosophila centrosomal map. 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.
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.
james_wakefield Details from cache as at 2022-01-21 10:52:47
Awards/Honorary fellowships
MRC ROPA (Realising Our Potential Award) Jan 2001- April 2002
EU TMR Award, 1999-2000
Wellcome Trust Prize Studentship, 1995-1999
Nuffield Foundation Summer Scholarship, 1994
Chair of Streatham Biosciences Education Team (Aug 2010 - July 2012)
Member of Biosciences Strategy Group (Aug 2010 - July 2012)
Member of CLES Education Strategy Group (Aug 2010 - July 2012)
Member of CLES Management Group (Aug 2010 - July 2012)
Member of EPSRC CDT working group (Jan 2013 - current)
Member of Living Systems Building working group (Sept 2012 - current)
Outside Exeter
Membership Secretary, British Society for Cell Biology (2013 - present)
Chair of LSI DTC Directorate, University of Oxford (Oct 2007 - Dec 2009)
Member of DTC Advisory Board, University of Oxford (Oct 2004 - Dec 2009)
Member of Divisional Education Committee, University of Oxford (Oct 2007 - Dec 2009)
Invited Guest Editor, Chromosome Research, July-Dec 2010
1.EMBO Drosophila Cell Division Cycle Meeting, Sept 2013. Invited talk
2. Gurdon Institute, Cambridge, May 2013. Invited talk
3.UK Drosophila Cell Developmental Biology Workshop, May 2013. Invited talk
4. Birkbeck College, London, April 2013. Invited talk
5.University of Cardiff, December 2012. Invited talk
6. University of Bath, December 2012. Invited talk
7.University of Reading, November 2012. Invited talk
8.Vice Chancellor's Conference, Anglia Ruskin University, September 2012. Invited talk
9. ASCB Annual Meeting, San Diego, USA, December 2009. Invited talk
10. UK Council for Graduate Education, Manchester, June 2009. Invited talk
11. University of Cambridge, September 2007. Invited talk
12. University of Rome, Italy, June 2008. Invited talk
13. EMBO Chromosome Segregation and Aneuploidy Meeting, Finland, June 2007. Invited talk
14. University of Edinburgh, September 2006. Invited talk
15. EMBO Drosophila Cell Cycle Meeting, Porto, Portugal, June 2006. Invited talk
16. University of Bristol, May 2006. Invited talk
17. EMBO Drosophila Cell Cycle Meeting, Siena, Italy, June 2001. Invited talk
External:
Principal Applicant, 3 year Project Grant, BBSRC (2013-2016) – "Microtubule Associated Proteins with roles in mitosis: A systems approach". Total funding – £472,000 FEC
Principal Applicant, 3 year Project Grant, Cancer Research UK (2010-2013) – "The role of the Chromosomal Passenger Complex in central spindle formation and cytokinesis" Total funding £455,000 FEC equivalent
Principal Applicant, 3 year Project Grant, The Wellcome Trust (2004-2007) – "Cellular and molecular analysis of ciottoli, a male meiotic mutant in Drosophila". Total funding £462,000 FEC equivalent
Principal Applicant, 3 year Project Grant, BBSRC (2003-2006) - "Identification of mitotic phospho-proteins in Drosophila". Total funding £535,000 FEC
Principal Applicant, Royal Society (2003-2004) - "Identification of mitotic phospho-proteins in Drosophila". Total funding £14,500 FEC equivalent
Internal (since 2010):
Principal Applicant, University of Exeter ISSF Wellcome Trust Seed Corn Fund (2012/13) – "Generation of affinity purified, fluorescently labelled antibodies to disrupt cell division". Total funding – £13,444 (non-FEC)
Principal Applicant, University of Exeter CLES Strategic Development Fund (2011/12) – "Using quantitative proteomics to understand cell division". Total funding – £19,250 (non-FEC)
Principal Applicant, University of Exeter Bridging the Gap Pilot Grant (2011/12) – "Symmetry breaking during cell division". With Peter Ashwin (Mathematics, Exeter). Total funding – £1,760 (non-FEC)
Principal Applicant, EMBO Workshop Fund (2013) - "University of Exeter / EMBO Drosophila Cell Division Cycle Meeting". Total funding - £36,500
The Dynamic Cell (BSCB Autumn Meeting). For Sept 2014
EMBO Drosophila Cell Division Cycle Meeting. For Sept 2013.
Forum for Scientific Method in Biology, University of Oxford, March 2009.
I currently teach the 1st Term Biology content of our multi-disciplinary, research-driven Natural Sciences Degree undergraduate programme.
Dr Pete Jones

