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.