Cellular modifiers of TDP-43 phase transition and cytoplasmic aggregation
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
- Therapeutic Development Branch, National Center for Advancing Translational Sciences, National Institutes of Health, United States
- iPSC Core, National Heart, Lung, and Blood Institute, National Institutes of Health, United States
- Functional Genomics Laboratory, National Center for Advancing Translational Sciences, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
A chemical genetic screen identifies TDP-43 phase modulators.
(A) Workflow of the chemical genetic screen. (B) Dose-dependent reduction of anisosome number by identified chemicals. DLD1 TDP-43 2KQ-Clover cells were treated with doxycycline to induce anisosomes for 24 hr and then treated with drugs at the indicated concentrations. At least 500 cells were imaged for anisosome count per condition. A nonlinear regression model was used to fit the data by GraphPad Prism.
Figure 1—figure supplement 1
The impact of different inhibitors on TDP-43 2KQ levels.
Parental DLD1 or Clover-TDP-43 2KQ cells were exposed to doxycycline for 24 hr and then treated with the indicated inhibitors for 6 hr. Cell lysates were analyzed by immunoblotting with GFP and p97 (loading control) antibodies. SPA, Spautin-1, 5 μM; BTZ, Bortezomib, 10 nM; TRP, Tripterin, 1 μM; GA, Geldanamycin, 10 μM; VLX, VLX-1570, 0.5 μM; VER, Verdinexor, 20 μM; PlaB, Pladienolide-B, 20 nM; ANS, Anisomycin, 200 nM; LMB, Leptomycin B, 200 nM; KPT, KPT-276, 15 μM. Note that VLX treatment caused a fraction of TDP43 to form detergent insoluble aggregates. The significance of this finding remains unclear. See Figure 1—figure supplement 1—source data 1 and 2 for original western blots.
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Figure 1—figure supplement 1—source data 1
PDF file containing original western blots for Figure 1—figure supplement 1, indicating no change in TDP-43 abundance in drug-treated cells.
- https://cdn.elifesciences.org/articles/110172/elife-110172-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Original files for western blot analysis are shown in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/110172/elife-110172-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
Two distinct types of TDP-43 phase modulators.
(A) DLD1 TDP-43 2KQ-Clover cells treated with doxycycline for 24 hr were further treated with the indicated compounds for 6 hr and imaged (SPA, Spautin-1, 5 μM; KPT, KPT-276, 15 μM; CP, CP-673451, 30 μM; TRP, Tripterin, 1 μM; BTZ, Bortezomib, 10 nM). Shown are single confocal z-section views. Scale bar, 5 μm. (B) Fluorescence recovery after photobleaching (FRAP) analyses of anisosomes after treatment with the indicated drugs for 3–5 hr. Circles indicate bleached areas. Shown are single confocal z-section views. Scale bars, 1 μm. (C, D) Quantification of the experiments represented in B. MFI, mean fluorescence intensity. N=5 anisosomes/condition. (E) Reverse FRAP analyses of anisosome dynamics. The areas indicated by the dashed lines were bleached. Shown are single confocal z-section views. Scale bars, 5 μm. (F) Quantification of the MFI loss over time in unbleached anisosomes as shown in E. 4–6 anisosomes were analyzed for each condition. Error bars in (C, D, F), SD. (G) Quantification of the initial rate of fluorescence loss of unbleached anisosomes as shown in E. ****p<0.0001; ***p<0.001 by unpaired Student’s t-test (two-tailed). Error bars, SD. The rate of fluorescence loss in the first 10 s after photobleaching was calculated. 4–6 anisosomes were analyzed for each condition.
Figure 2—figure supplement 1
HSP90 inhibitor Geldanamycin (GA) converts TDP-43 anisosome to a gel-like state.
