To validate the predictions that CABP7 is required for cytokinesis and that TOR1AIP1 is required for spindle assembly, we decided to carry out a functional analysis of these genes. Both genes represent true positives, as their RNAi phenotypes could be complemented by RNAi-resistant transgenes (). To test directly the phenotypic clustering predictions that the binucleation phenotype of CABP7 arose from a cytokinesis defect and that the chromosome alignment phenotype of TOR1AIP1 arose from spindle assembly problems, we performed high-resolution four-dimensional confocal microscopy with microtubule and chromosome markers. Indeed, CABP7 suppression caused cytokinesis failure after normal chromosome segregation, resulting in a single cell containing four centrosomes and two nuclei (). Knockdown of TOR1AIP1 caused spindle formation to fail in prometaphase, because centrosomes could form only weak mitotic asters and failed to establish a bipolar spindle or align chromosomes, leading to aberrant mitotic exit and cell death (). Thus, CABP7 and TOR1AIP1 are bona fide cytokinesis and spindle assembly genes, respectively, revealing a novel connection between calcium binding and cytokinesis on the one hand and nuclear membrane proteins and the assembly of the mitotic microtubule spindle on the other hand.
Functional analysis of spindle phenotypes
Secondary four-dimensional confocal imaging assays are currently not high-throughput methods. We therefore focused our four-dimensional confocal spindle assembly assays on knockdown experiments of genes with successful rescue experiments ( and Supplementary Movies 31
). Consistent with the predictions of gene function from the primary screen, prometaphase delays were explained by spindle assembly defects (AURKB, INCENP, TOR1AIP1
), whereas binucleated cells resulted from chromosome alignment and/or segregation problems that subsequently prevented cytokinesis because chromatin persisted in the area of the cleavage furrow19,20
); in other cases chromosome segregation was normal but cytokinesis appeared to be specifically affected and cells contained two nuclei and four centrosomes (PTGER2, ECT2, CABP7, C13orf23
). Binucleated cells either remained viable and ceased dividing (PTGER2
) or formed multipolar spindles that resulted in aberrant chromosome segregation in the next cycle which, when coupled to another failed cytokinesis, resulted in large polylobed nuclei (ECT2, CABP7, C13orf23
). Together this high-resolution assay for spindle formation, chromosome alignment and segregation demonstrates that the phenotypic predictions derived from the automatic mining of the primary genome-wide screen are valid. In addition, the analysis of phenotype development with high temporal resolution in single cells directly shows the causal relationship of different phenotypic classes. Thus, the detailed phenoprints of the primary RNAi screen provide mechanistic hypotheses for the observed phenotypes that can now be pursued in targeted biochemical and cell biological experiments for each gene. As exemplified by our imaging of the spindle microtubules, such future experiments should ideally complement the chromosome visualization assay of the primary screen with information about other key elements of the mitotic machinery, such as centrosomes, spindle microtubules and kinetochores.