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First published online 1 April 2003
doi: 10.1242/jcs.00400

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Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo

University of Dundee, MSI/WTB Complex, School of Life Sciences, Dow Street, Dundee DD1 5EH, UK

View larger version (79K): [in a new window]   Fig. 1. Stable cell lines expressing GFP-SMN show normal nuclear morphology. Three-dimensional projections of deconvolved serial sections showing cells from line GFP-SMNE10.3 (green) counterstained with antibodies to Sm proteins (A-C), coilin (D-F) and SIP1/Gemin2 (G-I). GFP-SMN co-localises in Cajal bodies with Sm proteins (arrows in C), coilin (arrows in F) and SIP1/Gemin2 (arrows in I). Sm-containing speckles are also visible (arrowheads in C). Bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 2. FP-SMN is expressed at low levels relative to endogenous SMN, interacts with SIP1/Gemin2 and does not affect cell cycle. (A) Total cell lysates from the parental HeLa cell line, line CFP-SMNE8.8 and parental HeLa cells transiently transfected with a plasmid expressing CFP probed with anti-SMN and anti-FP antibodies. All three cell lines give a strong band at 38 kDa with anti-SMN, representing endogenous SMN. Line CFP-SMNE8.8 shows an additional, much weaker, band at 68 kDa, representing stably expressed CFP-SMN. A band of the same size is detected in line CFP-SMNE8.8 using the anti-FP antibody, whereas parental HeLa cells transiently transfected with CFP alone give a single band at 30 kDa. (B) Co-immunoprecipitations from total cell lysates of line GFP-SMNE10.3 using anti-FP antibodies. Detection of products using anti-SMN antibodies demonstrates the presence of GFP-SMN in the input material and the immunoprecipitated material (anti-GFP lane). A duplicate blot probed with anti-SIP1/Gemin2 shows a clear enrichment of SIP1/Gemin2 in the immunoprecipitated fraction (anti-GFP lane). (C) FACS analysis of synchronised cell populations stained with propidium iodide shows a similar profile for lines GFP-SMNE18.6, CFP-SMNE12A and the parental HeLa cell line.

View larger version (38K): [in a new window]   Fig. 3. High-level expression of FP-SMN leads to cytoplasmic accumulation of SMN-complex proteins and disruption of nuclear structure. Three-dimensional projections of serial sections showing HeLa cells and MCF-7 cells expressing high levels of GFP-SMN following transient transfection. All cells show cytoplasmic accumulation of GFP-SMN (arrows in C, F and I). These accumulations also contain SIP1/Gemin2 (A-F) and Sm proteins (G-I). They do not contain TMG-capped RNA (J-L) or coilin (M-O). Many cells also show a large number of small nuclear bodies containing GFP-SMN, TMG-capped RNA and coilin (arrows in L and O). Bar, 10 µm.

View larger version (39K): [in a new window]   Fig. 4. Cytoplasmic accumulations of FP-SMN can be induced by expressing CFP-SMN in a GFP-SMN stable cell line. Three- dimensional projections of deconvolved serial sections of cells from line GFP-SMNE10.3 transiently transfected with a plasmid encoding CFP-SMN. An untransfected cell (A, bottom left) shows the characteristic SMN distribution of diffuse cytoplasmic signal and nuclear Cajal bodies. A transiently transfected cell (A top right, and B) shows accumulation of GFP-SMN and CFP-SMN in the cytoplasm (arrows). Note: with this combination of FP proteins, a small amount of bleed-through is seen from CFP-SMN into the GFP image, but no bleed-through is observed from GFP-SMN into the CFP channel. Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 5. Time-lapse analysis of interphase cells shows fusion and separation of GFP-SMN Cajal bodies and close interactions between bodies. A-C show 3D projections of serial sections of living cells from line GFP-SMNE10.3 taken from a time series. (A) Fusion of a small body (arrow) with a larger one. (B) Separation of a large body (arrow) into two smaller ones. (C) Close interaction between two bodies (arrow). The frequency of these and other events are shown in panel D.

View larger version (54K): [in a new window]   Fig. 6. SMN and coilin show different rates of flux through the Cajal body. (A) Single confocal sections of a cell from line YFP-coilinE1.1.1. The bleached area (yellow ring) contains a Cajal body (red arrow). The image series and the colour graph show recovery of the bleached body compared to the intensity of an unbleached body (green arrow) in the same nucleus. The greyscale graph shows averaged data from bleached CBs in three separate cells. (B) Single confocal sections of a cell from line GFP-SMNE10.3. The bleached area (yellow ring) contains a Cajal body (red arrow). The image series and the graph show recovery of the bleached body compared to the intensity of an unbleached body (green arrow) in the same nucleus and an unbleached body (blue arrow) in a neighbouring nucleus. The greyscale graph shows averaged data from bleached CBs in three separate cells. (C) Three-dimensional projection of series of confocal sections through a cell from line GFP-SMNE10.3. The bleached area (yellow box) contains a Cajal body (green arrow). The image series and the graph show recovery of the bleached body compared to the intensity of an unbleached body (green arrow) in the same nucleus and an unbleached body (blue arrow) in a neighbouring nucleus. (D) Three-dimensional projection of series of confocal sections through a cell from line GFP-SMNE10.3. The bleached area (yellow box) encompasses a large region of cytoplasm. The image series and graph show the progressive loss of signal from Cajal bodies in the bleached cell over successive bleaches (green, red and blue arrows) compared to a Cajal body in a neighbouring cell (pink arrow).

View larger version (78K): [in a new window]   Fig. 7. Dynamics of GFP-SMN and YFP-coilin through mitosis. (A) Three-dimensional projections of serial sections from a time series of line GFP-SMNE10.3 from prophase through to telophase. Mitotic Cajal bodies (MCBs) are arrowed. (B) GFP-SMNE10.3 through telophase. Large numbers of small SMN-positive bodies (arrows) appear in late telophase. (C) GFP-SMNE10.3 from telophase to G1. The SMN-positive bodies disappear as GFP-SMN begins to accumulate in nuclear Cajal bodies (arrows). (D) Three-dimensional projections of serial sections from a time series of line YFP-coilinE1.1.1 from prophase through to telophase. Mitotic Cajal bodies are arrowed. (E) YFP-coilinE1.1.1 in G1. Coilin-positive Cajal bodies are seen to form either distant from (arrow) or near to (arrowhead) nascent nucleoli.

View larger version (55K): [in a new window]   Fig. 8. SMN and coilin co-localise in mitotic Cajal bodies (MCBs) until telophase. Three-dimensional projections of deconvolved serial sections through cells of line GFP-SMNE10.3 in metaphase (A-C), anaphase (D-F), telophase (G-I) and cytokinesis (J-L) counterstained with anti-coilin antibodies (red). GFP-SMN and coilin co-localise until late telophase (arrows in C, F and I). This association is lost soon after the re-import of coilin into the forming nuclei as the daughter cells flatten out, leaving a large number of SMN-positive bodies in the cytoplasm (arrows in L). Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 9. Mitotic Cajal bodies (MCBs) contain Sm proteins and TMG-capped RNA. Three-dimensional projections of deconvolved serial sections through cells of line GFP-SMNE10.3 in mitosis counterstained with antibodies to Sm proteins (A-H) and TMG-capped RNA (I-L). The majority of SMN-positive bodies also contain Sm proteins and TMG-capped RNA, although some bodies contain SMN in the absence of Sm proteins (arrows in B) and some contain TMG-capped RNA in the absence of SMN (arrowheads in I). The overlays (D, H and L) show GFP-SMN in green and antibody staining in red. Bar, 10 µm.

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