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First published online 5 February 2008
doi: 10.1242/jcs.022772

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Homoeostasis between the GTPase Spg1p and its GAP in the regulation of cytokinesis in S. pombe

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

View larger version (34K): [in this window] [in a new window]   Fig. 1. Analysis of the steady-state levels of Cdc16p and Byr4p through the cell cycle. Cells of the genotypes indicated below were synchronised by cdc25-22 arrest-release. Cells were collected at the indicated times (minutes after release), fixed and stained with DAPI and Calcofluor. The percentage of cells in anaphase or septation was determined. Western analysis was performed on lysates of the different strains with the indicated antibodies. (A) cdc25-22 leu1::cdc16-HA(ura4+) cdc16+. (B) cdc25-22 byr4+. The three arrows to the right of the western blot in B indicate the different forms of Byr4p seen in mitosis (upper and lower) and the interphase form of the Byr4p (middle). The western blot on the right is part of the 60 minute protein sample treated with alkaline phosphatase (+P) or alkaline phosphatase in the presence of phosphatase inhibitors (M).

View larger version (58K): [in this window] [in a new window]   Fig. 2. Analysis of the steady-state level of Byr4p in SIN mutants. (A) The indicated mutants were grown in YE medium to exponential phase and incubated for 4 hours at 36°C. Protein extracts were analysed by western blotting with the indicated antibodies. (B) leu1::nmt1-spg1(ura4+) ura4-D18 cells were grown in EMM2 plus leucine in the presence of 2 µM thiamine (off) or without thiamine for 18 hours at 29°C (on) to induce spg1 expression. Cells overexpressing spg1 showed a multiseptated phenotype. Protein extracts were prepared, and western blots were probed with the indicated antibodies. The three arrows indicate the most rapidly migrating form, which predominates in cdc16-116, and two others that represent different states of phosphorylation. How these relate to the forms observed in Fig. 1 will be the subject of future experiments. (C) The mutant spg1-Q41A-HA was expressed from pREP3 in a spg1::ura4+ background, in the presence of 2 µM thiamine at 25°C, and cells were then shifted to 19°C for 9 hours or to 36°C for 5 hours. Cells were fixed and stained with DAPI and Calcofluor. Bar, 10 µm. (D) Protein extracts from the experiment shown in C, as well as from wild-type and spg1::ura4+ cells rescued by expression of either pREP41-cdc7 (without thiamine) or pREP3-spg1 (grown in the presence of thiamine) were analysed by western blotting with the indicated antibodies. The bottom panel shows the steady-state level of Spg1p-Q41A–HA at the indicated temperatures.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Byr4p is degraded if its interaction with Spg1p is compromised. (A) Protein extracts (EX) were prepared from the indicated strains and passed over a glutathione-agarose column. The flow-through (FT) and three successive eluates (E1, E2, E3) from the column were analysed using antibodies against Byr4p or GST. (B) Protein extracts were prepared from the indicated strains, grown at 25°C and western blots were probed with antibodies against Byr4p, GFP and tubulin. The relevant genotypes of the strains are shown above the panel. When two copies of spg1 are present, the first indicates the gene inserted into leu1, the second the state of the native spg1 locus. (C) Protein extracts from the indicated strain were analysed as described in A. (D) Cells from the indicated strains were shifted from exponential growth in YE medium at 25°C to 36°C for 4 hours before harvesting the cells. Protein extracts were prepared and western blots were probed with the indicated antibodies. The western blot on the right of panel D shows the mts3-1 sample untreated (E), treated with alkaline phosphatase (+P) or with alkaline phosphatase in the presence of phosphatase inhibitors (M). The arrows indicate the positions of the three forms as defined in Fig. 2A.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Analysis of the effects of increased expression of domains of byr4. (A) Expression of the byr4(1-595) fragment from the nmt1 promoter was induced by growth in the absence of thiamine for 20 hours at 25°C in EMM2 medium. Cells were fixed and stained with DAPI and Calcofluor. (B) The byr4(1-595) fragment was expressed from the nmt1 promoter as in A. Cells also expressed the indicated tagged GFP-SIN fusion protein. The GFP signals were photographed in living cells. (C) Expression of the byr4(595-665) fragment from the nmt1 promoter was induced by growth in the absence of thiamine for 20 hours at 25°C in EMM2 medium. Cells were fixed and stained with DAPI and Calcofluor. (D) The byr4(595-665) fragment was expressed from the nmt1 promoter as in C, in cells expressing the indicated tagged GFP-SIN fusion protein. The GFP signals were photographed in living cells. (E) Protein extracts were prepared from either wild-type cells grown at 25°C (left panel) or mts2-1 cells incubated at 36°C for 4 hours (right panel), in which expression of the indicated fragment of byr4 from the nmt1 promoter had been induced, as described above. One lane, showing the effects of a truncation not discussed in this paper, has been removed from the gel. Bar, 10 µm (panels A-D).

View larger version (31K): [in this window] [in a new window]   Fig. 5. Degradation of Byr4p requires Cdc16p. (A) Protein extracts were prepared from the indicated strains grown exponentially at 25°C or shifted to 36°C for 4 hours before harvesting. Extracts were analysed on a western blot probed with the indicated antibodies. Two lanes to the right of the spg1 sample have been deleted from the image of the gel as they show the effects of a mutant not discussed in this study. (B) Protein extracts were prepared from cdc16::u4+ mob1-R4, in which expression of the indicated fragment of byr4 from the nmt1 promoter had been induced for 20 hours at 25°C in EMM2 medium. Extracts were analysed on a western blot probed with the indicated antibodies. (C) Protein extracts were prepared from leu1::spg1-B8-GFP spg1::u4+ cells in which expression of the indicated fragment of byr4 from the nmt1 promoter had been induced for 16 hours at 25°C in EMM2 medium before shifting to 36°C for 5 hours. Extracts were analysed on a western blot probed with the indicated antibodies.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Analysis of the localisation of Spg1p-GFP in SIN mutant backgrounds. The indicated strains were observed at either 25°C or after a shift to 36°C for 4 hours. The GFP signals were photographed in living cells maintained at 36°C. Bars, 10 µm.