Description of the data and file structure

The dataset contains Tabulated_Data.zip, which includes the source data for figures in CSV and Prism formats. 

General information

• Associated publication: Copley et al., " Isoform-specific steric zippers drive aberrant assembly and mislocalization of shortened TDP-43," Science Advances (2026).
• File naming convention: Files are named by figure panel (e.g., Fig_1B_sTDP43_dCtail_ThT_kinetics.csv). The prefix indicates the figure number and panel, followed by a brief description of the data contents.
• File formats: Data are provided as .csv files for all figures for broad accessibility. Depending on the figure, data are additionally provided as .pzfx (GraphPad Prism) files; the latter includes graphs and statistical analyses.
• Rows: Unless otherwise noted, each row represents an independent experimental replicate. The first row of each file is a header row containing column labels.
• Missing values: Blank cells indicate that the measurement was not performed or the condition does not apply to that sample/construct. No special missing-value codes (e.g., "NA", "N/A", or "-999") are used.
• For in vitro aggregation assays, proteins were expressed with an MBP solubility tag cleaved by TEV protease to initiate aggregation; TEV conditions served as negative controls.

Glossary of Key Terms

For users who may be unfamiliar with the terminology and methods used in this study:

·       MBP (Maltose-Binding Protein) — A solubility tag fused to recombinant proteins to keep them soluble during expression. Cleavage of MBP by TEV protease removes the tag and induces aggregation of the liberated protein in vitro.

• flTDP-43 – Full-length TDP-43 (414 amino acids), the canonical isoform of the RNA-binding protein TDP-43, containing an N-terminal domain, two RNA-recognition motifs (RRMs), and a C-terminal prion-like domain (PrLD).
• sTDP-43 – Shortened TDP-43 (295 amino acids), a splice isoform in which most of the PrLD is replaced by an isoform-specific 18-amino acid C-terminal tail. Enriched in motor neurons and highly insoluble in ALS patient tissue.
• Clip34 — A 34-nucleotide RNA derived from a TDP-43 binding site, used here as a physiologically relevant RNA ligand to assess TDP-43 RNA-binding activity and aggregation suppression.
• (AC)17 — A synthetic 34-nucleotide alternating adenosine-cytosine RNA used as a non-specific control RNA that does not bind TDP-43, included to distinguish TDP-43-specific RNA effects from nonspecific charge-based interactions.
• AUC (Area Under the Curve) — The integral of a kinetic turbidity time-course over the 16 h assay window, used as a single summary value for total aggregation or phase separation.

·       IC50 — The concentration of RNA (µM Clip34) required to reduce aggregate area by 50%, derived from dose-response regression of microscopy data. Used as a measure of RNA potency in suppressing TDP-43 aggregation.

·       B_max — Maximum RNA binding capacity, derived from nonlinear regression of EMSA binding curves.

·       KD (apparent dissociation constant) — The protein concentration at which half-maximal RNA binding is achieved; a measure of RNA-binding affinity.

·       Hill slope — A parameter from cooperative binding models reflecting the steepness of the binding curve; values >1 suggest cooperativity.

• sTDP-43ΔC-tail – A truncated form of sTDP-43 lacking the 18-amino acid C-terminal tail.

·       sTDP-43(18aa) – A synthetic 18-amino acid peptide corresponding to the isoform-specific C-terminal tail of sTDP-43, used to test whether this sequence is sufficient to form amyloid fibrils in isolation.

·       sTDP-43(2Pmut-18aa) – A synthetic 18-amino acid peptide corresponding to the sTDP-43 C-terminal tail with proline substitutions at the positions equivalent to I281 and L291, designed to disrupt both clusters of steric zippers. Used as a non-fibrillizing control peptide.

·       Peptide fibrillization assay – An in vitro assay in which synthetic peptides are incubated with ThT and monitored by fluorescence over 44 h with orbital shaking, to assess the intrinsic amyloid-forming capacity of short sequence motifs.

