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First published online 25 November 2008
doi: 10.1242/jcs.034488

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Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress in S. pombe

CRUK Cell Division Laboratory, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK

View larger version (65K): [in this window] [in a new window]   Fig. 1. Osmotic stress generates SICS at cell tips and perturbs septation. (A) Wild-type cells grown to mid-log phase in YES media were fixed and processed for Calcofluor staining at the indicated times after the addition of sorbitol to a final concentration of 1.2 M. Sorbitol treatment induced SICS at one (cell 1) or both (cells 2 and 3) cell tips and, later, away from tips (arrow in inset; the boxed cell highlighted in the 120-minute panel is shown magnified 2x in the inset). Long cells containing one septum (asterisk in the 120-minute panel), or two or three septa (180-minute panel) appeared following sorbitol treatment. (B) Cells were fixed 60 minutes post-stress and stained with the indicated final concentrations of Calcofluor white. (C) Sorbitol-treated wild-type cells were fixed 180 minutes post-stress, and were stained with DAPI and Calcofluor (left-hand panel) and TRITC-phalloidin (right-hand panel) to localize chromatin and F-actin, respectively. (D,E) Wild-type cells were grown to mid-log phase and subjected to the indicated stresses. Cells were fixed and processed for Calcofluor-white staining 30 minutes (D,E) and 60 minutes (E) post-stress (n=200). Scale bars: 5 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 2. Kinetics of stress-induced Calcofluor-white staining patterns. (A) After growth in YES media at 30°C, wild-type cells were subjected to stress, processed as in Fig. 1 and the indicated features scored at the indicated time points. (B) Images of the 60-minute post-sorbitol sample showing septating cells with SICS at their tips (upper panel) or separating cells with septal remnants at one tip and SICS at the other tip (lower panel). (C,D) The indicated strains were processed and scored, as in A, at the indicated time points. Scale bars: 5 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Cell-length distribution following osmotic stress. Wild-type cells were grown to mid-log phase in YES media at 30°C, processed for Calcofluor staining at the indicated time points following imposition of stress and cell length at division was plotted in bins of 0.25 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Re-initiation of tip growth converts tip-associated SICS dots into rings around the cortex. (A,B) cdc10.v50 cells were grown to early-log phase in YES media and cell-cycle progression was arrested by incubation at 36°C for 3.5 hours before the imposition of osmotic stress by the addition of sorbitol to a final concentration of 1.2 M. (A) Samples were taken at the indicated time points; arrows indicate rings of SICS staining around the cell cortex. (B) Quantification of the appearance of SICS at cell tips and SICS within the cell in sorbitol-treated, G1-arrested cdc10.v50 cells and in an asynchronous population of wild-type cells growing at 36°C. (C-E) Wild-type cells grown to mid-log phase in YES media at 30°C were stained in fluorescent lectin before being returned to culture. Continued growth resulted in dark non-stained tips. (C) Wild-type cells showing lectin, Calcofluor (calc.) or both (merge: Calcofluor in red, lectin in green) stainings at the indicated times post-stress. (D) Quantitation of the kinetics with which cells with dark tip regions appeared in the cultures following the addition of an equal volume of either pre-warmed YES media (control) or YES media containing 2.4 M sorbitol (sorbitol). (E) Quantitation of the kinetics of the appearance of dark tip-growth regions in lectin-treated wild-type cells following the addition of an equal volume of either pre-warmed YES media containing 1.2 M KCl (KCl) or 2.4 M sorbitol (sorbitol). Scale bars: 5 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Modulation of the actin cytoskeleton in response to osmotic stress. (A-J) Wild-type cells were grown as described in Fig. 1A, fixed and stained with rhodamine-phalloidin to localize F-actin (B,C,E,F,H,I) or DAPI and Calcofluor to localize chromatin and septa/SICS, respectively (A,D,G), at the indicated time points after the addition of sorbitol to a final concentration of 1.2 M. Captured images are presented either unprocessed (B,E,H) or with identical degrees of image processing applied (C,F,I) such that the signal in the processed images of stressed cells (F,I) was similar to that seen in the unstressed, unprocessed image (B). Specifically, using the levels function in Adobe Photoshop CS2, the brightest pixel in the unprocessed image shown in H was mapped to the pure-white level. This adjustment was saved and then applied to the unprocessed images shown in B and E. A contrast adjustment of +20 was then applied to each image. This two-step processing resulted in the images shown in C, F and I. (I) Asterisks highlight cells with actin patches that were relocalized to cell tips. (J) Quantification of the appearance of cells with SICS at cell tips and the appearance of cells with actin delocalized from tips. Scale