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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01611

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RNA association and nucleocytoplasmic shuttling by ataxin-1

¹ McMaster University, HSC 4H45, Department of Biochemistry, Hamilton, Ontario, L8N 3Z5, Canada
² Department of Laboratory Medicine and Pathology and the Institute of Human Genetics, University of Minnesota, MN 55455-0213, USA

View larger version (65K): [in a new window]   Fig. 1. Ataxin-1 nuclear inclusions are dynamic and distinct from huntingtin exon1 polyglutamine aggregates. Three-dimensional (x,y,time) analysis of huntingtin and ataxin-1 nuclear inclusions by live-cell video microscopy. (A,B) Fluorescence images of eGFP-huntingtin exon1[Q138] and eGFP-ataxin-1[Q84] video sequences captured at 0.5-second intervals for 30 seconds at 37°C. (C,D,E,F) x,y,t voxel volume images of time sequences in orthogonal view; intensities thresholded to inclusions only in E and F. (G,H) Three-dimensional single representative inclusions of huntingtin and ataxin-1 with a dotted line as a straight reference. (I) A single ataxin-1 nuclear inclusion movement (see also Movie 1 in supplementary material). (J,K) Voxel volumes were rendered with an isometric surface projection to reveal a spiral structure indicating a spinning motion. Bars: (A-F) 10 µm, (G-K) 1 µm. J, K are presented as red (left eye) and cyan (right eye) stereo projections. Three dimensional x,y,t axis added for orientation reference.

View larger version (77K): [in a new window]   Fig. 2. Ataxin-1 nuclear inclusion formation and movement are not dependent on polyglutamine. (A-C) Fluorescence microscopy images of eGFP ataxin-1[Q2] (A), [Q26] (B) or [Q84] (C) from live-cell video observations at 37°C in HeLa cells. (D-F) Three dimensional x,y,t voxel and isosurface representation of ataxin-1 body movement in either Q2 (D), Q26 (E), or Q84 (F) context showing similar body movement. Bar: 10 µm. (See also Movies 3-5 in supplementary material.) D-F are presented as red-cyan stereo images.

View larger version (50K): [in a new window]   Fig. 3. Ataxin-1 sub-nuclear localization is dependent on RNA and transcription. Fluorescence micrographs of eGFP-ataxin-1[Q26]- or [Q84]-expressing NIH 3T3 cells. (A) panels A,E: no drug added; all other panels show the effects of the indicated drugs under the following conditions: panel B: 25 µg/ml -amanatin for 3 hours at 37°C; panels C,F: a 0.05 µg/ml actinomycin D at 37°C for 3 hours; panel B: 25 µg/ml -amanatin for 3 hours at 37°C; panel D: RNase for 1 hour; panel G: 0.5 µm colchicine for 3 hours; panel H, 5% DMSO for 1 hour; panels I, J, MG132 and cycloheximide respectively, for 1 hour. (B) panels A-D: live-cell microscopy of ataxin-1[Q84]-expressing cell over a time course of 2-4 hours of treatment with 25 µg/ml -amanatin, showing inclusion dispersal. (See also Movie 6 in supplementary material.) (C) SYTO Green total RNA staining of cells expressing mRFP-ataxin-1[Q84] (panels A-C). Arrows indicate the presence of RNA in nuclear ataxin-1 inclusions in panels A and B. Bar: 10 µm.

View larger version (47K): [in a new window]   Fig. 4. Ataxin-1 nuclear inclusions are distinct from other nuclear bodies. Live-cell fluorescent patterns of ataxin-1 (A,D,G,J) and ASF/SF2 (B), hnRNPA1 (E), Nup98 (H), or 14-3-3 (K) proteins fused to fluorescent protein variants. (M-O) Co-expression of eGFP-ataxin-1[Q2] (M) and mRFP-TAP/NXF1 (N) is shown by the merge image in O. Bar: 10 µm.

View larger version (101K): [in a new window]   Fig. 5. Ataxin-1 nuclear inclusions co-localize with the TAP/NXF1 mRNA export factor. Laser confocal microscopy images of eGFP-ataxin-1 and mRFP-TAP/NXF1 (A-D), huntingtin exon-1[Q138] and mRFP-TAP/NXF1 (E-H) proteins. (I-L) Immunofluorescence with anti-TAP primary antibody and Alexa 594 secondary antibody on fixed NIH3T3 cell transfected with eGFP-ataxin-1[Q84] to detect endogenous TAP protein. (M-O) Cells transfected with mRFP-TAP/NXF1 alone, displaying no punctate localization (as in F and J). Bar: 10 µm.

View larger version (36K): [in a new window]   Fig. 6. Ataxin-1 recruits TAP/NXF1 protein to nuclear inclusions as the result of cell heat-shock. Fluorescence micrographs of eGFP-ataxin-1[Q26] and mRFP-TAP/NXF1 in live cells after heat shock at 42°C for 5 minutes (A-C), followed by incubation for 1 hour at 37°C (I-J), with time course of TAP/NXF1 localization to ataxin-1 nuclear inclusions over 60 minutes shown in C-H. (K-M) Control of mRFP alone or expressed with eGFP-ataxin-1[Q26] after similar heat shock treatment. (N-P) Control of eGFP-PML protein co-expressed with TAP/NXF1-mRFP after heat shock as in panels A-J. Bar: 10 µm.

View larger version (70K): [in a new window]   Fig. 7. Wild-type ataxin-1 can shuttle to and from the nucleus. FRAP live cell shuttle assay. Laser confocal fluorescence over DIC overlay of either GFP-wild-type or -mutant ataxin-1 proteins in bikaryons treated with cycloheximide before (panels A,F) and after (panels C,H) photo bleaching the entire area of the bikaryon except one nucleus (mask area, panels B, G). Recovery of wild-type ataxin-1 is seen by 10 minutes (D,E), but no recovery of mutant ataxin-1 is seen even after 30 minutes (I,J). Bar: 10 µm.

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