• Steric zipper – A self-complementary β-sheet structure that forms the spine of amyloid fibrils. Predicted using the ZipperDB database, which scores the fibrillization propensity of each hexapeptide within a protein sequence.
• ThT (Thioflavin T) – A fluorescent dye that binds to amyloid fibrils and β-sheet-rich aggregates, used here to monitor aggregation kinetics in real time.
• Turbidity – Absorbance at 395 nm, used as a measure of light scattering by protein aggregates in solution.
• ΔThT / ΔTurbidity – The change in ThT fluorescence or turbidity signal between the final time point (t = 16 h) and the initial time point (t = 0), used as a summary measure of aggregation extent.
• Sedimentation analysis – Centrifugation-based fractionation of aggregation reactions into supernatant (soluble) and pellet (insoluble) fractions, quantified by SDS-PAGE densitometry. Values reported as a percentage of total protein in the input sample, which is not centrifuged.
• CellProfiler – Open-source image analysis software used to quantify the percentage of image area occupied by ThT-positive aggregates in fluorescence microscopy images.
• EMSA (Electrophoretic Mobility Shift Assay) – A gel-based assay used to measure protein–RNA binding. A fluorescently labeled RNA (Clip34) is incubated with increasing concentrations of protein; binding is detected as a shift in RNA migration.
• ALS (Amyotrophic Lateral Sclerosis) – A fatal neurodegenerative disease characterized by the loss of motor neurons, in which TDP-43 pathology (cytoplasmic aggregation) is observed in ~97% of cases.
• Primary cortical neurons – Neurons dissociated from embryonic rat cortices and cultured in vitro. Used here to assess the effects of sTDP-43 variants on neuronal survival, subcellular localization, and aggregation in a disease-relevant cell type.
• Longitudinal survival analysis – A live-imaging approach in which individual neurons are tracked over time (here, 10 days post-transfection) using automated fluorescence microscopy. Cell death is scored based on rounding of the soma, loss of mApple fluorescence, and degeneration of neuritic processes.
• mApple – A diffusely localized red fluorescent protein co-expressed in neurons as a morphological marker to identify and track individual cells over time.
• EGFP (Enhanced Green Fluorescent Protein) – Used as a tag fused to sTDP-43 variants to enable visualization of protein localization and aggregation state in live and fixed neurons.
• Nuclear/cytoplasmic (N/C) ratio – The ratio of fluorescence intensity in the nucleus versus the cytoplasm, used to quantify the subcellular localization of EGFP-tagged sTDP-43 variants. Values greater than 1 indicate predominantly nuclear localization.
• Leptomycin B (LMB) – A pharmacological inhibitor of the nuclear export receptor Exportin 1 (XPO1), used to test whether cytoplasmic mislocalization of sTDP-43 depends on active, receptor-mediated nuclear export.
• HEK293T cells – A human embryonic kidney cell line used for overexpression experiments assessing nuclear export, RNA splicing, and localization of sTDP-43 variants.

File-by-file descriptions

Figure 1

Fig_1B_sTDP43_dCtail_ThT_kinetics.csv

• ThT fluorescence kinetics (arbitrary units) from in vitro aggregation assays, standardized to 0 at t = 0
• 3 conditions: flTDP-43, sTDP-43, sTDP-43ΔC-tail (6 biological replicates each, reported as individual values)
• Time column in HH:MM:SS format, 1-min intervals over 16 h (961 rows × 19 columns)

Fig_1C_sTDP43_dCtail_delta_ThT.csv

• ΔThT values (ThT fluorescence at t = 16 h minus t = 0, arbitrary units) from in vitro aggregation assays
• 3 conditions: flTDP-43, sTDP-43, sTDP-43ΔC-tail (6 biological replicates each, one value per replicate per condition)

Fig_1D_sTDP43_dCtail_delta_turbidity.csv

• ΔTurbidity values (absorbance at 395 nm at t = 16 h minus t = 0) from in vitro aggregation assays
• 3 conditions: flTDP-43, sTDP-43, sTDP-43ΔC-tail (6 biological replicates each, one value per replicate per condition)

Fig_1F_sTDP43_dCtail_microscopy.csv

• Percentage of image area occupied by aggregates (%) from ThT fluorescence microscopy at t = 16 h, quantified using CellProfiler
• 3 conditions: flTDP-43, sTDP-43, sTDP-43ΔC-tail (5 biological replicates each, one value per replicate per condition representing the average of 3–6 images)

Fig_1H_sTDP43_dCtail_sedimentation.csv

• Percentage of protein in the supernatant fraction relative to input (%) from sedimentation analysis at t = 16 h, quantified by densitometry of Coomassie-stained SDS-PAGE gels
• 3 conditions: flTDP-43, sTDP-43, sTDP-43ΔC-tail (4 biological replicates each, one value per replicate per condition)

Prism files (data files with graphs and statistical analyses):

Fig_1BtoD_S1BtoD_sTDP43_dCtail_ThT_turbidity.pzfx

• GraphPad Prism file containing ThT kinetics, ΔThT, and ΔTurbidity data for both the +TEV (No RNA) and –TEV control conditions, corresponding to Fig. 1B–D and Fig. S1B–D
• Datasets included: ThT fluorescence kinetics over 16 h (x-axis in hours), ΔThT at t = 16 h, and ΔTurbidity (absorbance at 395 nm) at t = 16 h, for 3 conditions (flTDP-43, sTDP-43, sTDP-43ΔC-tail) with 6 biological replicates each.
• Includes graphs and statistical analyses as presented in Fig. 1B–D and Fig. S1B–D

Fig_1F_S1F_sTDP43_dCtail_microscopy.pzfx

• GraphPad Prism file containing microscopy aggregate quantification data for both Fig. 1F and Fig. S1F
• Two datasets: "No RNA" (+TEV, Fig. 1F) and "No TEV" (–TEV control, Fig. S1F), each with 3 conditions (flTDP-43, sTDP-43, sTDP-43ΔC-tail) and 5 biological replicates
• Includes graphs and statistical analyses as presented in Fig. 1F and Fig. S1F

Fig_1H_sTDP43_dCtail_sedimentation.pzfx

• GraphPad Prism file containing the same data as Fig_1H_sTDP43_dCtail_sedimentation.csv
• Includes graphs and statistical analyses as presented in Fig. 1H

Figure 2

Fig_2D_flTDP43_Clip34_avg_aggregate_area.csv

• Percentage of image area occupied by aggregates (%) from ThT fluorescence microscopy, quantified using CellProfiler, for flTDP-43 aggregation assays in the presence of increasing concentrations of Clip34 RNA or a control (AC)17 RNA
• 12 conditions: No RNA, Clip34 at molar ratios of 1:4, 1:6, 1:8, 1:10, 1:12, 1:16, 1:24, 1:32, 1:64, and 1:128 (RNA: protein), and (AC)17 at 1:4 (4 biological replicates each)

