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Files in this Data Supplement:
Table S1. Strains used in this study.
Fig. S1. Kinetics of the appearance of SICS at cell tips of wild-type cells grown in rich versus minimal media. Wild-type cells were grown to mid-log phase in either rich (YES) or supplemented minimal (EMM2) medium. Cells were treated with sorbitol to a final concentration of 1.2 M in the appropriate corresponding medium (by adding an equal volume of 2.4 M sorbitol in YES or EMM2), fixed, processed for calcofluor staining and the appearance of SICS at cell tips was plotted.
Fig. S2. Modulation of the microtubule cytoskeleton in response to osmotic stress. (A) Wild-type cells were grown to mid-log phase in YES and fixed and processed for immunofluoresence either before the addition of sorbitol (0 min) or at the indicated times after the addition of sorbitol. Microtubules were localized using the TAT1 anti-tubulin antibody (upper panels) and chromatin was localized using DAPI (lower panels). (B) Quantitation of the microtubule phenotypes seen in fixed sorbitol-treated cells (as shown in A). The number of interphase cells containing a microtubule bundle which was shorter than the diameter of the cell, together with the number of G2 cells with microtubule bundles oriented close to perpendicular to the long axis of the cell was determined for each time point (left-hand plot) as was the number of late G2 cells with at least one microtubule bundle of more than 2/3 the length of the cell (right hand plot). Scale bar: 5 µm.
Fig. S3. Sorbitol response in cdc2.33-arrested cells. (A,B) cdc2.33 cells were grown at 36°C for 2 hours prior to the addition of sorbitol to a final concentration of 1.2 M, then incubated at 36°C post-stress. (A) Representative images of cdc2.33-arrested cells 120 minutes post-stress. (B) Quantitation of septation and the appearance of SICS.
Fig. S4. The appearance of SICS in cells compromised in their ability to synthesize new cell-wall material. (A,B) Wild-type, pck1.Δ, and pck2.Δ cells were grown to mid-log phase in YES medium and were then treated with sorbitol to a final concentration of 1.2 M. Cells were fixed and processed for Calcofluor staining either immediately before stress or 25 minutes and 50 minutes post-stress. (A) Representative images of pck1.Δ (left-hand panel) and pck2.Δ cells (right-hand panel) taken 25 minutes after sorbitol treatment. The insets show the boxed regions magnified 2×. (B) Quantification of the total number of wild-type, pck1.Δ and pck2.Δ cells with SICS at cell tips either immediately before sorbitol treatment or 25 min and 50 min post-sorbitol treatment. (C) The appearance of SICS is dependent on the activity of (1-3)β-D glucan synthase. Quantification of the appearance of SICS 30 minutes after the addition of sorbitol to wild-type cells which had either been given a 10-minute pre-treatment or simultaneously treated with the indicated concentration of aculeacin A. Scale bar: 5 µm.
Movie 1. Microtubule dynamics are suppressed by sorbitol treatment. Movie showing microtubule dynamics imaged at high temporal resolution (one frame every 10 seconds) in cells expressing α-tubulin by the atb2.nmt81GFP allele at 30°C either before (left-hand movie) or immediately after (right-hand movie) sorbitol treatment.
Movie 2. Suppression and resumption of microtubule dynamics and remodelling of microtubule bundles following osmotic stress. Movie showing microtubule dynamics imaged at low temporal resolution (one frame every 3 minutes) in cells expressing α-tubulin by the atb2.nmt81GFP allele at 30°C. Cells were subjected to sorbitol treatment immediately after the second (3 minute) time point.
Movie 3. Suppression and resumption of microtubule dynamics in wild-type, spc1/sty1.Δ, wis1.DD and atf1.Δ cells. Movie showing microtubule dynamics of the indicated strains. Time is shown in the upper right-hand corner. The movies are aligned so that time point 0 is the point at which the cells experience the osmotic shock. Note that the recovery of dynamics in wis1.DD precedes that of wild type and the absence of recovery of the spc1/sty1.Δ and atf1.Δ cells.
Movie 4. Microtubule dynamics in spc1/sty1.Δ cells are suppressed by sorbitol treatment. Movie showing microtubule dynamics imaged at high temporal resolution (one frame every 10 seconds) in sty1.Δ cells expressing α-tubulin by the atb2.nmt81GFP allele at 30°C immediately after sorbitol treatment. Note the lack of recovery of in the majority of the cells in the field of view.
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