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First published online 17 October 2006
doi: 10.1242/jcs.03241

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Fission yeast Tor2 promotes cell growth and represses cell differentiation

Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain

View larger version (61K): [in a new window]   Fig. 1. Isolation of a temperature-sensitive allele of tor2+. (A) Wild-type (tor2+), tor2ts (tor2-51) and the heterozygous diploid (tor2+/tor2-51) strains were spotted onto yeast extract (YES) medium at 1:10 dilutions. The plates were incubated at different temperatures (25, 30, 32 and 36°C). (B) Nomarski photomicrographs of wild-type (tor2+) and tor2ts (tor2-51) colony cells plated on YES medium after incubation overnight at 25°C, transferred to the indicated temperatures and then incubated for 12 hours. (C) Wild-type (tor2+) and tor2ts (tor2-51) cells were plated on YES medium and incubated at 36°C for three days (left panel). The same plate was then incubated at 25°C for a further three days (right panel). Photographs of the plates are shown.

View larger version (64K): [in a new window]   Fig. 2. Tor2 promotes cell growth and is part of the TORC1 complex. (A) Wild-type (tor2+) and tor2-51 cells were grown in EMM to mid-log phase at 25°C, shifted to the restrictive temperature of 32°C, and samples were taken at the indicated times. RNA was extracted and northern blot performed and hybridised using probes against the ribosomal protein genes rps1102 and rpl1701. The rRNA in each sample, stained with Methylene Blue, is shown to verify equal loading of RNA in each lane. (B,C) Crude extracts were prepared from cells exponentially growing in rich medium. The cells expressed tagged proteins, Mip1-myc, Ste20-myc or Pop3-myc, and HA-Tor1 or HA-Tor2, as indicated. The extracts were immunoprecipitated (IP) with the indicated antibodies (, anti-) and the precipitates were subjected to SDS-PAGE and examined by western blot (WB). Immunoblots of the crude extracts are also shown.

View larger version (41K): [in a new window]   Fig. 3. Inactivation of Tor2 mimics nitrogen starvation. (A) Flow cytometry analysis of wild-type (tor2+) and tor2-51 cells at the indicated times after shifting a culture to the restrictive temperature of 32°C. The top panel represents cell size (measured as forward scatter, FSC) and the lower panel represents the DNA content. (B) Nomarski photomicrographs of h90 tor2-51 cells in YES medium at the same density after incubation at 25°C or 32°C for 24 hours. (C) Wild-type (tor2+) and tor2-51 cells were grown in EMM to mid-exponential phase at 25°C, then shifted to the restrictive temperature of 32°C, and samples were taken at the indicated times. RNA was extracted and northern blot performed and hybridised using probes against the indicated genes. cdc13 mRNA and ribosomal (rRNA) levels were used as loading controls.

View larger version (46K): [in a new window]   Fig. 4. Rum1 and Ste9 are required for G1 arrest but not for growth inhibition after Tor2 inactivation. (A) Flow cytometry analysis and Nomarski photomicrographs of h90 tor2+, h90 tor2-51, h90 tor2-51 rum1 and h90 tor2-51 ste9 cells incubated at 25°C or at 32°C for 8 hours and 24 hours, as shown. (B) The indicated strains were grown in EMM at 32°C for 24 hours. RNA was extracted and northern blot was performed and hybridised using probes against the ribosomal protein gene rps1102. The rRNA in each sample, stained with Methylene Blue, is shown to verify equal loading of RNA in each lane.

View larger version (23K): [in a new window]   Fig. 5. Overexpression of Tor2 inhibits sexual differentiation but not entry into the stationary phase. (A) h90 tor2+ and h90 nmt-tor2+ cells were grown in the presence or absence of thiamine for 18 hours, and then spotted onto malt extract plates with or without thiamine. These plates were incubated for 48 hours at 25°C and then stained with iodine vapour (left panel). Alternatively, a sample from each spot was resuspended in water at the same optical density and treated overnight with glusulase. Equal volumes were then plated on YES medium and the number of colonies was counted and represented as a plot (right panel). (B) Flow cytometry analysis of h90 tor2+ and h90 nmt-tor2+ cells after nitrogen starvation. Cells were grown to mid-exponential phase in minimal medium with or without thiamine for 18 hours to induce Tor2 overexpression. These cells were washed several times in minimal medium lacking nitrogen, and were nitrogen starved in the same medium in the presence or absence of thiamine. Samples were taken at the indicated times after nitrogen depletion. (C) Cell size distribution determined by flow cytometry analysis (measured as forward scatter, FSC) of the h90 tor2+ and h90 nmt-tor2+ cells as in (B). (D) h90 tor2+ and h90 nmt-tor2+ cells were grown to mid-exponential phase in minimal medium plus thiamine, then washed several times in minimal medium lacking thiamine prior to incubation in the same medium for 18 hours. They were then washed several times in minimal medium lacking thiamine and nitrogen, and nitrogen starved in the same medium for the indicated times. Samples were collected from which RNA was extracted, and a northern blot was performed and hybridised with probes against ste11+ and mei2+ genes. As a loading control, the amount of rRNA in each sample was estimated from its staining with Methylene Blue. (E) h90 tor2+, h90 nmt-tor2+ and h90 nmt-tor2+ pREP3x-ste11+ cells were grown to mid-exponential phase in the absence of thiamine for 18 hours, and then spotted onto malt extract plates. These plates were incubated for 48 hours at 25°C and then stained with iodine vapour.

View larger version (19K): [in a new window]   Fig. 6. Tor2 interferes with Mei2 function. (A) h+/h– tor2+/tor2+ and h+/h– nmt-tor2/tor2+ diploid cells were grown in the presence or absence of thiamine for 18 hours, and were then spotted onto malt extract plates with or without thiamine. Plates were incubated for 48 hours at 25°C and then stained with iodine vapour (left panel). Alternatively, a sample from each spot was resuspended in water at the same optical density and treated overnight with glusulase. Equal volumes were then plated on YES medium and the number of colonies was counted and represented as a plot (right panel). (B) The indicated strains were plated on minimal medium or minimal medium supplemented with thiamine and incubated at 32°C for three days. Photographs of the plates are shown. Lower panel, the indicated strains were grown at 25°C in minimal medium in the presence or absence of thiamine for 18 hours to mid-exponential phase (OD595=0.8-1), diluted to OD595=0.1, and then incubated in the same medium at 32°C. Growth curves at 32°C are shown. (C) Crude extracts were prepared from exponentially growing cells expressing tagged proteins, as indicated. They were subjected to IgG pull-down (TAP pull-down) with IgG-Sepharose beads. HA-Tor2 was detected by western blot (WB). Immunoblots of the crude extracts are also shown.

View larger version (13K): [in a new window]   Fig. 7. Tor2-regulated pathways in fission yeast: a working model. In the presence of nutrients (nitrogen), Tor2 actively represses mating and meiosis, and promotes ribosome biogenesis and cell growth. When nitrogen is limited, Tor2 is inactivated, cells stop growing, and cell differentiation is switched on.

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