Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Proc Natl Acad Sci U S A. 2013 Dec 3;110(49):19808-13. doi: 10.1073/pnas.1320013110. Epub 2013 Nov 19.
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.
动物有丝分裂纺锤体的组装依赖于中心体依赖和中心体非依赖的机制，但它们的相对贡献尚不清楚。在这里，我们通过在缺乏功能性中心体的果蝇 S2 细胞中进行全基因组 RNAi 筛选，研究了中心体非依赖的纺锤体组装途径的分子基础。该筛选鉴定了 197 个参与无中心体纺锤体组装的基因，其中 8 个基因以前没有描述过有丝分裂表型，产生有缺陷和/或短的纺锤体。所有 197 个基因在中心体存在的情况下也产生了 RNAi 表型，表明它们都不是完全选择性地用于无中心体途径。然而，当中心体不存在时，一组基因产生了一个选择性的极聚焦缺陷，表明中心体补偿了这种形状缺陷。另一组基因仅在中心体存在时才与多极纺锤体的形成特异性相关。我们进一步表明，染色体乘客复合物协调了有丝分裂纺锤体组装/维持所需的多个中心体非依赖过程。另一方面，尽管形成了富含染色体的 RanGTP 梯度，但 RCC1 耗尽的 S2 细胞，即染色体上的 Ran 鸟嘌呤核苷酸交换因子，建立了功能性的双极纺锤体。最后，我们表明，缺乏功能性中心体的细胞在染色体向赤道板迁移和后期起始时存在延迟，这可以用缺乏极逐出力来解释。总的来说，这些发现确立了中心体非依赖的纺锤体组装程序的组成性质，以及该程序如何适应动物体细胞中心体的存在/缺失。
