The protein kinase Slk1p is related to the SIN component Sid2p. (A) Schematic representation of Slk1p. The conserved kinase domain of 304 amino acids (162–465) is in the middle of Slk1p. The arrow indicates the lysine residue within the putative ATP binding site at position 191. (B) Alignment of the predicted amino acid sequence of the kinase domains of Slk1p with S. pombe Sid2p and S. cerevisiae Dbf2p and Dbf20p. Identical amino acids are highlighted in black and similar amino acids are shaded in gray. Slk1p shares 51, 50, and 50% amino acid sequence identity with Sid2p, Dbf2p, and Dbf20p, respectively. The arrow indicates the conserved lysine residue in the consensus ATP-binding site.

Slk1p-GFP localization to the SPB is dependent on the SIN scaffold proteins Sid4p and Cdc11p. (A) P_rad21-sid4-gfp is a strain (MBY4933) in which Sid4p expression is under the control of the promoter of the rad21 gene. Top panel, Sid4p-GFP levels were roughly of comparable intensity in all four SPBs in wild-type cells (MBY4972) undergoing meiosis II. Bottom panels, the level of Sid4p was dramatically reduced in two of the four SPBs present in meiosis II cells. Scale bar, 10 μm. (B) Meiotic sporulation phenotype resulting from reduction of Sid4p function. Shown are images of P_rad21-sid4 cells containing two-spored asci. DAPI staining of these cells (demarcated with dotted lines) reveals the presence of four nuclei. P_rad21-sid4 cells (MBY3507) were induced to undergo meiosis, fixed, and stained with DAPI. Arrows show asci with two spores. The dotted lines trace the outline of the indicated two-spored asci with four nuclei. Scale bar, 10 μm. (C) Quantification of the sporulation phenotypes in P_rad21-sid4 cells. Wild-type (MBY1238) and P_rad21-sid4 cells (MBY3507) were induced to undergo meiosis and sporulation on YPD plates for 2 d at 28°C. At least 500 tetranucleate cells were counted for each sample. (D) P_rad21-sid4 expressing Slk1p-GFP and Pcp1p-mCherry was induced to undergo meiosis on YPD plates. Top panel, Slk1p-GFP was detected in all four SPBs in wild-type cells (MBY4364) undergoing meiosis II. Bottom panel, Slk1p-GFP was detected in two or less SPBs in P_rad21-sid4 cells (MBY4884). The dotted lines in the Pcp1p-mCherry panel trace the outline of the cell. Scale bar, 10 μm. (E) Quantification of Slk1p-GFP during meiosis II in wild-type (MBY4364) and P_rad21-sid4 (MBY4884) cells. Cells were induced to undergo meiosis on YPD plates at 28°C for 16 h. At least 200 cells with four clearly discernable SPBs, marked by Pcp1p-mCherry, were counted for each sample. (F) Quantification of Slk1p-GFP during meiosis II in sid4-A1 (MBY4883) cells. Cells were induced to undergo meiosis on YPD plates at 28°C for 14 h. At least 100 cells with four clearly discernable SPBs, marked by Pcp1p-mCherry, were counted for each sample. (G) Quantification of Slk1p-GFP during meiosis II in cdc 11-123 cells (MBY4882). Cells were induced to undergo meiosis on YPD plates at 35°C for 10 h. At least 100 cells with four clearly discernable SPBs, marked by Pcp1p-mCherry, were counted for each sample.

