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Anastral spindle assembly and γ-tubulin in Drosophila oocytes

DOI: 10.1186/1471-2121-12-1

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Abstract:

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.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.Anastral spindles assemble without centrosomes by a pathway that is still not fully understood. In particular, the mechanism by which microtubule nucleation occurs has not been well defined. Although chromatin has been shown to play an essential role [1], the involvement of the microtubule nucleator, γ-tubulin, is still an open question. γ-Tubulin localizes to centrosomes and other microtubule organizing centers in mitosis and is essential for nucleating microtubules in organisms as diverse as yeast, Drosophila, Xenopus, humans, and higher plants [2-5]. γ-Tubulin is also found on spindle microtubules, where it has been proposed to nucleate microtubules for spindle maintenance by functioning in a chromatin-mediated nucleation pathway that augments the dominant pathway of nucleation by centrosomes [6,7].γ-Tubulin is present in cells as a large ring complex, γTuRC, comprising 12-13 γ-tubulin molecules a

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