(A) DLD1 cells were treated with doxycycline to induce anisosome formation and then treated with GA at 10 μM for 4 hr. Cells were stained with Hoechst (blue) and imaged by a confocal microscope. Scale bar, 5 μm. (B) A TDP-43-bearing anisosome (dashed circle) in a GA-treated cell was photobleached and then imaged. Scale bar, 1 μm. The graph shows the quantification. Error bars, SD (N=at least 5 anisosomes/condition). MFI, mean fluorescence intensity.
Figure 3
A genome-wide siRNA screen identifies modifiers of TDP43 phase behavior.
(A) The workflow of the siRNA genetic screen. GW, genome-wide; AS, anisosome; HT, high-throughput. (B) Pathway analysis of genes whose knockdown reduces anisosome number in cells. The relative anisosome count is indicated by both color and size of the nodes. Candidate genes were selected based on Z-score>2 after normalization to the siRNA negative control. (C) Pathway-enriched (KEGG) analysis of TDP-43 phase modifiers. (D) A list of identified genes linked to amyotrophic lateral sclerosis (ALS) in C.
Figure 4 with 1 supplement
Anisosome phase behavior is modulated by RNA splicing and protein translation.
(A) The splicing inhibitor Pladienolide-B (PlaB) reduces anisosome number in a dose-dependent manner. Representative images of anisosome-induced cells treated with DMSO (control), 5 nM, or 20 nM PlaB for 16 hr. Shown are maximum intensity projection views. Scale bar: 10 µm. (B) Quantification showing the number of anisosome (AS) per cell in TDP-43 2KQ-Clover cells treated with PlaB as indicated. *p<0.05, **p<0.01, ****p<0.0001 by ordinary one-way ANOVA and Dunnett’s multiple comparisons. n=3 independent experiments, and in each experiment, at least 30 randomly selected cells were analyzed. (C) Representative z-section confocal fluorescence recovery after photobleaching (FRAP) images of anisosomes in cells treated for 16 hr with DMSO or PlaB (20 nM). BB, before photobleaching, right after photobleaching (0 s), or 15 and 30 s after photobleaching (15 s and 30 s). Scale bar, 1 µm. (D) The graph shows the quantification of the remaining TDP-43 fluorescence (FL) in C. Error bars indicate mean ± SD, N=28 for control and 23 for PlaB-treated cells. MFI, mean fluorescence intensity. (E) Live-cell imaging of anisosome fusion in TDP-43 2KQ-Clover cells treated with 20 nM PlaB for 5 hr before tracking the fusion. Representative reconstructed 3D images from Video 1 showing fusion events indicated by arrows. Scale bar, 1 µm. (F) Representative maximum intensity projection views of anisosome-induced (24 hr) cells treated with DMSO (control) or anisomycin (ANS) (200 nM) for 16 hr. Scale bar, 10 µm. (G) Quantification of the number of anisosomes per cell in randomly selected images of DMSO-, cycloheximide (CHX)-, or ANS-treated cells. ****p<0.0001 by ordinary one-way ANOVA and Dunnett’s multiple comparisons. Each dot represents a randomly selected field with at least 20 cells counted from one of the 3 independent experiments. (H) Quantification of anisosome size in control or cells treated with CHX or ANS. ****p<0.0001 by ordinary one-way ANOVA and Dunnett’s multiple comparisons. N=3 independent experiments. AU, arbitrary unit. (I) As in C except that anisosome-induced cells were treated with CHX or ANS before photobleaching. Scale bar, 1 µm. (J) Quantification of fluorescence recovery in CHX- or ANS-treated cells vs the DMSO control. N=10 anisosomes/condition. Shaded bands in (D, J) represents error bars, SD.
Figure 4—figure supplement 1
The impact of splicing inhibitor and translation inhibitor on anisosomes.
(A) A violin plot showing the relative size distribution of anisosomes (AS) in DLD1 cells treated with Pladienolide-B at the indicated concentration for 16 hr. ****p<0.0001 by one-way ANOVA. N=3 independent biological repeats. (B) DLD1 cells were treated with doxycycline to induce anisosome formation and then treated with cycloheximide (CHX) at 20 μg/mL or DMSO as a control for 16 hr. Cells were stained with Hoechst (blue) and imaged by a confocal microscope. Scale bar, 5 μm.
