Supplemental Material for NCB01024SRR

Cajal body dynamics and association with chromatin are ATP-dependent.

Melpomeni Platani, Angus I. Lamond, and Jason R. Swedlow

Movies from Figures

Figure_1.mov

HeLaGFP-coilin cell transfected with histone H2B-YFP (red). GFP signal is in green. Each frame is a maximum intensity projection and is separated by 3 minutes.

Figure_3_NaAz.mov: HeLaGFP-coilin cell transfected with histone H2B-YFP treated with sodium azide for 30’ before imaging. GFP signal is in green H2B-YFP is in red. Each frame is a maximum intensity projection and is separated by 3 minutes.

Supplementary Data—NCB01024SRR

I. Residual Cajal bodies after DNAse I Digestion Associate with Chromatin

Purpose: To ascertain the fate of Cajal bodies after enzymatic removal of chromatin.

Method: HeLaGFP-coilin cells were cultured on coverslips as described in the manuscript, then permeablised in 0.05% Triton X-100 in Buffer A (10 mM PIPES pH 7.0, 80 mM KCl, 20 mM NaCl, 0.5 mM EGTA, 2 mM EDTA, 0.2 mM spermine, 0.5 mM spermidine), a polyamine-containing buffer used to maintain chromatin structure after cell permablization1,2. In independent experiments we did not detect any change in Cajal body fluorescence or number during this permeablization. After 30 sec, cells were washed with TMS buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM CaCl2, 1 mM MgSO4, 0.27 M sucrose, 0.2 mM spermine, 0.5 mM spermidine, 0.01% Triton X-100) or with the same buffer containing 500 U/ml DNAse I (Worthington). Independent experiments revealed no detectable proteolysis of nuclear proteins with this enzyme. Coverslips were then incubated at 37oC for 1 hour, then washed with Buffer A, fixed with freshly prepared 3.7% CH2O in Buffer A, stained with 0.1 µg/ml DAPI for 10 minutes and then mounted in nitrogen-purged 0.5% p-phenylene diamine in Tris buffered glycerol. 3D images were collected on a DeltaVision restoration microscope (Applied Precision, Inc.) by collecting optical sections spaced by 0.2 µm, and restored using a constrained iterative deconvolution algorithm. The images shown are maximum intensity projections through 2.4 µm slices through the data stack. Images were transferred to Adobe Illustrator for layout.

For counting Cajal bodies, images were imported into a server running the Open Microscopy Environment (http://www.openmicroscopy.org). Cajal bodies were identified automatically using the findSpots program3 and counted using the SQL interface in OME.

Results: We compared the localization and content of DNA and GFP-coilin in the HeLaGFP-coilin cell line before and after DNAse I treatment. We note that this treatment is not a nuclear matrix preparation, as it does not contain the salt or detergent extractions required to reveal the nuclear matrix. The removal of DNA from briefly permeablized cells was confirmed by the loss of about 90% of fluorescence detected by DAPI staining as well as the presence of diffuse, fibrous DAPI stained material in the area surrounding the nucleus. We interpret this material as DNA that has been partially digested, but completely released from the cells.

We noted a 4-fold loss in GFP fluorescence in cells treated with DNAse I compared to those incubated in control buffer. Separate experiments have confirmed that coilin is not proteolysed in our protocol (data not shown), so we believe this represents the loss of coilin protein from nuclei during DNA digestion. We still detect residual GFP-containing structures that resemble Cajal bodies, suggesting that these remaining Cajal bodies have lost a substantial portion their GFP-coilin, probably through exchange with their surroundings. Our previous work demonstrated that GFP-coilin can exchange between Cajal bodies and the nucleoplasm3.

We recorded 3D images of the cells incubated with control buffer or DNAse I and examined the spatial relationships of DNA and Cajal bodies. Supplemental Figure 1 shows a 2.4 µm slice through the 3D volume of control (Supplemental Information Fig.1a) and DNAse I-treated (Supplemental Information Fig.1b) cells. Cells treated with DNAse I showed a small number of Cajal bodies, but these were always associated with residual chromatin. To quantify the loss of Cajal bodies due to DNAse I digestion, we counted Cajal bodies (including both larger and smaller classes of Cajal bodies3) in DNAse I treated and control cells (Supplemental Information Fig.1c). This revealed a significant loss of Cajal bodies after DNAse I digestion. Note that the cell in Supplemental Information Figure 1b is a rare one that shows a relatively high number of residual Cajal bodies. This image was chosen to highlight the association of Cajal bodies and chromatin.

II. Comparison of CB Diffusion with Previous Measurements of Nuclear Diffusion

Nuclear diffusion measurements are available for a number of macromolecules. In general, nuclear diffusion constants are less than that measured in aqueous solution, either because of steric interactions or because of binding events similar to what we have observed here4. Diffusion constants of nuclear macromolecules range between 10-7 – 10-9 cm2/sec5-7. A spherical particle the size of a CB in aqueous solution is expected to have a diffusion constant of 8.9 x 10-9 cm2/sec. If we include empirical measurements of nuclear viscosity8, we obtain a predicted CB diffusion constant of 1.1 x 10-9 cm2/sec. Our measurements of CB diffusion constants in living nuclei are significantly smaller (10-12 – 10–11 cm2/sec), but higher than those measured for specific gene loci or bulk chromatin 9,10, consistent with our observation that CBs switch between being chromatin-associated and diffusing through the nucleoplasm.

Figure Legend

Supplemental Information Figure 1

(a & b) Fixed HeLaGFP-coilin cells incubated at 37oC for 1 hour in either control buffer (a) or DNAse I (b). DNAse digestion leaves residual Cajal bodies (green) that always associate with undigested DNA (red). Scale bar, 5 µm.
(c) To quantify the number of Cajal bodies after DNAse I digestion, images of 15 cells for each treatment were collected and the number of Cajal bodies determined. The graph shows the mean number of Cajal bodies in each nucleus; error bar shows the standard deviation for each measurement.

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