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First published online 27 November 2002
doi: 10.1242/jcs.00211

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Control of Cajal body number is mediated by the coilin C-terminus

Karl B. Shpargel, Jason K. Ospina, Karen E. Tucker, A. Gregory Matera^* and Michael D. Hebert

Department of Genetics, Center for Human Genetics and Program in Cell Biology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH 44106-4955, USA
Present address: Department of Biochemistry, University of Mississippi Medical Center, Jackson, MS 39216-4505, USA

View larger version (62K): [in a new window]   Fig. 1. Deletion of the coilin self-association domain affects epitope recognition. (A) Schematic of human coilin showing the location of the self-interaction/CB-targeting domain (Hebert and Matera, 2000), two nuclear localization signals (NLS), nucleolar localization signal (NoLS) (Hebert and Matera, 2000) and the RG box that mediates interaction with SMN (Hebert et al., 2001). The regions of coilin that were used to generate anti-coilin antibodies R288 (Andrade et al., 1993) and R508 (Chan et al., 1994) are also indicated. (B) Coilin lacking the self-association domain is not recognized by anti-coilin Ab R508. HeLa cells transfected with myc-tagged coilin lacking the first 93 amino acids were subject to staining with anti-myc (left panels) or anti-coilin (right panels) antibodies.

View larger version (63K): [in a new window]   Fig. 2. Coilin mutants display unregulated nuclear body formation. Upper panels, HeLa cells were transfected with GFP fused to the N-terminus of coilin (left), GFP fused to the C-terminus of coilin (middle) or a GFP-tagged truncation of coilin (1-481, right). The GFP-coilin(1-481) foci colocalized with other CB markers (SMN and fibrillarin) when transfected into HeLa cells (data not shown). Note that several out-of-focus dots appear blurred. In the lower panels, deletion of the nuclear localization signals (residues 106-234) in the GFP-coilin background results in exclusively cytoplasmic localization without foci in HeLa cells (left). Deletion of the NLSs in the coilin-GFP background results in a similar pattern, with the exception that nuclear foci are detected. Although the bulk of the fluorescence was cytoplasmic, two populations of cells were observed, some with faint nuclear foci and others with brighter foci (middle). When transfected into coilin knockout MEFs, this same coilinNLS-GFP construct localized solely to the cytoplasm (right).

View larger version (74K): [in a new window]   Fig. 3. Coilins from different species display variation in nuclear body formation. HeLa cells (left two columns) or mouse embryonic cells lacking endogenous coilin (right two columns) were transfected with FP-tagged human, mouse or frog coilin. Representative cells of both low and high expressers are shown. The nucleus is defined by a dotted line for MEF cells transfected with FP-frog coilin.

View larger version (12K): [in a new window]   Fig. 4. Schematic of human and mouse coilin. Chimeric constructs were generated by use of a conserved HindIII site, essentially swapping the amino acids downstream of this site. Numbers of foci per cell and percentages of cells with foci upon transfection into coilin-knockout MEFs are displayed to the right of the representative construct.

View larger version (62K): [in a new window]   Fig. 5. Mutations in mouse coilin affect nuclear body formation. (A) Alignment of the C-termini of human, mouse and frog coilin. The HindIII site used in generating the coilin chimeras is shown. (B) Summary of the mutations generated in this study. All constructs are fused to the C-terminus of GFP, with the exception of human coilin-GFP (FL-GFP). The constructs were transfected into MEF cells lacking endogenous coilin (Tucker et al., 2001) and scored for their ability to generate foci. Note that human constructs coilin-GFP and (1-481) as well as mouse construct (1-477) produce numerous unregulated foci. (C) HeLa cells were transfected with GFP-tagged wild-type mouse coilin or mutations of mouse coilin. Endogenous coilin (right column) was detected by using an antibody specific to human coilin (R508). Arrows mark the transfected cells, with concurrent disruption of Cajal bodies (right panels). Arrowheads show non-transfected cells, which display normal coilin localization in CBs and the nucleoplasm (right column). Foci formed by GFP-mouse coilin-SST to DDT were able to recruit SMN and fibrillarin, whereas loss of coilin foci upon overexpression of GFP-mouse coilin-SST to AGA also correlated with a loss of SMN foci (data not shown).

View larger version (73K): [in a new window]   Fig. 6. Ectopically expressed SMN produces numerous SMN foci. (A) HeLa cells expressing high levels of myc-tagged SMN were visualized by anti-myc antibodies (green). Coilin was detected in the same cell with R508 (in red), and the merged image is shown (right panel). Arrows denote Cajal bodies, which contain both SMN and coilin, whereas arrowheads mark some of the SMN gems, which are present both in the cytoplasm and the nucleus. The bottom panels display some of the large SMN foci that are observed upon high expression of SMN. (B) MEF coilin-knockout cells transiently transfected with myc-SMN were detected by anti-myc antibodies. Arrowheads demarcate some of the foci, including large aggregates, formed in high expressing cells (right).

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