Fig_2E_sTDP43_Clip34_avg_aggregate_area.csv

• Percentage of image area occupied by aggregates (%) from ThT fluorescence microscopy, quantified using CellProfiler, for sTDP-43 aggregation assays in the presence of increasing concentrations of Clip34 RNA or a control (AC)17 RNA
• 12 conditions: No RNA, Clip34 at molar ratios of 1:4, 1:6, 1:8, 1:10, 1:12, 1:16, 1:24, 1:32, 1:64, and 1:128 (RNA: protein), and (AC)17 at 1:4 (4 biological replicates each; one missing value in the (AC)17 1:4 condition)

Fig_2F_flTDP43_Clip34_aggregate_area_regression.csv

• Percentage of image area occupied by aggregates (%) for flTDP-43, plotted against Clip34 RNA concentration (µM) for dose-response regression analysis (Fig. 2F)
• First column contains Clip34 RNA concentration (µM); columns 2–5 contain individual replicate values (4 biological replicates) at each concentration (11 concentration points, including 0 µM No RNA)

Fig_2G_sTDP43_Clip34_aggregate_area_regression.csv

• Percentage of image area occupied by aggregates (%) for sTDP-43, plotted against Clip34 RNA concentration (µM) for dose-response regression analysis (Fig. 2G)
• First column contains Clip34 RNA concentration (µM); columns 2–5 contain individual replicate values (4 biological replicates) at each concentration (11 concentration points, including 0 µM No RNA)

Fig_2H_flTDP43_sTDP43_Clip34_aggregate_area_IC50s.csv

• IC50 values (µM Clip34 RNA) derived from dose-response regression of aggregate area data for flTDP-43 and sTDP-43 (Fig. 2H)
• 2 conditions: flTDP-43 and sTDP-43 (4 biological replicates each, one IC50 value per replicate fit)

Prism file (data files with graphs and statistical analyses):

Fig_2DtoH_flTDP43_sTDP43_Clip34_microscopy.pzfx

• GraphPad Prism file containing all microscopy-based aggregate quantification data underlying Fig. 2D–H
• Includes: average aggregate area (%) for flTDP-43 and sTDP-43 across Clip34 RNA titration conditions (Fig. 2D–E), dose-response regression data for flTDP-43 and sTDP-43 (Fig. 2F–G), and IC50 values derived from individual replicate fits (Fig. 2H)
• Includes graphs, dose-response curve fits, and statistical analyses as presented in Fig. 2D–H

Figure 3

Fig_3A_sTDP43_Ctail_zipperDB.csv

• ZipperDB fibrillization scores relevant to residues of the sTDP-43 C-terminal tail; 18 amino acids, S273–S290, as displayed in Fig. 3A. Each value is calculated for a hexapeptide, so residues that cannot serve as the first position of a hexapeptide (i.e., the final five residues of the C-terminal tail, 291-295) are not assigned individual scores.
• 2 rows × 18 columns: first row contains residue identifiers (single-letter amino acid code + position number); second row contains the corresponding ZipperDB score (kcal/mol; more negative values indicate higher predicted fibrillization propensity)

Fig_3B_sTDP43_5G_Ctail_zipperDB.csv

• ZipperDB fibrillization scores relevant to residues of the sTDP-43(5G) C-terminal tail; 18 amino acids, S273–S290, in which T289 and S290 (as well as residues not shown: L291, K292, V293) are substituted with glycines (G289-293) to disrupt the second predicted steric zipper cluster, as displayed in Fig. 3B. Each value is calculated for a hexapeptide, so residues that cannot serve as the first position of a hexapeptide (i.e., the final five residues of the C-terminal tail, 291-295) are not assigned individual scores.
• 2 rows × 18 columns: first row contains residue identifiers; second row contains ZipperDB scores (kcal/mol)

Fig_3C_sTDP43_I281P_L291P_Ctail_zipperDB.csv

• ZipperDB fibrillization scores relevant to residues of the sTDP-43(2Pmut) C-terminal tail, carrying proline substitutions at I281 and L291 to disrupt both predicted steric zipper clusters, as displayed in Fig. 3C
• 2 rows × 19 columns: first row contains residue identifiers (mutated positions labeled I281P and L291P); second row contains ZipperDB scores (kcal/mol); blank cells indicate positions where ZipperDB does not return a score (proline hinders β-strand formation; hexapeptides with proline in the first or last position are not assigned scores)

Fig_3D_sTDP43_I281P_Ctail_zipperDB.csv

• ZipperDB fibrillization scores relevant to residues of the sTDP-43(I281P) C-terminal tail, carrying a single proline substitution at I281 to disrupt only the first predicted steric zipper cluster, as displayed in Fig. 3D
• Same format as Fig. 3A–C: 2 rows × 18 columns (residue identifiers and ZipperDB scores in kcal/mol); blank cells indicate no score returned for hexapeptides with proline in the first or last position

