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. 2011 Jan 5:12:1.
doi: 10.1186/1471-2121-12-1.

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

Sharyn A Endow  1 , Mark A Hallen
Affiliations

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

Sharyn A Endow et al. BMC Cell Biol. .

Abstract

Background: Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral Drosophila oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.

Results: We show, for the first time, using a bright GFP fusion protein and live imaging, that the Drosophila maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.

Conclusions: The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.

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Figures

Figure 1
Figure 1
γ-Tubulin localizes to oocyte MI spindles. (A) MI spindle of a late stage 13 or (B) stage 14 oocyte expressing γTub37C-GFP (γTub, left) and Ncd-mRFP1 (Ncd, right). (C) MI spindles of a late stage 13 oocyte (left) or stage 14 oocytes (middle, right) expressing γTub37C-GFP but not Ncd-mRFP1. γ-Tubulin is present in the MI spindle midzone and at the spindle poles, including pole bodies (arrows). Bar, 3 μm.
Figure 2
Figure 2
MI spindles in γTub37C APL10 mutant oocytes. γTub37C APL10 mutant oocytes contained bipolar MI spindles, many of which were normal, resembling those in wild-type (WT) oocytes, although some were frayed (arrow) or had split poles (not shown). Spindles visualized by Ncd-GFP. Bar, 3 μm.
Figure 3
Figure 3
Mutant γTub37C in oocytes. γTub37C E116R-GFP, mutated in a conserved γTub37C residue, is present in the cytoplasm, but does not localize to oocyte MI spindles (left). Dark regions (arrows) correspond to the condensed MI chromosomes [23]. Spindles detected with Ncd-mRFP1 (right). Bar, 3 μm.
Figure 4
Figure 4
γTub37C APL10 mutant embryos. (A) Anaphase I arrest. (B) Anastral spindles associated with MII metaphase chromosomes (inset) from an embryo containing 6 or more anastral spindles and a polar body with 1N chromosomes. (C) Astral cycle 1 mitotic spindle containing a haploid set of chromosomes. (D) Two small astral spindles associated with interphase or prometaphase nuclei from an embryo containing ~20 astral spindles (~cycle 6) and two pairs of MII-like anastral spindles without central spindle bodies. (E) Wild-type spindles. Top, anaphase II spindle with central spindle pole body (large arrow) and segregating haploid chromosome sets (inset); middle, cycle 1 mitotic spindle containing two haploid chromosome sets (inset); bottom, cycle 6 mitotic spindle. Tubulin, red; Ncd-GFP, green; DNA, blue. Bars, 5 μm.
Figure 5
Figure 5
Fluorescence photobleaching analysis of γTub37C in MI spindles. (A) Prebleach (PreB), bleach and recovery images from FRAP assays of γTub37C-GFP in the oocyte MI spindle (left) and cytoplasm (right). Time, s. ROI (yellow circles; radius, w = 1.062 μm). The MI spindle (left) is outlined to show its boundaries (pink lines). Bar, 2 μm. (B) Mean normalized fluorescence over time (n = 21), corrected for photobleaching during the assays. For comparison, only the data for the large ROI are shown (spindle, green; cytoplasm, grey). Inset, fits to new reaction-diffusion FRAP recovery model that assumes a conical bleach profile (spindle, purple; cytoplasm, cyan).
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