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Flp1, a fission yeast orthologue of the S. cerevisiae CDC14 gene, is not required for cyclin degradation or rum1p stabilisation at the end of mitosis

Nathalie Cueille^1,*, Ekaterina Salimova^1,*, Veronica Esteban^2,*, Miguel Blanco^2,*, Sergio Moreno², Avelino Bueno² and Viesturs Simanis^1,

¹ Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, 1066 Epalinges, Switzerland
² Instituto de Microbiologia Bioquimica, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
^* These authors contributed equally to this paper
Author for correspondence (e-mail: viesturs.simanis{at}isrec.unil.ch )

View larger version (103K): [in a new window]   Fig. 1. Alignment of S. cerevisiae CDC14-related proteins. Black boxes indicate identity in all proteins, grey boxes indicate a related amino acid in three or more proteins. The following amino acids were considered interchangeable for the purposes of this alignment: K/R, S/T, I/L/V, N/Q, D/E, F/Y. When two different amino acids were present three times each, all are on a grey background, with three in white and three in black. A box indicates that the two yeast proteins have a common conserved amino acid that is not present in the other proteins. The C-terminal extension of the C. elegans protein is not shown.

View larger version (39K): [in a new window]   Fig. 2. Characterisation of flp1::kanMX6 cells. (A) The flp1::kanMX6 allele structure. The location of the fragment used to probe the Southern blot shown in B is indicated, as are the positions of the EcoRI sites that give rise to the fragments observed in the autoradiograph. Correct replacement of the flp1+ gene gives rise to a 2.4 kb fragment. (B) Southern blot of DNA prepared from wild-type and flp1::kanMX6 haploid cells. Note the absence of the 3.3 kb band in the flp1::kanMX6 cells. (C,D) flp1::kanMX6 cells were grown in YE at 25°C to mid-exponential phase (C) and shifted to 36°C for 5 hours (D). (C) Cell 1 is a postmitotic, binucleate cell. (D) Cell 1 is multicompartmented; cell 2 has septated once, and then undergone at least one more mitosis without septating; cell 3 has septated centrally, but both nuclei are located on the same side of the septum; and cell 4 shows one of its nuclei bisected by the developing division septum.

View larger version (42K): [in a new window]   Fig. 3. flp1p is phosphorylated during mitosis. The strain cdc25-22 flp1-HAc was synchronised by arrest-release and protein samples were prepared from cells at the indicated times. Note that a slower-migrating form of flp1p-HA appears during mitosis. The black arrowhead (B) indicates the rapidly migrating, interphase form of the protein, the grey arrowhead indicates the form present in G2 cells. (A) The progression through mitosis after release from the cdc25-22 block, as determined by fixing cells at each time point, and staining with DAPI and Calcofluor. (B) Western blot of protein samples prepared at the indicated times, probed with 12CA5 and TAT-1, to reveal flp1p-HA and -tubulin, respectively. (C) flp1p-HA was immunoprecipitated from protein samples prepared at the indicated times. After washing, the 45 minute immunoprecipitate was divided in half and treated with alkaline phosphatase (+), or not (-). A western blot of these protein samples was probed with 12CA5 to reveal the position of flp1p-HA.

