Drosophila Mitotic Genes Figure 9. Mitotic phenotypes observed in the CC1 and CC3 phenoclusters. In merges, DNA is blue and tubulin green. (A) colchicine/ hypotonic-treated metaphase chromosomes (c-meta); metaphase (meta), anaphase (ana) and telophase (telo) observed after RNAi for Mcm3 (CC1). Note the relative lack of sister chromatid cohesion in the heterochromatic regions of c-metaphase chromosomes, as well as difficulties in chromosome congression and segregation. (B) C-metaphase (c-meta) and mitotic figures in Top2 (CC3) RNAi cells. Metaphase chromosomes are poorly condensed, leading to problems in chromosome separation during ana/telophase. Scale bars, 5 mm. doi:10.1371/journal.pgen.1000126.g009 mally elongated and irregularly condensed, suggesting a defect in chromosome shortening. In RNAi cells for these genes, chromo- some congression and segregation were also affected, consistent with previously published results (Figure 9B; Table S6). Genes Required for Spindle Assembly RNAi for 33 genes caused defects in spindle structure that were apparent as early as prophase or the beginning of prometaphase. Most of these genes (29/33) can be grouped in three broad phenoclusters (SA1, SA2 and SA3); although the phenotypes associated with the remaining 4 genes do not resemble each other, we assign them to a single miscellaneous group (SA4) for convenience (Figure 3). Inactivation of the 18 genes in the SA1 group resulted in the formation of bipolar spindles that were significantly shorter than control spindles. Knockdowns of most of these genes also caused poorly focused spindle poles, monopolar spindles, hypercontracted chromosomes and defects in chromo- some congression and segregation (Figures 3, 10; Figures S11 and S12). The monopolar spindles observed in these RNAi cells might not reflect defective centrosome separation at prophase, but instead be a consequence of the instability of short bipolar spindles. This is suggested by previous observations of Orbit/ Mast-depleted S2 cells. Live imaging of these cells has shown that the centrosomes of bipolar minispindles often collapse towards each other during prometaphase to form a monopolar spindle [47]. The excessive chromosome contraction is the likely outcome of a delayed progression through mitosis and could be responsible for a partial impairment of kinetochore function, resulting in defective chromosome congression and segregation. The SA1 phenocluster includes the b-tubulin gene b-tub56B, 4 genes that encode MT-interacting proteins [Map60/CP60, Eb1, Minispindles (Msps) and Mars/HURP], the mitotic kinase gene ik2, and 3 genes (CG4865, CG14781 and CG17293) that encode proteins of unknown function (Table S6). The remaining 9 genes of the SA1 group are involved in either transcription or translation. CG8950 encodes a PolII transcription factor; tho2 specifies a component of the conserved THO complex, which couples splicing and mRNA export (Table S6). Trip1/eIF3-S2, CG8636/eIF3-S4, Int6/eIF3-S6, eIF3-p66 and eIF3-S10 encode different subunits of the highly conserved eukaryotic translation initiation factor 3, while Nnp-1 and CG1234 are involved in ribosome biogenesis or maturation (Table S6). The Int6 gene is a frequent integration site of the MMTV virus in mouse mammary tumors and its silencing leads to mitotic defects in human cells (Table S6). In addition to short spindles, Int6-depleted cells displayed two unusual phenotypic traits: a severe undercondensation of the pericentric regions of the chromosomes and abnormally long astral MTs in telophase figures (Figure 10A). Horse tail-like telophase asters were also observed in msps and CG8950 RNAi cells (Figure 3 and Figure S11). The SA2 phenocluster includes only 5 genes: CG11881 and CG16969, that encode proteins of unknown function; Grip75 and c tubulin 23C, that specify components of the gamma tubulin ring complex; and NippedA, that encodes a subunit of the conserved TRRAP complex implicated both in transcriptional regulation and DNA repair (Table S6). Interestingly, loss of the TRRAP complex affects gene expression at mitotic stages [48]. Downregulation of the genes in the SA2 group results in spindles with a low MT density and poorly focused poles (Figure 11 and Figure S13). Aberrant spindles with low MT density have previously been observed in S2 cells depleted of gamma tubulin ring components [49]. RNAi cells for the SA2 genes were also defective in chromosome congression and sister chromatid separation, just as those of the CS3 phenocluster (Figure 11). This phenotypic profile suggests that the products of the SA2 genes are required for the stability of spindle MTs and for their interaction with the kinetochores. RNAi for the 6 the genes in the SA3 phenocluster resulted in anastral and poorly focused spindles with normal MT density (Figure 12 and Figure S14). These genes encode the DSas-4 protein required for centriole duplication; the PCM component Centroso- min (Cnn) required for MT nucleation; the CG17826 product homologous to the C. elegans centrosomal protein Spd2; and Abnormal spindle (Asp), a protein that associates with both the centrosomes and the MT minus ends (Table S6). The other two genes in the SA3 group encode the NiPp1 inhibitor of protein PLoS Genetics | www.plosgenetics.org 11 July 2008 | Volume 4 | Issue 7 | e1000126