Dynamics of Meu14p and Psy1p in wild-type and slk1Δ asci. (A) Localization of GFP-Psy1p in wild-type and slk1Δ asci. Wild-type cells (MBY3664) and slk1Δ cells (MBY3699) expressing GFP-Psy1p were induced to undergo meiosis, fixed, and stained with DAPI. Green panel reveals GFP-Psy1p localization, whereas the red panel reveals the nuclear architecture. Several examples of failure of encapsulation of nuclei with in spore membranes in slk1Δ are shown. (B) Quantification of normal and abnormal encapsulation of nuclei within spores of wild-type (MBY3664) and slk1Δ cells (MBY3699). (C) Time-lapse imaging of Meu14p-GFP in wild-type (MBY3975) and slk1Δ cells (MBY4492). In both cases, Meu14p assembles into rings. Although constriction of Meu14p rings leads to four Meu14p-containing structures in wild-type cells (Arrows), more than four (and up to eight) Meu14p-containing structures are detected upon constriction of Meu14p rings in slk1Δ cells (arrows). Scale bar, 10 μm. (D) Time-lapse fluorescence microscopy of GFP-Psy1p in wild-type and slk1Δ cells. Top panel, wild-type cells (MBY3664). The rest of the panels show imaging of slk1Δ cells (MBY3699). In all cases, Psy1p localizes initially and assembles cup-shaped structures. In some cases (I; 13/40 cells imaged) Psy1p localization and sporulation proceeded normally, but generated smaller spherically shaped spores. In other cases (II; 27/40 cells imaged) Psy1p and sporulation were abnormal and led to the defective spore formation. Scale bar, 10 μm. (E) Quantification of the time taken for the assembly of the spore membranes after meiosis II in wild-type (MBY3664; n = 15) and slk1Δ cells (MBY3699; n = 21).

Increased expression of psy1 suppresses the sporulation defect of sid2-250 slk1Δ double mutant cells. (A) Bright field microscopy of meiotic sid2-250 slk1Δ cells carrying the indicated plasmids. Expression of psy1 or slk1 was induced on minimal medium at 24°C for 5 d. Scale bar, 10 μm. (B) Sporulation efficiency of the transformants. Transformants were scored for the percentage of asci that contained present spores. (C) Quantification of spore germination of the transformants carrying the indicated plasmids. Vegetative cells were cultured to midlog phase in liquid minimal medium supplemented with 25 μM thiamine, concentrated to OD₅₉₅ = 1.0 and then spotted on MM complete without leucine plates to allow sporulation. The sporulated cells were collected and digested with NEE-154 glusulase, and 2000 spores were plated on a YES plate and incubated for 5 d at 24°C.

Slk1p localizes to the SPBs and the spore periphery during meiosis. (A) Visualization of Slk1p-GFP and the SPB marker Pcp1p-mCherry. (i) No detectable signal of Slk1p-GFP during vegetative growth. (ii) No detectable signal of Slk1p-GFP during horsetail stage of meiosis. (iii) Slk1p-GFP colocalizes with Pcp1p-mCherry on the two SPBs during meiosis I. (iv) Slk1p-GFP colocalizes with Pcp1p-mCherry on the four SPBs during early meiosis II. (v) Slk1p-GFP moves gradually from SPBs to the FSM during late meiosis II. (vi) Slk1p-GFP is distributed on the FSM at the end of meiosis II. (vii) Slk1p-GFP signal is not observed in mature spores. Slk1p-GFP/Pcp1p-mCherry–expressing cells (MBY4364) were cultured on YPD plates to induce meiosis. Scale bar, 10 μm. (B) Time-lapse imaging of redistribution of Slk1p-GFP from the SPB to the spore periphery. Arrows show that Slk1p-GFP localizes to the SPBs. Scale bar, 5 μm. (C) Observation of live cells (MBY5210) expressing mCherry-Atb2p and Slk1p-GFP, during the first meiotic division (i), metaphase II (ii), anaphase II (iii), and sporulation (iv). The tubulin marker mCherry-Atb2p was expressed under the control of nmt1 promoter from the plasmid (pREP1-mCherry-atb2) on MM lacking leucine plates with 5 μM thiamine. Scale bar, 10 μm.