View larger version (48K): [in a new window]   Fig. 4. Localisation of flp 1p. (A) Living cells expressing flp 1p-GFP were photographed, and images were processed as described in Materials and Methods. (B) Cells expressing flp 1p-GFP were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI and flp 1p-GFP was detected with rabbit antiserum to GFP, followed by FITC-conjugated goat-anti rabbit serum. (C) Cells expressing flp 1p-HA were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI; flp 1p-HA was detected with 12CA5 followed by CY3-conjugated goat anti-mouse serum; DNA was stained with DAPI; and the nucleolar antigen fibrillarin was detected using affinity-purified rabbit antibodies against S. cerevisiae nop 1p followed by FITC-conjugated goat-anti-rabbit serum. Note that while the nucleolar staining overlaps in the merge, the dot corresponding to the spindle pole body does not. (D) Cells expressing flp 1p-HA were fixed and processed for indirect immunofluorescence. DNA was stained with DAPI; the spindle pole antigen sad 1p was detected with rabbit anti-sad 1p, followed by FITC-conjugated goat anti-rabbit serum; and flp 1p-HA was detected with 12CA5, followed by CY3-conjugated goat anti-mouse serum. (E) nda3-KM311 cells expressing flp 1p-GFP were incubated for 5 hours at 20°C, fixed and processed for indirect immunofluorescence. flp 1p-GFP was detected with rabbit antiserum to GFP followed by FITC-conjugated goat anti-rabbit serum, while F-actin was detected using Rhodamine-conjugated Phalloidin. (F) nda3-KM311 cells expressing flp 1p-GFP were incubated for 5 hours at 20°C, fixed and processed for indirect immunofluorescence. flp 1p-GFP was detected with rabbit antiserum to GFP followed by FITC-conjugated goat anti-rabbit serum, while DNA was detected using DAPI. (G) Living cells expressing flp 1p-GFP were treated with Latrunculin A for 10 minutes, then photographed. Images were processed as described in Materials and Methods. i indicates an interphase cell in image 1; the arrow indicates the location of the spindle pole body in the interphase cell. Image 2 shows the disorganised dot-like staining observed in mitotic cells, and image 3 shows a pair of interphase cells that have been treated with LatA. Note that the nucleolar and spindle pole body staining are both still present.

View larger version (51K): [in a new window]   Fig. 5. Localisation of flp 1p-GFP in SIN mutants. Spores were inoculated into minimal medium containing adenine and leucine, and allowed to germinate (except D1-4). The localisation of flp 1p-GFP was examined in multinucleate cells. (A) cdc7 null allele. (1) Interphase, binucleate cell: note that flp 1p-GFP is present in the nucleolus and on the spindle pole body. (2) Cell undergoing a second mitosis: note the presence of a single medial ring and staining along the spindle. (B) cdc14 null allele. (1) Binucleate cell, early in second mitosis: note the presence of the medial ring, and the spindle staining. (2) Multinucleate interphase cell: note that flp 1p-GFP is present in the nucleolus and on the spindle pole body. (C) spg1 null allele. (1) Mitotic cell: note the presence of two medial rings. (2) Mitotic cell: note the presence of staining along the short spindle and on the spindle pole bodies. (3) Multinucleate interphase cell: note that flp 1p-GFP is present in the nucleolus and on the spindle pole body. (Inset) Binucleate cell early in the second mitosis: note the spindle staining. (D) cdc16 null allele, germinating cells were fixed and processed for indirect immunofluorescence. flp 1p-GFP was detected with rabbit antiserum against GFP (2,4); DNA was detected with DAPI (1,3). Images 1-4 are type I cells. Note that there is no discrete nucleolar or spindle pole body staining of flp 1p-GFP. Spotty aggregates were sometimes observed (arrows). Image 5 is a type II cell, nucleolar and spindle pole body staining of flp 1p-GFP is observed. (E) sid2-1 cdc16::ura4+ flp 1-GFPc cells were grown at 25°C, and the GFP fluorescence was observed in living cells. The aggregate observed in cell 2 is shown by the arrow. Images 1 and 3 are phase-contrast views of the cells shown in images 2 and 4, respectively.

View larger version (83K): [in a new window]   Fig. 6. Genetic interactions between the SIN and flp 1. (A) Cells were grown in YE medium at 25°C and shifted to 36°C for 5 hours, fixed and stained with DAPI and Calcofluor. (B) Cells were grown at 29°C, then shifted for 8 hours to 25°C or for 5 hours to 36°C, before fixation and staining with DAPI and Calcofluor. (C,D) flp 1::kanMX6 leu 1-32 and leu 1-32 (WT) strains were transformed to leucine prototrophy with pREP3-spg 1 (C) or pREP41-byr4 (D). Expression of the genes was induced for 18 hours at 25°C, then cells were fixed and stained with DAPI and Calcofluor.

