Figure 11. Mitotic phenotypes observed in the SA2 phenoclus- ter. In merges, DNA is blue and tubulin green. (A) Mitotic figures with low microtubule density showing defective chromosome segregation after RNAi for NippedA. (B) Disorganized spindles with low microtubule density in Grip75 RNAi cells; note that the ana/telophase-like spindles contain chromosomes with unseparated sister chromatids. (C) Staining for Cenp-C (red) shows that the chromosomes of the ana/telophase-like figures of Grip75 RNAi cells comprise both sister kinetochores. Scale bar, 5 mm. doi:10.1371/journal.pgen.1000126.g011 Genes Required for Chromosome Condensation, Spindle Formation, and/or Cytokinesis We identified 6 genes required for both chromosome conden- sation and spindle formation, which can be subdivided into two phenoclusters (SC1 and SC2). The SC1 phenocluster includes the three components of the chromosome passenger complex (Incenp, Drosophila Mitotic Genes Ial/Aurora B and Borealin), as well as chromatin assembly factor 1 (Caf1). Consistent with previous studies, downregulation of these proteins resulted in elongated and poorly condensed chromo- somes, disorganized spindles, defective chromosome congression and segregation, and frequent failures in cytokinesis (Figure 3 and Figure S16; Table S6). The SC2 group includes Polo kinase and the Drosophila homolog of the Myb transcriptional activator (Figure 3 and Table S6). In polo RNAi cells, the chromosomes were fuzzy and irregularly condensed, while in Myb-depleted cells the chromosomes were overcontracted and swollen with no resolution between sister chromatids. Dowregulation of either of these two proteins disrupts MT-kinetochore interactions, leading to failures of chromosome congression and sister chromatid separation (Figure 3 and Figure S16). Genes Required for Cytokinesis The RNAi phenotypes of the 7 genes required for cytokinesis (Figure 3) have been described previously in greater detail (Table S6). They can be subdivided into two phenoclusters (CY1 and CY2). Inactivation of the genes in the CY1 group [fascetto (feo), racGAP50, pavarotti (pav) and pebble (pbl)] results in early cytokinetic defects in both the central spindle and the contractile ring. In contrast, ablation of the CY2 genes [anillin (ani), citron kinase (sti) and twinstar (tsr)] does not affect either central spindle or contractile ring assembly, but it does disrupt the final stages of cytokinesis (Figure 3 and Table S6). Discussion Our RNAi screen for mitotic genes differs from those previously performed in two important ways. First, we used a bioinformatic approach to focus our experiments on a group of genes that was enriched in mitotic functions. Second, we analyzed potential mitotic phenotypes not only by examining cells stained for tubulin and DNA, but also by looking at colchicine-treated chromosome preparations. Since this latter technique allows the analysis of well spread metaphase chromosomes with excellent cytological resolu- tion, we were able to identify 44 genes required to prevent spontaneous chromosome breakage, most of which have not previously been implicated in the maintenance of chromosome integrity. The human orthologs of some of these genes may play roles in carcinogenesis, as shown for many genes required for chromo- some stability [51,52]. In addition, examination of colchicine- arrested metaphases led to the detection of phenotypes such as precocious sister chromatid separation (CS2 phenocluster) and a lack of sister chromatid cohesion in the heterochromatic regions (CC1 phenocluster). These phenotypic traits allowed us to distinguish between genes required for proper chromosome segregation, permitting their assignment to different functional groups. Although previous RNAi screens were not designed to detect subtle changes in chromosome structure, they identified many genes involved in spindle assembly, chromosome segregation and cytokinesis [1–10]. Of particular interest is a comparison between our screen and a recent genome-wide screen performed by Goshima and coworkers in S2 cells [10] (see Text S1 and Table S7 for details). Goshima et al. used automated microscopy to identify 189 genes required for spindle assembly and chromosome alignment at metaphase. Remarkably, 38% of these 189 genes are included in the first 1000 genes of our consensus co-expression list and 50% in the first 2000. We identified 98 genes involved in the same processes, 30 of which were not found in the Goshima et al. screen. However, we failed to detect 17 genes that elicited RNAi phenotypes in their screen. Together, these results further PLoS Genetics | www.plosgenetics.org 13 July 2008 | Volume 4 | Issue 7 | e1000126