Data supporting Genetically encoded lysine photocage for spatiotemporal control of TDP-43 nuclear import

Here, we demonstrate spatiotemporal control over the nuclear import of TDP-43 by installing a photocage (ortho-nitrobenzyl ester) on a single lysine residue (K84) through amber codon suppression in HEK293T cells.

Scheme 1:

· Schematic overview of the site of photocage addition in TDP-43

· Schematic of photocage cleavage

Figure 1:

· Diagram of proposed mechanism

· Cell image of HEK293T cells transfected with pCMV-TDP-43 mRuby

· Cell image of HEK293T cells transfected with pCMV-TDP-43 K84ONBK

· Associated Hoechst stains

Figure 2:

· Tilescan of TDP-43 mRuby expressing cells showing a variety of expression levels

· Tilescan of TDP-43 K84ONBK expressing cells showing a variety of expression levels

· Timelapse of TDP-43 K84ONBK expressing cells

· Western blot using an anti-TDP-43 antibody and associated Ponceau S stain for different expression constructs

Figure 3:

· Timelapse of K84ONBK expressing cells post light exposure

· Data associated with the quantification of nuclear / cytosolic ratio post light exposure as plotted in Figure 3B

Figure 4:

· Images of cells expressing K84ONBK pre and post exposure with varying intensities of light.

· Data used to quantify the final nuclear / cytosolic ratio for each condition as shown in Figure 4B.

Figure 5:

· Images of cell exhibiting either a nuclear, cytosolic diffuse, cytosolic liquid, or cytosolic solid phenotype that were used for FRAP experiments. Example images of pre-bleaching, time zero, and post-bleaching for each condition are shown.

· Data used to quantify the fluorescence recovery after photobleaching shown in Figure 5B.

Figure 6:

· Cell images of cotransfection with K84ONBK-mRuby and TDP-43-mCerulean. Cells transfected with either plasmid or both together are shown.

· Colocalized pixels between the mRuby and mCerulean channels for each condition.

Supplementary Figure 1:

· Lower magnification view of the cells shown in Figure 1 B and the associated Hoechst stain.

Supplementary Figure 2:

· TDP-43 K84ONBK mRuby expressing cells co-stained with an antibody for the stress granule marker G3BP1.

Supplementary Figure 3:

· Cell images taken before and after irradiating a single cell with 355 nm light.

Supplementary Figure 4:

· Pre and 11 hour post photoconversion performed on a cell with puncta.