Supplementary Figure Legends
Figure S1: Spore formation competence of conditional SIN mutants, and characterisation of
rad21P-SIN alleles, analysis of meiotic localisation of SIN proteins in the mob1-R4 mutant and
rad21P-mob1 allele.
Panel A: Diploids carrying the indicated SIN mutant in a pat1-114 background were shifted to 32°C
for 3 hours to induce meiosis; one plate was then incubated further at 25°C, while the other was
maintained at 32°C. After 2 days, the plates were exposed to iodine vapour until the control had
stained dark brown. Panel B: The indicated strains were induced to undergo meiosis, and the
presence of spores and nuclei was examined by combined bright-field/DAPI staining. Note that
spores are present in rad21p-sid2, but not in the rad21p-sid2 mug27-D strains. Panel C: Upper
panels: Cells carrying the indicated rad21p-SIN alleles in a pat1-114 background were induced to
undergo meiosis, and protein extracts were prepared from vegetatively (V) growing cells and
compared with an extract made 3h after induction of meiosis (M). The filter was probed with 12CA5
(anti-HA). α−tubulin serves as a control. Lower panels: Cells carrying the indicated rad21p-SIN alleles
were mated and examined after 2 days at 25°C; asci were stained with DAPI. Note the absence of
spore formation. Panel D: The indicated tagged alleles were examined in meiotic mob1-R4 cells. For
each GFP-tagged allele, the left panel is a horsetail stage cell, the middle panel is meiosis I and the
right panel is meiosis II. Panel E. Each GFP-tagged allele is expressed in the rad21P-mob1
background; the left panel is a meiosis I cell, the right panel is a meiosis II cell. Note that the
localisation of the SIN proteins is not perturbed by the absence of mob1p. The scale bar corresponds
to 10 µm.
Figure S2: Analysis of SIN protein localisation in mitosis, meiosis and meiotic mutants.
Panel A: Localisation of cdc7p-GFP and GFP-sid1p in mitotic cells of the indicated genotype. Note the
presence of SPB-associated signals, whether the cell is mono- or bi-nucleated. Panel B: The indicated
strains were grown to mid-exponential phase in YE medium. Note the presence of the tagged GFP
proteins on all SPBs, independent of cell cycle stage. Panel C: Cells were self-mated: (a) horsetail
stage (b) meiosis I (c) meiosis II; note the presence of spg1-GFP on the SPB at all stages of meiosis.
Panel D: A self-mating of the indicated strain was photographed after staining with DAPI. A
DAPI/transmission image of a typical ascus is shown. Panel E: Cells were self-mated: (a) horsetail
stage (b) meiosis I (c, d) meiosis II; note the presence of spg1p-GFP on the SPB at all stages. Panel F:
Cells were self-mated and imaged: (a) horsetail stage (b) meiosis I (c) meiosis II; note the presence of
a GFP signal in (a) and (b), but not in (c). Panel G, the micrographs show a horsetail stage cell (a), a
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meiosis I cell with a spindle (b) and a binucleate cell without a spindle (c) that has completed meiosis
I, but not formed a detectable meiosis II spindle. Note the absence of GFP-byr4p in (c). The scale bar
corresponds to 10 µm.
Figure S3: Analysis of SIN protein levels during meiosis in wild-type and mutant backgrounds
Panels A to K: Cells bearing the indicated mutant (left side of western blots) in a pat1-114
background, were induced to undergo meiosis by incubation at 32°C. The time above each lane
indicates the number of hours after induction of meiosis. The graph indicates mono (1n), bi-(2n) and
tetranucleated (4n) cells; x-axis; time (h), y-axis; percentage. Western blots were probed to reveal
the indicated proteins. The mitotic cyclin cdc13p and α-tubulin served as controls.
Figure S4: Analysis of SIN protein levels in meiosis
Panels A, B, C and E: Cells bearing the indicated mutant (left side of western blots), of the indicated
tagged SIN protein (right hand side) in a pat1-114 background, were induced to undergo meiosis by
incubation at 32°C. Westerns were probed with an antibody to detect the tag. Graphs and western
blots are labelled as described for figure S3. Panel D, an immunoprecipitate from 5h after induction
of meiosis was treated with phosphatase as described in materials and methods. Note the partial
down-shift of cdc11p-HA, indicating that some of the slower-migrating forms are the result of
phosphorylation.
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