redpajama_set_name: RedPajamaCommonCrawl


text:
Алекса́ндр Васи́льевич Бороду́лин:
 Бородулин, Александр Васильевич (1906—1980) — фрезеровщик Ленинградского металлического завода, Герой Социалистического Труда.
 Бородулин, Александр Васильевич (1921—1984) — советский инженер-конструктор, организатор производства.

redpajama_set_name: RedPajamaWikipedia


text:
Home Chevron icon It indicates an expandable section or menu, or sometimes previous / next navigation options. News
WATCH: Meet the last family practicing this 400-year-old Indian art form
Dylan Barth
Rogan is an ancient Indian art form that has been practiced for 400 years.
The Khatri family has practiced Rogan art for eight generations, and they believe they're the last ones keeping the tradition alive in India.
The rise of industrial textiles in the 1980s put many artisans out of work. And the COVID-19 pandemic has threatened the business as tourism has dried up.
View more episodes of Business Insider Today on Facebook.
The palm of the hand is the essential tool for getting the ancient painting technique of Rogan just right.
Rogan art is believed to have come to India from Persia around 400 years ago. The designs were once common on ceremonial clothing and bed covers throughout India.
Today, the Khatri family says they are the only artisans left in the world practicing this traditional craft.
The base of the paint is castor oil, and it's what gives the art its name — Rogan means oil in Farsi. The oil is heated over a fire for two days until it becomes a honey-like texture. Then, it's combined with crushed pigment to create the paint.
The painting process involves just two tools: a metal stylus and a hand. Abdul Gaffur Khatri, the eldest of the family business, swirls the paint in his hand to create heat and thin it out. He then floats the threat of paint over the fabric using the metal stylus.
While he has an idea of the final design in his mind, the painting process is completely freehand.
"The perfect thread out of this color, which can run on cloth, is in the mind of the artist," Khatri told Business Insider. "It is all God's gift. It cannot be done by everyone or by any machine."
Khatri can finish some pieces in five days, while more complex designs can take him up to two years. Larger wall art pieces can cost over $1,000.
A metal stylus and one's hand are the only things needed to create Rogan art.
Prakhar Jain for Business Insider Today
The Khatri family normally sell their pieces to tourists in the town of Bhuj, about 25 miles from the small village of Nirona, where they work. This year, the pandemic put a strain on their peak season.
"I can tell you that we have incurred losses because tourists didn't come, and this business depends on them. There's no other source. So what do we do?" Khatri told Business Insider, "Now we are just waiting and watching to see what will happen next."
The struggle feels familiar — the rise of mass produced textiles in the 1980s forced many artisans to drop their crafts and look for more lucrative work. Khatri made the hard decision to leave the family business and search for opportunities in Mumbai.
"In 1980 I decided that I will not waste my life on this art," Khatri told Business Insider, "And there were some arguments in the family due to that because at that time sending a kid from a village to Mumbai was a big deal."
Khatri returned to his village just a few years later after his family plead with him to carry on the tradition.
"I remember at that time I promised him that I'll dedicate my whole life to this art," Khatri told Business Insider. "And now I can say that promise is complete."
Since then, the Khatri family has brought rogan art to the international level. They've earned over 10 awards for their work, and India's Prime Minister even gifted one of their pieces to President Barack Obama in 2014.
Now, the family uses social media and education to spread awareness about this rare art form. Since starting a program in 2010 to grow and diversify the village's Rogan artists, they've trained around 300 women, 20 of whom went on to work for the family.
Though they've since had to lay off those employees due to the pandemic, they plan to hire them back when business returns to normal.