Fig_3E_sTDP43_L291P_Ctail_zipperDB.csv

• ZipperDB fibrillization scores relevant to residues of the sTDP-43(L291P) C-terminal tail, carrying a single proline substitution at L291 to disrupt only the second predicted steric zipper cluster, as displayed in Fig. 3E.
• Same format as Fig. 3A–D: 2 rows × 18 columns (residue identifiers and ZipperDB scores in kcal/mol); blank cell at G286 indicates no score returned for that hexapeptide with proline in the last position (L291P)

Fig_3G_Ctail_peptide_ThT_kinetics.csv

• ThT fluorescence kinetics (arbitrary units) from in vitro peptide fibrillization assays, standardized to 0 at t = 0
• 6 conditions: sTDP-43(18aa) C-terminal tail peptide and sTDP-43(2Pmut-18aa) double proline mutant peptide, each tested at 3 concentrations (5 µM, 10 µM, 16 µM), with 3 biological replicates per concentration (18 columns total)
• Time column in HH:MM:SS format, 2-min intervals over 44 h

Fig_3H_Ctail_peptide_delta_ThT.csv

• ΔThT values (ThT fluorescence at t = 44 h minus t = 0, arbitrary units) from in vitro peptide fibrillization assays
• 6 conditions: sTDP-43(18aa) and sTDP-43(2Pmut-18aa) peptides each at 5 µM, 10 µM, and 16 µM (3 biological replicates each, one value per replicate per condition)

Prism files (data files with graphs and statistical analyses):

Fig_3AtoE_sTDP43_zipperDB.pzfx

• GraphPad Prism file containing all ZipperDB fibrillization score data underlying Fig. 3A–E
• Five datasets: sTDP-43 C-tail (Fig. 3A), sTDP-43(5G) (Fig. 3B), sTDP-43(I281P/L291P) (Fig. 3C), sTDP-43(I281P) (Fig. 3D), and sTDP-43(L291P) (Fig. 3E), each with 18–19 residue positions (S273–S290/L291P) and corresponding ZipperDB scores (kcal/mol)
• Includes graphs as presented in Fig. 3A–E

Fig_3G_3H_Ctail_peptides_ThT.pzfx

• GraphPad Prism file containing all peptide ThT fibrillization data underlying Fig. 3G–H
• Includes: ThT kinetics over 44 h for sTDP-43(18aa) and sTDP-43(2Pmut-18aa) peptides at 5, 10, and 16 µM (Fig. 3G), and ΔThT values at t = 44 h for all conditions (Fig. 3H)
• Includes graphs and statistical analyses as presented in Fig. 3G–H

Figure 4

Fig_4B_sTDP43_I281PL291P_5G_ThT_kinetics.csv

• ThT fluorescence kinetics (arbitrary units), standardized to 0 at t = 0, from in vitro aggregation assays comparing sTDP-43 C-tail zipper mutants
• 3 conditions: sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G), with 5 biological replicates each, except 4 biological replicates for sTDP-43 (14 data columns plus one blank column); time column in HH:MM:SS format, 1-min intervals over 16 h

Fig_4C_sTDP43_I281PL291P_5G_delta_ThT.csv

• ΔThT values (ThT fluorescence at t = 16 h minus t = 0, arbitrary units) from in vitro aggregation assays comparing sTDP-43 C-tail zipper mutants (Fig. 4C)
• 3 conditions: sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) (5 biological replicates each; one missing value in the sTDP-43 condition)

Fig_4D_sTDP43_I281PL291P_5G_delta_turbidity.csv

• ΔTurbidity values (absorbance at 395 nm at t = 16 h minus t = 0) from in vitro aggregation assays comparing sTDP-43 C-tail zipper mutants (Fig. 4D)
• 3 conditions: sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) (5 biological replicates each; one missing replicate in the sTDP-43 condition)

Fig_4F_sTDP43_I281PL291P_5G_microscopy.csv

• Percentage of image area occupied by aggregates (%) from ThT fluorescence microscopy at t = 16 h, quantified using CellProfiler, comparing sTDP-43 C-tail zipper mutants (Fig. 4F)
• 3 conditions: sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) (5 biological replicates each; one missing replicate in the sTDP-43 condition)

Fig_4H_sTDP43_I281PL291P_5G_sedimentation.csv

• Percentage of protein in the supernatant fraction (S/I %, sedimentation) following centrifugation at t = 16 h, comparing sTDP-43 C-tail zipper mutants (Fig. 4H)
• 3 conditions: sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) (4 biological replicates each)

Prism files (data files with graphs and statistical analyses):

Fig_4BtoD_S5BtoD_sTDP43_I281PL291P_5G_ThT_turbidity.pzfx

• GraphPad Prism file containing ThT kinetics, ΔThT, and ΔTurbidity data for both the +TEV (No RNA) and –TEV control conditions for sTDP-43 C-tail zipper mutants, corresponding to Fig. 4B–D and Fig. S5B–D
• Datasets include ThT fluorescence kinetics over 16 h, ΔThT at t = 16 h, and ΔTurbidity at t = 16 h for sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) (4 or 5 biological replicates each)
• Includes graphs and statistical analyses as presented in Fig. 4B–D and Fig. S5B–D

