A genome–wide screen to identify genes controlling the rate of entry into mitosis in fission yeast

Abstract

We have carried out a haploinsufficiency (HI) screen in fission yeast using heterozygous deletion diploid mutants of a genome-wide set of cell cycle genes to identify genes encoding products whose level determines the rate of progression through the cell cycle. Cell size at division was used as a measure of advancement or delay of the G2-M transition of rod-shaped fission yeast cells. We found that 13 mutants were significantly longer or shorter (greater than 10%) than control cells at cell division. These included mutants of the cdc2, cdc25, wee1 and pom1 genes, which have previously been shown to play a role in the timing of entry into mitosis, and which validate this approach. Seven of these genes are involved in regulation of the G2-M transition, 5 for nuclear transport and one for nucleotide metabolism. In addition we identified 4 more genes that were 8–10% longer or shorter than the control that also had roles in regulation of the G2-M transition or in nuclear transport. The genes identified here are all conserved in human cells, suggesting that this dataset will be useful as a basis for further studies to identify rate-limiting steps for progression through the cell cycle in other eukaryotes.

Noisy Cell-Size-Correlated Expression of Cyclin B Drives Probabilistic Cell-Size Homeostasis in Fission Yeast

“…Thus, we propose that the nuclear accumulation of Cyclin/CDK complexes is size dependent and could serve as a molecular correlate of cell size in the P(Div) model. This proposal is consistent with the fact that a number of nuclear transport factors have been implicated in cell-size control at mitotic entry [ 29 , 30 ].…”

“…Cdc13 is one of few cell-cycle proteins that has been shown to be haploinsufficient, with a Cdc13 heterozygous deletion diploid dividing ∼17% longer [ 30 ] than WT. The dosing of Cdc13 level against cell size has not been performed, and to investigate this, we engineered a strain expressing Cdc13-sfGFP controlled by a new inducible promoter modulated using anhydrotetracycline (tet) ( Figure S3 ), which features high maximum expression and a linear dose response over an extended range of expression.…”

A systematic genetic screen identifies essential factors involved in nuclear size control

“…152 potential aberrant N/C ratio mutants were identified and subject to a secondary round of screening as described for the primary screen. The previously described 5300 control strain in which the KanR deletion cassette is inserted in pseudogene SPAC212.05c was used as a control for this and subsequent screening stages [34]. 60 potential N/C aberrant ratio mutants, identified in both primary and secondary screens, were carried forward for tertiary screening.…”

“…The haploid wild type control used for this screen was derived from the previously described 5300 heterozygous diploid control strain in which the KanR deletion cassette is inserted in pseudogene SPAC212.05c [34]. It is of note that this wild type control strain, in which the Ish1-yEGFP construct is integrated into the deletion cassette deleting the 5300 pseudogene, displayed an N/C ratio of 0.05 in this screen, with other strains distributed around this value (Fig 2A).…”

A systematic cell size screen uncovers coupling of growth to division by the p38/HOG network inCandida albicans

“…Subsequent analyses of many of these size mutants at a single cell level have suggested that the critical cell size at Start may depend on growth rate in G1 phase and/or on cell size at birth (31, 32). Visual screens of S. pombe haploid and heterozygous deletion collections for size phenotypes also revealed dozens of novel size regulators, many of which altered size in a genetically additive fashion (33, 34). A large number of genes appear to influence size in metazoan species.…”