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First published online 8 April 2008
doi: 10.1242/jcs.023812

Journal of Cell Science 121, 1383-1392 (2008)
Published by The Company of Biologists 2008
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Slk1 is a meiosis-specific Sid2-related kinase that coordinates meiotic nuclear division with growth of the forespore membrane

Livia Pérez-Hidalgo, Ana Elisa Rozalén, Cristina Martín-Castellanos and Sergio Moreno*

Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain


Figure 1
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  		Fig. 1. slk1 encodes a serine-threonine protein kinase similar to S. pombe Sid2. (A) Schematic representation of the Slk1 domains. The kinase domain is represented in black and the AGC-kinase C-terminal domain is represented in grey. The amino acid positions of these domains are indicated. (B) Sequence comparison of Slk1 and related proteins. Sequence alignment was generated using CLC Free Workbench 4.0.3 software (CLC Bio, Aarhus, Denmark). Shading was performed with the Boxshade 3.21 program at the http://www.ch.embnet.org/server. Identical amino acids are highlighted in black and similar amino acids are highlighted in grey. Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens. (C) The phylogenetic tree was constructed using CLC Free Workbench 4.0.3 software according to the UPGMA algorithm.

 

Figure 2
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  		Fig. 2. slk1 expression is meiosis-specific. (A) Northern-blot analysis of slk1 expression in a pat1-driven meiosis. Strain h–/h– pat1-114/pat1-114 (S964) was induced to enter synchronous meiosis and total RNA was extracted at the indicated times. Samples were processed and blotted, and filters were hybridized with a slk1 probe. As a loading control, 18S rRNA stained with Methylene Blue is shown. exp, exponential growth; meiS, pre-meiotic S-phase; MI, meiosis I; MII, meiosis II. (B) The same experiment as in A, but in this case strain h–/h– pat1-114/pat1-114 slk1-GFP/slk1-GFP (S1706) harbouring a functional version of slk1 tagged with GFP was used, and protein was extracted at the indicated times and processed for western blot. Slk1-GFP was detected with anti-GFP antibodies. Tubulin levels are shown as a loading control. (C) FACS analysis of samples taken during the experiment shown in A. Pre-meiotic S-phase occurs between 1.5 and 2 hours. Spores generated are released during sample processing, and appear as a 1C peak between 6 and 9 hours. (D) Meiosis progression of the experiment shown in A was monitored by DAPI staining. White square, one nucleus; triangle, two nuclei (meiosis I); circle, three to four nuclei (meiosis II); black square, spores. At least 300 cells were counted for each time-point.

 

Figure 3
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  		Fig. 3. slk1 is required for spore formation. (A) The slk1{Delta} mutant produces asci with small spores. Wild-type (S1478) and slk1{Delta} (S1883) h90 homothallic strains were incubated on sporulation media (MEA) for 2 days, and Nomarski microphotographs were taken. Scale bar: 10 µm. (B) The slk1{Delta} mutant proceeds through meiotic divisions with normal kinetics. Meiosis progression in pat1-114 diploid strains S964 (wild type) and S1566 (slk1{Delta}) was monitored by DAPI staining. Percentages of cells with one nucleus (green square), two nuclei (red triangle; meiosis I) and three to four nuclei (blue circle: meiosis II) are shown.

 

Figure 4
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  		Fig. 4. slk1 is required for proper engulfment of the nuclei by the forespore membrane at the end of meiosis. Homothallic h90 strains S1478 (wild-type) and S1883 (slk1{Delta}) carrying the plasmid pREP81-Psy1-GFP were sporulated on MEA plates at 25°C. (A) A percentage of slk1{Delta} cells show defects in nucleus engulfment by the forespore membrane, as revealed by Psy1-GFP fluorescence. Three representative cells are shown. Left panel: bright-field images. Central panels: Hoechst and GFP fluorescence. Right panel: merge. (B) Frequency of abnormal forespore membrane formation in the slk1{Delta} mutant. Cells are classified according to the number of defective spores. In class I, all nuclei are engulfed by the forespore membranes; class II, one forespore membrane fails to engulf a nucleus; class III, there are two defective forespore membranes; class IV, there are three defective forespore membranes; class V, there are four defective forespore membranes; class VI, there are fewer than four forespore membranes; and class VII, forespore membrane formation is incomplete. Means and standard deviations of three independent experiments are presented. In each experiment, at least 200 cells were counted. (C) Time-lapse experiment showing forespore membrane growth in a slk1{Delta} strain (S1883) carrying the plasmid pREP81-Psy1-GFP. Cells were sporulated on MEA plates and DNA was stained with Hoechst. Stacks of five images separated by 1 µm were taken every 5 minutes. GFP (green) and Hoechst (red) merged images were generated with ImageJ. Arrows mark the abnormal engulfment of DNA by the forespore membrane in one spore. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. Forespore membrane growth is defective in the slk1{Delta} mutant. (A) Time-lapse experiments showing forespore membrane growth in wild-type (S1478) and slk1{Delta} (S1883) strains carrying the plasmid pREP81-Psy1-GFP. Cells were sporulated on MEA plates and DNA was stained with Hoechst (blue). Stacks of eight images separated by 0.5 µm were taken every 2 minutes. Deconvolution of images and maximal projections were obtained using Deltavision software. GFP and Hoechst merged images were generated with ImageJ. Scale bars: 4 µm. (B) Forespore membrane (FSM) growth stops prematurely in slk1{Delta} cells. The FSM length of wild-type and slk1{Delta} spores shown in A was measured, and means and standard deviations are represented.