Fig_4F_S5F_sTDP43_I281PL291P_5G_microscopy.pzfx

• GraphPad Prism file containing microscopy aggregate quantification data for both the +TEV (No RNA) and –TEV control conditions for sTDP-43 C-tail zipper mutants, corresponding to Fig. 4F and Fig. S5F
• Two datasets: +TEV (Fig. 4F) and –TEV (Fig. S5F), each with 3 conditions (sTDP-43, sTDP-43(I281P/L291P), sTDP-43(5G)) and 4 or 5 biological replicates
• Includes graphs and statistical analyses as presented in Fig. 4F and Fig. S5F

Fig_4H_sTDP43_I281PL291P_5G_sedimentation.pzfx

• GraphPad Prism file containing the same sedimentation (S/I %) data as Fig_4H_sTDP43_I281PL291P_5G_sedimentation.csv
• Includes graphs and statistical analyses as presented in Fig. 4H

Figure 5

Fig_5B_sTDP43_I281P_L291P_ThT_kinetics.csv

• ThT fluorescence kinetics (arbitrary units), standardized to 0 at t = 0, from in vitro aggregation assays comparing single and double proline substitution mutants of sTDP-43 (Fig. 5B)
• 4 conditions: sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P), with 4 biological replicates each (16 data columns total); time column in HH:MM:SS format, 1-min intervals over 16 h

Fig_5C_sTDP43_I281P_L291P_delta_ThT.csv

• ΔThT values (ThT fluorescence at t = 16 h minus t = 0, arbitrary units) from in vitro aggregation assays comparing single and double proline substitution mutants of sTDP-43 (Fig. 5C)
• 4 conditions: sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P) (4 biological replicates each)

Fig_5D_sTDP43_I281P_L291P_delta_turbidity.csv

• ΔTurbidity values (absorbance at 395 nm at t = 16 h minus t = 0) from in vitro aggregation assays comparing single and double proline substitution mutants of sTDP-43 (Fig. 5D)
• 4 conditions: sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P) (4 biological replicates each)

Fig_5F_sTDP43_I281P_L291P_microscopy.csv

• Percentage of image area occupied by aggregates (%) from ThT fluorescence microscopy at t = 16 h, quantified using CellProfiler, comparing single and double proline substitution mutants of sTDP-43 (Fig. 5F)
• 4 conditions: sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P) (4 biological replicates each)

Fig_5H_sTDP43_I281P_L291P_sedimentation.csv

• Percentage of protein in the supernatant fraction (S/I %, sedimentation) following centrifugation at t = 16 h, comparing single and double proline substitution mutants of sTDP-43 (Fig. 5H)
• 4 conditions: sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P) (3 biological replicates each)

Prism files (data files with graphs and statistical analyses):

Fig_5BtoD_S6BtoD_sTDP43_I281P_L291P_ThT_turbidity.pzfx

• GraphPad Prism file containing ThT kinetics, ΔThT, and ΔTurbidity data for both the +TEV (No RNA) and –TEV control conditions for the single and double proline substitution mutants of sTDP-43, corresponding to Fig. 5B–D and Fig. S6B–D
• Datasets include ThT fluorescence kinetics over 16 h, ΔThT at t = 16 h, and ΔTurbidity at t = 16 h for sTDP-43, sTDP-43(I281P), sTDP-43(L291P), and sTDP-43(I281P/L291P) (4 biological replicates each)
• Includes graphs and statistical analyses as presented in Fig. 5B–D and Fig. S6B–D

Fig_5F_S6F_sTDP43_I281P_L291P_microscopy.pzfx

• GraphPad Prism file containing microscopy aggregate quantification data for both the +TEV (No RNA) and –TEV control conditions for the single and double proline substitution mutants of sTDP-43, corresponding to Fig. 5F and Fig. S6F
• Two datasets: +TEV (Fig. 5F) and –TEV (Fig. S6F), each with 4 conditions (sTDP-43, sTDP-43(I281P), sTDP-43(L291P), sTDP-43(I281P/L291P)) and 4 biological replicates
• Includes graphs and statistical analyses as presented in Fig. 5F and Fig. S6F

Fig_5H_sTDP43_I281P_L291P_sedimentation.pzfx

• GraphPad Prism file containing the same sedimentation (S/I %) data as Fig_5H_sTDP43_I281P_L291P_sedimentation.csv
• Includes graphs and statistical analyses as presented in Fig. 5H

Figure 6

Fig6A_S7A-C.csv is a large, single-cell longitudinal survival/imaging dataset from a longitudinal fluorescence microscopy survival assay in primary neurons. Here is a full description of the file's structure and content:

Experimental Design

This is a single-cell-resolution longitudinal imaging dataset for Figs. 6A and S7A–C of the manuscript. Each row represents one individual neuron (well/cell track) imaged over up to 10 consecutive time points. Primary neurons express fluorescent fusion proteins (EGFP alone as a control, or sTDP-43-EGFP and variants).

