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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01585

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The novel fission yeast (1,3)ß-D-glucan synthase catalytic subunit Bgs4p is essential during both cytokinesis and polarized growth

¹ Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Salamanca, 37007 Salamanca, Spain
² Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto 8-9-1, Kobe 658-8501, Japan

View larger version (78K): [in a new window]   Fig. 1. bgs4+ cwg1+and orb11+ are the same gene. (A) bgs4+ suppresses the lethal thermosensitive phenotype of cwg1-1 mutant cells. Wild-type (leu1-32 h–) and cwg1-1 (cwg1-1 leu1-32 h–) cells transformed with the empty plasmid pAL-KS+ or the plasmid pAL-bgs4+ (pJG1) and grown at 28°C were streaked out on EMM plates and incubated at 37°C for 4 days. (B) bgs4+ suppresses the in vitro GS defect of cell extracts from cwg1-1 cells grown at 37°C. The same cells as in A were grown on EMM + 1.2 M sorbitol liquid medium at 37°C and the GS activity of cell extracts was analyzed at 30°C. Values in parentheses are the specific activity average, calculated from three independent extracts. (C) Both homozygous cwg1-1/cwg1-1 and heterozygous cwg1-1/bgs4 diploids present the same thermosensitive spherical morphology. Phase-contrast and Calcofluor white (CW)-stained UV micrographs of log-phase cells grown on EMM liquid medium at 37°C for 12 hours. (D) bgs4+ suppresses the thermosensitive morphology of orb11-59 cells. Wild-type and orb11-59 cells transformed with the indicated plasmids were grown at 37°C for 24 hours and visualized as in C. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 2. Absence of bgs4+ promotes lysis in both germinating spores and growing cells. (A) bgs4 spores are able to germinate but lyse before their first cell division. Tetrads from bgs4+/bgs4 strain were dissected on YES and YES + 1.2 M sorbitol medium and incubated at 28°C for 1 and 2 days, respectively. Photographs of a tetrad representative for each case, with two bgs4+ colonies and two germinated bgs4 spores lysing before cell division, are shown. Arrows indicate the germination site, with cell projections swelling up (+Sorbitol) and finally lysing (–Sorbitol). (B) bgs4+ shut-off promotes cell growth arrest after 14 hours of growth on EMM + thiamine medium. Sorbitol only partially protects the bgs4 cells and delays the arrest of cell growth to 17 hours. Log-phase control and bgs4 p81X-bgs4+ cells were grown on EMM or EMM + 1.2 M sorbitol liquid media at 32°C, either in the absence (–T, induced) or in the presence (+T, repressed) of thiamine. Cell growth was monitored after 12 or 15 hours of growth with thiamine, depending on the absence or the presence of sorbitol in the medium, respectively. (C) bgs4+ shut-off promotes cell lysis and release of cytoplasmic material from either the growing pole or the septum region. Log-phase bgs4 p81X-bgs4+ cells grown on EMM + T liquid medium at 32°C for 18 hours were visualized as in Fig. 1C. (D) bgs4+ shut-off induces a dramatic decrease in GS activity. Log-phase bgs4 p81X-bgs4+ cells grown on EMM ± T as in B were collected at the indicated times and assayed for GS activity as in Fig. 1B. (E) bgs4+ shut-off produces actin delocalization and depolymerization prior to cell lysis. bgs4 p81X-bgs4+ cells grown on EMM + T as in C were collected at the indicated times, fixed, and stained with rhodamine-conjugated phalloidin. Bar, 5 µm.

View larger version (53K): [in a new window]   Fig. 3. bgs4+overexpression produces defects in cell integrity. (A) bgs4+ overexpression causes cell growth arrest, which is delayed in the presence of sorbitol. Log-phase wild-type and bgs4 p3X-bgs4+ cells were grown and monitored after 18 hours of growth in the absence of thiamine as in Fig. 2B. (B) Rounded and elongated morphology of bgs4+-overexpressing cells. Log-phase bgs4 p3X-bgs4+ cells were grown on EMM liquid medium at 32°C and visualized at the indicated times as in Fig. 1C. (C) bgs4+ overexpression induces actin delocalization in rounded cells. bgs4 p3X-bgs4+ cells grown on EMM liquid medium at 32°C for 24 hours were stained for actin visualization as in Fig. 2E. (D) bgs4+ overexpression produces a gradual reduction in GS activity. Log-phase bgs4 p3X-bgs4+ cells grown on EMM as in A were collected at the indicated times and assayed for GS activity as in Fig. 1B. Bar, 10 µm.

View larger version (118K): [in a new window]   Fig. 4. Bgs4p localizes to the growing regions: one or both poles, medial ring and septum. (A) CW staining and GFP-Bgs4p localization throughout the mitotic cell cycle. Early log-phase cells (GFP-bgs4+ bgs4), grown on YES liquid medium at 28°C, were visualized for CW staining and GFP fluorescence. CW was added at 50 µg/ml followed by immediate examination of the cells. A total of 436 cells from 41 different images were ordered and analyzed. Cells representative of each cell cycle step were selected and ordered to show a cell cycle progression. (B) Magnification of CW staining and GFP-Bgs4p localization to the medial ring and along the plasma membrane during septum formation. (C) Bgs4p localizes to the medial ring later than Bgs1p. CW staining and GFP-Bgs1p or GFP-Bgs4p localization during mitosis and cytokinesis. GFP-bgs1+ bgs1 and GFP-bgs4+ bgs4 cells were grown as in A, ethanol-fixed, and analyzed for GFP and CW/Hoechst staining. A total of 132 GFP-Bgs1p- and 76 GFP-Bgs4p-expressing mitotic cells, from 35 and 16 different images respectively, were ordered and analyzed. Cells representative of the anaphase to the cytokinesis process were selected and ordered to show a progression. Bar, 2 µm (A,C); 1 µm (B).

