Skip to main content
. 2021 Sep 7;10:e67605. doi: 10.7554/eLife.67605

Figure 7. Expressing TDP-43 mutants with an altered cooperative binding mRNA in HeLa cells leads to the formation of mRNA-poor TDP-43 condensates in small fraction of cells.

(a) Subcellular distribution of wild-type TDP-43 or G146A mutant. Cycloheximide treatment was used to dissociate stress granules in the cell cytoplasm, when indicated in the figure. Wild-type TDP-43 is generally homogenously distributed in the cytoplasm but can be also found in cytoplasmic mRNA-rich stress granules (yellow head-arrows). G146A mutant is also generally homogenously distributed in the cytoplasm but can be found in brilliant condensates in the cytoplasm and G146A condensates do not colocalize with mRNAs (white head-arrows). Scale bar: 40 µm. Representative images of larger areas are shown in Figure 7—figure supplement 1b. (b) Violinplots representing TDP-43 (anti-HA) and mRNA (in situ hybridization with poly(dT) probes) fluorescence intensity in the cytoplasmic granules/aggregates detected under indicated conditions. Wild-type TDP-43 or Q213A mutant, a negative control, can be recruited in mRNA-rich stress granules that disappeared after cycloheximide treatment. On the other hand, K140A/T141A and G146A mutants are located in dense cytoplasmic condensates poorly enriched in mRNAs. Interestingly, K140A/T141A and G146A are not sensitive to cycloheximide. Cytoplasmic granules/aggregates were detected automatically by using Cell Profiler. n: number of granules/aggregates detected. p<0.05*; p<0.01** (paired two-sample t-test).

Figure 7—source data 1. Expressing TDP-43 mutants with an altered cooperative binding to mRNA in HeLa cells leads to the formation of mRNA-poor TDP-43 condensates in small fraction of cells (See legend of Figure 7b).

Figure 7.

Figure 7—figure supplement 1. Cytoplasmic and nuclear G146A condensates are not stress granules.

Figure 7—figure supplement 1.

(a) Hela cells were co-transfected with indicated HA-tagged TDP-43 (G146A or wild type) and Sam68-GFP. Cells were then treated with hydrogen peroxide to promote the appearance of nuclear G146A condensates (blue arrow). We noticed the presence of Sam68 nuclear bodies (orange arrow). However, Sam68 is not recruited in G146A condensates in the nucleus. (b) Left-panel: Large scale images of wild-type TDP-43 and G146A expressed in HeLa cells. Right panel: zoom in on indicated areas (pink rectangles). Most of the cells display a homogenous distribution of wild-type TDP-43 or G146A in the cytoplasm and nucleus. However, in some cells, we can notice the recruitment of TDP-43 in mRNA-rich stress granules while G146A is rather associated to smaller and brighter condensates that are not enriched in mRNA. Cycloheximide makes TDP43-rich stress granules disappeared but not G146A condensates. Scale bar: 40 µm.