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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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Functional acentrosomal spindles form from MTs nucleated within the nucleus and at kinetochores. (a) Sequence showing MT regrowth in a wild-type spermatocyte expressing both β-tubulin56D::EGFP and Aurora B::mCherry to label MTs and kinetochore position, respectively. Prior to the UV pulse, no MTs are detected in the low magnification or boxed and zoomed panels. After the pulse, MTs emanate from the centrosomes (*) and form throughout the cytoplasm. Within the nucleus MTs appear at the persisting membranes, in the nucleoplasm and directly adjacent to the kinetochores. These latter MTs first appear as foci but rapidly assume a ‘v’ shape as they extend away from the kinetochore led by their vertices (arrows follow formation and extension of a single example). Non-kinetochore nucleated MTs undergo similar movements. Both populations can fuse into larger more intricate structures. (b) Sequence from an increased duration recording of an EGFP-tagged tubulin expressing cell following MT regrowth. In this cell, a functional, multi-polar spindle forms. Although one centrosome is proximal to the nucleus (*), it does not appear to contribute substantial numbers of MTs and instead travels along the acentrosomal half-spindle towards its pole. As the spindle matures (1590), k-fibres are observed (arrowheads) which eventually shorten (2070–2400) similar to those in anaphase controls. Time is in seconds relative to the UV pulse. All images are z-projections. Bars are 10 and 5 μm in low magnification and zoomed panels, respectively.
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RSOB140047F2: Functional acentrosomal spindles form from MTs nucleated within the nucleus and at kinetochores. (a) Sequence showing MT regrowth in a wild-type spermatocyte expressing both β-tubulin56D::EGFP and Aurora B::mCherry to label MTs and kinetochore position, respectively. Prior to the UV pulse, no MTs are detected in the low magnification or boxed and zoomed panels. After the pulse, MTs emanate from the centrosomes (*) and form throughout the cytoplasm. Within the nucleus MTs appear at the persisting membranes, in the nucleoplasm and directly adjacent to the kinetochores. These latter MTs first appear as foci but rapidly assume a ‘v’ shape as they extend away from the kinetochore led by their vertices (arrows follow formation and extension of a single example). Non-kinetochore nucleated MTs undergo similar movements. Both populations can fuse into larger more intricate structures. (b) Sequence from an increased duration recording of an EGFP-tagged tubulin expressing cell following MT regrowth. In this cell, a functional, multi-polar spindle forms. Although one centrosome is proximal to the nucleus (*), it does not appear to contribute substantial numbers of MTs and instead travels along the acentrosomal half-spindle towards its pole. As the spindle matures (1590), k-fibres are observed (arrowheads) which eventually shorten (2070–2400) similar to those in anaphase controls. Time is in seconds relative to the UV pulse. All images are z-projections. Bars are 10 and 5 μm in low magnification and zoomed panels, respectively.

Mentions: Following the UV pulse, MTs appeared in multiple cellular regions: at the centrosomes, within the cytoplasm, and around and within the former nucleus (n = 12 cells; figure 2a). As noted previously [26], some of these nuclear MTs re-grew off of the membranes surrounding the nuclear compartment. We further observed prominent nucleation in this nucleoplasm and directly at the kinetochores. The maturation of one kinetochore-derived MT structure is detailed in figure 2a (arrows; electronic supplementary material, video S4). We found that nucleation initiated rapidly, and within 4 s of the UV pulse tubulin foci appeared directly adjacent to 80% of the Aurora B signals (n = 58 kinetochores). Kinetochore-nucleated MTs assumed a short half-spindle-like ‘v’ shape that elongated and moved away at 1.1 ± 0.2 μm min−1 (n = 16), often coalescing into more elaborate structures. Imaging of EB1::EGFP and Aurora B::mCherry indicated that, as with control centrosome-derived spindles, kinetochore-nucleated half-spindles were polarized with their minus ends focused into poles and their dynamic plus ends nearest to the chromosome (electronic supplementary material, figure S1). It is noteworthy that a similar population of short kinetochore-bound MTs has also been detected by electron microscopy in wild-type prometaphase [23,24]. Our live cell work now accounts for the origin these naturally occurring MTs and further reveals that they can promote spindle assembly. The v-shaped morphology of meiotic kinetochore-derived MT structures varies from those reported for mitosis, which appear as a single unified bundle (e.g. [21,35,36]). It is possible that this difference reflects the geometry of the source kinetochores. Unlike mitotic cells, each primary spermatocyte kinetochore is a compound structure composed of two partially resolved and closely positioned sister chromatid kinetochores [37]. We propose that MTs are independently nucleated at each of these adjacent sites. These elongate and rapidly organize and fuse together in a manner analogous to that of the nucleoplasmic MTs described earlier and similar to the motor protein-mediated mechanisms reported for mitosis [35,38,39].Figure 2.