When a dog frightens mama opossum, Baby-O flings from her mama's back and begins an exciting adventure.
Our local wildlife rescue organization, Shasta Wildlife Rescue and Rehabilitation (SWRR), holds an Annual Open House and Baby Shower event at Anderson River Park, in Anderson, CA. Educational animals will be on hand and there'll be activities for children. Tim and I will be there as well.
My first introduction to SWRR was over 20 years ago when a turkey vulture fledgling took up residence in our henhouse. A few years later I discovered a mama opossum roadkill with live babies still in her pouch along our little country road. In each instance, SWRR volunteers gathered the young and cared for them until they were able to make it on their own in the wild.
Shasta Wildlife Rescue volunteer feeds an orphaned baby opossum.
A few years back, Karlene Stoker (SWRR volunteer), asked if I would write a story about a baby opossum. Karlene does a lot of opossum rehabilitation in her role as a volunteer. She figured if I could make turkey vultures sympathetic in No Place for Ugly Birds, then a sympathetic story about a baby opossum wouldn't be too far of a stretch.
Thus, Oh No! Baby-O was conceived. Oh, No! Baby-O has it's official release/birthday later this summer (August 2015). But today, there's a special surprise at the SWRR Open House and Baby Shower. A limited number of advanced copies will go on sale as a fundraiser for SWRR! Isn't that exciting!
So, if you're in the area, come on by. Don't forget your Orphaned Baby Animal Shower gift. Here's a list.
Why bring a shower gift?
First, this is one of the ways SWRR is supported.
Second, all persons bringing gifts will be entered into a drawing for this piece of original art.
Baby Opossum in Dogwood Blossom – Donated original painting for Shasta Wildlife Rescue and Rehabilitation.
The drawing will be held at 1 p.m.
I have a new post up over at Children's Book Academy… I'd love to know what you think.
A Child's Take on the Digital vs Print Debate with Mary A Livingston – Children's Book Academy.
The Children's Book Academy is offering a spectacular course for children's writers.
And today's the last day for Early Bird registration!


Size: 37 x 8 x 13 H"
This beautiful Orange Colored Coral Trout is complete with hooks to hang. The coral trout is a species of fish in the Serranidae family. Native to the western Pacific Ocean, its natural habitat includes open seas and coral reefs. This item is NEW, painstakingly hand crafted and hand painted with pride in the Philippines using only the finest quality materials. Truly whimsical home decor masterpieces which offer a variety of functional uses and a broad appeal to collectors of every type. These treasures bring character to any home, office, business or commercial institution. Check out other great items from LM Treasures.


Texas Best Preowned is proud to offer the industry's best auto warranty program. Most cars we sell include a 3 month/5000 mile warranty at no extra charge. For peace of mind we also offer a selection of extended auto warranty programs through Preferred Warranties.
What do I do if my car breaks down or won't start?
No problem. You're covered for up to $75 of emergency assistance for towing, dead battery and more - even flat tires or running out of gas. Just call our 24-hour toll-free number, and keep your money in your pocket. Before being towed, call the PWI Claims Department for a nearby shop.
Call us.We'll make sure your problem is covered, and tell you what to do next. Our phones are staffed during traditional business hours, and our answering service is available to take messages 24 hours a day. Or visit us online to open a claim, and we'll call you back during business hours.
Yes. We'll be glad to work with them, and our experienced claims managers can make sure that the repairs are made right and the costs are fair. If you don't have a favorite shop, we can probably refer you to one in our network of quality repair centers.
We make every effort to pay the shop directly so you keep your money in your pocket. If you choose a shop that won't accept direct payment, we'll reimburse you promptly.
Less than a week. Once the repair is finished and we've received the paperwork, your claim will be processed and mailed within five working days. Guaranteed!
What if something breaks when I'm out of town?
With our vast network, we'll probably be able to find a quality repair center for you anywhere in the country.
How do I get a Preferred Warranty?
What has made Preferred Warranties Inc. one of the fastest growing Extended Service Contract providers in the U.S.?
Dependability. Preferred Warranties are available only through select dealerships. They've proven that they know cars, and that they deal in quality, dependable pre-owned vehicles. But whether a car is new or used, foreign or domestic, things break. This is why the warranty you buy is every bit as important as the vehicle you buy. And this is why Preferred Warranties are being chosen by more and more car buyers every day. When something breaks, Preferred Warranties has you covered. If the problem area is listed in the Preferred Warranty plan you've chosen, you can count on quality repairs and fast, hassle-free payment. Period. You get peace-of-mind. We get the best advertising there is: the recommendations and loyalty of satisfied customers and dealers.
Claims paid in 5 days-guaranteed!
FROM A QUALITY COMPANY THAT GOES THE EXTRA MILE.
A winner of the Northeastern Pennsylvania Better Business Bureau's "Torch Award for Marketplace Ethics"
WARRANTIES & SERVICE THAT GO THE EXTRA MILE.


