Anap-based labeling enables visualization of TDP-43 and G3BP1.

A, B. HeLa cells expressing G3BP1-Anap and TDP-43-Anap under basal conditions or 250 μM sodium arsenite treatment. Anap and antibody signals are shown in blue and green, respectively; for merged channels, Anap was pseudo-colored red. Scale bars: 10 µm (overview), 3 µm (zoom). C, D. FRAP of G3BP1-Anap, G3BP1-GFP, and TDP-43-Anap, following 250 μM sodium arsenite treatment. One granule from each of three independent cells was selected and photobleached for FRAP analysis. ROI signal intensities are displayed in rainbow RGB (red-high, blue-low). Scale bars: 5 µm (cells), 1 µm (ROI). E. Comparison of HeLa cells expressing TDP-43-Anap and TDP-43-YFP under basal conditions or 250 μM sodium arsenite treatment. Here, Anap labeling and YFP labeling yield a blue signal and a yellow to green signal, respectively. F, G. Relative fluorescence recovery of each time point after photobleaching for G3BP1-Anap, G3BP1-GFP, TDP-43-Anap and TDP-43 ΔNLS-YFP. Here, error bars with filled area are used for data presentation. FRAP recovery curves were compared using two-way ANOVA. H. Immunoblotting of wild-type and Anap labeled G3BP1. The mouse anti-G3BP1 antibody was used. KO: G3BP knock out; F337Stop/Anap: the expression of G3BP1F337TAG via Anap labeling without or with the addition of Anap. I. Colocalization of G3BP1-Anap with TIA-1. The mouse anti-G3BP1 antibody and rabbit anti-TIA-1 antibody were used. Anap, G3BP1-Ab, and TIA-1-Ab signals are shown in blue, grey and green, respectively; for merged channels, Anap was pseudo-colored red. Scale bars: 10 µm (overview), 3 µm (zoom). J. Colocalization of TDP-43-Anap with G3BP1. The mouse anti-G3BP1 antibody and rabbit anti-TDP-43 antibody were used. Anap, TDP-43-Ab, and G3BP1-Ab signals are shown in blue, grey, and green, respectively; for merged channels, Anap was pseudo-colored red. Scale bars: 10 µm (overview), 3 µm (zoom). K. Immunoblotting of wild-type TDP-43, TDP-43-Anap and PFKP proteins in TDP-43 knockout HeLa cells. The Rabbit anti-TDP-43 antibody and rabbit anti-PFKP antibody were used. KO: TDP-43 knockout; V100Stop/Anap: the expression of TDP-43V100TAG via Anap labeling without or with the addition of Anap. L. The expression levels of PFKP in TDP-43 knockout HeLa cells expressing wide-type TDP-43 or TDP-43-Anap. One-way ANOVA was used to compare the levels among groups. M. Survival of TDP-43 knockout HeLa cells expressing TDP-43-Anap after treatment with 12.5 μM sodium arsenite for 24h. Calcien EM staining was used to quantify cell survival, and the relative survival rate was calculated as the ratio of sodium arsenite-treated to untreated cells for each group. OE: overexpression of TDP-43. One-way ANOVA was used to compare levels among groups. N. Survival of iTDPKO inducible mouse ES cells expressing TDP-43-Anap. Here, cell counting-Lite 2.0 Luminescent cell viability assay kit was used to detect the survival rate of ES cells. TDP-43 knockout was induced by 4-HT (300ng/ml) for 5 days. The relative survival rate= 4-HT induction/DMSO for each group. One-way ANOVA was used to compare levels among groups. Colocalization threshold analysis was performed in Fiji/ImageJ to calculate the Pearson correlation coefficient (R) for each region of interest (A, B, I, J). The X and Y axes represent the fluorescence intensity values of the red and green channels, respectively. When signals are colocalized, pixels with high intensity in one channel correspond to high intensity in the other, forming a diagonal distribution. In contrast, non-colocalized signals cluster along the axes. A higher R value indicates a greater degree of colocalization. Scale bar, 3 μm. All quantitative data (F, G, L, M, N) are shown as mean ± SEM. ***P = 0.0001; ****P < 0.0001; n.s., not significant.

Controls for Anap-based labeling of TDP-43 and G3BP1 in HeLa cells.

A. Controls for TDP-43-Anap labeling. Four experimental conditions were tested: (1) HeLa cells expressing both the TAG-mutated TDP-43 plasmid and the Anap incorporation system in the presence of Anap (TDP-43-Anap); (2) cells expressing both plasmids without Anap (TDP-43-Stop); (3) cells cultured with Anap only; and (4) cells expressing the Anap incorporation system with the addition of Anap. Signals for TDP-43-Anap, TDP-43 antibody staining, and G3BP1 antibody staining are shown in blue, grey, and green, respectively. The Anap signal is pseudo-colored red in the merged images. B. Controls for G3BP1-Anap labeling. The same four conditions were tested: (1) cells expressing the TAG-mutated G3BP1 plasmid and the Anap incorporation system in the presence of Anap (G3BP1-Anap); (2) cells expressing both plasmids without Anap (G3BP1-Stop); (3) cells cultured with Anap only (+ Anap only); and (4) cells expressing the Anap incorporation system with the addition of Anap (Anap system+Anap). Signals for G3BP1-Anap, G3BP1 antibody staining, and TIA-1 antibody staining are shown in blue, grey, and green, respectively. The Anap signal is pseudo-colored red in the merged images. Scale bars, 40 μm.

Anap labeling of TDP-43 and G3BP1 in neurons.

A, C. Primary mouse cortical neurons expressing G3BP1-Anap and TDP-43-Anap under basal conditions or 250 μM sodium arsenite treatment. Cells were stained with anti-G3BP1 (human-specific) or anti-TDP-43 (human-specific) antibodies with chicken anti-Tuj1 as a neuron marker. Signals: Anap (blue, pseudo-colored red in merged images), antibody (green), Tuj1 (gray). Scale bar, 10 µm. B, D. The colocalization level of each region of interest for G3BP1 and TDP-43. Colocalization threshold analysis was performed in Fiji/ImageJ to calculate the Pearson correlation coefficient (R) for each region of interest. The X and Y axes represent the fluorescence intensity values of the red and green channels, respectively. When signals are colocalized, pixels with high intensity in one channel correspond to high intensity in the other, forming a diagonal distribution. In contrast, non-colocalized signals cluster along the axes. A higher R value indicates a greater degree of colocalization. Scale bar, 1 μm.

Schematic of the Anap labeling system for G3BP1 and TDP-43 using genetic code expansion.

Briefly, two plasmids were required to express the protein with site-specific Anap incorporation, one for Anap incorporation and one for the mutated protein of interest (TAG introduction). As the plasmids were transfected into cells, the orthogonal Anap-tRNA synthetase would charge the Anap, a fluorescent amino acid, to its cognate tRNA, and the tRNA would incorporate the Anap site-specifically into the protein of interest in response to the TAG stop codon. Cells expressing Anap-labeled TDP-43 and G3BP1 were subsequently imaged by confocal microscopy, either after fixation or in live-cell conditions. Upon stress conditions, TDP-43-Anap redistributes to the cytoplasmic inclusions, and G3BP1-Anap assembles into stress granule.