Manipulation of yeast strains (tagging with fluorescent proteins, marker insertions, gene deletions, and promoter substitution using the CUP1-1
promoter) was performed as described previously (Janke et al., 2004
). The strains generated during the course of this work are listed in .
Sporulation and spore counting
Highly standardized sporulation conditions were used, and we confirmed that our protocol was able to reproduce results when performed on different days. It essentially followed the method described previously by Alani et al. (1990)
. In brief, the strains were thawed from glycerol −80°C stocks, grown on YP–glycerol plates for 2 d, streaked to single colonies on YPD plates, and grown for 2 d. Single colonies were used to inoculate 25 ml YPD cultures in a 100-ml flask and were grown for 30 h (230 rpm at 30°C). Presporulation growth was performed in 1% YP–acetate for 13.5 h (1:50 inoculation of 400 ml in a 2-liter flask at 230 rpm, 30°C, and good aeration). Cells were washed once with water (1 vol at RT and 2,000 rpm for 3 min) and distributed in 50-ml aliquots into 250-ml flasks with sporulation medium (water with the indicated amount of KAc [wt/vol]; added from a sterile filtered stock solution) at a cell density of 1 OD600
cells/ml). Sporulation was performed without sealing the flasks for >24 h at 230 rpm and 30°C. Thereupon, aliquots of the cells were fixed with 70% ethanol, washed with water, and resuspended in 60% glycerol/water containing 1 μg/ml Hoechst 33342.
Spore counting was performed using stacks of images acquired from Hoechst 33342–stained samples. We used a microscope (IRBE; Leica) equipped with a 63× NA 1.4 oil objective (Leica), a camera (CoolSNAP HQ; Photometrics), and a DAPI filter set (Chroma Technology Corp.). The pictures were recorded using Metamorph software (Molecular Devices). Maximum projections of the Hoechst 33342 images were superimposed with the phase-contrast image using Metamorph software. G0 and nonads (meiosis but no spores) were discriminated based on Hoechst 33342 staining. This spore-counting method was essential to reliably discriminate all of the different species. Counting using only phase-contrast microscopy led to significant systematic errors. Sporulation efficiency was calculated as follows: [(% tetrads × 4) + (% triads × 3) + (% dyads × 2) + % monads]/4.
For live cell imaging, cells were adhered with Concanavalin A on small glass bottom Petri dishes (MaTek). All live cell experiments were performed at RT. Live cell imaging () was performed on an imaging system (DeltaVision Spectris; Applied Precision) equipped with GFP and Cy3 filters (Chroma Technology Corp.), a 60× NA 1.4 oil immersion objective (plan Apo, IX70; Olympus), softWoRx software (Applied Precision), and a CoolSNAP HQ camera. For the experiment shown in F, sporulating cells were inspected on a microscope (IRBE; Leica) equipped with a plan Apo 100× NA 1.4 oil objective (Leica), a CoolSNAP HQ camera, and DAPI, CFP, YFP, and Cy3 filter sets (Chroma Technology Corp.). FRAP was either performed on a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.; ) or on a wide-field epifluorescence microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) equipped with a laser scanner for photobleaching using a high aperture 63× NA 1.2 water immersion objective (C-Apochromat; Carl Zeiss MicroImaging, Inc.; A). The wide-field microscope was necessary to visualize Mpc54p-GFP at SPBs in cells before meiosis II as a result of the lower amounts of this protein at SPBs. Quantification of videos (; and 4 A) was performed using MetaMorph software and maximum projections of the videos. Quantification of the experiment in (B and C) was performed using LSM 510 software (Carl Zeiss MicroImaging, Inc.). Conversion of file formats from 12 to 8 bit was performed using Metamorph software. Photoshop (Adobe) was used to mount the images and to produce merged color images. No image manipulations other than contrast, brightness, and color balance adjustments were used.
For electron microscopy and analysis of the overexpression of MP genes ( D), a diploid strain containing one copy of MPC54
, and SPO74
under control of the CUP1-1
promoter (strain YCT851) was induced with 10 μm CuSO4
(added 4 h after induction of sporulation on 0.3% acetate). A sample was taken at a time point in which mixed populations of cells in meiosis I and meiosis II were maximally enriched (5.5 h) and were processed for electron microscopy using osmium tetroxide fixation as described previously (Knop and Strasser, 2000
). Samples were visualized on an electron microscope (Biotwin CM-120; Philips) using a CCD camera (DualVision; Gatan). Strong overexpression of all three proteins as compared with wild-type cells was validated using Western blotting.
