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Different mechanisms of cell polarisation in vegetative and shmooing growth in fission yeast

Cancer Research UK London Research Institute, Cell Cycle Laboratory, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

View larger version (37K): [in a new window]   Fig. 1. Cyr1sxa2 strain in the presence of pheromone. 3 µg/ml of P factor was added at time 0 and samples were taken every hour. (A) Cells were stained with calcofluor and the sepation index was monitored. (B) The cells were imaged with a phase microscope mounted with a Hamamatsu camera and cell length was measured using NIH Image software. (C) Cells were fixed in ethanol and processed for FACS analysis. (D) The cell wall was stained with 1:1000 (from a 5 mg/ml stock in water) FITC-lectin (red), for 10 minutes; the lectin was then washed out and the cells were allowed to grow for 8 hours in the presence or absence of pheromone. Cells were then stained with calcoflour (green), which stains growing ends. The areas in red have not grown since the lectin pulse, whereas the areas in green have.

View larger version (49K): [in a new window]   Fig. 2. Protein levels during a pheromone time course. (A) Western blot of total cell extracts from a pheromone time course probed with anti-Tea1, anti-GFP (for Tea2GFP) and anti-Tip1 antibodies. New forms of protein appearing after pheromone addition are marked by arrows. (B) Cells were shifted to 36°C in the presence or in the absence of pheromone for 4 hours. Cells at 36°C in the presence of pheromone arrest in G1 but do not activate the shmooing growth mode (Fig. 7B,C). Cells were then released at 25°C to allow synchronous progression into shmooing growth. Samples were taken at time 0, after 4 hours at 36°C and after 1 hour release and western blots were carried out on total cell extracts for Tea1p. (C) Native cell extracts were made from vegetatively growing cells and treated with -phosphatase in the absence () or in the presence (+inh) of phosphatase inhibitors. An untreated sample is run as a control (C). Western blots were probed for Tea1p. The lower dephosphorylated form, which increases after phosphatase treatment, is marked by an arrow.

View larger version (111K): [in a new window]   Fig. 3. Tea1p, Tip1p and Tea2p partially delocalise in the presence of pheromone. (A) tip1YFP-tea1GFP (B) and tip1YFP-tea2GFP strains were used to determine colocalisation in vivo. Cells were grown in minimal medium without or with pheromone for 5 (A) or 7 (B) hours. Pictures were taken with a confocal microscope using YFP and CFP filters, which allow excitation and detection of each fluorophore separately. The more narrow ends, marked by an asterisk, are known to be growing from previous calcofluor and actin stainings. The arrows in A indicate the Tea1GFP dots co-localising with Tip1YFP in the absence of pheromone but not in the presence of pheromone. In B the arrows indicate the Tea2GFP dots co-localising with Tip1YFP both in the presence and absence of pheromone. Scale bar, 3µm. (C) Images of a tea2GFP strain in the YFP and CFP channels, showing there is no bleed through in the YFP channel. Bar, 5 µm. (D) To confirm the localisation in a wild-type background, we mated an h90 Tea2GFP overnight on glutamate plates and photographed mating cells. Bar, 3 µm. (E) Images of Tea1GFP, Tip1YFP and Tea2GFP in the absence or after 6 hours in the presence of pheromone. Bar, 3 µm.

View larger version (74K): [in a new window]   Fig. 4. Tea1 localisation in the presence of pheromone in G2 and G1. A cdc25-22cyr1sxa2 strain was arrested in G2 at 36°C for 90 minutes; pheromone was added and cells were incubated for a further 2 hours; cells were then released into cell cycle progression at 25°C in the continued presence of pheromone. Samples were taken at three different time points: (A) after cells had been arrested in G2 at 36°C in the absence of pheromone; (B) after having been incubated with pheromone for 2 hours at 36°C; and (C) after cells had been allowed to enter G1 at 25°C in the continued presence of pheromone. Cells from each time point were treated with MBC, a microtubule depolymerising drug, to totally depolymerise microtubules. The drug was then washed out and microtubules were allowed to repolymerise for 50 seconds. Cells were fixed in methanol and processed for tubulin and Tea1p immunofluorescence. (D,E) The same experiment was repeated with a cdc25-22cyr1sxa2tea1GFP strain, timepoints a, b and c correspond to points A, B and C. (D) Cells were fixed in methanol and processed for tubulin while Tea1GFP was imaged live on a confocal microscope (E; see Materials and Methods for details). (F) Cells from C (after the release into G1 in the presence of pheromone) were also immunostained for Tip1p and tubulin. Bars, 5 µm.

View larger version (34K): [in a new window]   Fig. 6. Actin relocalisation in the mutants strains. 3 µg/ml of P factor was added at time 0. (A) Samples were taken every 30 minutes, fixed in formaldehyde for 40 minutes and stained for actin with rhodamine phalloidin. Actin localisation was scored for each time point. (B) Samples were taken every 2 hours, fixed in ethanol and processed for FACS analysis. (C) h90 wild-type and mutant strains were grown overnight in full nitrogen and low glucose medium (10 g/l) to 2x107 cells/ml. Cells were then spun and re-suspended in low glucose, nitrogen-free medium at 1x106 cells/ml to induce mating and fused cells were scored every hour. Experiments were repeated at least three times and at least 200 cells were scored for each time point. Standard deviation was calculated and error bars are shown.

View larger version (22K): [in a new window]   Fig. 5. Microtubules in morphological mutants. Cells were treated with 3 µg/ml P factor for 9 hours, fixed in methanol and then stained with anti-tubulin antibody. All strains are cyr1sxa2. Bar, 5 µm.

View larger version (54K): [in a new window]   Fig. 7. Ability of the cells to shmoo in the presence of drugs. (A) Cells were treated with 3 µg/ml of P factor for 4 hours at 36°C. Cells were then shifted to 25°C in the presence of 200 µg/ml of LatA or DMSO, as a control, and visualised under the light microscope. 3 µg/ml of P factor was added to cells at 25°C and at 36°C. (B) Samples were taken every 2 hours, fixed in ethanol and processed for FACS. (C) After 8 hours, cells were fixed in formaldehyde and stained for actin. (D) Cells were arrested at 36°C in the presence of P factor for 4 hours; 25 µg/ml of MBC or DMSO, as a control, were added and the cells were shifted to 25°C to induce shmooing. Samples were taken every 30 minutes, fixed in formaldehyde, stained with rhodamine phalloidin to visualise actin and scored for actin localisation. Times shown are calculated from the shift to 25°C. (E) Cells treated with MBC were fixed in methanol at the end of the time course and processed for tubulin immunofluorescence to verify that no microtubules were present. Bar, 5 µm.

View larger version (39K): [in a new window]   Fig. 8. Pom1 behaviour in the presence of pheromone. 3 µg/ml of P factor was added at time 0 to a cyr1sxa2pom1HA strain (A) or a cyr1sxa2pom1GFP strain (B). (A) Western blot of total cell extracts probed with anti-HA antibody to visualise Pom1p. (B) Pom1 GFP in live cells at time zero and after 6.5 hours in pheromone. The arrow indicates the longest shmoo, which shows Pom1GFP diffused throughout the cell. 3 µg/ml of P factor was added at time 0 to a pom1cyr1sxa2 cell. (C) Samples were taken every 2 hours, fixed in ethanol and processed for FACS analysis. (D) Samples were taken every 30 minutes, fixed in formaldehyde, stained with rhodamine phalloidin to visualise actin and scored for actin localisation. (E) The first 2 hours of the time course for two independent experiments (I and II) are plotted together with a wildtype control to show the rise in delocalised actin and the reduction in monopolar actin for pom1.