JCS023812 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

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• Supplemental Figure S1 -

  Fig. S1. sid2-250 and orb6-25 phenotypes are not enhanced by the loss of slk1⁺ in the mitotic cycle. (A) Wild-type (S1485), slk1Δ (S1925), sid2-250 (S1926), sid2-250 slk1Δ (S1928), orb6-25 (S1917), and orb6-25 slk1Δ (S1921) were grown at permissive temperature of 25°C. Serial dilutions were spotted onto YES plates, and incubated at the indicated temperatures (25°C, 32°C and 36°C). (B) Wild-type (S1485), slk1Δ (S1925), sid2-250 (S1926), and sid2-250 slk1Δ (S1928) cells were grown at the permissive temperature (25°C) until exponential growth, and shifted to 36°C. Samples were taken at 25°C and 2 and 5 hours after the shift to 36°C, stained with DAPI and calcofluor, and photographed. Bar, 10 µm. (C) orb6-25 (S1917) and orb6-25 slk1Δ (S1921) were grown at the permissive temperature of 25°C in YES, and shifted to the semi-restrictive temperature of 33°C, and to the restrictive temperature of 36°C. Samples were taken after 5 hours of incubation at each temperature, stained with DAPI and calcofluor, and photographed. Bar, 10 µm.

• Supplemental Figure S2 -

  Fig. S2. Activation of the SIN pathway occurs normally in the slk1Δ mutant. Strains carrying GFP-tagged alleles of sid1 and cdc7 were sporulated on MEA plates at 25°C and photographed. Bar, 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Overexpression of slk1⁺ cannot complement the growth defect of sid2-250 and orb6-25 ts mutants. Wild-type (S1347), sid2-250 (S1927) and orb6-25 (S1918) strains carrying plasmids pREP3X, pREP3X-slk1⁺ or pREP41-N-EGFP-slk1⁺ were grown in MM plus thiamine until exponential phase at the permissive temperature (25°C). Cells were washed, serially diluted, and spotted onto MM plates with or without thiamine. Plates were incubated at 25°C, 32°C and 36°C.

• Supplemental Figure S4 -

  Fig. S4. Spindle dynamics during meiosis is not affected by the loss of slk1⁺. Time-lapse experiments showing forespore membrane growth and spindle assembly and disassembly in wild-type (S1930) and slk1Δ (S1929) strains carrying the plasmid pREP81-Psy1-GFP. Cells were sporulated on MEA plates, and DNA was stained with Hoechst. Stacks of 8 images separated 0.5 µm were taken every 2 minutes. Deconvolution of images and maximal projections were obtained using Deltavision software, and GFP and Hoechst merged images were generated with ImageJ. Bars, 4 µm.

• Movie 1
• Movie 2 -

  Movies 1 and 2. Forespore membrane growth is defective in the slk1Δ mutant. Time-lapse video corresponding to Fig. 5 shows that forespore membrane growth is defective in the slk1Δ mutant. Cells carrying plasmid pREP81-Psy1-GFP were sporulated on MEA plates at 25°C, and DNA was stained with Hoechst. Stacks of 8 images separated 0.5 µm were recorded at 2-minute intervals during 40 minutes in a Deltavision RT microscope. Movie 1: Wild type. Movie 2: slk1Δ.