Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which a... more Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a po...
Representing the dynamic nature of biological processes is a challenge. This article describes a ... more Representing the dynamic nature of biological processes is a challenge. This article describes a collaborative project in which the authors – a philosopher of biology, an artist and a cell biologist – explore how best to represent the entire process of cell division in one connected image. This involved a series of group Drawing Labs, one-to-one sessions, and discussions between the authors. The drawings generated during the collaboration were then reviewed by four experts in cell division. We propose that such an approach has value, both in communicating the dynamic nature of biological processes and in generating new insights and hypotheses that can be tested by artists and scientists.
Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimeth... more Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice...
Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing ... more Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of morgana (mora) causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Morgana localizes to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT super-complex, and with additional well-known Hsp90 co-chaperones. Acute inhibition of Morgana function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds microtubules directly and promotes microtubule polymerization in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role.
Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which a... more Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator, γ-Tubulin Ring Complex (γ-TuRC), within the cell. A conserved MT-associated protein complex, Augmin, recruits γ-TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed ‘branching’ MT nucleation. Here, we purify endogenous, GFP-tagged Augmin and γ-TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates γ-TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-γ-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branching MT nucleation. It also provides a po...
Representing the dynamic nature of biological processes is a challenge. This article describes a ... more Representing the dynamic nature of biological processes is a challenge. This article describes a collaborative project in which the authors – a philosopher of biology, an artist and a cell biologist – explore how best to represent the entire process of cell division in one connected image. This involved a series of group Drawing Labs, one-to-one sessions, and discussions between the authors. The drawings generated during the collaboration were then reviewed by four experts in cell division. We propose that such an approach has value, both in communicating the dynamic nature of biological processes and in generating new insights and hypotheses that can be tested by artists and scientists.
Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimeth... more Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice...
Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing ... more Morgana/CHORDC1/CHP1 is a highly conserved CHORD (Cysteine and Histidine Rich Domain) containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of morgana (mora) causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Morgana localizes to mitotic spindles and co-purifies with the Hsp90-R2TP-TTT super-complex, and with additional well-known Hsp90 co-chaperones. Acute inhibition of Morgana function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds microtubules directly and promotes microtubule polymerization in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role.
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Papers by James Wakefield