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First published online 12 May 2009
doi: 10.1242/jcs.044040

Journal of Cell Science 122, 1872-1881 (2009)
Published by The Company of Biologists 2009
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Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

Scoty M. Hearst1, Andrew S. Gilder1, Sandeep S. Negi1, Misty D. Davis1, Eric M. George1, Angela A. Whittom1, Cory G. Toyota1, Alma Husedzinovic2, Oliver J. Gruss2 and Michael D. Hebert1,*

1 Department of Biochemistry, The University of Mississippi Medical Center, Jackson, MS 39216, USA
2 Zentrum fur Molekulare Biologie der Universitat Heidelberg, DKFZ-ZMBH Alliance 69120 Heidelberg, Germany


Figure 1
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  		Fig. 1. Known coilin phosphoresidues and mutations. Schematic of human coilin showing the locations of the self-association domain and RG box. Below is the C-terminal sequence from residue 562 to the end of the protein, residue 576. Residues shown to be phosphorylated by tandem MS/MS analysis are indicated in the schematic (T122, S271, S272, T303, S489) or by asterisks in the sequence (S566, S568, T570, S571, S572, T573). Mutations of these six C-terminal residues to alanine (C6A) or glutamate/aspartate (C6D/E, denoted in subsequent text as C6D) are shown. The OFF and ON coilin mutants contain mutations of all 11 phosphorylated residues.

 

Figure 2
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  		Fig. 2. Coilin phosphomutants impact localization. (A) HeLa cells expressing wild-type (WT) GFP-coilin or GFP-coilin mutants (C6A, C6D, OFF or ON) were fixed and GFP (green), SMN (red) and DAPI (blue) signals were detected. The right column shows the overlay of all three images (Merge). Some CBs are labeled with arrows. Arrowheads indicate nucleolar staining observed in cells expressing C6A and OFF. Scale bars: 10 µm. (B) Quantification of WT and coilin mutant localization in HeLa cells. At least 100 cells were counted for each construct. (C) Quantification of WT and coilin mutant localization in coilin-knockdown HeLa cells. At least 50 cells were counted for each construct.

 

Figure 3
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  		Fig. 3. Coilin phosphomimics reduce coilin self-association. HeLa cells were transfected with wild-type (WT) GFP-coilin or mutants thereof (C6A, C6D, OFF, ON), followed by immunoprecipitation with anti-GFP antibodies and western blotting with anti-coilin antibodies. TCL, total cell lysate; IP, immunoprecipitation. The positions of GFP-tagged and endogenous coilin are shown. (B) Immunoprecipitation reactions are shown for WT, OFF and ON coilin constructs. Lane 4 is a negative control in which cells were transfected with empty GFP vector and the lysate was treated as described above.

 

Figure 4
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  		Fig. 4. FRAP analysis of wild-type and phosphomutants of coilin. FRAP analysis was performed on Cajal bodies in cells expressing wild-type GFP-Coilin as well as cells expressing ON, OFF, C6A and C6D mutants. After alignment, recovery curves were generated by double normalization and the time to half maximal recovery (T50) was calculated for each. The mean ± s.e. value of T50 from at least five cells is displayed.

 

Figure 5
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  		Fig. 5. IEF of endogenous coilin from HeLa and WI-38 cells. (A) Lysate from HeLa cells was untreated or treated with CIP, followed by IEF (pH 7-10 IPG strip), SDS-PAGE and western blotting. Coilin was detected using anti-coilin antibodies. (B) Interphase HeLa cell lysate was subjected to IEF (pH 5-8 IPG strip), SDS-PAGE and western blotting. Coilin and β-tubulin were detected on the same blot using appropriate antibodies. (C) Lysate from interphase WI-38 cells was subjected to IEF (pH 5-8 IPG strip), SDS-PAGE and western blotting, followed by the detection of coilin and β-tubulin. (D) Mitotic HeLa cell lysate was subjected to IEF (pH 5-8 IPG strip), SDS-PAGE and western blotting, followed by the detection of coilin and β-tubulin. In panels E and F, HeLa and WI-38 extracts were treated as described for B and C, except that β-tubulin and SMN were detected. Representative gels are shown. Note that interphase HeLa coilin contains more phosphoisoforms (arrows) than found in WI-38, and coilin is hyperphosphorylated in WI-38 (arrow) relative to HeLa.

 

Figure 6
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  		Fig. 6. Quantitative PCR analysis of coilin, SMN and PPM1G expression in HeLa and WI-38 cell lines and dephosphorylation of coilin by PPM1G. (A) Coilin, SMN and PPM1G expression levels relative to β-actin are shown. HeLa values for each message of interest are normalized to 100%. Error bars represent percentage error about the mean. The difference between relative coilin levels in HeLa compared with WI-38 is not significant (P=0.25). However, there is a significant decrease in the relative expression levels of SMN (P=0.0023) and PPM1G (P=0.000058) in WI-38 compared with HeLa cells. (B) Lysate from mitotic HeLa cells was untreated or treated with CIP or recombinant His-tagged PPM1G, followed by SDS-PAGE, western blotting and detection of coilin using appropriate antibodies.

 

Figure 7
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  		Fig. 7. Expression of PPM1G in WI-38 cells induces CBs. (A) WI-38 cells expressing YFP-PPM1G were fixed and YFP (green), coilin (red) and DAPI (blue) signals were detected. The right column shows the overlay of all three images (Merge). The majority of YFP-PPM1G-expressing cells (70%) did not have CBs (row a), but 30% of cells exhibited CBs (rows b and c, arrows). Approximately half of the cells with CBs strongly overexpressed YFP-PPM1G, which accounted for 14% of the 36 cells scored (row c, note also cytoplasmic localization of YFP-PPM1G). In this particular cell, a nuclear aggregate of YFP-PPM1G was detected (arrowhead) next to a CB (arrow). Scale bars: 2 µm. (B) GFP-coilin, GFP-SmB and GFP alone expression in WI-38 cells. Merged images are shown and an arrow indicates the location of a CB in a cell expressing GFP alone. Scale bar: 2 µm. (C) Induction of CBs in WI-38 cells. The constructs shown were transfected into WI-38 cells. After 24 hours, the cells were processed and scored for CBs. The percentage of cells with CBs is shown. Note that only cells with nuclear YFP-PPM1G (or inactive-PPM1G) were scored and, for all constructs, high-expressing cells were excluded. Cells counted: YFP-PPM1G active=132, YFP-PPM1G inactive=128, GFP-coilin=50, GFP-SmB=31 and GFP alone=44.

 

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© The Company of Biologists Ltd 2009