Correlation of MP formation with the age of the SPBs involved
We fused an RFP with a retarded formation of the fluorophore of ~2–6 h (RedStar; Knop et al., 2002
) to the integral SPB component Spc42p (Donaldson and Kilmartin, 1996
). This allowed the discrimination of SPBs from all three generations (oldest SPB, intermediate SPB [formed before meiosis I], and two new SPBs [formed before meiosis II]; ). (DsRed that was used previously to discriminate SPBs from different generations in yeast [Pereira et al., 2001
; Nickas et al., 2004
] has a maturation time of the fluorophore of >10 h and, therefore, was not suitable for this application). For live cell imaging of MP assembly, eqFP611 was used as the RFP (Janke et al., 2004
). It exhibits properties similar to those of RedStar.
The antibody specific for Spo74p was produced with bacterially expressed 6HIS-Spo74p and was affinity purified. All of the other antibodies have been described previously (Knop and Strasser, 2000
Simulation of SNC
Assumptions for the digitization module (assumption A; see supplemental material) are listed as follows: (1) Initially, the free Mpc54p monomers and the Mpc70p/Spo74p heterodimers are homogeneously distributed in the cytoplasm. Diffusion is fast and readjusts a homogeneous distribution in the cytoplasm. Therefore, crystal growth is not diffusion limited. (2) The sizes of the initial crystal seeds at the four different SPBs are different. (3) Mpc54p monomers and Mpc70p/Spo74p heterodimers are incorporated into the crystals at the SPBs if they are both present in some spatial region within some short period of time. (4) A larger crystal provides more binding sites than a smaller one and, therefore, incorporates more protein. Thus, a larger crystal depletes the cytoplasmic pools more rapidly than a smaller one. (5) The crystal size is limited by the size of the SPB. (6) If the crystal size reaches a certain threshold level, the crystal is considered to be a fully functional MP.
Assumptions for the simulation of populations (sporulation profile; assumptions B and C; see supplemental material) are listed as follows: (7) The functional relationship between the number of Mpc54p, Mpc70p, and Spo74p proteins in the cell and acetate concentration (acetate → protein function) can be approximated by the experimentally derived functional relationship between sporulation efficiency and acetate concentration. (8) Because of variations within the population, the cellular response of cells in a cell population to a given acetate concentration (i.e., the amount of Mpc54p/Mpc70p/Spo70p) varies according to a second symmetric two-parameter distribution. (9) The acetate concentration available to individual cells in the population is described by a simple symmetric two-parameter distribution (e.g., Gaussian). This accounts for the asynchronicity of the population with regard to progression through meiosis. Cells that perform MP assembly earlier than others—because they performed the meiotic divisions faster—have more acetate available as a result of the simultaneous consumption of acetate by the population.
The simulations were performed using Mathematica software (version 5.0; Wolfram Research Inc.). In the first step, we designed a set of differential equations that models the crystal growth according to assumptions 1–5 (). To account for assumption 1, we implemented the homogeneously distributed amounts of Mpc54p monomers and Mpc70p/Spo74p heterodimers as time-dependent functions (Mpc54[t] and Mpc70Spo74[t]) that describe the available amounts of protein in the cytoplasm. We defined four time-dependent functions (Crystal1[t]–Crystal4[t]) that describe the crystal growth at up to four potential SPBs. To account for assumption 3, the differential equations describing the transition from spore-free to spore-containing cells contain the product of Mpc54[t] and Mpc70Spo74[t]. Assumption 4 is included by a positive feedback in the crystal growths that is proportional to the size of the corresponding crystal. Assumption 5 is implemented by a saturation function that is characterized by the two parameters of slope and maximum crystal size (saturation). The basic differential equations are shown in .
Basic differential equations derived from assumptions 1–5. See Materials and methods.
Initial conditions for the set of differential equations are the four different initial crystal seeds (assumption 2) and the two initial amounts of Mpc54p monomer and Mpc70p/Spo74p dimer in the cytoplasm. We calculated the initial amounts of proteins for an interval of acetate concentration using the function from assumption 7. This function (generally termed acetate → protein function, mathematically defined as Mpc54Metabolism[c] for Mpc54p monomers and Mpc70Spo74Metabolism[c] for Mpc70p/Spo74p heterodimers; see supplemental material) is characterized by three parameters: an offset, the steepness, and the maximum amplitude. The set of nonlinear differential equations was solved numerically and iteratively for the entire interval of acetate concentrations.