View larger version (46K): [in a new window]   Fig. 7. flp 1p function is not required for the accumulation or degradation of either rum 1p pr cdc 13p, or the dephosphorylation of ste9p in G1. (A,B,C) cdc25-22 or cdc25-22 flp 1::kanMX6 cells were synchronised by arrest-release, and protein samples were prepared at the indicated times. Western blots were probed with antiserum recognising rum 1p, cdc 13p and -tubulin. (A) Western blot for rum 1p, cdc 13p and -tubulin. Asn indicates asynchronous population. (B) Samples were removed at intervals, and cells were fixed, and stained with DAPI and Calcofluor. The graphs show the mitotic index and septation index at the indicated times after release from the cdc25-22 arrest. (C) Exponentially growing cdc 13-myc 13c and cdc 13-myc 13c flp 1::kanMX6 cells were fixed and indirect immunofluorescence was used to detect cdc 13p (mAb 9E10, followed by CY3-conjugated goat anti-mouse serum), in mitotic cells and septating cells. Note that cdc 13p is destroyed and reappears with similar kinetics in the two strains. (D,E) cdc 10-129 and cdc 10-129 flp 1::kanMX6 cells were grown at 25°C to mid-exponential phase in minimal medium and were shifted to 36°C for 4 hours to block the cells in G1, and then returned to 25°C by rapid agitation in an ice-water bath. Samples were taken at the indicated times, and the ste9p phosphorylation level was examined by western blotting. (E) Samples were removed from the each culture at the indicated times, and the percentage of G1 cells in each culture was determined by FACS analysis. The asterisk in D indicates the position of the loading control, -tubulin, which was detected using TAT-1.

View larger version (44K): [in a new window]   Fig. 8. Increased expression of flp 1+ arrests cells in G2. A single copy of the flp 1+ gene was integrated at the leu 1 locus of wild-type cells. Expression of flp 1+ was induced by incubation in medium without thiamine at 25°C. Samples were removed at intervals, processed for FACS analysis, and fixed and stained with Calcofluor and DAPI. (A) Uninduced control cells (top), cells 20 hours after induction (bottom). (B) FACS analysis of uninduced (top) and induced (bottom) cells. The position of the G1 peak was established by incubating cells in 12 mM hydroxyurea (HU) for 3 hours. -N are cells grown in medium lacking a nitrogen source. (C) leu 1-32 cells were transformed to leucine prototrophy with a REP3 plasmid expressing flp 1p(C286S). Expression was induced for 24 hours at 25°C, cells were fixed, and stained with DAPI and Calcofluor. (D) leu 1-32 cells were transformed to leucine prototrophy with a pREP3-flp 1+. Expression was induced (-T) and samples were removed at the indicated times thereafter. Protein extracts were prepared and western blots probed for the indicated antigens. For cdc25p, the two arrows indicate the position of the upper (hyper-phosphorylated) and lower bands. A portion of culture resuspended in medium containing thiamine served as a control. (E) leu 1-32 cdc25-HA3 was transformed with either pREP3 or pREP3-flp 1+ and expression was induced for 20 hours at 25°C. Protein samples were prepared and a western blot was probed with 12CA5. The samples were run in adjacent lanes, on the same gel.

View larger version (25K): [in a new window]   Fig. 9. Mitotic control mutants have altered sensitivity to increased expression of flp1+. A single, nmt1-promoter-controlled copy of flp1+ was introduced into the leu1 locus and crossed into the indicated strain backgrounds. Expression of flp1+ was induced and cell number was determined at intervals thereafter. Cells were grown at 25°C. (A) The effect of increased flp1+ expression in wee1-6 and cdc25::ura4+ wee1-50. Induced images were taken 26 hours (approximately 6 generations) after induction. (B) Cell number increase after induction of flp1+. Cell numbers are expressed in arbitrary units. Cell numbers at 16 hours were approximately 106 ml-1. (C,D) Induction of flp1+ in dominant activated alleles of cdc2.

View larger version (50K): [in a new window]   Fig. 10. Genetic interactions between flp1::kanMX6 and mitotic control mutants. Cells of the indicated genotype were grown at 25°C in YE and a part of the culture was shifted to 36°C for 5 hours. Cells were fixed and stained with DAPI and Calcofluor.

View larger version (30K): [in a new window]   Fig. 11. flp1+ functions in the S. pombe morphology checkpoint. Cells of the indicated genotype were grown at 25°C in YE, then shifted for 5 hours to 36°C before fixation. (A,B) DAPI and Calcofluor staining of cells 5 hours after shift to 36°C. (C) The percentage of cells with one, two or more than two nuclei. Cells were scored as 2 or >2 nuclei, whether they were septated or not, as long as there was no sign of cleavage of the septum.