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First published online 25 May 2004
doi: 10.1242/jcs.01155

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The nucleolus is involved in mRNA export from the nucleus in fission yeast

Takashi Ideue¹, Abul Kalam Azad^1,*, Jun-ichi Yoshida², Tadao Matsusaka³, Mitsuhiro Yanagida⁴, Yasumi Ohshima¹ and Tokio Tani^2,

¹ Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan
² Department of Biological Science
³ Department of Environmental Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan
⁴ Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

View larger version (74K): [in a new window]   Fig. 1. Phenotypes of the ptr11 mutant. (A) The cut phenotype observed in ptr11-1. ptr11-1 and wild-type 972 cells cultured at 37°C for 4 hours were stained by DAPI. The undivided nucleus was cleaved by a septum in ptr11-1. (B) ptr11-1 cells cultured at 26°C were shifted to 37°C for 4 hours and then subjected to in situ hybridization with the oligo dT probe. Poly(A)+ RNA accumulated in the cut phenotype nuclei at 37°C in ptr11-1. (C) ptr11-1 has no apparent defects in protein transport. The plasmid expressing Pap1p fused with GFP was introduced into cells and then localization of the expressed fusion protein was examined in the absence (a,b) or presence (c,d) of LMB at 37°C.

View larger version (88K): [in a new window]   Fig. 2. In situ hybridization of poly(A)+ RNA in mutants with the cut phenotype. After shifting to 37°C for 2 hours, each indicated mutant was subjected to in situ hybridization with the biotin-labeled oligo dT probe. Photographs were taken with the same exposure time.

View larger version (45K): [in a new window]   Fig. 3. Poly (A)+ mRNA accumulated in the anucleolate nuclei in ptr4-1 and ptr11-1. Cells cultured at 26°C were shifted to 37°C for 4 hours and then triple stained using the oligo dT probe, DAPI and anti-fibrillarin antibody (see Materials and Methods).

View larger version (101K): [in a new window]   Fig. 4. Electron microscopic analysis of ptr11-1. The ptr11-1 cells were cultured at 37°C for 4 hours, fixed and embedded in Quetol-651. Thin sections were examined under an electron microscope. Part of a cut phenotype cell with the cleaved nuclei is shown. Arrowheads denote electron-dense materials. No, the nucleolus. Bar, 1 µm.

View larger version (54K): [in a new window]   Fig. 5. In situ hybridization of poly(A)+ RNA in nuc1-632. The mutant cells were cultured at 26°C to the midlog phase and shifted to 37°C for 4 hours. The cells were then subjected to triple staining analysis using the oligo dT probe, DAPI and anti-fibrillarin antibody.

View larger version (106K): [in a new window]   Fig. 6. Cellular distribution of TBP mRNA transcribed from the intron-containing gene (A) or intron-less cDNA (B) in ptr11-1 and nuc1. Wild-type 972, ptr11-1 or nuc1 cells harboring the TBP-expressing plasmid was cultured at 26°C without thiamine for 16 hours, then maintained at 26°C or shifted to 37°C for 6 hours. The cells were subsequently subjected to in situ hybridization with the Cy3-labeled TBP probe specific for the mRNA expressed from the plasmid. The first and third columns show the intracellular distribution of the TBP mRNA and the second and fourth columns show cells stained with DAPI in the corresponding fields.

View larger version (59K): [in a new window]   Fig. 7. spMex67p-GFP accumulates in the nucleolar region in the heat-shocked cells. (A) The wild-type S. pombe cells expressing spMex67p-GFP from the weakest nmt1 promoter were grown at 30°C and either maintained at 30°C (left panels) or shifted to 42°C for 3 or 30 minutes (middle and right panels). After staining with Hoechst 33342, the distribution of spMex67p-GFP was observed with a fluorescence microscope. Insets in the left panels show enlarged pictures of the nucleus. In merged images, red denotes DNA and green denotes spMex67p-GFP. (B) Wild-type cells expressing Cut15p-GFP using the native cut15 promoter were grown at 30°C (left panel) or heat shocked at 42°C for 30 min (right panel). Intracellular distribution of Cut15p-GFP was not changed after the heat shock treatment, whereas spMex67p-GFP accumulates rapidly in the nucleolar region under the same conditions.

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