Sir William Dunn School of Pathology, University of Oxford , South Parks Road, Oxford OX1 3RE , UK.
Biol Open. 2012 May 15;1(5):422-9. doi: 10.1242/bio.2012596. Epub 2012 Mar 6.
Dgp71WD/Nedd1 proteins are essential for mitotic spindle formation. In human cells, Nedd1 targets γ-tubulin to both centrosomes and spindles, but in other organisms the function of Dgp71WD/Nedd1 is less clear. In Drosophila cells, Dgp71WD plays a major part in targeting γ-tubulin to spindles, but not centrosomes, while in Xenopus egg extracts, Nedd1 acts as a more general microtubule (MT) organiser that can function independently of γ-tubulin. The interpretation of these studies, however, is complicated by the fact that some residual Dgp71WD/Nedd1 is likely present in the cells/extracts analysed. Here we generate a Dgp71WD null mutant lacking all but the last 12 nucleotides of coding sequence. The complete loss of Dgp71WD has no quantifiable effect on γ-tubulin or Centrosomin recruitment to the centrosome in larval brain cells. The recruitment of γ-tubulin to spindle MTs, however, is severely impaired, and spindle MT density is reduced in a manner that is indistinguishable from cells lacking Augmin or γ-TuRC function. In contrast, the absence of Dgp71WD leads to defects in the assembly of the acentrosomal female Meiosis I spindle that are more severe than those seen in Augmin or γ-TuRC mutants, indicating that Dgp71WD has additional functions that are independent of these complexes in oocytes. Moreover, the localisation of bicoid RNA during oogenesis, which requires γ-TuRC function, is unperturbed in Dgp71WD(120) mutants. Thus, Dgp71WD is not simply a general cofactor required for γ-TuRC and/or Augmin targeting, and it appears to have a crucial role independent of these complexes in the acentrosomal Meiosis I spindle.
Dgp71WD/Nedd1 蛋白对于有丝分裂纺锤体的形成是必不可少的。在人类细胞中，Nedd1 将 γ-微管蛋白靶向中心体和纺锤体，但在其他生物体中，Dgp71WD/Nedd1 的功能不太清楚。在果蝇细胞中，Dgp71WD 在将 γ-微管蛋白靶向纺锤体方面发挥主要作用，但不在中心体，而在非洲爪蟾卵提取物中，Nedd1 作为一种更普遍的微管（MT）组织者，可以独立于 γ-微管蛋白发挥作用。然而，这些研究的解释受到以下事实的复杂化，即分析的细胞/提取物中可能存在一些残留的 Dgp71WD/Nedd1。在这里，我们生成了一个缺乏除编码序列最后 12 个核苷酸外的所有序列的 Dgp71WD 缺失突变体。Dgp71WD 的完全缺失对幼虫脑细胞中 γ-微管蛋白或 Centrosomin 向中心体的招募没有可量化的影响。然而，γ-微管蛋白向纺锤体 MT 的招募严重受损，纺锤体 MT 密度以与缺乏 Augmin 或 γ-TuRC 功能的细胞无法区分的方式降低。相比之下，Dgp71WD 的缺失导致无中心体的雌性减数分裂 I 纺锤体的组装缺陷比 Augmin 或 γ-TuRC 突变体更严重，表明 Dgp71WD 具有与这些复合物在卵母细胞中独立的其他功能。此外，在 Dgp71WD(120) 突变体中，卵母细胞中需要 γ-TuRC 功能的bicoid RNA 的定位未受到干扰。因此，Dgp71WD 不仅仅是 γ-TuRC 和/或 Augmin 靶向所需的一般辅助因子，而且在无中心体的减数分裂 I 纺锤体中，它似乎具有独立于这些复合物的关键作用。
