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First published online 4 March 2003
doi: 10.1242/jcs.00377

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The endo-ß-1,3-glucanase eng1p is required for dissolution of the primary septum during cell separation in Schizosaccharomyces pombe

¹ Instituto de Microbiología Bioquímica, Departamento de Microbiología y Genética, CSIC/ Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
² Department of Genetics, University of Debrecen, PO Box 56, 4010 Debrecen, Hungary

View larger version (22K): [in a new window]   Fig. 1. eng1p is an endo-ß-1,3-glucanase. (A) Schematic representation of the S. cerevisiae (ScEng1p) and S. pombe endo-ß-1,3-glucanases (Speng1p). The structure of each protein is shown at the same scale (indicated at the top as the number of amino acids), with a gray rectangle indicating the conserved region between the proteins. A black box in the N-terminal region indicates the predicted secretory signal sequence, while triangles mark the position of putative N-glycosylation sites. White boxes represent Ser/Thr-rich regions (indicated by S/T) or the Thr-rich domain (marked with a T). (B) ß-glucanase activity against laminarin (ß-1,3-glucan) in cells (strain h20) transformed with plasmid pAB10 (carrying Pnmt1-eng1) or vector alone (pREP3X). Cells were grown for 16 hours in the presence (white bars) or absence (black bars) of thiamine to induce the expression of the eng1+ gene. (C) Enzymatic activity of cells overexpressing eng1+ against pustulan (ß-1,6-glucan) or p-nitrophenyl-ß-D-glucoside (PNPG).

View larger version (87K): [in a new window]   Fig. 2. Microscopic appearance of wild-type and eng1 mutant cells. Wild-type (h20) or the isogenic eng1 mutants cells (YAB14) were grown in rich medium (YES), washed, and stained with Calcofluor. Photographs of differential interference contrast microscopy (DIC) or Calcofluor-stained cells (CF) are shown.

View larger version (89K): [in a new window]   Fig. 3. Time-lapse DIC images of wild-type (A) and eng1 mutant cells (B). Black arrowheads mark the position of the previous septum while white arrowheads indicate the new septum formed before dissolution of the previous one. Numbers indicate the hours elapsed. (C) Scanning electron microscopy of wild-type (panels 1) and eng1 cells (panel 2). Bars, 2 µm.

View larger version (154K): [in a new window]   Fig. 4. Electron microscopy ultrastructure of wild-type (A) and eng1 mutant septa (B,C) during the separation process. p, primary septum; s, secondary septum. Arrows mark the remnants of the primary septum that has not been dissolved in mutant cells. Bars, 0.5 µm.

View larger version (117K): [in a new window]   Fig. 5. eng1p protein is synthesized periodically during the cell cycle. cdc25-22 mutant cells carrying the eng1-GFP fusion allele integrated in the chromosome were arrested by incubation at the restrictive temperature (37°C) for 4 hours and then transferred to the permissive temperature (25°C) to monitor the localization of the fusion protein. Numbers indicate minutes after transfer to the permissive temperature.

View larger version (115K): [in a new window]   Fig. 6. eng1p localizes to the septum. (A) Wild-type cells containing the eng1-GFP fusion allele were grown to early-log phase and stained with Calcofluor before microscopic observation. Calcofluor fluorescence (CF), GFP fluorescence and the overlay of both images (Merge) are shown. (B) Three-dimensional reconstruction of eng1p localization. Cells containing the eng1-GFP fusion allele were observed under a confocal microscope and z-sections of 0.3 µm were taken. The image was reconstructed using LSM510 software.

View larger version (33K): [in a new window]   Fig. 7. eng1+ expression and ß-1,3-glucanase activity vary periodically during the cell cycle. Synchrony was induced by arrest-release of cdc25-22 mutants and samples were taken at the indicated time points (minutes) for RNA extraction (A) or ß-glucanase activity determinations (B). (A) RNA was probed with specific probes for eng1+ or ura4+. The graph represents the anaphase index () or septation index (•) at each time point. In this experiment, the peak of septum formation occurred at 70-90 minutes. (B) ß-glucanase activity was assayed using laminarin as substrate in samples from a cdc25-22 (upper panel) or cdc25-22 eng1 (lower panel) mutant strains. The septation index for each strain is indicated (•). The anaphase index was determined by counting the percentage of anaphase cells (cells with two nuclei and without a septum) after DAPI staining. The septation index was determined by counting the percentage of cells with septum after calcofluor staining.

View larger version (67K): [in a new window]   Fig. 8. ace2+ is required for cell separation. (A) Expression of eng1+ and act1+ genes in wild-type (lane 1), ace2-null mutants (cells lacking the SPAC6G10.12C ORF, lane 2), or cells containing a deletion of SPAC4G8.13c (lane 3). (B) Morphology of the mutant cells grown in rich medium to early-log phase. Wild-type (1) and mutants lacking SPAC4G8.13c (2) show a normal morphology, while ace2 mutants (3) have a clear mycelial and branched phenotype, indicating severe cell separation defects.

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