Key Metadata Columns

• experiment: experiment ID (e.g., MD90)
• well.id: plate well address and unique cell identifier
• group: construct expressed — EGFP, sTDP43-EGFP, sTDP43(I281PL291P)-EGFP, sTDP43(I281P)-EGFP, or sTDP43(mNES)-EGFP
• last_tp / last_time: last imaging time point and time (in hours, up to 239 h / ~10 days) — used for survival analysis
• death_cause: cause of cell disappearance (e.g., unfound) or NA if cell survived
• censored / event: standard survival analysis flags (TRUE = censored/survived, FALSE/1 = death event)

Fluorescence Measurements (per time point, .1–.10)

For each of up to 10 time points, the file records per-cell fluorescence statistics in both channels:

• RFP.mean.1–.10: mean RFP fluorescence intensity at each time point, used as a morphological/viability marker (mApple)
• GFP.mean.1–.10: mean GFP fluorescence intensity at each time point, reflecting the expression level of the EGFP-tagged construct
• norm1: the GFP.mean fluorescence value at time point 1, normalized to the median GFP.mean value at time point 1 from the negative control (EGFP) specific to each experiment; used to normalize construct expression level across cells

Biological Groups

The group column encodes the five constructs being compared in Figs. 6A and S7A–C:

• EGFP — cytoplasmic GFP control (n=1169)
• sTDP43-EGFP — wild-type sTDP-43 fused to EGFP (n=927)
• sTDP43(I281PL291P)-EGFP — double proline substitution at I281 and L291 (n=1014)
• sTDP43(I281P)-EGFP — single proline substitution at I281 (n=977)
• sTDP43(mNES)-EGFP — sTDP-43^5G variant carrying mutations in the C-terminal tail that disrupt the predicted nuclear export signal; equivalent to sTDP-43^5G described in other figures of this dataset (n=878)

These data underlie the Kaplan–Meier survival curves shown in Fig. 6A (and supplemental Figs. S7A–C), where neuronal survival is compared across sTDP-43 variants relative to sTDP-43 WT and EGFP controls.

Fig_6B_Clip34_EMSA_Bmax.csv

• CSV file containing B_max (maximum RNA binding capacity) values from Clip34 RNA EMSA binding curve fits for Fig. 6B
• Four columns: flTDP-43, sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G); 3 replicates for flTDP-43 and sTDP-43, 4 replicates for the mutant and 5G constructs
• Values are in arbitrary fluorescence units (~1.4–2.2 × 10⁷); sTDP-43 shows lower B_max relative to the other constructs

Prism files (data files with graphs and statistical analyses):

Fig_6B_S8EtoG_Clip34_EMSA.pzfx

• GraphPad Prism file containing the Clip34 RNA EMSA binding curve data for Fig. 6B and Fig. S8E–G
• Includes the full nonlinear binding curve data (fluorescence signal vs. protein concentration) for flTDP-43, sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G), from which the B_max values in Fig_6B_Clip34_EMSA_Bmax.csv were derived
• Contains graphs and statistical analyses as presented in Fig. 6B and supplemental Fig. S8E–G

Figure S1

Fig_S1B_No_TEV_sTDP43_dCtail_ThT_kinetics.csv

• CSV file containing ThT fluorescence kinetics time-course data (–TEV / No TEV control condition) for the sTDP-43 ΔC-tail constructs, corresponding to Fig. S1B
• Parallel to Fig_1B_sTDP43_dCtail_ThT_kinetics.csv (the +TEV condition), but here TEV protease was omitted, so the MBP solubility tag is not cleaved, and aggregation is not induced.
• Time points collected over 16 h with 5 biological replicates per condition; serves as the negative control, demonstrating aggregation is TEV-dependent

Fig_S1C_No_TEV_sTDP43_dCtail_delta_ThT.csv

• CSV file containing the endpoint ΔThT fluorescence values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43 ΔC-tail constructs, corresponding to Fig. S1C
• Direct –TEV counterpart to Fig_1C_sTDP43_dCtail_delta_ThT.csv (+TEV condition); 5 biological replicates per construct

Fig_S1D_No_TEV_sTDP43_dCtail_delta_turbidity.csv

• CSV file containing endpoint Δturbidity values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43 ΔC-tail constructs, corresponding to Fig. S1D
• Direct –TEV counterpart to Fig_1D_sTDP43_dCtail_delta_turbidity.csv (+TEV condition); 5 biological replicates per construct

Fig_S1F_No_TEV_sTDP43_dCtail_microscopy.csv

• CSV file containing fluorescence microscopy aggregate quantification data (–TEV / No TEV control condition) for sTDP-43 ΔC-tail constructs, corresponding to Fig. S1F
• Direct –TEV counterpart to Fig_1F_sTDP43_dCtail_microscopy.csv (+TEV condition); 5 biological replicates per construct

Figure S3

Fig_S3B_flTDP43_Clip34_sedimentation.csv

• CSV file containing sedimentation (S/I %) data for full-length TDP-43 (flTDP-43) across a Clip34 RNA titration, corresponding to Fig. S3B
• Eight columns: No RNA, Clip34 1:4, Clip34 1:8, Clip34 1:16, Clip34 1:32, Clip34 1:64, Clip34 1:128, and (AC)17 1:4 (negative control RNA); 4 biological replicates per condition

Fig_S3D_sTDP43_Clip34_sedimentation.csv

• CSV file containing sedimentation (S/I %) data for sTDP-43 across a Clip34 RNA titration (with (AC)17 negative control), corresponding to Fig. S3D
• Parallel to Fig_S3B_flTDP43_Clip34_sedimentation.csv but using the sTDP-43 construct; includes 8 conditions (No RNA, Clip34 1:4 through 1:128, (AC)17 1:4) with 4 biological replicates each (except 3 replicates for (AC)17 1:4)