Slk1p is required for proper sporulation. (A) Bright field images of asci. Wild-type (MBY1238), slk1Δ (MBY1850), and slk1K191R (MBY 4436) cells were sporulated on YPD plates for 2 d at 28°C and were observed by differential interference contrast microscopy. Scale bar, 10 μm. (B) Quantification of spore diameter in wild-type cells (MBY1238) or slk1Δ (MBY1850). Cells were induced to undergo meiosis and sporulation on YPD plates for 2 d at 28°C. Values are the mean ± SD. At least 200 spores were counted for each sample. (C) Quantification of spore number per ascus. At least 500 tetranucleate cells were counted for each sample. (D) Germination efficiency of wild-type, slk1Δ and slk1K191R spores. Asci formed on YPD plates for 2 d were randomly chosen and grown on YES solid medium after micromanipulation at 30°C. Colonies formed after 3 d of incubation at 30°C were counted. At least 200 asci were selected from each sample. (E) Slk1pK191R-GFP can localize to SPB during meiosis. The dotted lines indicate the outline of the asci. Pcp1p-mCherry was used as a marker of the SPB. Scale bar, 10 μm. (F) Quantification of localization patterns of Slk1p-GFP and Slk1p K191R-GFP during meiosis II. Cells expressing Slk1p-GFP (MBY 4364) and Slk1pK191R-GFP (5118) were induced to undergo meiosis on YPD plates at 28°C for 16 h. At least 200 cells with four clearly discernable SPBs, marked by Pcp1p-mCherry, were counted for each sample. (G) Thin-section EM images of the ascospores (white arrowhead) of wild-type cells. Scale bar, 1 μm. (H) Electron microscopy of ascospores of slk1Δ cells (MBY1850) to show anucleate spores and naked nuclei. Black arrows show small but normally encapsulated ascospores. Black arrowheads show naked nuclei. The white arrow points to an anucleate spore. Nuclei are marked by N. Scale bar, 1 μm.

Overlapping role for Slk1p and Sid2p in sporulation. (A) Synthetic genetic interaction between sid2-250 and slk1Δ mutants. Left panel, proper spores are formed in sid2-250 cells (MBY4363) at 24°C. Middle Panel, aberrant spores are formed in slk1Δ cells (MBY1850). Right panel, no spores are formed in sid2-250 slk1Δ cells (MBY4355). Scale bar, 10 μm. (B) Iodine vapor staining of slk1Δ, sid2-250, and sid2-250 slk1Δ cells. A heterothallic wild-type strain (h⁻) and a homothallic wild-type strain (h⁹⁰) were used as negative and positive control, respectively. (C) Time-lapse imaging of Meu14p-GFP in sid2-250 slk1Δ cells (MBY4594). Initially Meu14p assembles into rings in these cells. However, constriction of Meu14p rings leads to more than four (and up to eight) Meu14p-containing structures (arrows). Scale bar, 10 μm. (D) Time-lapse fluorescence microscopy of GFP-Psy1p in sid2-250 slk1Δ cells (MBY4377). Psy1p localizes initially and assembles cup-shaped structures. However, spore membrane expansion was abnormal leading to defective sporulation. Scale bar, 10 μm. (E) EM images of the ascospores of sid2-250 slk1Δ cells (MBY4355) show anucleate spore wall and naked nuclei. The black arrowheads show naked nuclei. White arrows show misshapen anucleate spore wall. Nuclei are marked by N. Cells were cultured on YPD plates for 2 d at 24°C to induce meiosis. Scale bar, 1 μm.

Genetic interactions between spo3-gfp and slk1 mutants. (A) Bright-field images of asci from strains of indicated genotypes. spo3-gfp (MBY3663), slk1Δ (MBY1850) and slk1Δ spo3-gfp (MBY 3698) cells were sporulated on YPD plates at 28°C for 2 d and were observed by phase contrast microscopy. Scale bar, 10 μm. (B) EM images of the ascospores of slk1Δ spo3-gfp cells (MBY3698) show anucleate spore wall and naked nuclei. The black arrowheads show naked nuclei. White arrows show misshapen anucleate spore wall. Nuclei are marked by N. Scale bar, 1 μm. (C) Schematic representation of data described in the study.