text:
Video of PZ timeline update 2015 HQ
PZ Cussons is a multinational consumer goods business. We manufacture and distribute some of the most familiar household brands in the world, from Imperial Leather to Cussons Baby and Morning Fresh. We operate in four core categories – personal care, home care, food and nutrition and electricals. Worldwide we employ over 5000 people in Africa, Asia and Europe & North America.
PZ Cussons' history dates back to the 1800's , when the minds of two extraordinary people – George Paterson and George Zochonis – met in Sierra Leone and embarked on a journey of exploration and opportunity. Find out more about our unique history.
We're an entrepreneurial, international company, committed to enhancing lives of consumers, with quality, value and innovation. Find out more about our vision, mission, and the values that are alive in our people and the way we do business every day.
Meet our super team of Executive and Non Executive Directors. Passionate experts, yet always keen to learn, discover and innovate, they lead by inspiring example.
Ashanti Region:
Continental (Kumasi)
M Osei Akoto (Kumasi)
Kofi Gyimah (Kumasi)
Obek Ent P(ankrono Ashanti)
Yahweh Is Glory (Obuasi)
Brong-Ahafo Region:
PZ Cussons is incredibly proud to be the business behind some of the world's best known and best loved brands. We believe in doing good business and leading by example on a global scale. In short, we think differently to your average multinational and it shows in our continued success.


text:
Q: Can the text content of an element be read in Angular without reading from the DOM? I need to be able to use translated strings in code in my Angular application, but with the i18n tools not yet up to the task, I implemented a slightly hacky version that takes advantage of Angular's existing i18n capabilities, with the intention of phasing out my solution for native Angular as its i18n capabilities catch up to my needs (should be a 5.x release, fingers crossed).
Basically, I have a TranslationDirective that reads text from the DOM, and emits events when the text changes:
@Directive({
  selector: '[myAppTranslation]'
})
export class TranslationDirective implements AfterViewChecked, OnChanges {
  /**
   * dependencies takes an array of the values needed to calculate the output translation
   * we use this for change detection, to minimize the DOM interaction to only when it is necessary
   */
  @Input() dependencies: any[];
  isDirty = true;
  @Input() messageKey: string;
  message: string;
  @Output() messageUpdated = new EventEmitter<TranslationEvent>();

  constructor(public el: ElementRef) {}

  /**
   * sets the translated message and triggers the TranslationEvent
   */
  setMessage() {
    const message = (this.el.nativeElement.textContent || '').trim();
    const oldMessage = (this.message || '');
    if (oldMessage !== message) {
      this.message = message;
      this.isDirty = false;
      this.triggerTranslationEvent();
    }
  }

  ngOnChanges() {
    this.isDirty = true;
  }

  ngAfterViewChecked() {
    if (this.isDirty) {
      this.setMessage();
    }
  }

  /**
   * triggers the messageUpdated EventEmitter with the TranslationEvent
   */
  triggerTranslationEvent() {
    // need to delay a tick so Angular doesn't throw an ExpressionChangedAfterItHasBeenCheckedError
    setTimeout(() => {
      const event = new TranslationEvent(this.messageKey, this.message);
      this.messageUpdated.emit(event);
    });
  }
}

export class TranslationEvent {
  constructor(public messageKey: string, public message: string) {}
}

which gets used like this:
<span
  myAppTranslation
  i18n
  [dependencies]="[today]"
  [messageKey]="todaysDateKey"
  (messageUpdated)="setTodaysDateTranslation($event)"
>
  Today is {{today | date:'short'}}
</span>

Since the strings to be translated all reside in the templates, Angular's xi18n tool reads them just fine, and the Angular compiler will replace them with the translated strings.
This is functional, but its not great. I suspect that there is a timing bug just waiting to bite me that just hasn't manifested yet. There is an inefficient and slow write-to-DOM-read-from-DOM cycle that would be really nice to eliminate.
I'd like to be able to eliminate one source of problems by bypassing the DOM if I can at all avoid it. Does Angular keep an in-memory copy of the contents of an element that is accessible without going through the DOM? If that's possible, can I avoid writing the translation element to the DOM entirely?