Figure 5 with 1 supplement
XPO1 regulates anisosome liquid-to-solid transition.
(A) Pharmacological inhibition of XPO-1 with Leptomycin B (LMB, 200 nM) reduces the number of anisosomes. The graph on top indicates the experimental design. Arrows show cells with enlarged anisosomes. Scale bar, 10 µm. (B) Quantification of anisosome numbers per cell in experiments represented by A. Error bars indicate mean ± SD; ****p<0.0001 by ordinary one-way ANOVA and Dunnett’s multiple comparisons. n=3 independent experiments, each with duplicated controls. (C) LMB dose-dependently reduces anisosome number. Anisosomes were induced in TDP-43 2KQ-Clover cells followed by treatment with LMB at the indicated concentrations for 16 hr. The histogram shows the distribution of cells (50–90 cells/condition) by anisosome count. (D) Fluorescence recovery after photobleaching (FRAP) experiments demonstrate that anisosomes remain in a liquid phase following LMB treatment (200 nM, 16 hr). Anisosome-induced (24 hr) cells were treated with DMSO or LMB for 16 hr and then photobleached at the indicated areas. Scale bar, 1 µm. (E) Quantification of the FRAP experiment in D. N=18 for control and 16 anisosomes for LMB-treated cells. (F) Time-lapse confocal microscopy detects anisosome fusion after LMB (200 nM, 5 hr) treatment in TDP-43 2KQ-Clover cells. Arrows indicate fusion events. Scale bar, 1 µm. (G) Overexpression of mCherry-tagged XPO-1 in TDP-43 2KQ-Clover cells induces cytoplasmic TDP-43 puncta. TDP-43 2KQ-Clover cells (green) transfected with mCherry-XPO1 (red) were stained with DAPI (blue) to label nuclei (dashed lines). Cells were imaged 48 hr post-transfection. Panels 1, 2 show a representative confocal section, while panels 3–6 show reconstructed 3D views. The position and volume of anisosomes were also presented in magenta in a surface-rendered view (SRV) in panel 5. The arrow in panel 3 indicates an example of cytoplasmic TDP-43 aggregate. Scale bar, 10 µm. (H) Quantification of the percentage of cells showing cytosolic TDP-43 puncta in XPO-1-positive (pos) and -negative (neg) cells in 8 randomly selected fields each with at least 20 cells from 3 independent experiments. ****p<0.0001 by two-tailed unpaired Student’s t-test. (I) FRAP-based confocal imaging reveals the transition of anisosomes into a gel-like state upon XPO-1 overexpression. Scale bar, 1 µm. (J) Anisosome formation changes endogenous XPO-1 localization. TDP-43 2KQ-Clover before or after anisosome induction were stained with anti-XPO-1 antibodies (red) and DAPI (blue). Scale bar, 10 µm. (K) Quantification of nuclear endogenous XPO-1 mean fluorescence intensity (MFI) in individual cells (indicated by dots) before or after anisosome induction. ****p<0.0001 by two-tailed unpaired Student’s t-test. N=58 cells for uninduced and 63 cells for induced condition in 2 independent repeats. (L) A schematic model depicting how the nuclear XPO-1 activity influences TDP-43 liquid-to-solid phase transition. AS, anisosome.
Figure 5—figure supplement 1
mCherry overexpression has no effect on anisosomes.
DLD1 cells were transfected with a mCherry-expressing plasmid and then induced for TDP-43 expression for 24 hr before imaging. The arrowheads indicate mCherry-positive cells that have only nucleus-localized anisosomes. Scale bar, 10 μm.
Figure 6
The inhibition of XPO-1 stabilizes anisosomes in an RNA-dependent manner.
