Drosophila Mitotic Genes Figure 6. Mitotic phenotypes observed in the CS3 phenocluster. In merges, DNA is blue and tubulin green. (A) Top panels, metaphase- and late anaphase-like figures from U2af50 (splicing factor) RNAi cells. Bottom panels, telophase-like figures from cid RNAi cells. Arrowheads point to chromosomes comprised of both sister chromatids. (B) Cyclin B distribution in control (top panels) and in CG3058 (splicing factor) RNAi cells (bottom panels). The Cyclin B-stained cells correspond to the mitotic figures at their left. Scale bar 5 mm. doi:10.1371/journal.pgen.1000126.g006 initiation of cytokinesis, can occur in the absence of sister chromatid separation. RNAi for the 9 genes in the CS4 group resulted in a pseudo metaphase-arrest phenotype (Figures 2, 8A, and Figure S6). Most dsRNA-treated cells with spindles of metaphase shape displayed apparently normal kinetochore fibers and normal chromosome congression. However, we also observed many mitotic figures with elongated ana/telophase-like spindles and unsegregated chromo- somes at the center of the cell. In these peculiar mitotic figures, the centromeres of most chromosomes had congressed to the middle of the spindle, while the chromosomes arms were parallel to the spindle axis with the telomeres pointing towards the spindle poles. In addition, in many cells with long telophase-like spindles, the chromosomes stuck at the cell equator displayed variable degrees of decondensation, as through they were undergoing the decondensation process that occurs during normal telophase (Figure 8A and Figure S6). Finally, in most ana/telophase-like figures, Cyclin B remained high, as observed in RNAi cells for the CS3 genes (data not shown). The CS4 phenocluster includes the Separase and three rows (thr) genes, which encode interacting proteins required for sister chromatid separation at anaphase (Figure 2 and Table S6). Previous studies have shown that embryonic cells of thr mutants display metaphase arrest with congressed chromosomes, followed by an irregular extension of the spindle without chromosome segregation and by chromosome decondensation [39,40]. This phenotype is fully comparable to that we observed in S2 cells after thr downregulation by RNAi. The CS4 group also includes the CyclinB gene and Otefin, a gene encoding a non-conserved protein that may interact with lamin (Table S6). All the remaining genes in the group are involved in RNA metabolism: one specifies a putative transcription factor while the others encode conserved splicing factors (Table S6). The genes included in the CS4 phenocluster are likely to be required for sister chromatid separation at the anaphase onset. We propose that upon inactivation of these genes, the opposing forces exerted by the MTs attached to the sister kinetochores keep the centromeres aligned at the metaphase plate. At the same time, however, the same forces that mediate the poleward motion of acentric fragments act on the chromosome arms, orienting them parallel to the spindle axis. Our observations also suggest that the latter forces can occasionally prevail over those exerted by the kinetochore fibers, so that some chromosomes leave the metaphase plate and move towards the poles with unseparated chromatids. The finding that RNAi cells for the CS4 genes undergo spindle elongation and chromosome deconden- sation while arrested in a metaphase-like state provides further support for the view that in S2 cells telophase events do not require sister chromatid separation. RNAi for the 9 genes in the CS5 phenocluster resulted in defective chromosome congression at metaphase and abnormal chromosome segregation at anaphase (Figure 2). Knockdowns of the expression of most of these genes caused a partial metaphase arrest characterized by extremely contracted chromosomes. However, even though sister chromatid separation did occur in most of the cases, ana/telophases were severely defective. The segregating chromatids were highly contracted and the two chromatid sets remained close to each other in many cells (Figure 8B and Figure S7). These unusual ana/telophases resemble very early anaphase figures, which are quite rare in untreated cells. These observations suggest that chromosome movement towards the poles is partially impaired in RNAi cells, resulting in delayed and irregular chromosome segregation. The CS5 phenocluster includes ida/APC5 and CG11419/APC10, that encode two subunits of the APC complex; and fizzy (fzy)/Cdc20, whose product regulates APC/C activity (Table S6). This phe- PLoS Genetics | www.plosgenetics.org 8 July 2008 | Volume 4 | Issue 7 | e1000126