A: It looks like a lot of your complexity is because you want to support dynamic text - that the text might change at run-time. I don't think you need to do that, because i18n text needs to be static so that:

*

*It can be extracted at compile-time.

*It can be translated in advance, and you have all the translations stored at a later compile-time.
Your example "Today is ..." is a perfect example of untranslatable text. :-) You should change it to just have "Today is" be a text chunk by itself, and then have the date displayed with the current locale outside of that span.

redpajama_set_name: RedPajamaStackExchange


text:
Q: Perl delete similar strings in an array I am new to Perl programming.
I need to find and delete partially matching strings in an array.
For example in my array there are strings:
@array = qw(abcd.txt abcdeff.txt abcdweff.txt abcdefrgt.txt);

I just want the first abcd.txt to be saved to the array and to delete the rest (which are similar in the first 4 characters) i.e. so that it will print only abcd.txt when @print "@array"; is called.

A: my %seen;
@array = grep !$seen{ substr($_,0,4) }++, @array;


Many Envato Market items can be described as "what you see is what you get", for example, a photo from PhotoDune or background image from GraphicRiver. These are straightforward and easy for customers to use. However, complex items like themes and plugins often require detailed documentation and comprehensive FAQs alongside the item as customers are more likely to need help with the features and functionality. There is no one better placed to provide that assistance than you, the author who created the item!
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The customer also has the option to purchase an additional six months at this time (support upgrade) or extend or renew support at a later time. Therefore, your obligated support period may extend beyond the first six months. Refer to this article for more information about support extensions and renewals.
Sales will appear on your statement as separate line items for the item price and any support components that are purchased. This article provides a complete guide to understanding your statements.
To avoid any confusion between you and your customers about your support obligations, our item support policy clearly outlines what is and what is not included.
Answer technical questions about features and functionality.
Help with third-party assets sold with the item (eg. bundled plugins).
You are not required to install or customize your items or assist with hosting or third party software.
As a minimum, all items (supported and unsupported) must work as described, be protected against major security concerns, and have version upgrades (at the author's discretion).
Before selling items on ThemeForest and CodeCanyon, please read the policy in detail so that you have a clear understanding of your obligations to your customers.


April 24, 2005 - The first successsfully cloned dog was born on this day. Snuppy was created through an elaborate process that involved taking skin cells from an adult Afghan hound and implanting them inside the fertile eggs of 123 surrogate moms.
Snuppy was the only pup to survive the experiment and by all accounts he is still going strong.
The controversial veterinarian and researcher who brought Snuppy to life has since founded a company that clones dogs for about $100,000 a pop. According to one report he had cloned over 700 dogs as of 2015.


Not a sheep: Why Jeremy Corbyn can't implement the full IHRA definition of Antisemitism?
Why Jeremy Corbyn can't implement the full IHRA definition of Antisemitism?
'The Mail on Sunday has unearthed a previously unpublicised video showing him speaking at a pro- Palestine meeting in 2012 which included the reading of a poem that said the 'victims of Auschwitz' were now the 'torturers'.
More here http://www.dailymail.co.uk/news/article-6027151/Corbyn-allegedly-called-Dame-Louise-Ellman-Honourable-Member-Tel-Aviv.html but obviously not on the Labour Party's propaganda arm, the BBC.
I wonder how many BBC 'journalists' would also fall foul of the IHRA definition of anti-Semitism?


They have dragon in their name/title (Half-Dragon doesn't count because it is hyphenated). Baby Dragon and Thousand Dragon would count.
They have dragon in their subtitle, AND have the D&D Monster banner. Lord of D. doesn't count toward this because he doesn't have the Monster banner even though on some cards, he has dragon in his subtitle.


