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Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila.

Savoian MS, Glover DM - Open Biol (2014)

Bottom Line: Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density.Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells.Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing Site, Cambridge CB2 3EH, UK m.s.savoian@massey.ac.nz.

ABSTRACT
Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

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Augmin decorates meiotic kinetochores but not spindles and promotes nuclear MT formation. (a) Augmin distribution in metaphase spermatocytes as shown by Dgt5 and Dgt6 staining. Augmin localizes to the centrosomes (arrows) but does not concentrate on the spindle's MTs. Zoomed panels reveal that Augmin forms foci at the centromeres (CID). (b) MTs depolymerization does not prevent recruitment of Augmin to the centrosomes (arrows) or at centromeres (corresponding zoomed panels) indicating that it is a kinetochore component. (c) Sequences showing MT regrowth in wild-type, wacΔ 12 hemizygous or γ-tubulin23Cpi homozygous mutants, each expressing β-tubulin::EGFP. The wac mutation does not affect nucleation in the cytoplasm or the activity of the centrosomes while severely hindering nuclear MT formation. Downregulation of γ-tubulin compromises all MT nucleation events. After a delay, only a few MTs appear at the centrosomes or in the nuclear region. Time is in seconds from the UV pulse. (d) Kinetic profiles of average tubulin fluorescence density at the centrosomes and within the nuclei of wild-type, wac and γ-tubulin mutant cells during MT regrowth. Fluorescence quantification is in arbitrary units (arb. units). The plots confirm Wac's nucleus-confined functions and the necessity of γ-tubulin in MT formation throughout the cell. See text for details. All images except (a) are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.
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RSOB140047F3: Augmin decorates meiotic kinetochores but not spindles and promotes nuclear MT formation. (a) Augmin distribution in metaphase spermatocytes as shown by Dgt5 and Dgt6 staining. Augmin localizes to the centrosomes (arrows) but does not concentrate on the spindle's MTs. Zoomed panels reveal that Augmin forms foci at the centromeres (CID). (b) MTs depolymerization does not prevent recruitment of Augmin to the centrosomes (arrows) or at centromeres (corresponding zoomed panels) indicating that it is a kinetochore component. (c) Sequences showing MT regrowth in wild-type, wacΔ 12 hemizygous or γ-tubulin23Cpi homozygous mutants, each expressing β-tubulin::EGFP. The wac mutation does not affect nucleation in the cytoplasm or the activity of the centrosomes while severely hindering nuclear MT formation. Downregulation of γ-tubulin compromises all MT nucleation events. After a delay, only a few MTs appear at the centrosomes or in the nuclear region. Time is in seconds from the UV pulse. (d) Kinetic profiles of average tubulin fluorescence density at the centrosomes and within the nuclei of wild-type, wac and γ-tubulin mutant cells during MT regrowth. Fluorescence quantification is in arbitrary units (arb. units). The plots confirm Wac's nucleus-confined functions and the necessity of γ-tubulin in MT formation throughout the cell. See text for details. All images except (a) are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.

Mentions: Our live cell observations of wac mutants indicated that Augmin is important for meiotic spindle formation. Because no detailed study of Augmin in male meiosis has been reported, we began characterizing the contributions of the complex by defining its localization using antibodies against two of its subunits, Dgt5 [11] and Dgt6 [21] (figure 3a). As expected from their interaction as part of the hetero-octamer, the two proteins show indistinguishable immuno-localizations, and we subsequently refer to them interchangeably as Augmin. Staining of metaphase cells was marked by a high background with punctae throughout the cell. Despite this, the signal sometimes appeared elevated in the region of the spindle. Although we noted some recruitment to the centrosomes as previously reported [21] (figure 3a; arrows), we were unable to detect any distinct co-localization with the k-fibres or other MTs. This staining pattern differs from the pronounced and homogeneous decoration of the spindle by Augmin that is characteristic of mitotic metaphase in vertebrate [13,18,20,40,41] and Drosophila tissue culture cells ([10,11,14,17,21]; see below). It also differs from the polar association found in Drosophila meiotic oocytes [14,42]. We consistently observed Augmin foci proximal to all of the centromeres we examined (n = 80). However, the signal could be difficult to discern from the background of nearby nucleoplasmic granules. We therefore treated cells with colcemid to depolymerize MTs and determine if the Augmin accumulations were at kinetochores, on MT ends or adjacent aggregates. We found that Augmin was present at centrosomes and centromeres in all cases (n = 80; figure 3b). Augmin has also been previously reported to bind to mitotic kinetochores in Drosophila tissue culture cells. In contrast to our findings, those studies indicated that kinetochore association is MT-dependent [21]. We do not think that our kinetochore localization following colcemid treatment is due to the retention of depolymerization resistant MTs, as Augmin is also detected in early prometaphase cells prior to the onset of spindle assembly (electronic supplementary material, figure S2). From these observations, we conclude that Augmin exhibits a male meiosis-specific distribution. Unlike the other systems examined to date, it is not enriched on any region of the spindle. Furthermore, it binds kinetochores irrespective of MT attachment status. These data suggest specialized functions and regulatory mechanisms in this cell type.Figure 3.


Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila.

