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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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Meiotic spindles form through centrosomal, acentrosomal and Wac-mediated pathways. (a–c) Time-lapse sequences of spindle formation in the indicated genetic backgrounds visualized using β-tubulin::EGFP expression. (a) Wild-type spindle formation initiates as centrosome-nucleated astral MTs penetrate into the nucleus (180; arrow). These increase in number and organize into bundles (180–960), some of which are k-fibres (arrowheads) as revealed by their contact with chromosome-based fluorescence ‘ghosts’. The chromosomes align at the spindle equator where they remain at metaphase (1500), shortly before anaphase entry. (a′) Fixed wild-type metaphase cell showing the distribution of the centromeric protein CID/CENP-A as a marker for kinetochore position, MTs and DNA. Arrowheads denote k-fibres. (b) A centrosome inactivated, asl2/asl3 mutant. Neither centrosomes nor asters are detected. Spindle assembly is first observed with the nucleation of a few MTs within the nucleus. Progressively more MTs appear that interact (480–960) to establish larger structures. These are poorly organized, but exhibit k-fibre-like MT bundles (1860; arrowheads). (b′) Fixation and staining of asl2/asl3 cells confirms that k-fibres (arrowheads) form in the absence of centrosomes. (c) Spindle assembly in a wac Δ12 hemizygous mutant. Like the wild-type, centrosomal MTs invade the nucleus (0–180; arrows). However, these require protracted periods to organize into a spindle with recognizable albeit non-robust k-fibres (960; arrowhead). As the k-fibres mature (arrowheads) the chromosomes assume an equatorial position where they remain throughout metaphase (3300). (c′) Fixation and staining of wac mutant cells confirms that this protein and by extension the Augmin complex are not needed for k-fibre formation (arrowheads) or normal spindle morphology. Time is in seconds relative to the onset of spindle formation. All images are z-projections. Bars are 10 μm except in zoomed fixed images where they are 2 μm.
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RSOB140047F1: Meiotic spindles form through centrosomal, acentrosomal and Wac-mediated pathways. (a–c) Time-lapse sequences of spindle formation in the indicated genetic backgrounds visualized using β-tubulin::EGFP expression. (a) Wild-type spindle formation initiates as centrosome-nucleated astral MTs penetrate into the nucleus (180; arrow). These increase in number and organize into bundles (180–960), some of which are k-fibres (arrowheads) as revealed by their contact with chromosome-based fluorescence ‘ghosts’. The chromosomes align at the spindle equator where they remain at metaphase (1500), shortly before anaphase entry. (a′) Fixed wild-type metaphase cell showing the distribution of the centromeric protein CID/CENP-A as a marker for kinetochore position, MTs and DNA. Arrowheads denote k-fibres. (b) A centrosome inactivated, asl2/asl3 mutant. Neither centrosomes nor asters are detected. Spindle assembly is first observed with the nucleation of a few MTs within the nucleus. Progressively more MTs appear that interact (480–960) to establish larger structures. These are poorly organized, but exhibit k-fibre-like MT bundles (1860; arrowheads). (b′) Fixation and staining of asl2/asl3 cells confirms that k-fibres (arrowheads) form in the absence of centrosomes. (c) Spindle assembly in a wac Δ12 hemizygous mutant. Like the wild-type, centrosomal MTs invade the nucleus (0–180; arrows). However, these require protracted periods to organize into a spindle with recognizable albeit non-robust k-fibres (960; arrowhead). As the k-fibres mature (arrowheads) the chromosomes assume an equatorial position where they remain throughout metaphase (3300). (c′) Fixation and staining of wac mutant cells confirms that this protein and by extension the Augmin complex are not needed for k-fibre formation (arrowheads) or normal spindle morphology. Time is in seconds relative to the onset of spindle formation. All images are z-projections. Bars are 10 μm except in zoomed fixed images where they are 2 μm.

Mentions: We began our examination of spermatocyte spindle formation by characterizing the contributions of the centrosome. For this, we performed time-lapse imaging of spermatocytes expressing β-tubulin56D::EGFP (enhanced green fluorescent protein) to label MTs [34]. In wild-type cells, spindle formation initiates at prometaphase as centrosome-derived astral MTs penetrate into the former nuclear volume—a compartment that remains partially delineated throughout division by the persisting layers of membrane associated with the nuclear envelope (figure 1a; electronic supplementary material, video S1) [23,24,28]. At this time, the chromosomes, often detectable as ‘ghosts’ against the background fluorescence, begin to move. Progressively more MTs extend from the polar regions and organize into kinetochore fibres (k-fibres; arrowheads), bundles that link each kinetochore to the spindle. This process repeats until all of the chromosomes align at metaphase (figure 1a′). We next examined MT generation in the acentrosomal pathway by studying mutants trans-heterozygous for asterless (asl2/asl3), which encodes a centriolar protein. Fixed preparations of these cells previously revealed that they lack centrosomes but retain the ability to build spindle-like structures [27]. When we examined living cells, we found disorganized MTs throughout the cytoplasm and, following prometaphase onset, also in the nucleus where they surrounded the chromosomes. The latter MTs increased in density and became loosely organized over time to form multi-polar spindle-like structures (n = 10 cells; figure 1b; electronic supplementary material, video S2). Although we were unable to assess the functionality of these formations, at least some of their MTs assembled into k-fibres (figure 1b,b′; arrowheads). Together our data confirm that spermatocytes engage centrosome and centrosome-independent MT nucleation and spindle assembly mechanisms.Figure 1.