bar: 5 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Modulation of the microtubule cytoskeleton in response to osmotic stress. GFP–-tubulin was expressed by the atb2.nmt81GFP allele in the indicated strains at 30°C. Osmotic stress was applied by the addition of sorbitol in filter-sterilized EMM2 to a final concentration of 1.2 M. (A) Each panel shows, from left to right, a 20-second-interval series of maximal projections of untreated wild-type cells (upper panels) or cells filmed immediately after being subjected to osmotic stress (lower panels, see also supplementary material Movie 1). (B) Each panel shows, from top left to bottom right, a 3-minute-interval series of maximal projections of wild-type cells. Sorbitol was added immediately after the second (3 minute) time point (arrow). In the image boxed in red (the 36-minute time point, top right), the microtubule bundles have fragmented into a number of short microtubules occurring at a variety of angles to the long axis of the cell. Later panels show that the microtubule cytoskeleton then gradually recovers its pre-stress distribution (see also supplementary material Movie 2). (C,D) Each panel shows a maximal projection of either sty1. cells (C) or wis1.DD cells (D) taken immediately before treatment (0 sec.; left panels) or 100 seconds after treatment with pre-warmed media (control; right panels) or with pre-warmed media containing 2.4 M sorbitol (sorbitol; lower right panels) (see also supplementary material Movies 3 and 4).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Calcofluor-white staining patterns following osmotic stress in cells lacking the osmotic-stress-activated kinases Ssp1 or Pmk1. YES cultures of the indicated strains were grown, subjected to osmotic stress and processed for Calcofluor staining (A-D) or DAPI staining (E), as described for Fig. 1. Scale bar: 5 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 8. SICS, tip growth and cell morphology in sorbitol-treated cells lacking the SRP MAP kinase Sty1. (A) sty1. cells that were processed, as described in Fig. 1A, at the indicated times are shown. (B,E) Wild-type and sty1. cells grown as in A were fixed and processed for Calcofluor staining to monitor SICS appearance at cell tips (B) and cell morphology (E). (C,D) Wild-type, sty1., atf1. and srk1. cells were coated with fluorescent lectin at t=0 to monitor tip growth at the indicated time points. (D) Representative images of sty1. cells that were coated with fluorescent lectin immediately after sorbitol treatment, returned to sorbitol medium for 300 minutes and then stained with Calcofluor. Calcofluor staining, top; lectin staining, middle; merged images (lectin in green, Calcofluor in red), bottom. (F) Wild-type and 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. The number of cells with morphology defects was determined by fixation and Calcofluor staining to generate the plot shown. (G) Quantification of the types of aberrant phenotype seen at the time points at which the occurrence of cells with defective morphology is highest in sorbitol-treated sty1. cells [210 minutes post-stress (E)] and cdc2.33 cells [120 minute post-stress (F)]. Scale bars: 5 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 9. SICS, tip growth and cell morphology in sorbitol-treated cells lacking the polarity factors Tea1 or Wsh3. (A-G) Wild type, tea1. and wsh3. cells were either processed as described for Fig. 1A to determine the kinetics of the appearance of SICS at cell tips (A,B,E) and to analyze cell morphology (C,D,E), or processed with fluorescent lectin for the analysis of tip growth as described for Fig. 4C (F,G). The arrow in the right-hand panel of E indicates a wsh3. cell with SICS at one tip (the end that was growing prior to the addition of sorbitol) that has re-initiated growth at a site on the cell cortex. (F) Quantification of the kinetics of the appearance of cells with dark tip regions in lectin-coated exponentially growing strains following sorbitol treatment. Note that tea1. and wsh3. cells re-initiate growth with the same kinetics as wild-type cells. (G) Pulsed lectin staining of wsh3. cells. Top panel, Calcofluor; middle, lectin; lower, merged (lectin in green, Calcofluor in red). The highlighted cell (star) has SICS at one tip (the growing tip prior to the addition of sorbitol) and has re-initiated tip growth (dark non-lectin-stained region) at the opposite (previously non-growing) tip. (H) tea1.pkGFP cells were grown to mid-log phase in YES media, fixed and processed for anti-Pk immunofluorescence either immediately before stress (upper panel) or 20 minutes post-stress (lower panel). (I) Maximum projections of wsh3.tdTomato cells either immediately before stress (upper panel) or 21 minutes after the addition of sorbitol to a final concentration of 1.2 M (lower panel). Scale bars: 5 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Kinetics of stress-induced Calcofluor-white staining patterns in mutant cells with constitutively activated (black line) or constitutively downregulated (red line) Plo1 kinase. Wild-type, plo1.S402A and plo1.S402E cells were grown and processed as described in Fig. 1A, and the appearance of SICS at cell tips was plotted as a function of time.

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