Fig 1.
Spontaneous diploidization of SPB mutants such as cdc31-2 at the permissive temperature.
(A) Wild-type (SLJ7819) and cdc31-2 mutant (SLJ10777) cells containing GFP-Tub1 (white) and Spc42-mCherry (magenta) were generated with a pURA3-CDC31 plasmid. After growth on 5-FOA at 23°C to select for loss of the plasmid, the cdc31-2 mutant spontaneously diploidize despite the formation of bipolar spindles. A representative image from each is shown along with the cell outline (dashes), and the percentage of large budded cells for with bipolar, monopolar or multipolar/broken spindles was quantitated (n>150). Bar, 2 μm. DNA content was assayed by flow cytometry. The biphasic peaks in wild-type cells represent cells with G1 (1N) and G2/M (2N) DNA content. At 23°C, cdc31-2 mutants have diploid DNA content (2N and 4N). (B) Schematic of pathway to diploidization in cdc31-2. Cells containing cdc31-2 do not undergo the typical cell division of haploids (gray box, dashed arrows). Instead, due to a defect in chromosome segregation, haploid (1N) cells undergo an aberrant cell division to produce a diploid (2N) and aploid (0N) cell. The diploid cell does not have the same defect as haploids, resulting in successful propagation. Because of this, we suspect that a suppressor mutation is acquired.
Fig 2.
Screen for suppressors of cdc31-2 diploidization.
(A) Suppressors of the cdc31-2 increase-in-ploidy were isolated following mutagenesis of SLJ6749 (MATα cdc31-2 CAN1::KANMX trp1Δ::KANMX cyh2 LYP1 ura3-1 his3-11,15 ade2-1 pURA3-CDC31) to ~50% viability using EMS. Loss of the pURA3-CDC31 covering plasmid was selected using 5-FOA; strains without a suppressor will spontaneously diploidize as shown in Fig 1 while those with a suppressor will remain haploid. (B) Haploid (1N) or diploid (2N) strains can be mated to a haploid to form diploid (2N) or triploid (3N) cells. The viability of meiotic products is high from diploids (88.2%, n = 40 tetrads) compared to triploids (6.2%, 40 tetrads). Using this property, suppressors of diploidization were selected by mating to SLJ6750 (MATa CDC31 can1Δ::STE2pr-HIS3MX CYH2 lyp1Δ::HYGMX ura3-1 trp1-1 his3-11,15 ade2-1) on YPD + G418 + Hyg. Following sporulation, haploid selection was carried out using SD-His-Lys-Arg+canavanine+thialysine+cycloheximide. (C) From ~100,000 EMS mutagenized cells, 61 possible suppressors were identified, and 54 were confirmed to be haploids in a secondary screen of the original mutagenized colonies by flow cytometric analysis of DNA content. Of these, 43 appeared to have mutations in the covering plasmid that allowed for growth. The remaining 11 suppressors were analyzed by tetrad dissection to ensure that suppression segregates 2:2 through at least two crosses to SLJ6121 (MATa cdc31-2 can1Δ::STE2pr-HIS3MX TRP1 CYH2 ura3-1 his3-11,15 ade2-1 pURA3-CDC31). An example of flow cytometry data from one hit is shown. (D) Coverage ratio of all 16 yeast chromosomes in the haploid suppressors (ems7, ems9, or ems11) relative to the diploid control (EMS7, EMS9, EMS11). Other single nucleotide polymorphisms and insertions/deletion polymorphisms identified in the haploid suppressors are listed in S1 Table. (E) Quantitative PCR was performed on all 11 suppressors to determine the mean copy number of all 16 chromosomes relative to a wild-type, with chromosome XV plotted in red. Error bars, standard deviation from the mean.
Fig 3.
An extra copy of the cdc31-2 gene is necessary and sufficient to suppress IPL.
(A) To test if an extra copy of cdc31-2 is necessary to suppress IPL, one copy of the cdc31-2 locus was deleted in cells with a chromosome XV disome homozygous for cdc31-2, as illustrated in the schematic. cdc31-2 ChXV(cdc31-2Δ::KANMX) are predicted to form diploids with two extra copies of chromosome XV, however, qPCR and PCR analysis suggests that they revert to a diploid (2N) karyotype due to chromosome loss, as indicated. (B-C) The DNA content by flow cytometry (top) (B) and growth (C) of wild-type (SLJ7249), cdc31-2 (SLJ809), the chromosome XV cdc31-2 disome (SLJ7106, cdc31-2 2xChXV(cdc31-2)) and the deletion ((SLJ7111, cdc31-2 ChXV(cdc31-2Δ::KANMX)) that contain pURA3-CDC31 were compared after growth in SD-Ura or 5-FOA at the indicated temperatures. Quantitative PCR was also used to verify the karyotype of strains from 5-FOA compared to a haploid control (bottom). Chromosome XV is plotted in purple. Error bars, standard deviation from the mean. (D) To test if an extra copy of cdc31-2 is sufficient to suppress IPL, one additional copy of cdc31-2 was inserted into the HIS3 locus on chromosome XV. (E-F) The DNA content (E) and growth (F) of wild-type (SLJ7249), cdc31-2 (SLJ809) and cdc31-2 with an empty vector, wild-type CDC31 or cdc31-2 at HIS3 (SLJ13092, SLJ13093 or SLJ13094) were analyzed after growth in SD-Ura or 5-FOA at the indicated temperatures. Quantitative PCR was used to verify the karyotype of strains from 5-FOA compared to a haploid control.
Fig 4.
Scaling of SPB components with ploidy.
(A) To determine levels of Cdc31 and cdc31-2, western blotting was performed using anti-Cdc31 antibody on strains containing the indicated number of gene copies of endogenously expressed CDC31 or cdc31-2. Pgk1 served as a loading control. A representative western blot is shown along with quantitation from two replicates. Error, SEM. (B) Schematic of the SPB showing the location of the core, luminal ring and half-bridge. (C) Side and top-down views of the SPB from haploids and diploids along with dimensions reported from EM measurements. Assuming the SPB is round and the bridge elliptical and limited to a single protein layer, theoretical scaling factors can be calculated. Based on dimensions calculated from EM measurements, which are shown, a single layer of protein in the SPB core would be 2.11 times larger in diploids. The half-bridge is thought to be a monolayer of constant length in both haploids and diploids, however, its width may scale. Two potential models for scaling of the luminal ring are depicted: a continuous scaling, where components increase proportionally to the circumference of the SPB core (1.45-fold); or radial dilation, where the amount of components do not increase. (D) Levels of fluorophore tagged protein derivatives expressed from endogenous loci in haploids or homozygous diploids were determined by quantitative imaging (see S3 Fig). For each protein, levels in haploid cells were normalized to 1. Errors, SEM with N>300 for each sample.
Table 1.
Ploidy level and function of spb ts alleles at 23°C in W303.
Fig 5.
Dosage and IPL in other SPB mutants.
(A) Schematic of SPB duplication pathway from an unduplicated SPB to duplicated side-by-side SPBs. Mutants defective in initiation, maturation and insertion of the new SPB have been isolated; shown are alleles required at each step that also exhibit IPL at 23°C (see Table 1). (B) To test if an extra copy of these mutant genes is sufficient to suppress IPL, one additional copy was inserted into the HIS3 locus on chromosome XV as in Fig 3D, 3E and 3F. The growth and DNA content of wild-type, mutant and mutant with an empty vector, wild-type gene or mutant gene at HIS3 were analyzed after growth in SD-Ura or 5-FOA at the indicated temperatures.