Fig_S3E_flTDP43_sTDP43_no_RNA_turbidity_kinetics.csv

• CSV file containing turbidity kinetics time-course data (No RNA condition) for both flTDP-43 and sTDP-43, corresponding to Fig. S3E
• Paired time-series columns for flTDP-43 and sTDP-43 measured over 16 h with 4 biological replicates each; serves as the No RNA baseline control for the Clip34 RNA-stimulated turbidity experiments

Fig_S3F_flTDP43_Clip34_turbidity_AUC.csv

• CSV file containing turbidity area-under-the-curve (AUC) values for flTDP-43 across a Clip34 RNA titration, corresponding to Fig. S3F
• Columns represent the same RNA concentration series as in the sedimentation files, with additional concentrations (No RNA, Clip34 1:4 through 1:128, (AC)17 1:4) with 4 biological replicates each; AUC integrates the full turbidity kinetics curves from Fig_S3E to quantify total aggregation over the 16 h time course

Fig_S3G_sTDP43_Clip34_turbidity_AUC.csv

• CSV file containing turbidity AUC values for sTDP-43 across a Clip34 RNA titration, corresponding to Fig. S3G
• Direct sTDP-43 counterpart to Fig_S3F_flTDP43_Clip34_turbidity_AUC.csv; same RNA concentration series (No RNA, Clip34 1:4 through 1:128, (AC)17 1:4) with 4 biological replicates each (except 3 replicates for (AC)17 1:4)

Prism files (data files with graphs and statistical analyses):

Fig_S3B_S3D_flTDP43_sTDP43_Clip34_sedimentation.pzfx

• GraphPad Prism file containing sedimentation (Supernatant/Input %) data for both flTDP-43 and sTDP-43 across a Clip34 RNA titration, corresponding to Fig. S3B (flTDP-43) and Fig. S3D (sTDP-43)
• Two datasets: flTDP-43 (Fig. S3B) and sTDP-43 (Fig. S3D), each with 8 conditions: No RNA, Clip34 at protein:RNA molar ratios of 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128, and (AC)₁₇ at 1:4 (negative control RNA); 4 biological replicates per condition. Note: the sTDP-43 dataset is missing one replicate value in the (AC)₁₇ 1:4 condition (n=3 for that condition)
• Values are reported as percentage of total protein in the supernatant fraction (Supernatant/Input %), normalized per replicate
• Includes graphs and statistical analyses as presented in Fig. S3B and Fig. S3D

Fig_S3EtoG_flTDP43_sTDP43_Clip34_turbidity.pzfx

• GraphPad Prism file containing turbidity kinetics and turbidity AUC data for flTDP-43 and sTDP-43 across a Clip34 RNA titration, corresponding to Fig. S3E (turbidity kinetics, No RNA), Fig. S3F (flTDP-43 turbidity AUC), and Fig. S3G (sTDP-43 turbidity AUC)
• Three datasets:
  • Turbidity kinetics (No RNA condition) — absorbance at 395 nm over 16 h for flTDP-43 and sTDP-43 with no RNA; 4 biological replicates each; time points at 1-min intervals in HH:MM:SS format (Fig. S3E)
  • Standardized turbidity AUC — flTDP-43 — AUC values integrated over the 16 h turbidity time course for flTDP-43 across 12 conditions: No RNA, Clip34 at protein:RNA molar ratios of 1:4, 1:6, 1:8, 1:10, 1:12, 1:16, 1:24, 1:32, 1:64, and 1:128, and (AC)₁₇ at 1:4; 4 biological replicates per condition (Fig. S3F)
  • Standardized turbidity AUC — sTDP-43 — same RNA concentration series as flTDP-43 AUC above; 4 biological replicates per condition, with one missing replicate in the (AC)₁₇ 1:4 condition (n=3) (Fig. S3G)
• AUC values are standardized (normalized) per replicate
• Includes graphs and statistical analyses as presented in Figs. S3E–G

Figure S5

Fig_S5B_No_TEV_sTDP43_I281PL291P_5G_ThT_kinetics.csv

• CSV file containing ThT fluorescence kinetics time-course data (–TEV / No TEV control condition) for sTDP-43(I281P/L291P) and sTDP-43(5G) constructs, corresponding to Fig. S5B
• Direct –TEV counterpart to Fig_4B_sTDP43_I281PL291P_5G_ThT_kinetics.csv (+TEV condition); time points collected over 16 h with 4 or 5 biological replicates per construct

Fig_S5C_No_TEV_sTDP43_I281PL291P_5G_delta_ThT.csv

• CSV file containing endpoint ΔThT fluorescence values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43(I281P/L291P) and sTDP-43(5G) constructs, corresponding to Fig. S5C
• Direct –TEV counterpart to Fig_4C_sTDP43_I281PL291P_5G_delta_ThT.csv (+TEV condition); 4 or 5 biological replicates per construct

Fig_S5D_No_TEV_sTDP43_I281PL291P_5G_delta_turbidity.csv

• CSV file containing endpoint Δturbidity values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43(I281P/L291P) and sTDP-43(5G) constructs, corresponding to Fig. S5D
• Direct –TEV counterpart to Fig_4D_sTDP43_I281PL291P_5G_delta_turbidity.csv (+TEV condition); 4 or 5 biological replicates per construct

