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First published online 10 October 2006
doi: 10.1242/jcs.03220

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S. pombe FEAR protein orthologs are not required for release of Clp1/Flp1 phosphatase from the nucleolus during mitosis

Chun-Ti Chen^1,*, Marie-Pierre Peli-Gulli^2,*, Viesturs Simanis² and Dannel McCollum^1,

¹ Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
² Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland

View larger version (36K): [in a new window]   Fig. 1. Clp1p nucleolar release in cells carrying mutations in FEAR pathway homologs. sid2-250 clp1-GFP cells (A) or sid2-250 clp1-GFP cells carrying the spo12 (B), plo1-25 (C), alp7 (D) or cut1-205 (E) mutations were grown at 25°C, then synchronized by centrifugal elutriation. Cells were then shifted to 36°C. Samples were fixed every 20 minutes in methanol, stained with DAPI, and scored for number of nuclei and Clp1p-GFP localization. At least 100 cells were scored for each time point. Inset images show Clp1p-GFP signal in cells of each strain with Clp1p-GFP released (*) or not released (#).

View larger version (38K): [in a new window]   Fig. 2. Overproduction of Plo1p and Spo12p do not cause release of Clp1p. cdc25-22 clp1-GFP cells carrying either a plasmid expressing plo1+ or spo12+ from the thiamine-repressible nmt1 promoter were grown at 25°C in the absence of thiamine for 12 hours. The cells were then shifted to 36°C for an additional 4 hours in the absence of thiamine to inactivate the Cdc25-22 mutant protein and arrest cells in G2 phase. The cells were then fixed, stained with DAPI, and representative DAPI, GFP and merged images are shown. Note that the nmt1 promoter does not become active until 12 hours after removal of thiamine.

View larger version (19K): [in a new window]   Fig. 3. Meiosis and spore formation in clp1 cells. (A,B) Diploid cells of the genotype h+/h+ ade6-M210 /ade6M-216 pat1-114 /pat1-114 and h+/h+ ade6-M210 /ade6M-216 pat1-114 /pat1-114 clp1::kanMX6/clp1::kanMX6 were grown to mid-exponential phase and then transferred to minimal medium without ammonium chloride to starve cells in G1. Cells were inoculated into complete medium at 33°C to induce meiosis. Samples were fixed at intervals and the number of nuclei per cell was determined. The key shown in A also applies to B. (C,D) Wild-type h+ and h– cells were mated on minimal medium lacking ammonium chloride. Cells were taken from the mating mixture and the percentage of complete asci (C) and number of nuclei per meiotic cell were determined (D).

View larger version (50K): [in a new window]   Fig. 4. Segregation of a nucleolar marker (Nuc1p-GFP) in clp1 cells. Time-lapse series of wt and clp1 cells expressing the nucleolar marker Nuc1p-GFP and the spindle pole body marker Cdc11p-GFP. Stacks of 11 z-sections of 0.5 µm were taken at 30-second intervals and projected as 2D images. Cells are shown at the indicated times. The first time SPBs labeled with Cdc11p-GFP appeared as separate dots was defined as time zero.

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