What treatments are suitable for Kidney Failure patients with 17% Kidney Function? For kidney failure patients, losing kidney function is the most common symptom for them. There is no doubt that kidney damage due to some causative factors make the kidney damaged, then more and more function will lose without effective treatments. Call for ONLINE DOCTOR.
How to stop the development of kidney function lose?
For kidney failure patents which only have 17% kidney function that urgent priority is to protect the rest kidney function. That need to stop the development of kidney function lose.
As a matter of fact, as long as the kidney damage repaired in some degree, that the development of kidney damage will be stopped and kidney function will be restored. The mainly reason for kidney damage is due to the capillaries in the kidney blocked and necrosis, leading to kidney damage, kidney function decline.
How to clean the blood vessels and repair kidney damage?
Clean the blood vessels need to dilate blood vessels and clean the garbage to keep them smoothly. Thus will make the kidneys have blood through, then necessary nutrition will support for kidneys to work. Thus will make the kidney cells revitalized, the damage will be repaired effectively.
Micro-Chinese Medicine Hot Compress Therapy have the functions of dilation blood vessels and cleaning wastes in blood. It is able to help kidney failure patients repair kidney damage and improve kidney function in huge degree. Thus will give a new chance for kidney failure patients which only have 17% kidney function at present.
If you want to get more information about Micro-Chinese Medicine Hot Compress Therapy for improving kidney function and kidney failure, please leave a message for us, or send us an email. We will try our best to help you.


text:
Scott Rice •
It Was a Dark and Stormy Night •
Maybe Professor Scott Rice just got tired of correcting term papers that murdered the English language, defied common sense, and read like they'd been constructed by Rube Goldberg. Whatever the reason, there came a day in 1983 when he launched the Bulwer-Lytton Fiction Contest, challenging scribes everywhere to concoct the opening sentence of the world's worst novel. (According to the Bulwer-Lytton website, www stands for "wretched writers welcome.")
Rice's mailbox was flooded with entries, proving that there's as much competition to be a terrible writer as there is to be a terrific one. He's gathered the best—or rather the worst—from that first contest here for our entertainment. Chapters titled "Sister Marie in the Mud-Wrestling Pit," "The Boulevards of Laredo," and "Toddy Tworf the Morpeth Dwarf" hold plenty to keep you howling for hours.
A sampling of the shorter submissions:
"The leg, he is fractured," he said in broken English.
Bunny Berryman belied his name; he was a sour cop with a sour disposition and a face like two-week-old milk forgotten on the back porch step, and he knew it—he also knew his mother had given him a better name than Bunny, but he'd been called Bunny for so long he'd forgotten exactly what it was.
"Totally grody to the max," murmured Patty as wimpy Max Grody slouched across the Valley through the dark, stormy night, grabbed her, and like, you know, gagged her with a spoon.
And my personal favorite:
Just beyond the Narrows the river widens.