 

Figure 6
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  		Fig. 6. Genetic interaction between slk1 and spo3 alleles. (A) spo3-S3 and spo3-GFP alleles enhance the sporulation defect caused by the deletion of slk1. Homothallic wild-type (S1478), slk1{Delta} (S1883), spo3-S3 (S1734), slk1{Delta} spo3-S3 (S1889), spo3-GFP (S1458) and slk1{Delta} spo3-GFP (S1888) strains were sporulated on MEA plates, and DIC images were taken after 2 days of incubation at 25°C. (B) MEA plates from the experiment shown in A were stained with iodine vapour after incubation at 25°C for 3 days. Staining is slightly reduced in the slk1{Delta} single mutant, but it is completely abolished in the double mutants. (C) Psy1-GFP fails to encapsulate the nuclei in slk1{Delta} spo3-S3 cells. Mutant cells carrying the plasmid pREP81-Psy1-GFP were sporulated on MEA plates at 25°C, stained with Hoechst and photographed. Merged images are shown in the right column. (D) Forespore membranes are unable to engulf the nuclei in slk1{Delta} spo3-GFP cells. Forespore membranes were visualized by Spo3-GFP fluorescence and DNA was stained with Hoechst. Merged images are shown in the right column. Scale bars: 10 µm.

 

Figure 7
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  		Fig. 7. Slk1-GFP localizes to the spindle pole body and the forespore membrane during meiosis. (A) A homothallic strain harbouring the slk1-GFP allele under its own promoter (S1717) was incubated on MEA for 24 hours at 25°C. Meiotic cells were observed under the fluorescence microscope and photographed. Stacks of four images were taken every 2.5 minutes. Deconvolution of images and maximal projections were obtained using Deltavision software. Notice that Slk1 (arrow) moves from the spindle pole body to the forespore membrane (t=0-10 minutes). After 12.5 minutes, Slk1-GFP localizes to the forespore membrane. (B) A homothallic strain expressing Slk1-GFP under the nmt1(41) promoter (S1931) was incubated on MEA for 24 hours at 25°C and photographed. (Ba) Slk1 (arrow) localizes to the spindle pole body in metaphase II cells. (Bb, Bc) At anaphase II, Slk1 extends from the spindle pole body to the forespore membrane. (Bd) After anaphase II, Slk1 localizes to the forespore membrane. Note that, in the mitotic cells shown in the fields photographed, Slk1 localizes to the nucleolus when overexpressed. Scale bars: 4 µm.

 

Figure 8
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  		Fig. 8. Genetic interactions between slk1 and SIN mutants. (A) sid2-250 and cdc7-24 alleles enhance the sporulation defect caused by the deletion of slk1. Homothallic sid2-250 (S1884), cdc7-24 (S1886), slk1{Delta} sid2-250 (S1885) and slk1{Delta} cdc7-24 (S1887) strains were sporulated on MEA plates, and DIC images were taken after 2 days of incubation at 25°C. Note that double mutants fail to sporulate at the permissive temperature. (B) MEA plates from the experiment shown in A were stained with iodine vapour and photographed. (C) In slk1{Delta} sid2-250 cells, forespore membrane growth around the nuclei is defective. h90 slk1{Delta} sid2-250 cells carrying the plasmid pREP81-Psy1-GFP were sporulated on MEA plates for 2 days at 25°C, stained with Hoechst and photographed. Arrows highlight the abnormal distribution of DNA. (D) Time-lapse experiment showing forespore membrane growth in a sid2-250 slk1{Delta} strain (S1885) carrying plasmid pREP81-Psy1-GFP. Cells were sporulated on MEA plates and DNA was stained with Hoechst. Stacks of five images separated by 1 µm were taken every 5 minutes. GFP (green) and Hoechst (red) merged images were generated with ImageJ. The arrow marks a nucleus that is being cut by the forespore membrane upon closure. Scale bars: 10 µm.

 

Figure 9
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  		Fig. 9. sid2+ overexpression rescues the sporulation defect caused by deletion of slk1. (A) Schematic representation of the P41nmt1-sid2 construction. Endogenous sid2 was under the control of the P41nmt1 promoter, repressed by thiamine. (B) Homothallic strain slk1{Delta} P41nmt1-sid2 (S1890) was spotted onto MEA (sid2 expression high) and MEA+thiamine (sid2 expression low). DIC images of asci were taken after 2 days of incubation at 25°C. As controls, images of wild-type (S1478) and slk1{Delta} (S1883) asci are shown. Note that sid2+ overexpression improves the sporulation of slk1{Delta}, but sid2 shut-off does not enhance the sporulation defect of the slk1{Delta} mutant, indicating that sid2 is still expressed in the presence of thiamine. Scale bars: 10 µm. (C) The MEA plates of the experiment shown in B were stained with iodine vapour after 2 days of incubation at 25°C.

 

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© The Company of Biologists Ltd 2008