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First published online 20 June 2006
doi: 10.1242/jcs.03025

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The Schizosaccharomyces pombe septation initiation network (SIN) is required for spore formation in meiosis

Andrea Krapp, Philippe Collin, Adisa Cokoja^*, Sandra Dischinger, Elena Cano and Viesturs Simanis

Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland

View larger version (67K): [in a new window]   Fig. 1. Localisation of the SIN proteins during meiosis. Strains of opposite mating types expressing chromosomally GFP-tagged alleles of Sid4p (A), Cdc11p (B), Spg1p (C), Cdc16p (D), Cdc7p (E), Sid1p (F), Mob1p (G) or Sid2p (H) were mated on EMM-NH4Cl plates at 25°C for 20 hours. Cells were then resuspended in EMM-NH4Cl containing DAPI and visualised under a fluorescence microscope. The columns show cells in the horsetail stage, binucleate cells and tetranucleate cells. In cases where a DAPI image is shown, the DAPI is on the right, the GFP-fluorescence on the left. Bar, 10 µm.

View larger version (34K): [in a new window]   Fig. 2. The SIN proteins are required during meiosis. (A) pat1-114/pat1-114 (control) and pat1-114/pat1-114 homozygous for the indicated SIN temperature-sensitive mutation were replica-plated onto EMM-NH4Cl, shifted to 34°C for 3 hours and then shifted back to 25°C (25°C), or kept at 34°C for 24 hours (34°C). Plates were then exposed to iodine vapour to stain spore walls. (B) pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 were induced to sporulate at 33°C during 24 hours, fixed and stained with DAPI. Bar, 10 µm. Notice the absence of spores in the pat1-114 cdc11-136 mutant. (C) pat1-114/pat1-114 (control) and pat1-114/pat1-114 cells homozygous for the indicated SIN temperature-sensitive mutation were induced to sporulate at 34°C for 20 hours or for 3 hours and then shifted back to 25°C. Cells were then fixed, stained with DAPI and the percentage of tetranucleate cells containing spores was determined.

View larger version (18K): [in a new window]   Fig. 3. cdc11-136 is defective in spore formation. (A) Nitrogen-starved pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells were shifted to 34°C for the indicated time and then shifted back to 25°C. Cells were fixed and the percentage of asci that contained spores was determined by phase-contrast microscopy. (B) Kinetics of meiosis in pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells. Starved cells were induced to sporulate at 33°C, and a portion of the culture was fixed at the indicated time and stained with DAPI. Meiotic cells were classified according to the number of nuclei per cell. , mononucleate cells; , binucleate cells; , tetranucleate cells.

View larger version (45K): [in a new window]   Fig. 4. cdc11-136 diploid cells progress normally through the horsetail and the first meiotic division. (A) FACS analysis of diploid pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells growing vegetatively (M), starved in EMM-NH4Cl during 16 hrs at 25°C (M-N) and induced to sporulate at 33°C for 7 hours (+ NH4) were analysed for their DNA content as described in materials and methods. (B) Microscopy of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing the chromosomally GFP-tagged histone hht2 allele. In the left panel, two images of the same cell are shown, taken 5 minutes apart. (C,D) Observation of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing GFP-Atb2p under the control of its own promoter from a plasmid, during the horsetail stage (C) or the first meiotic division (D). Images were recorded at 1-minute intervals. The time after the first images (t=0) is indicated. (E) Live cell microscopy of pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing chromosomally GFP-tagged Pcp1p, to visualise the SPB. The left panel represents the horsetail stage. The position of the SPB is indicated by the white arrow. The right panel shows the first meiotic division. The time (in minutes) after the first image is indicated on each panel. Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 5. cdc11-136 diploid cells progress normally through the second meiotic division. (A) Observation of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing GFP-Atb2p under the control of its own promoter from a plasmid after 6 hours at 34°C. The time in minutes after the first images is indicated on the panels. (B) Observation of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing GFP-Hht2p, the nuclear envelope protein Nup107p-GFP, or the kinetochore Nuf2p-GFP. Cells were incubated for 6 hours at 34°C. Bars, 10 µm.

View larger version (39K): [in a new window]   Fig. 6. Abnormal forespore membrane formation in the cdc11-136 mutant. (A) Observation of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing either spo15p-GFP from a plasmid, or chromosomally GFP-tagged alleles of cut12. Cells were incubated for 6 hours at 34°C. (B) Observation of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing either spo3p-GFP or psy1p-GFP from a plasmid. Cells were incubated for 6 hours at 34°C. (C) pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing Psy1p-GFP were fixed after 6 hours at 34°C as described in Materials and Methods. Forespore membranes and chromatin were examined by using anti-GFP antibody (green) and DAPI (blue), respectively. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 7. The SIN is required for spore formation. (A) Microscopy of living pat1-114/pat1-114 and pat1-114/pat1-114 cdc11-136/cdc11-136 cells expressing chromosomally tagged Cdc7p-GFP (left) or Sid1p-GFP (right). Cells were induced to undergo meiosis by incubation for 6 hours at 34°C. (B) Analysis of the kinetics of meiosis in pat1-114/pat1-114, pat1-114/pat1-114 spg1-B8/spg1-B8 and pat1-114/pat1-114 sid1-C14/sid1-C14 cells. Starved cells were induced to sporulate at 34°C, and a portion of the culture was fixed at the indicated time and stained with DAPI. Meiotic cells were classified by the number of nuclei per cell. , mononucleate cells; , binucleate cells; , tetranucleate cells. (C) cdc2-N22 cells of opposite mating types expressing Cdc7p-GFP were mated on EMM-NH4Cl plates at 25°C for 24 hours. Cells were then resuspended in EMM-NH4Cl and GFP fluorescence was examined. Bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 8. Ectopic activation of the SIN leads to septation during meiosis. (A) pat1-114/pat1-114 diploid cells carrying a plasmid expressing Spg1 under the control of the nmt1 promoter were grown in minimal medium lacking thiamine for 4 hours at 25°C and then starved in EMM-NH4Cl for 16 hrs at 25°C. Meiosis was induced by incubation at 34°C for 6 hours, when the cells were fixed and stained with DAPI and Calcofluor. The control was cells carrying an empty nmt1 expression vector. (B) Cells were treated as in A, but samples were taken at different time points after induction of meiosis. The septation index in the total population was determined and subdivided into categories of cells containing one, two or four nuclei. (C) Cells were treated as in A. The septation index among the cells containing fournuclei was determined and subdivided into the following three categories: (1) asci containing two nuclei on each side of the division septum (septation occurred at or after MI); (2) asci containing four nuclei on the same side of the division septum (septation occurred before or after MI); (3) multiseptated asci. Bars, 10 µm.