text:
The Full Wiki
More info on John Henry Raney
John Henry Raney: Wikis
Note: Many of our articles have direct quotes from sources you can cite, within the Wikipedia article! This article doesn't yet, but we're working on it! See more info or our list of citable articles.
Categories: 1849 births > 1928 deaths > Members of the United States House of Representatives from Missouri
John Henry Raney (September 28, 1849 - January 23, 1928) was a U.S. Representative from Missouri.
Born in Gravelton, Missouri, Raney attended Union School, Des Arc, Missouri, and Woods School, Virginia Settlement, Missouri. He served as judge of the county court of Wayne County 1880-1882. He studied law. He was admitted to the bar in 1881 and commenced practice at Greenville, Missouri. He also engaged in agricultural pursuits and as a stock raiser. He served as prosecuting attorney of Wayne County 1882-1888. He was an unsuccessful candidate for election in 1888 to the State house of representatives. He served as delegate to all Republican State conventions 1884-1927. He served as delegate to the Republican National Convention in 1892. He was one of the board of regents of the State normal school, Cape Girardeau, Missouri from 1893 to 1895.
Raney was elected as a Republican to the Fifty-fourth Congress (March 4, 1895-March 3, 1897). He was an unsuccessful candidate for reelection in 1896 to the Fifty-fifth Congress. He resumed the practice of law in Piedmont, Missouri. He was an unsuccessful candidate for circuit judge of the twenty-first judicial district in 1898. He served as again prosecuting attorney of Wayne County in 1921 and 1922. He died near Patterson, Missouri, January 23, 1928. He was interred in the Masonic Cemetery, Piedmont, Missouri.
John Henry Raney at the Biographical Directory of the United States Congress
John Henry Raney at Find a Grave
Categories: 1849 births | 1928 deaths | Members of the United States House of Representatives from Missouri
Up to date as of November 16, 2009
RANEY, John Henry - Biographical Information
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text:
Hugo Baptista : disciplina del delirio
Silva, Carlos
In this text, critic Carlos Silva places emphasis on Hugo Baptista's skill as a painter. Baptista's work is characterized by a lyrical use of color and an expressive approach to painterly material. It is with good reason, Silva asserts, that Baptista was awarded two major prizes in 1959, one at the Salón Anual de Arte Venezolano (Museo de Bellas Artes de Caracas) and one (the grand prize) at the Salón Arturo Michelena (Ateneo de Valencia, state of Carabobo).
Venezuelan art critic Carlos Silva (1937–2002) wrote this text on the occasion of Disciplina del delirio, an exhibition of work by painter Hugo Baptista (1935–2005) held at Galería Dimaca in Caracas in 1998. The text contains anecdotes about the artist's life as well as critical assessments of his work supported by figures like Adriano González León and Perán Erminy, both of whom were close to the El Techo de la Ballena group in the sixties. Silva's text provides an overview of Baptista's work; approaching to past events—including his first solo show at the Museo de Bellas Artes, held in 1959—as a way to introduce the current works by the artist in this exhibition, describing, for instance, the series of boats and cranes.
Disciplina del delirio. -- Caracas, Venezuela : Galeria Dimaca, 1998
Baptista, Hugo, 1935-
exhibitions (events)
painters (artists)
Silva, Carlos. "Hugo Baptista : disciplina del delirio." In Disciplina del delirio, 3-10. Caracas, Venezuela: Galería Dimaca, 1998.
CINAP. Centro de Informacion Nacional de Artes Plásticas. Galería de Arte Nacional, Plaza Los Museos, Los Caobos, Caracas.
Reproduced with the permission of Maritza de Silva, Caracas, Venezuela
Fundación Mercantil, Caracas
Gabriela Quero


text:
Gilad Atzmon, of The Blockheads, performs at Peterborough Jazz Club
By Zoe Myall
zoe.myall@iliffepublishing.co.uk
International saxophonist and clarinetist, Gilad Atzmon, performs live with his quartet at Peterborough Jazz Club at The New Theatre in The Broadway, on Sunday (November 10), at 7.30pm.
As a member of The Blockheads, who performed 'Hit Me With Your Rhythm Stick', Gilad also recorded and performed with their front man Ian Dury, as well as with Robbie Williams, Sinead O'Connor and Paul McCartney.
In 2014, he recorded with Pink Floyd and is featured on their latest album The Endless River (Anisina).
Jazz star Gilad Atzmon. (20944396)
Atzmon's album, Exile, was the BBC jazz album of the year in 2003, and in 2013 his Metropolis album was voted top CD of the year by the Jazz Journal Critics' Poll.
His 'fiery, fluent playing and remarkable technique' are said to have established him as one of the most talented saxophonists on the world stage.
He performs a mix of classic jazz standards and originals with an energy and enthusiasm that is dubbed 'hugely infectious'.
Tickets for the performance cost £15 and are available from The New Theatre on 01733 852992 or from the website at: www.newtheatre-peterborough.com
Also coming up at The New Theatre is The Fawlty Towers themed dinner show on Friday, November 15, at 7.30pm.
Join Basil and his team as they open the notorious dining room at the Fawlty Towers Hotel to the public for a night of theatrical mayhem 'like no other'.
You'll enjoy a three-course meal with some of the gang's most iconic TV moments recreated in uproarious live action – with you as a much honoured guest.
There's a Ghost Hunt event on Friday, November 22, at 8pm and Fame, The Musical from Monday, November 25, until Saturday, 30. This production of Fame stars Keith Jack from Any Dream Will Do, Joseph and the Amazing Technicolor Dreamcoat and Jorgie Porter from Hollyoaks and Dancing On Ice.
The New Theatre Fame Masterclass for children aged 12-18 on November 30 has now sold out.
ALSO IN WHAT'S ON:
Rob Beckett on tour with his show Wallop!
Long Sutton festive fair 2019 is a reason to 'eat, drink and be merry'
Stags and Hens - The Remix, on stage at the South Holland Centre in Spalding
Zoe Myall


