POLKADOTS Are Foci of Functional Interactions in T-Cell Receptor–mediated Signaling to NF-B

Supplemental Material

This article contains the following supporting material:

• Movie 1 - Formation and decay of Bcl10 POLKADOTS. CH12 B cells were loaded with 250 μg/ml conalbumin and allowed to settle to the bottom of a poly-D-lysine coated chamber slide for 10 min on a stage heated to 37°C. Following addition of D10 T cells expressing Bcl10-GFP, imaging commenced upon T cell/B cell conjugate formation. Video shows Bcl10-GFP (green) overlayed on the Nomarski image (grayscale). Images were collected every 30 s over an imaging time course of 3 hr. Periodic focal plane adjustments were made to keep the activated POLKADOT in focus. Video is at 3 fps. Bar is 5 μM.

• Movie 2 - A significant fraction of Bcl10 is stably incorporated in POLKADOTS. D10 T cells expressing Bcl10-PA-GFP+MALT1-mKO were mixed with antigen-loaded (250μg/ml conalbumin) CH12 B cells. Cells were added to a poly-D-lysine coated chamber slide on a stage heated to 37°C. Conjugates were allowed to form for 20 min, and T cells with POLKADOTS were located via imaging MALT1-mKO fluorescence. The video shows MALT1-mKO (red), Bcl10-PA-GFP (green) and DIC (blue). Images were collected every 8s with a 1-5 min gap every 10 frames. The time course spans the first 30 min immediately after multi-photon activation of a single POLKADOT. Video is at 3 fps. Bar is 5 μM.

• Movie 3 - Cytoplasmic Bcl10 is freely diffusible. D10 T cells expressing Bcl10-PA-GFP+MALT1-mKO were mixed with CH12 B cells. Cells were added to a poly-D-lysine coated chamber slide on a stage heated to 37°C. Conjugates were allowed to form for 20 min, and T cells were located via imaging MALT1-mKO fluorescence. The video shows MALT1-mKO (red) and Bcl10-PA-GFP (green). Images were collected every 9s. The time course spans the first 2 min immediately after cytoplasmic activation. Video is at 2 fps. Box shows activated region. Bar is 5 μM.

• Movie 4 - Video 3 shown with the DIC (blue) channel added.

• Movie 5 - Cytoplasmic Bcl10 is in rapid equilibrium with POLKADOTS-associated Bcl10. D10 T cells expressing Bcl10-PA-GFP+MALT1-mKO were mixed with antigen-loaded (250 μg/ml conalbumin) CH12 B cells. Cells were added to a poly-D-lysine coated chamber slide on a stage heated to 37°C. Conjugates were allowed to form for 20 min, and T cells with POLKADOTS were located via imaging MALT1-mKO fluorescence. The video shows MALT1-mKO (red) and Bcl10-PA-GFP (green). Images were collected every 13s. The time course spans the first 2 min immediately after cytoplasmic activation. Video is at 2 fps. Box shows activated region. Bar is 5 μM.

• Movie 6 - Video 5 shown with the DIC (blue) channel added.

• Figure 1 - NF-κB assays of Bcl10, PKCθθ, RIP2, CARMA1 and MALT1 constructs. Data show indistinguishable activation of an NF-κB luciferase reporter by unmodified wild-type signaling proteins vs. CFP or YFP fusions, demonstrating that the fluorescent protein tags do not interfere with signaling function (in all cases, t-tests were performed, and p-values indicated no significant difference between unmodified and fluorescent protein tagged signaling proteins). Additionally, the CARD mutant of Bcl10 (G78R), which is known to have severely impaired NF-κB activation, shows minimal enhancement of luciferase activity, as expected. Values are expressed as fold-activation relative to the NF-κB reporter plasmid alone. Error bars are SEM.

• Figure 2 - Bcl10-PA-GFP is activated only within the desired ROI. D10 T cells expressing Bcl10-PA-GFP were fixed, and PA-GFP was activated with 405 nm laser excitation. The excitation ROI (red box) encompassed approximately half of the cell. Bar is 5 μM.

• Figure 3 - Bcl10-CFP overexpression levels. The No Antigen samples from Figure 4C were run on an SDS-PAGE gel with an equivalent amount of cell extract from the D10 parental cell line. Labeling of cell lines is as in Fig. 4, except for the parental cell line (D10). Following western blotting with a rabbit anti-Bcl10 antibody, the levels of Bcl10-CFP were compared to the endogenous level of Bcl10 in the parental cell line. The blot was stripped and reprobed with a goat anti-β-actin (Actin) antibody. Note that degradation products of Bcl10-CFP may overlap with the endogenous Bcl10 band in cell lines expressing this fusion protein. Thus, the overexpression levels are relative to the endogenous Bcl10 expression level in the D10 parental cell line (defined as 1.0). Overexpression values were also normalized to relative β-actin expression. N/A, not applicable.

• Figure 4 - Control FRET data. (A) Confocal microscopy and FRET images for D10 T cells expressing Bcl10-CFP+non-fused YFP, 60 min post-conjugation to conalbumin-loaded (250μg/ml) CH12 B cells. Images show minimal FRET. Bar is 5 μM. (B) Calculations of %N-FRET for whole D10 T cells expressing non-fused CFP+non-fused YFP or Bcl10-CFP+non-fused YFP. CH12 B cell stimulation is as in (A). Percent N-FRET was calculated for whole cells or for Bcl10-CFP POLKADOTS, as indicated. The maximal FRET detected in these control experiments (0.5%) was used as the threshold for detection of significant FRET for experiments in Figures 3 and 6. Data are means +/- SEM for 50-100 cells.

• Figure 5 - POLKADOTS colocalize with membrane-bound structures. D10 T cells expressing Bcl10-CFP (blue) and MALT1-YFP (yellow) were stimulated with antigen-pulsed CH12 B cells for 60min. Cellular membranes were stained with CellTrace Bodipy TR methyl ester (red) for 10 minutes before fixation and confocal imaging.