In the second step, we derived the steady-state crystal sizes from the numerical solution of the differential equations. Corresponding to assumption 6, all crystals with a size above a given threshold (30% of the maximum crystal size, defined by the saturation parameters) were regarded as spores. We defined SporesSum[c] as the function that represents the number of spores resulting from this digitization step depending on the acetate concentration c. Assumption 8 was implemented by convolving this function with the Gaussian distribution that describes the variation of available acetate. As a result, we obtained the statistical appearances of the five possible spore configurations (0–4 spores) depending on the acetate concentration c.
Finally, we iterated the steps of solving the differential equations and calculating the statistical appearances for a certain interval of maximum amplitudes of Mpc54Metabolism[c] and Mpc70Spo74Metabolism[c]. Considering assumption 9, the statistical appearances were weighted using the Gaussian distribution for the maximum amplitudes that are introduced to model the cellular response. This yielded the theoretical sporulation profile.
Different dominant markers (hphNT1
; Janke et al., 2004
) were introduced next to the centromeres of chromosome V between ORFs YER001w
in a diploid homothallic yeast strain (SK1 background; resulting strains YCT918 and YCT919). The heterozygous CEN5-hph
strain (YCT930) was selected on Hygromycin B/G418 plates upon the mating of spores of strains YCT918 and YCT919. YCT930 was then used to delete one copy of either CDC5
(ORF YMR001c) or PRE3
(ORF YJL001w) with the natNT2
marker (Janke et al., 2004
). After sporulation in liquid medium containing either 0.1 or 0.01% acetate medium, 107
asci were spotted on a YPD plate. Unsporulated cells were killed by ether treatment (Guthrie and Fink, 1991
). The ascii were incubated on YPD plates for 18 h. The cells were collected and spread on YPD plates (100–150 colonies per plate) and grown for 2 d. The colonies were assayed for the presence of all three markers simultaneously (kan+, hph+, and nat+) as well as for only two or one of the markers by replica plating. 400–600 colonies were evaluated for each sample. Colonies, which contained cells with all three markers, were considered to derive from cells (with respect to the deletion of the essential gene) that were formed by mating of nonsister spores. Cells containing the nat+ marker (which marks the deletion of the essential gene) but only the kan+ or hph+ marker in addition were considered to be the result of mating upon germination but not between nonsister spores. Colonies containing the kan+ and/or the hph+ marker but not the nat+ marker were considered to originate from other types of mating.
A homothallic YJM145/SK1 (Kane and Roth, 1974
; McCusker et al., 1994
) hybrid strain was generated by mating spores of strain YCT925 (YJM145 background) that contained one CENV-hphNT1
integration with spores of strain YCT918 that contained one CENV-kanMX
integration and selection on Hygromycin B/G418 plates. Upon sporulation of the resulting strain under low acetate concentration (0.01%), a population of heterozygous diploids was generated through the isolation of 80 dyads by micromanipulation (of which 88% did form a colony). For the generation of homozygous diploids, 40 dyads were dissected (spore survival frequency was 68%). Both species were grown on YPD plates for 2 d and independently pooled in water. Equal amounts of the cells (each 5 × 105
cells) were mixed and grown at 30°C in 400 ml YPD for 24 h (~12–13 generations). For subsequent rounds, 106
cells were transferred to a new flask and grown for another 12–13 generations. The composition of the culture was analyzed in the beginning of the experiment and after each round of growth for the content of hph+ or kan+ (homozygous diploids) or hph+ and kan+ cells (heterozygous diploids).
Calculation of the fraction of ORFs in the yeast genome with significant centromere linkage
Significant centromere linkage can be observed up to ~35 cM away from the centromere (Sherman and Wakem, 1991
), whereas the total yeast genome covers ~4,500 cM (Mortimer et al., 1992
). With 16 chromosomes present in yeast and a total of 5,792 annotated protein-encoding ORFs (www.yeastgenome.org
), an estimated 1,440 ORFs are within the CEN
Statistical significance of centromere linkage for groups of genes
A shows that essential genes are overrepresented close to the centromeres. 70/317 genes found within a 20-kbp distance to a centromere are essential. In comparison, 1,032/5,773 yeast genes are essential. This overrepresentation of essential genes is significant at a P = 0.03 according to a hypergeometric test.
Online supplemental material
Videos 1 and 2 show the transition from anaphase meiosis I to metaphase meiosis II and correspond to (A and C). Supplemental material provides the Mathematica files for the simulation () and provides a description of the three parts of the simulation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200507168/DC1