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

Savoian MS, Glover DM - Open Biol (2014)

Functional acentrosomal spindles form from MTs nucleated within the nucleus and at kinetochores. (a) Sequence showing MT regrowth in a wild-type spermatocyte expressing both β-tubulin56D::EGFP and Aurora B::mCherry to label MTs and kinetochore position, respectively. Prior to the UV pulse, no MTs are detected in the low magnification or boxed and zoomed panels. After the pulse, MTs emanate from the centrosomes (*) and form throughout the cytoplasm. Within the nucleus MTs appear at the persisting membranes, in the nucleoplasm and directly adjacent to the kinetochores. These latter MTs first appear as foci but rapidly assume a ‘v’ shape as they extend away from the kinetochore led by their vertices (arrows follow formation and extension of a single example). Non-kinetochore nucleated MTs undergo similar movements. Both populations can fuse into larger more intricate structures. (b) Sequence from an increased duration recording of an EGFP-tagged tubulin expressing cell following MT regrowth. In this cell, a functional, multi-polar spindle forms. Although one centrosome is proximal to the nucleus (*), it does not appear to contribute substantial numbers of MTs and instead travels along the acentrosomal half-spindle towards its pole. As the spindle matures (1590), k-fibres are observed (arrowheads) which eventually shorten (2070–2400) similar to those in anaphase controls. Time is in seconds relative to the UV pulse. All images are z-projections. Bars are 10 and 5 μm in low magnification and zoomed panels, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042853&req=5

RSOB140047F2: Functional acentrosomal spindles form from MTs nucleated within the nucleus and at kinetochores. (a) Sequence showing MT regrowth in a wild-type spermatocyte expressing both β-tubulin56D::EGFP and Aurora B::mCherry to label MTs and kinetochore position, respectively. Prior to the UV pulse, no MTs are detected in the low magnification or boxed and zoomed panels. After the pulse, MTs emanate from the centrosomes (*) and form throughout the cytoplasm. Within the nucleus MTs appear at the persisting membranes, in the nucleoplasm and directly adjacent to the kinetochores. These latter MTs first appear as foci but rapidly assume a ‘v’ shape as they extend away from the kinetochore led by their vertices (arrows follow formation and extension of a single example). Non-kinetochore nucleated MTs undergo similar movements. Both populations can fuse into larger more intricate structures. (b) Sequence from an increased duration recording of an EGFP-tagged tubulin expressing cell following MT regrowth. In this cell, a functional, multi-polar spindle forms. Although one centrosome is proximal to the nucleus (*), it does not appear to contribute substantial numbers of MTs and instead travels along the acentrosomal half-spindle towards its pole. As the spindle matures (1590), k-fibres are observed (arrowheads) which eventually shorten (2070–2400) similar to those in anaphase controls. Time is in seconds relative to the UV pulse. All images are z-projections. Bars are 10 and 5 μm in low magnification and zoomed panels, respectively.
Mentions: Following the UV pulse, MTs appeared in multiple cellular regions: at the centrosomes, within the cytoplasm, and around and within the former nucleus (n = 12 cells; figure 2a). As noted previously [26], some of these nuclear MTs re-grew off of the membranes surrounding the nuclear compartment. We further observed prominent nucleation in this nucleoplasm and directly at the kinetochores. The maturation of one kinetochore-derived MT structure is detailed in figure 2a (arrows; electronic supplementary material, video S4). We found that nucleation initiated rapidly, and within 4 s of the UV pulse tubulin foci appeared directly adjacent to 80% of the Aurora B signals (n = 58 kinetochores). Kinetochore-nucleated MTs assumed a short half-spindle-like ‘v’ shape that elongated and moved away at 1.1 ± 0.2 μm min−1 (n = 16), often coalescing into more elaborate structures. Imaging of EB1::EGFP and Aurora B::mCherry indicated that, as with control centrosome-derived spindles, kinetochore-nucleated half-spindles were polarized with their minus ends focused into poles and their dynamic plus ends nearest to the chromosome (electronic supplementary material, figure S1). It is noteworthy that a similar population of short kinetochore-bound MTs has also been detected by electron microscopy in wild-type prometaphase [23,24]. Our live cell work now accounts for the origin these naturally occurring MTs and further reveals that they can promote spindle assembly. The v-shaped morphology of meiotic kinetochore-derived MT structures varies from those reported for mitosis, which appear as a single unified bundle (e.g. [21,35,36]). It is possible that this difference reflects the geometry of the source kinetochores. Unlike mitotic cells, each primary spermatocyte kinetochore is a compound structure composed of two partially resolved and closely positioned sister chromatid kinetochores [37]. We propose that MTs are independently nucleated at each of these adjacent sites. These elongate and rapidly organize and fuse together in a manner analogous to that of the nucleoplasmic MTs described earlier and similar to the motor protein-mediated mechanisms reported for mitosis [35,38,39].Figure 2.

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