(A) A schematic diagram of the semi-permeabilized cell assay. SLO, streptolysin O. (B) TDP-43 in anisosomes becomes soluble after cell permeabilization (indicated by NucSpot-positive staining in magenta). Note: The intensity of NucSpot dye correlates with the permeabilization time. Dashed circles highlight cells permeabilized early during the incubation. The cells have fewer anisosomes. Scale bars, 5 μm. (C) TDP-43 mean fluorescence intensity (MFI) displays an inverse correlation with the NucSpot dye intensity in B. AU, arbitrary unit. N=52 cells. (D) Small TDP-43-positive puncta were reformed after the incubation of permeabilized DLD1 cells with cow liver cytosol in the presence of ATP-regenerating system (ARS) and GTP (100 μM) (ATP/GTP) at 37°C for 40 min. Cells incubated with cytosol without ATP/GTP or with phosphate-buffered saline (PBS) plus ATP/GTP serve as controls. Cells were fixed and stained with Hoechst (blue) to label the nuclei. Scale bars, 5 μm. (E) Quantification of the TDP-43 mean fluorescence intensity (MFI) in cells as shown in D. Error bars, SD. ****p<0.0001; ***p<0.001 by unpaired Student’s t-test. N=at least 35 randomly selected cells representing two independent experiments. (F) LMB treatment (200 nM, 16 hr) stabilizes anisosomes. Dashed circles indicate permeabilized cells. Scale bars, 5 μm. (G) RNase T1 treatment destabilizes anisosomes in DLD1 cells pre-treated with LMB (200 nM, 16 hr). DLD1 cells induced for TDP-43 expression were treated with LMB (200 nM, 16 hr). Cells were permeabilized in the absence or presence of RNase T1 for 40 min before imaging. Images shown in this figure are maximum intensity projection views of confocal z-sections. Scale bars, 5 μm.
Figure 7
Inhibition of XPO-1 mitigates TDP-43 hyperphosphorylation in TDP-43 K181E organoids.
(A) Confocal fluorescence imaging reveals significant reduction in phosphorylated TDP-43 (p-TDP43) in K181E/K181E organoids following KPT-276 treatment. Organoids of the indicated genotypes at day 87 were treated with DMSO as a control, or with KPT-276 (20 nM) for 35 days. Organoids were fixed, sectioned, and stained with antibodies against TUJ1, a neuronal marker (magenta), TDP-43 (green), and p-TDP-43 (red). Data represents organoids from two individual batches. (B, C, D) Quantification of p-TDP-43 puncta mean volume (B), total TDP-43 intensity (C), and the percentage of cells with reduced nuclear TDP-43 (D) from experiments represented by A. For B, p-TDP-43-positive puncta were segmented from collected 3D images for mean puncta volume calculation. Error bars indicate mean ± SEM. *p<0.05, ****p<0.0001, ns, not significant, by one-way ANOVA. n=3 organoids per condition.
Videos
Video 1
PlaB treatment induces anisosome fusion.
Video 2
Blocking XPO-1-mediated nuclear export induces anisosome fusion.
Additional files
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Supplementary file 1
A chemical genetic screen identified compounds that reduce anisosome numbers.
- https://cdn.elifesciences.org/articles/110172/elife-110172-supp1-v1.xlsx
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Supplementary file 2
A genome-wide siRNA screen identified potential anisosome regulators.
- https://cdn.elifesciences.org/articles/110172/elife-110172-supp2-v1.xlsx
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Supplementary file 3
Top 110 genes confirmed from a secondary screen as modulators of anisosome dynamics.
- https://cdn.elifesciences.org/articles/110172/elife-110172-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110172/elife-110172-mdarchecklist1-v1.pdf
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Source data 1
Excel tables used to make graphs in the study.
- https://cdn.elifesciences.org/articles/110172/elife-110172-data1-v1.xlsx
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Cellular modifiers of TDP-43 phase transition and cytoplasmic aggregation
eLife 15:RP110172.
https://doi.org/10.7554/eLife.110172.4