View larger version (59K): [in a new window]   Fig. 5. Bgs4p localization during septation depends on medial ring formation and positioning, but the SIN proteins may be dispensable for its localization to the medial ring and septum. GFP-bgs4+ bgs4 mutant cells were grown as in Fig. 4A, shifted to 32°C (cdc3-6, cdc15-140 and cdc16-116) for 4 hours or to 37°C (mid1-366, cdc14-118) for 3-6 hours, and visualized as in Fig. 4A, except cdc14-118 cells, which were also analyzed for nucleus staining as in Fig. 4C. Cells representative of the different mutant phenotypes are shown. In mid1-366, cells with overlapping and non-overlapping CW (arrowhead) and GFP-Bgs4p (arrow) fluorescence are shown. In cdc14-118, cells representative of the phenotype at different times are shown, including cells displaying a partial septum (arrowhead) synthesized before they expressed the mutant phenotype, some of them with GFP-Bgs4p (arrow) localized to the partial septum structure. Bar, 2 µm.

View larger version (28K): [in a new window]   Fig. 6. Bgs4p localization depends on polarity establishment proteins and on the actin cytoskeleton. (A) Bgs4p localizes to the altered growing poles and septa in the end-marker mutants tea1-1, tea2-1 and tea2-1 cdc11-119. (B) Bgs4p localizes all around the cell membrane and septum in the actin mutant cps8-188. GFP-bgs4+ bgs4 mutant cells were grown as in Fig. 4A, shifted to 37°C for 6 hours (cps8-188 and tea2-1 cdc11-119) or 8 hours (tea1-1 and tea2-1), and visualized as in Fig. 4A. Bar, 2 µm.

View larger version (77K): [in a new window]   Fig. 7. Polarized F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, poles and septum, but not for its stable maintenance at the growing zones. (A) Maintenance of Bgs4p to the medial ring and poles does not require the F-actin function. Early log-phase cells (RFP-bgs4+ bgs4 crn1+-GFP) grown on YES liquid medium at 28°C were transferred to YES medium containing Lat A (100 µM) or an equal volume of DMSO as control and incubated at 28°C for 15 minutes. Cells grown in the presence of DMSO displayed the same RFP-Bgs4p and Crn1p-GFP localization as initial control cells (data not shown). The Lat A-treated cells were washed, transferred to fresh YES medium, and incubated at 28°C for 3 hours. Cells were collected at the indicated times, fixed, and analyzed for RFP (Bgs4p) and GFP (actin-associated Crn1p) fluorescence. (B) The F-actin cytoskeleton is needed for Bgs4p relocalization to the growing regions. Cells grown as in A were exposed to stress conditions (high temperature and the absence of nitrogen) for 2 hours to induce Bgs4p and F-actin patch delocalization. Then, the cells were transferred to YES medium and incubated at 28°C for 2 hours in the presence of Lat A or DMSO (–Lat A control). Cells were collected at the indicated times and analyzed as in A. (C) The F-actin cytoskeleton is necessary for Bgs4p delocalization from the growing zones upon stress treatment. Cells were grown and treated with Lat A as in A to induce actin depolymerization and to maintain Bgs4p polarization. Then, the cells were washed and exposed to stress conditions (absence of nitrogen) in the presence of Lat A or DMSO (–Lat A control). Cells were collected at the indicated times and analyzed as in A.

View larger version (71K): [in a new window]   Fig. 8. Bgs4p is present and localized to all sites of cell wall synthesis during sexual differentiation. Homothallic GFP-bgs4+ bgs4 h90 cells grown on EMM at 28°C up to the early stationary phase were transferred onto SPA plates and incubated at 28°C. Samples were collected at 3, 5, 8, 24 and 48 hours, resuspended in EMM medium containing CW (50 µg/ml), and visualized as in Fig. 4A. A total of 435 mating cells and zygotes from 86 different images were ordered and analyzed. Cells and zygotes representative of each mating and sporulation step were selected and ordered to show a sexual phase progression. Bar, 2 µm.

View larger version (70K): [in a new window]   Fig. 9. Bgs4p localizes to the mating projections in an ordered mating type-dependent sequence, appearing first in P (h+) cells and later in M (h–) cells. Early-logarithmic phase cells (GFP-bgs4+ bgs4 h– and h+, and RFP-bgs4+ bgs4 h– and h+) grown on EMM at 28°C were collected, washed and mixed in equal cell amounts (h+ GFP-bgs4+ x h– RFP-bgs4+ and h+ RFP-bgs4+ x h– GFP-bgs4+). Cell mixtures were transferred onto SPA plates and incubated at 28°C. Samples were collected at 30-minute intervals from 0 to 5 hours, ethanol-fixed, and examined for GFP and RFP fluorescence. Merged images of GFP (green) and RFP (red) fluorescence are shown. Arrowheads show the first time point at which h+ cells develop mating projections and localize Bgs4p (GFP or RFP). Arrows show the first time point at which mating projections and localized Bgs4p (GFP or RFP) are detected in h– cells.

View larger version (44K): [in a new window]   Fig. 10. Bgs4p localizes to the different sites of cell wall growth during spore germination. The homothallic GFP-bgs4+ bgs4 h90 strain was grown and sporulated for 7 days as in Fig. 8. The spores were collected and incubated in YES liquid medium at 28°C. Samples were taken at 3, 7, 9 and 11 hours, and examined as in Fig. 4A. A total of 252 spores from 42 different images were ordered and analyzed. Spores illustrative of each germination step were selected and ordered to show a germination progression. Bar, 2 µm.

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