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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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Polo kinase differentially regulates Augmin distribution and spindle morphology in mitosis and male meiosis. (a) Comparison of spindle morphology in control and BI 2536 Plk1 family-specific inhibitor treated mitotic tissue culture cells. In control cells, Augmin homogeneously coats the spindle and localizes to the centrosomes. BI 2536 treatment results in a characteristic loss of k-fibres and spindle attachment as revealed by MT and CID distribution. Augmin is almost entirely excluded from the spindle but remains at the centrosomes. (b) Identical BI 2536 treatment of spermatocytes leads to increased MT density near the spindle poles with a concomitant loss of k-fibres causing the chromosomes to lay loose on the spindle (zoomed panels). (c) BI 2536 treatment mis-localizes Augmin in spermatocytes. The complex becomes aberrantly recruited to the MT-rich portions of the spindle. The signal is greatest near the poles and extends towards the equator. It is no longer observed at the centrosomes or kinetochores (CID; zoomed panels). (d) Polo inhibition alters γ-tubulin distribution. BI 2536 treatment redirects the protein from the centrosomes to the polar regions corresponding to the enhanced MT density. (e) Wac activity is required for BI 2536-mediated spindle and localization phenotypes. Treatment of wac mutants with BI 2536 does not dramatically increase spindle MT numbers or affect γ-tubulin's centrosome exclusive placement. (f) Augmin and γ-tubulin distribution following Polo downregulation in polo1 hemizygous mutants. As with BI 2536-treated spermatocytes, Polo inactivation through mutation results in Augmin and γ-tubulin assuming pronounced spindle distributions that extend from the poles towards the equator. See text for details. All images are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.
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RSOB140047F4: Polo kinase differentially regulates Augmin distribution and spindle morphology in mitosis and male meiosis. (a) Comparison of spindle morphology in control and BI 2536 Plk1 family-specific inhibitor treated mitotic tissue culture cells. In control cells, Augmin homogeneously coats the spindle and localizes to the centrosomes. BI 2536 treatment results in a characteristic loss of k-fibres and spindle attachment as revealed by MT and CID distribution. Augmin is almost entirely excluded from the spindle but remains at the centrosomes. (b) Identical BI 2536 treatment of spermatocytes leads to increased MT density near the spindle poles with a concomitant loss of k-fibres causing the chromosomes to lay loose on the spindle (zoomed panels). (c) BI 2536 treatment mis-localizes Augmin in spermatocytes. The complex becomes aberrantly recruited to the MT-rich portions of the spindle. The signal is greatest near the poles and extends towards the equator. It is no longer observed at the centrosomes or kinetochores (CID; zoomed panels). (d) Polo inhibition alters γ-tubulin distribution. BI 2536 treatment redirects the protein from the centrosomes to the polar regions corresponding to the enhanced MT density. (e) Wac activity is required for BI 2536-mediated spindle and localization phenotypes. Treatment of wac mutants with BI 2536 does not dramatically increase spindle MT numbers or affect γ-tubulin's centrosome exclusive placement. (f) Augmin and γ-tubulin distribution following Polo downregulation in polo1 hemizygous mutants. As with BI 2536-treated spermatocytes, Polo inactivation through mutation results in Augmin and γ-tubulin assuming pronounced spindle distributions that extend from the poles towards the equator. See text for details. All images are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.

Mentions: If as in mitosis [10–12,14,16–19,21] Augmin functions in spermatocytes as an extra-centrosomal γ-tubulin targeting factor, then reduced Wac or γ-tubulin should lead to overlapping phenotypes. We found that loss of Wac did not prevent γ-tubulin23C, the sole γ-tubulin isoform expressed in testes [43], from accumulating at the centrosomes (electronic supplementary material, figure S3a; figure 4e); nor did the depletion of γ-tubulin preclude Augmin from loading at this now diminished structure (electronic supplementary material, figure S3b). When challenged in the MT re-growth assay the γ-tubulin mutants behaved like those downregulated for Wac with only feeble numbers of MTs appearing within the nuclear compartment. In addition, few MTs appeared at any cellular location including the centrosomes (n = 12 cells; figure 3c; electronic supplementary material, figure S6). The extent of this reduction in MT generation was surprising given that mutants of γ-tubulin and other core components of the MT nucleating γ-tubulin Ring Complex routinely form astral MT arrays of normal appearance that cap the ends of unstable but bipolar spindles [30,33] (electronic supplementary material, figure S4a). We find that spindle and kinetochore interactions are regularly obscured in these mutants. K-fibres, which in wild-type cells appear as MT bundles terminating at the kinetochore, are often difficult to detect, while kinetochores commonly contact an MT's lateral surface (electronic supplementary material, figure S4b). Thus, although untreated γ-tubulin mutants display large numbers of MTs, their nucleation potential and ability to form functional spindles is severely compromised.Figure 4.


