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First published online 2 July 2003
doi: 10.1242/jcs.00634

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In yeast, the pseudohyphal phenotype induced by isoamyl alcohol results from the operation of the morphogenesis checkpoint

¹ Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
² Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK

View larger version (82K): [in a new window]   Fig. 1. Bud formation is uncoupled from nuclear division in diploid cells treated with IAA. An exponentially growing heterozygous CDC3/CDC3-GFP strain was incubated in YEPD (1% Difco yeast extract, 2% Difco-Bacto Peptone and 2% glucose) plus 0.5% IAA. Samples were removed at 1-hour intervals and stained with DAPI. The samples were examined with DIC microscopy to record overall appearance, and fluorescence microscopy to visualise CDC3-GFP (green) or nuclei (blue). (A) Filaments representative of the changes observed. The time of sampling is indicated in each panel. Arrows in the 1- and 2-hour samples indicate examples of multiple septin patches; the solid arrow in the 8-hour sample (right) indicates a branch forming and the open arrow (left) a double septin ring. Scale bar is 5 µm. The figure does not show examples from all the time points analysed in B and C. (B) The number of buds produced per filament over the 8 hours examined. Over 100 filaments were analysed for each time point. (C) The number of nuclei per filament. Filaments were categorised as indicated, according to whether they had nuclei in a large cell at either end of the filament or a nucleus within the chain of small buds. (D) At the indicated time points, samples were withdrawn and fixed with 2.5% w/v formaldehyde and briefly sonicated. Cell number was determined, counting each clump or filament as a single cell. Filled circles, no IAA; open circles, plus IAA.

View larger version (58K): [in a new window]   Fig. 2. IAA uncouples bud formation from nuclear division in haploid cells. Exponentially growing haploid 1287h cells were added to YEPD containing 0.5% IAA and incubated for 24 hours. (A) Cells stained with DAPI. (B) DIC images of the cells shown in A. Open arrow, wide filaments; solid arrow, narrow filaments; solid arrows with barbed tails, filaments with a mixture of wide and narrow compartments. (C) Cells were treated as above for 8 hours and Cdc11 visualised by immunocytofluorescence using an anti-Cdc11 antibody as described previously (Sudbery, 2001). (D) The same cells as in C, stained with DAPI. Septin rings form in the absence of nuclear division. (E) Cells were grown for 16 hours in YEPD plus 0.5% IAA, treated with zymolyase and stained with DAPI. Cells that contain nuclei separate after the zymolyase treatment. Scale bars: 10 µm (A,B,E); 5 µm (C,D).

View larger version (63K): [in a new window]   Fig. 3. The response to IAA is dependent on SWE1. (A) A haploid 1278h MAT strain (wild type; wt) and a swe1 derivative were incubated for 16 hours in YEPD alone or YEPD plus 0.5% IAA, and photographed using DIC optics. No filaments were produced by the SWE1 mutant upon IAA treatment. Scale bar: 10 µm. (B) Flow cytometry traces showing relative DNA content in untreated 1278h cells, and cells exposed to 0.5% IAA for the indicated times. The proportion of cells in G1 and G2 was determined for each time point and the proportion of cells in G2 in the treated (open symbols) and untreated (closed symbols) cultures is plotted against time.

View larger version (24K): [in a new window]   Fig. 6. Tyrosine phosphorylation of Cdc28 is Swe1 but not Slt2-dependent. Cultures of the indicated genotype were grown to mid-log phase and treated with 0.5% IAA. At the indicated times, total protein extracts were obtained by TCA precipitation and probed by western blotting with anti-phosphotyro-Cdc28 (anti-pY19) and anti-active Slt2 antisera. The membrane was then stripped and probed with anti-PSTAIR antisera. Levels of activated Slt2 and phosphotyro-Cdc28 were determined using the signal from the lower band (Cdc28) in the anti-PSTAIR panel as a loading control. Values on the ordinate are the ratio of each experimental signal to its loading control.

View larger version (25K): [in a new window]   Fig. 4. Cells treated with IAA show polarisation of the actin cytoskeleton. Wild-type haploid cells were treated with 0.5% IAA. After 4 hours cells were fixed and stained with TRITC-conjugated phalloidin. The figure shows a collage of typical cells. Scale bar: 5 µm.

View larger version (64K): [in a new window]   Fig. 5. Filament formation depends on SLT2. (A) Appearance of slt2 cells incubated for 16 hours in YEPD or YEPD plus 0.5% IAA. Scale bar: 10 µm. (B) Samples of wild-type cultures in the log-phase of growth were taken at the indicated times after addition of 0.5% IAA. Total protein extracts were probed by western blotting with anti-active (p44/42) Slt2 antisera. Values on the ordinate are the ratio of each experimental signal to its loading control.

View larger version (100K): [in a new window]   Fig. 7. The slt2 filamentation defect is rescued by mih1. Cells of the indicated genotype were incubated in YEPD or YEPD plus 0.5% IAA for 16 hours. Scale bar: 20 µm.

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