Do you confirm to own McDougall Insurance & Financial - Bancroft?
Do you own McDougall Insurance & Financial - Bancroft?
Do you own McDougall Insurance & Financial - Bancroft? Get reviews alert from SafeToDoBusiness.
The McDougall group in Arnprior is in a small town but has big city services. When I was involved in an accident they were very prompt and knowledgeable about getting me back on the road while my car was being repaired. Big shout out to Scott Ferrier for his expertise.
We have been using Bristor insurance for many years for Auto and Home and the prices are great. The customer service that we get is incredible. We deal with Shannon LeBlanc and she has been excellent.
Been dealing with Brister Group Winchester for at least the past 5 years and the service has been excellent. Our broker William Barclay is very helpful and helped us save money with the best possible insurance quotes and goes above and beyond with his customer service!
I've been dealing with Brister Insurance for over 10 years, for both my auto and home insurance. My representative go to, Shannon LeBlanc, is highly knowledgeable, courteous, and thorough. I had some issues with my car insurance where Shannon went above and beyond what anyone would normally expect from an insurance broker. I highly recommend Brister Insurance on pricing and customer service.
I have been a client of McDougall Insurance for just over a year now. At the beginning of the summer I was in an accident that wrote my car off. I called my broker Mike Cunha right away and he was just leaving for the day but told me to call back as soon as things were settled. When I called back Mike had left all my info with his colleagues and they were waiting for me! They helped me talk to the right people and get everything done quickly and efficiently! I have never had such great service!! :) A big thank you to everyone at McDougall and especially Mike for all the help! I would definitely recommend McDougall to all my friends and family!
Thanks for all your help, Kristen! You certainly made getting insurance an effortless process!
Was very happy with the service Debbie provided when I purchased my truck and needed my first insurance policy. Everything was explained and she made sure I had all the coverages I needed to fully insure my vehicle.
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I recently bought my first home and was entirely new to home insurance. Thank you to the girls in Picton for all your help and making the process so stress free.
My spouse and I utilized Brister Insurance when our previous insurance company gave us an insane quote for our home purchase. We contacted Shannon Leblanc at Brister Insurance since they were local, and began the process. Shannon was able to secure us Home Insurance for $1000 less per year than what our previous insurer wanted. We completed everything online through email eliminating the need to drive to their office or play telephone tag. This was really convenient for us as I work nights. I also got my motorcycle insurance through Shannon based on my pleasant experience with my home policy. Many insurance companies and brokers will only treat you like a policy number, where Shannon will treat you how a customer should be treated - with respect and courtesy. I highly recommend this brokerage for anyone in Ontario. From motorcycle insurance to home policies, no other company can compete with the quality of service you will receive from Brister Insurance.
Dealt with Michel Bruyère. Very patient and knowledgeable! Ended up saving moneynona better insurance coverage for my motorcycle. Highly recommend.
Great service! Love their welcoming and trustworthy personalities. Very amazing business and would recommend to everyone.
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The friendly and knowledgeable staff at Bruyere Insurance is always available to help us. Any insurance need we have, they are always happy to help. I recommend Bruyere Insurance Brokers.
Dealing with McDougall in Trenton has been great. Great personable staff. Honest people that will guide you through the proper product for you.
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I have had my auto and home insurance with Darlene Hughes of McDougall Ins. for about 12 yrs. and have been completely satisfied . Darlene has done an exceptional job for me in every way and is always there to answer any question I may have. I would highly recommend McDougall Ins. to anyone. Wayne Booklin Ont.
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Brister Insurance provides quality service for all our insurance needs including building, commercial liability, bond renewal and cargo marine.
Please refer to my comments under Morrisburg office of Brister Insurance Brokers Ltd.
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