Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells
Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells
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果蝇 S2 细胞中参与中心体独立有丝分裂纺锤体组装的基因
DOI：
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发表时间：
2013
影响因子：
11.1
通讯作者：
H. Maiato
中科院分区：
文献类型：
--
作者：
Sara Moutinho;N. Stuurman;Olga Afonso;M. Hornsveld;P. Aguiar;Gohta Goshima;R. Vale;H. Maiato
有丝分裂纺锤体是一种主要由微管组成的结构，在细胞分裂过程中指导DNA的分离。在动物体细胞中，中心体（微管成核结构）位于有丝分裂纺锤体极附近。然而，生殖细胞缺乏中心体，如果中心体功能受损，甚至体细胞也可以进行细胞分裂。本研究报告了果蝇细胞的全基因组RNAi筛选基因参与纺锤体组装的功能中心体的情况下。结果表明，纺锤体组装途径与中心体和无中心体涉及一组基本相同的基因，证明了中心体独立的纺锤体组装的组成性质。然而，当中心体缺失时，某些基因敲除显示出不同的表型，从而揭示了纺锤体如何适应中心体的存在或缺失。动物有丝分裂纺锤体的组装依赖于中心体依赖性和中心体非依赖性机制，但它们的相对贡献仍然未知。在这里，我们研究了中心体独立的纺锤体组装途径的分子基础，通过在缺乏功能中心体的果蝇S2细胞中进行全基因组RNAi筛选。这个屏幕确定了197个基因参与acentrosomal纺锤体组装，其中8个没有以前描述的有丝分裂表型和生产缺陷和/或短纺锤体。当中心体存在时，所有197个基因也产生RNAi表型，这表明没有一个基因对无中心体途径具有完全选择性。然而，当中心体不存在时，一个基因子集产生了极聚焦的选择性缺陷，这表明中心体弥补了这种形状缺陷。另一个基因的子集是专门与多极纺锤体的形成只有当中心体存在。我们进一步表明，染色体乘客复杂的编排多个中心体独立的有丝分裂纺锤体组装/维护所需的过程。另一方面，尽管形成了染色体富集的RanGTP梯度，S2细胞耗尽RCC 1，鸟嘌呤核苷酸交换因子Ran的染色体上，建立功能性双极纺锤体。最后，我们发现，没有功能中心体的细胞有一个延迟的染色体congression和后期发病，这可以解释缺乏极性弹射力。总体而言，这些研究结果建立了一个中心体独立的纺锤体组装程序的组成性质，以及该程序是如何适应动物体细胞中中心体的存在/不存在。
Significance The mitotic spindle, a structure composed primarily of microtubules, guides the segregation of DNA during cell division. In somatic animal cells, centrosomes (microtubule nucleating structures) reside near the mitotic spindle poles. However, germ cells lack centrosomes, and even somatic cells can execute cell division if centrosome function is compromised. This study on Drosophila cells reports a whole-genome RNAi screen for genes involved in spindle assembly in the absence of functional centrosomes. The results show that spindle assembly pathways with and without centrosomes involve an essentially identical set of genes, demonstrating the constitutive nature of centrosome-independent spindle assembly. However, certain gene knockdowns show distinct phenotypes when centrosomes are absent, thus revealing how spindles adapt to the presence or absence of centrosomes. Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.
DOI：
10.1083/jcb.201202135
发表时间：
2012-08-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Courtois A;Schuh M;Ellenberg J;Hiiragi T
通讯作者：
Hiiragi T
影响因子：
10.5
作者：
Kwon, Mijung;Godinho, Susana A.;Pellman, David
通讯作者：
Pellman, David