Savoian MS, Glover DM - Open Biol (2014)

Augmin decorates meiotic kinetochores but not spindles and promotes nuclear MT formation. (a) Augmin distribution in metaphase spermatocytes as shown by Dgt5 and Dgt6 staining. Augmin localizes to the centrosomes (arrows) but does not concentrate on the spindle's MTs. Zoomed panels reveal that Augmin forms foci at the centromeres (CID). (b) MTs depolymerization does not prevent recruitment of Augmin to the centrosomes (arrows) or at centromeres (corresponding zoomed panels) indicating that it is a kinetochore component. (c) Sequences showing MT regrowth in wild-type, wacΔ 12 hemizygous or γ-tubulin23Cpi homozygous mutants, each expressing β-tubulin::EGFP. The wac mutation does not affect nucleation in the cytoplasm or the activity of the centrosomes while severely hindering nuclear MT formation. Downregulation of γ-tubulin compromises all MT nucleation events. After a delay, only a few MTs appear at the centrosomes or in the nuclear region. Time is in seconds from the UV pulse. (d) Kinetic profiles of average tubulin fluorescence density at the centrosomes and within the nuclei of wild-type, wac and γ-tubulin mutant cells during MT regrowth. Fluorescence quantification is in arbitrary units (arb. units). The plots confirm Wac's nucleus-confined functions and the necessity of γ-tubulin in MT formation throughout the cell. See text for details. All images except (a) are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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RSOB140047F3: Augmin decorates meiotic kinetochores but not spindles and promotes nuclear MT formation. (a) Augmin distribution in metaphase spermatocytes as shown by Dgt5 and Dgt6 staining. Augmin localizes to the centrosomes (arrows) but does not concentrate on the spindle's MTs. Zoomed panels reveal that Augmin forms foci at the centromeres (CID). (b) MTs depolymerization does not prevent recruitment of Augmin to the centrosomes (arrows) or at centromeres (corresponding zoomed panels) indicating that it is a kinetochore component. (c) Sequences showing MT regrowth in wild-type, wacΔ 12 hemizygous or γ-tubulin23Cpi homozygous mutants, each expressing β-tubulin::EGFP. The wac mutation does not affect nucleation in the cytoplasm or the activity of the centrosomes while severely hindering nuclear MT formation. Downregulation of γ-tubulin compromises all MT nucleation events. After a delay, only a few MTs appear at the centrosomes or in the nuclear region. Time is in seconds from the UV pulse. (d) Kinetic profiles of average tubulin fluorescence density at the centrosomes and within the nuclei of wild-type, wac and γ-tubulin mutant cells during MT regrowth. Fluorescence quantification is in arbitrary units (arb. units). The plots confirm Wac's nucleus-confined functions and the necessity of γ-tubulin in MT formation throughout the cell. See text for details. All images except (a) are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.
Mentions: Our live cell observations of wac mutants indicated that Augmin is important for meiotic spindle formation. Because no detailed study of Augmin in male meiosis has been reported, we began characterizing the contributions of the complex by defining its localization using antibodies against two of its subunits, Dgt5 [11] and Dgt6 [21] (figure 3a). As expected from their interaction as part of the hetero-octamer, the two proteins show indistinguishable immuno-localizations, and we subsequently refer to them interchangeably as Augmin. Staining of metaphase cells was marked by a high background with punctae throughout the cell. Despite this, the signal sometimes appeared elevated in the region of the spindle. Although we noted some recruitment to the centrosomes as previously reported [21] (figure 3a; arrows), we were unable to detect any distinct co-localization with the k-fibres or other MTs. This staining pattern differs from the pronounced and homogeneous decoration of the spindle by Augmin that is characteristic of mitotic metaphase in vertebrate [13,18,20,40,41] and Drosophila tissue culture cells ([10,11,14,17,21]; see below). It also differs from the polar association found in Drosophila meiotic oocytes [14,42]. We consistently observed Augmin foci proximal to all of the centromeres we examined (n = 80). However, the signal could be difficult to discern from the background of nearby nucleoplasmic granules. We therefore treated cells with colcemid to depolymerize MTs and determine if the Augmin accumulations were at kinetochores, on MT ends or adjacent aggregates. We found that Augmin was present at centrosomes and centromeres in all cases (n = 80; figure 3b). Augmin has also been previously reported to bind to mitotic kinetochores in Drosophila tissue culture cells. In contrast to our findings, those studies indicated that kinetochore association is MT-dependent [21]. We do not think that our kinetochore localization following colcemid treatment is due to the retention of depolymerization resistant MTs, as Augmin is also detected in early prometaphase cells prior to the onset of spindle assembly (electronic supplementary material, figure S2). From these observations, we conclude that Augmin exhibits a male meiosis-specific distribution. Unlike the other systems examined to date, it is not enriched on any region of the spindle. Furthermore, it binds kinetochores irrespective of MT attachment status. These data suggest specialized functions and regulatory mechanisms in this cell type.Figure 3.

Bottom Line: Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density.Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells.Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Cambridge, Downing Site, Cambridge CB2 3EH, UK m.s.savoian@massey.ac.nz.

ABSTRACT
Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

Show MeSH