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

Savoian MS, Glover DM - Open Biol (2014)

Polo kinase differentially regulates Augmin distribution and spindle morphology in mitosis and male meiosis. (a) Comparison of spindle morphology in control and BI 2536 Plk1 family-specific inhibitor treated mitotic tissue culture cells. In control cells, Augmin homogeneously coats the spindle and localizes to the centrosomes. BI 2536 treatment results in a characteristic loss of k-fibres and spindle attachment as revealed by MT and CID distribution. Augmin is almost entirely excluded from the spindle but remains at the centrosomes. (b) Identical BI 2536 treatment of spermatocytes leads to increased MT density near the spindle poles with a concomitant loss of k-fibres causing the chromosomes to lay loose on the spindle (zoomed panels). (c) BI 2536 treatment mis-localizes Augmin in spermatocytes. The complex becomes aberrantly recruited to the MT-rich portions of the spindle. The signal is greatest near the poles and extends towards the equator. It is no longer observed at the centrosomes or kinetochores (CID; zoomed panels). (d) Polo inhibition alters γ-tubulin distribution. BI 2536 treatment redirects the protein from the centrosomes to the polar regions corresponding to the enhanced MT density. (e) Wac activity is required for BI 2536-mediated spindle and localization phenotypes. Treatment of wac mutants with BI 2536 does not dramatically increase spindle MT numbers or affect γ-tubulin's centrosome exclusive placement. (f) Augmin and γ-tubulin distribution following Polo downregulation in polo1 hemizygous mutants. As with BI 2536-treated spermatocytes, Polo inactivation through mutation results in Augmin and γ-tubulin assuming pronounced spindle distributions that extend from the poles towards the equator. See text for details. All images 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
Show All Figures
getmorefigures.php?uid=PMC4042853&req=5

RSOB140047F4: Polo kinase differentially regulates Augmin distribution and spindle morphology in mitosis and male meiosis. (a) Comparison of spindle morphology in control and BI 2536 Plk1 family-specific inhibitor treated mitotic tissue culture cells. In control cells, Augmin homogeneously coats the spindle and localizes to the centrosomes. BI 2536 treatment results in a characteristic loss of k-fibres and spindle attachment as revealed by MT and CID distribution. Augmin is almost entirely excluded from the spindle but remains at the centrosomes. (b) Identical BI 2536 treatment of spermatocytes leads to increased MT density near the spindle poles with a concomitant loss of k-fibres causing the chromosomes to lay loose on the spindle (zoomed panels). (c) BI 2536 treatment mis-localizes Augmin in spermatocytes. The complex becomes aberrantly recruited to the MT-rich portions of the spindle. The signal is greatest near the poles and extends towards the equator. It is no longer observed at the centrosomes or kinetochores (CID; zoomed panels). (d) Polo inhibition alters γ-tubulin distribution. BI 2536 treatment redirects the protein from the centrosomes to the polar regions corresponding to the enhanced MT density. (e) Wac activity is required for BI 2536-mediated spindle and localization phenotypes. Treatment of wac mutants with BI 2536 does not dramatically increase spindle MT numbers or affect γ-tubulin's centrosome exclusive placement. (f) Augmin and γ-tubulin distribution following Polo downregulation in polo1 hemizygous mutants. As with BI 2536-treated spermatocytes, Polo inactivation through mutation results in Augmin and γ-tubulin assuming pronounced spindle distributions that extend from the poles towards the equator. See text for details. All images are z-projections. Bars are 10 μm except in zoomed panels where they are 2 μm.
Mentions: If as in mitosis [10–12,14,16–19,21] Augmin functions in spermatocytes as an extra-centrosomal γ-tubulin targeting factor, then reduced Wac or γ-tubulin should lead to overlapping phenotypes. We found that loss of Wac did not prevent γ-tubulin23C, the sole γ-tubulin isoform expressed in testes [43], from accumulating at the centrosomes (electronic supplementary material, figure S3a; figure 4e); nor did the depletion of γ-tubulin preclude Augmin from loading at this now diminished structure (electronic supplementary material, figure S3b). When challenged in the MT re-growth assay the γ-tubulin mutants behaved like those downregulated for Wac with only feeble numbers of MTs appearing within the nuclear compartment. In addition, few MTs appeared at any cellular location including the centrosomes (n = 12 cells; figure 3c; electronic supplementary material, figure S6). The extent of this reduction in MT generation was surprising given that mutants of γ-tubulin and other core components of the MT nucleating γ-tubulin Ring Complex routinely form astral MT arrays of normal appearance that cap the ends of unstable but bipolar spindles [30,33] (electronic supplementary material, figure S4a). We find that spindle and kinetochore interactions are regularly obscured in these mutants. K-fibres, which in wild-type cells appear as MT bundles terminating at the kinetochore, are often difficult to detect, while kinetochores commonly contact an MT's lateral surface (electronic supplementary material, figure S4b). Thus, although untreated γ-tubulin mutants display large numbers of MTs, their nucleation potential and ability to form functional spindles is severely compromised.Figure 4.

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