Drosophila Mitotic Genes Figure 5. Mitotic phenotypes observed in the CS1 and CS2 phenoclusters. In all merged figures, DNA is blue and tubulin green. (A) Colchicine/hypotonic-treated metaphase chromosomes (c-meta) from control (ctr) cells; metaphase (meta), anaphase (ana) and telophase (telo) from untreated S2 cells. (B) Unreplicated chromosomes and impaired chromosome migration towards the spindle poles in cells treated with dup dsRNA (CS1). (C) Precocious sister chromatid separation and defective chromosome segregation observed after RNAi for dmt (CS2). Scale bars, 5 mm. doi:10.1371/journal.pgen.1000126.g005 embryonic cells of dup mutants, suggesting that dup is required for both DNA replication and the checkpoint that prevents mitosis until completion of S-phase [30,31]. RNAi for the 5 genes in the CS2 group resulted in precocious sister chromatid separation, lack of chromosome congression to the cell equator at metaphase, and unequal or otherwise abnormal sister chromatid segregation (Figures 5C and S1). Four of the genes included in this CS2 phenocluster (bub1, bub3, zw10 and rod) are well known components of the spindle checkpoint machinery (Table S6). The other gene, dalmatian (dmt) has never been implicated in this checkpoint. However, since studies in C. elegans have clearly shown that genes with similar RNAi phenotypes are often required for a common process [26,32–34], we propose that dmt might play a role in the spindle checkpoint. Inactivation of the 18 genes in the CS3 phenocluster (Figure 2) resulted in a peculiar mitotic phenotype. The chromosomes of metaphase-like figures were not connected to the spindle poles by bundles of kinetochore microtubules (MTs) and thus never congressed to the equator of the spindle. In addition to metaphase-like spindles, the RNAi cells of the CS3 phenocluster also showed many elongated ana/telophase spindles. However, these spindles contained chromosomes with unseparated sisters chromatids; these chromosomes usually appeared to segregate to the poles at random (Figures 6, 7 and S2–S4). Some of these peculiar ana/telophase-like figures displayed both a central spindle and an actin-based contractile ring (Figure S5). However, most of these structures were morphologically irregular and were thus probably unable to mediate cytokinesis. To define their phenotype in more detail, RNAi cells for the CS3 genes were stained for the checkpoint proteins ZW10 and BubR1 and also for the cell cycle marker Cyclin B. In most ana/ telophase-like figures, Cyclin B was still high, whereas in control cells it was degraded during anaphase and absent from telophases (Figure 6B). In the metaphase-like RNAi figures, ZW10 did not exhibit any streaming towards the cell poles as occurs in normal metaphases (Figure 7A), consistent with a defect in microtubule attachments to the kinetochore [35]. Moreover, the ana/ telophase-like figures showed strong ZW10 and BubR1 centro- meric signals; these signals were mostly absent from control ana/ telophase chromosomes (Figure 7A and data not shown). Finally, the chromosomes of the ana/telophase-like cells displayed two centromeric spots after staining for the kinetochore marker Cenp- C (Figure 7B). These findings confirm that the chromosomes at the poles of the ana/telophase spindles seen in the CS3 phenocluster are indeed comprised of both sister chromatids. The CS3 phenocluster includes the CG9938/Hec1/Ndc80, CG8902/Nuf2 and CG1558/Nsl1 genes, which encode interacting components of the Drosophila kinetochore [36,37], as well as cid/ Cenp-A, that encodes the Drosophila centromere-specific histone H3 variant [17,18]. Of the remaining 14 genes in the CS3 group, one specifies a conserved product of unknown function (CG8233) and 13 encode highly conserved splicing factors (Table S6). The RNAi phenotypes of the genes in the CS3 phenocluster suggest that their products are required for proper kinetochore-microtubule inter- actions. We propose that in the absence of these interactions, the spindle checkpoint remains engaged and sister chromatid separation does not occur. The high levels of Cyclin B and the lack of ZW10 streaming in CS3 RNAi cells are both consistent with this hypothesis [38]. We further posit that the chromosomes are driven to the spindle poles by the same forces that act on the acentric chromosome fragments. As the chromosomes move towards the poles, the spindle elongates so as to resemble an ana/telophase spindle; some of these spindles manage to assemble a defective central spindle and attempt to undergo cytokinesis. Collectively, these results provocatively indicate that in S2 cells typical telophase events, such as central spindle assembly and PLoS Genetics | www.plosgenetics.org 7 July 2008 | Volume 4 | Issue 7 | e1000126