Fig_S5F_No_TEV_sTDP43_I281PL291P_5G_microscopy.csv

• CSV file containing fluorescence microscopy aggregate quantification data (–TEV / No TEV control condition) for sTDP-43(I281P/L291P) and sTDP-43(5G) constructs, corresponding to Fig. S5F
• Direct –TEV counterpart to Fig_4F_sTDP43_I281PL291P_5G_microscopy.csv (+TEV condition); 4 or 5 biological replicates per construct

Figure S6

Fig_S6B_No_TEV_sTDP43_I281P_L291P_ThT_kinetics.csv

• CSV file containing ThT fluorescence kinetics time-course data (–TEV / No TEV control condition) for sTDP-43(I281P) and sTDP-43(L291P) single-mutant constructs and double mutant construct sTDP-43(I281P/L291P), corresponding to Fig. S6B
• Direct –TEV counterpart to Fig_5B_sTDP43_I281P_L291P_ThT_kinetics.csv (+TEV condition); time points collected over 16 h with 4 biological replicates per construct

Fig_S6C_No_TEV_sTDP43_I281P_L291P_delta_ThT.csv

• CSV file containing endpoint ΔThT fluorescence values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43(I281P) and sTDP-43(L291P) single-mutant constructs and double mutant construct sTDP-43(I281P/L291P), corresponding to Fig. S6C
• Direct –TEV counterpart to Fig_5C_sTDP43_I281P_L291P_delta_ThT.csv (+TEV condition); 4 biological replicates per construct

Fig_S6D_No_TEV_sTDP43_I281P_L291P_delta_turbidity.csv

• CSV file containing endpoint Δturbidity values (t = 16 h) for the –TEV (No TEV) control condition for sTDP-43(I281P) and sTDP-43(L291P) single-mutant constructs and double mutant construct sTDP-43(I281P/L291P), corresponding to Fig. S6D
• Direct –TEV counterpart to Fig_5D_sTDP43_I281P_L291P_delta_turbidity.csv (+TEV condition); 4 biological replicates per construct

Fig_S6F_No_TEV_sTDP43_I281P_L291P_microscopy.csv

• CSV file containing fluorescence microscopy aggregate quantification data (–TEV / No TEV control condition) for sTDP-43(I281P) and sTDP-43(L291P) single-mutant constructs and double mutant construct sTDP-43(I281P/L291P), corresponding to Fig. S6F
• Direct –TEV counterpart to Fig_5F_sTDP43_I281P_L291P_microscopy.csv (+TEV condition); 4 biological replicates per construct

Figure S8

Fig_S8E_Clip34_EMSA_curves.csv

• CSV file containing raw EMSA (electrophoretic mobility shift assay) fluorescence values (calculated based on band signal) across a protein concentration titration (0–16 µM) for four TDP-43 constructs — flTDP-43, sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G) — corresponding to Fig. S8E
• 15 rows (14 protein concentrations) × 16 columns (3 replicates for flTDP-43 and sTDP-43; 4 replicates for sTDP-43 mutants); used to generate the binding curves and derive the Bmax values reported in Fig_6B_Clip34_EMSA_Bmax.csv

Fig_S8F_Clip34_EMSA_KD.csv

• CSV file containing apparent Kd values (µM) derived from the Clip34 EMSA binding curves for four TDP-43 constructs — flTDP-43 (n=3), sTDP-43 (n=3), sTDP-43(I281P/L291P) (n=4), and sTDP-43(5G) (n=4) — corresponding to Fig. S8F
• Companion to Fig_S8E_Clip34_EMSA_curves.csv and Fig_6B_Clip34_EMSA_Bmax.csv; Kd values are fit from individual replicate binding curves and plotted to compare RNA-binding affinity across constructs

Fig_S8G_Clip34_EMSA_hill_slope.csv

• CSV file containing Hill slope values derived from the Clip34 EMSA binding curves for flTDP-43, sTDP-43, sTDP-43(I281P/L291P), and sTDP-43(5G), corresponding to Fig. S8G
• Companion to Fig_S8F_Clip34_EMSA_KD.csv; 3 or 4 replicates per construct; Hill slopes are fit per replicate alongside Kd values and assess the cooperativity of RNA binding for each construct

Figure S9

FigS9B.csv

• CSV file containing nuclear/cytoplasmic (N/C) fluorescence ratio measurements for four EGFP-tagged constructs — EGFP, NLS/NES (a nuclear localization signal/nuclear export signal reporter used as a positive control for LMB-induced nuclear accumulation), EGFP-sTDP43, and EGFP-tail — each treated with vehicle or leptomycin B (LMB), corresponding to Fig. S9B
• Eight columns (4 constructs × ±LMB); per-cell N/C ratios are reported with ~30–38 cells per condition, used to assess nuclear export dependence of each construct

Sharing/Access information

Copley et al., " Isoform-specific steric zippers drive aberrant assembly and mislocalization of shortened TDP-43." Science Advances (2026).

Code/software

No custom code or scripts are included in this deposit. Statistical analyses and curve fitting were performed in GraphPad Prism (version 8 or later; GraphPad Software, San Diego, CA). Data were plotted and exported from Prism; CSV files were generated from the corresponding Prism projects. Prism files (.pzfx) require GraphPad Prism version 8 or later to open. CSV files can be opened with any spreadsheet application or text editor.