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

Savoian MS, Glover DM - Open Biol (2014)

Meiotic spindles form through centrosomal, acentrosomal and Wac-mediated pathways. (a–c) Time-lapse sequences of spindle formation in the indicated genetic backgrounds visualized using β-tubulin::EGFP expression. (a) Wild-type spindle formation initiates as centrosome-nucleated astral MTs penetrate into the nucleus (180; arrow). These increase in number and organize into bundles (180–960), some of which are k-fibres (arrowheads) as revealed by their contact with chromosome-based fluorescence ‘ghosts’. The chromosomes align at the spindle equator where they remain at metaphase (1500), shortly before anaphase entry. (a′) Fixed wild-type metaphase cell showing the distribution of the centromeric protein CID/CENP-A as a marker for kinetochore position, MTs and DNA. Arrowheads denote k-fibres. (b) A centrosome inactivated, asl2/asl3 mutant. Neither centrosomes nor asters are detected. Spindle assembly is first observed with the nucleation of a few MTs within the nucleus. Progressively more MTs appear that interact (480–960) to establish larger structures. These are poorly organized, but exhibit k-fibre-like MT bundles (1860; arrowheads). (b′) Fixation and staining of asl2/asl3 cells confirms that k-fibres (arrowheads) form in the absence of centrosomes. (c) Spindle assembly in a wac Δ12 hemizygous mutant. Like the wild-type, centrosomal MTs invade the nucleus (0–180; arrows). However, these require protracted periods to organize into a spindle with recognizable albeit non-robust k-fibres (960; arrowhead). As the k-fibres mature (arrowheads) the chromosomes assume an equatorial position where they remain throughout metaphase (3300). (c′) Fixation and staining of wac mutant cells confirms that this protein and by extension the Augmin complex are not needed for k-fibre formation (arrowheads) or normal spindle morphology. Time is in seconds relative to the onset of spindle formation. All images are z-projections. Bars are 10 μm except in zoomed fixed images where they are 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB140047F1: Meiotic spindles form through centrosomal, acentrosomal and Wac-mediated pathways. (a–c) Time-lapse sequences of spindle formation in the indicated genetic backgrounds visualized using β-tubulin::EGFP expression. (a) Wild-type spindle formation initiates as centrosome-nucleated astral MTs penetrate into the nucleus (180; arrow). These increase in number and organize into bundles (180–960), some of which are k-fibres (arrowheads) as revealed by their contact with chromosome-based fluorescence ‘ghosts’. The chromosomes align at the spindle equator where they remain at metaphase (1500), shortly before anaphase entry. (a′) Fixed wild-type metaphase cell showing the distribution of the centromeric protein CID/CENP-A as a marker for kinetochore position, MTs and DNA. Arrowheads denote k-fibres. (b) A centrosome inactivated, asl2/asl3 mutant. Neither centrosomes nor asters are detected. Spindle assembly is first observed with the nucleation of a few MTs within the nucleus. Progressively more MTs appear that interact (480–960) to establish larger structures. These are poorly organized, but exhibit k-fibre-like MT bundles (1860; arrowheads). (b′) Fixation and staining of asl2/asl3 cells confirms that k-fibres (arrowheads) form in the absence of centrosomes. (c) Spindle assembly in a wac Δ12 hemizygous mutant. Like the wild-type, centrosomal MTs invade the nucleus (0–180; arrows). However, these require protracted periods to organize into a spindle with recognizable albeit non-robust k-fibres (960; arrowhead). As the k-fibres mature (arrowheads) the chromosomes assume an equatorial position where they remain throughout metaphase (3300). (c′) Fixation and staining of wac mutant cells confirms that this protein and by extension the Augmin complex are not needed for k-fibre formation (arrowheads) or normal spindle morphology. Time is in seconds relative to the onset of spindle formation. All images are z-projections. Bars are 10 μm except in zoomed fixed images where they are 2 μm.
Mentions: We began our examination of spermatocyte spindle formation by characterizing the contributions of the centrosome. For this, we performed time-lapse imaging of spermatocytes expressing β-tubulin56D::EGFP (enhanced green fluorescent protein) to label MTs [34]. In wild-type cells, spindle formation initiates at prometaphase as centrosome-derived astral MTs penetrate into the former nuclear volume—a compartment that remains partially delineated throughout division by the persisting layers of membrane associated with the nuclear envelope (figure 1a; electronic supplementary material, video S1) [23,24,28]. At this time, the chromosomes, often detectable as ‘ghosts’ against the background fluorescence, begin to move. Progressively more MTs extend from the polar regions and organize into kinetochore fibres (k-fibres; arrowheads), bundles that link each kinetochore to the spindle. This process repeats until all of the chromosomes align at metaphase (figure 1a′). We next examined MT generation in the acentrosomal pathway by studying mutants trans-heterozygous for asterless (asl2/asl3), which encodes a centriolar protein. Fixed preparations of these cells previously revealed that they lack centrosomes but retain the ability to build spindle-like structures [27]. When we examined living cells, we found disorganized MTs throughout the cytoplasm and, following prometaphase onset, also in the nucleus where they surrounded the chromosomes. The latter MTs increased in density and became loosely organized over time to form multi-polar spindle-like structures (n = 10 cells; figure 1b; electronic supplementary material, video S2). Although we were unable to assess the functionality of these formations, at least some of their MTs assembled into k-fibres (figure 1b,b′; arrowheads). Together our data confirm that spermatocytes engage centrosome and centrosome-independent MT nucleation and spindle assembly mechanisms.Figure 1.

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
Related in: MedlinePlus