RNA-binding proteins (RBPs) containing prion-like domains (PLDs) enter nuclear bodies when specific signaling pathways are inhibited by chemicals.

(a) Two working models of how RBPs with PLDs enter nuclear bodies. In model 1, nuclear bodies recruit RBPs in their native states. In model 2, nuclear bodies sequester RBPs in their soluble misfolded oligomer states. (b) Structure of CuET, domain architecture of TDP-43, and how CuET treatment induces the formation of the TDP-43 granular structures. (c) Immunofluorescence imaging of cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) and treated with CuET for 0, 1 or 3 h using anti-PML (red). Scale bar = 10 µm. (d), (e) Airyscan imaging (d) and intensity profile analysis (e) of cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) and treated with CuET for 1 h. PML nuclear bodies were labeled with anti-PML (red) by immunofluorescence. Scale bar = 5 µm. (f) Structure of AdOx, domain architectures of FUS and TAF15, and how AdOx treatment induces the formation of the FUS and TAF15 granular structures. (g), (h) Immunofluorescence imaging of cells stably expressing either FUS–SNAPf (g) or SNAPf–TAF15 (h) (labeled by SNAP-Cell Oregon Green, green) using anti-NPM1 (red) with or without AdOx pretreatment. Scale bar = 10 µm. (i), (j) Airyscan imaging (i) and intensity profile analysis (j) of cells stably expressing FUS–SNAPf (labeled by SNAP-Cell Oregon Green, green) with AdOx pretreatment. The nucleoli were labeled with anti-NPM1 (red) by immunofluorescence. Scale bar = 5 µm.

Triple-color imaging assay suggests that TDP-43 remains in its native state when initially entering PML nuclear bodies by transient proteotoxic stress, while prolonged proteotoxic stress causes TDP-43 to misfold in the nuclear bodies.

(a) Structures of SNAP-tag substrates used in triple-color imaging assay. (b) In triple-color imaging assay using AggTag method, proteins in native states, soluble oligomer states, and insoluble aggregate states can be distinguished by the activations of P1 and P2 fluorescence. (c) CuET treatment leads to proteotoxic stress in cells. Western blot image showing an accumulation of poly-ubiquitylated proteins upon treatment with CuET. (d) Triple-color imaging assay results for TDP-43 during proteotoxic stress. Confocal fluorescence images of P0 (cyan), P1 (red), and P2 (green) in cells stably expressing SNAPf–TDP-43 upon treatment with CuET for different time periods. Scale bar = 10 µm. (e) Schematic illustration of the triple-color imaging assay result for TDP-43. TDP-43 remains in its native state when initially entering PML nuclear bodies by transient proteotoxic stress, while prolonged proteotoxic stress causes TDP-43 to misfold after TDP-43 enters the nuclear bodies.

Fluorescence lifetime imaging assays using P3 and P4 estimate local micropolarity and microviscosity changes of TDP-43 entering PML nuclear bodies during proteotoxic stress by CuET.

(a) Structure of P3 and its polarity-dependent lifetime. As the local micropolarity decreases, the lifetime of P3 increases. (b) Linear plot of dielectric constant as a function of P3 lifetime. (c) Representative fluorescence lifetime images of P3 in cells stably expressing SNAPf–TDP-43 at different time points of CuET treatment. Scale bar = 10 µm. (d) Dot plot of dielectric constant values of TDP-43 per cell at different time points of CuET treatment. (e) Combined histograms of dielectric constant values of TDP-43 at different time points of CuET treatment. (f) Structure of P4 and its viscosity-dependent lifetime. As the local microviscosity increases, the lifetime of P4 increases. (g) Linear plot of viscosity as a function of P4 lifetime. Y-axis is shown in log scale. (h) Representative fluorescence lifetime images of P4 in cells stably expressing SNAPf–TDP-43 at different time points of CuET treatment. Scale bar = 10 µm. (i) Dot plot of viscosity values of TDP-43 per cell at different time points of CuET treatment. (j) Combined histograms of viscosity values of TDP-43 at different time points of CuET treatment.

Triple-color and fluorescence lifetime imaging assays suggest that FUS and TAF15 enter the nucleolus in their native states upon methyltransferase inhibition by AdOx.

(a) Confocal fluorescence images of P0 (cyan), P1 (red), and P2 (green) in cells stably expressing FUS–SNAPf with or without AdOx pretreatment. Scale bar = 10 µm. (b) Representative fluorescence lifetime images of P3 in cells stably expressing FUS–SNAPf with or without AdOx pretreatment. Scale bar = 10 µm. (c) Dot plot of dielectric constant values of FUS per cell with or without AdOx pretreatment. (d) Combined histograms of dielectric constant values of FUS with or without AdOx pretreatment. (e) Representative fluorescence lifetime images of P4 in cells stably expressing FUS–SNAPf with or without AdOx pretreatment. Scale bar = 10 µm. (f) Dot plot of viscosity values of FUS per cell with or without AdOx pretreatment. (g) Combined histograms of viscosity values of FUS with or without AdOx pretreatment. (h) Confocal fluorescence images of P0 (cyan), P1 (red), and P2 (green) in cells stably expressing SNAPf–TAF15 with or without AdOx pretreatment. Scale bar = 10 µm. (i) Representative fluorescence lifetime images of P3 in cells stably expressing SNAPf–TAF15 with or without AdOx pretreatment. Scale bar = 10 µm. (j) Dot plot of dielectric constant values of TAF15 per cell with or without AdOx pretreatment. (k) Combined histograms of dielectric constant values of TAF15 with or without AdOx pretreatment. (l) Representative fluorescence lifetime images of P4 in cells stably expressing SNAPf–TAF15 with or without AdOx pretreatment. Scale bar = 10 µm. (m) Dot plot of viscosity values of TAF15 per cell with or without AdOx pretreatment. (n) Combined histograms of viscosity values of TAF15 with or without AdOx pretreatment.

Heat shock proteins mediate the localization of TDP-43 into PML nuclear bodies to prevent its degradation under proteotoxic stress.

(a) TDP-43 enters PML nuclear bodies together with HSPA1A upon treatment with CuET. Immunofluorescence imaging of cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) and treated with CuET for 0, 1 or 3 h using anti-HSPA1A (red). Scale bar = 10 µm. (b), (c) Airyscan imaging (b) and intensity profile analysis (c) of cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) and treated with CuET for 1 h. EBFP–HSPA1A (cyan) was co-expressed and PML nuclear bodies were labeled with anti-PML (red) by immunofluorescence. Scale bar = 5 µm. (d) Confocal fluorescence images of cells co-expressing EGFP–TDP-43 (green) and SNAPf–DNAJA1 (labeled by SNAP-Cell TMR Star, red) before and after treatment with CuET. (e) Confocal fluorescence images of cells co-expressing EGFP–TDP-43 (green) and SNAPf–DNAJA2 (labeled by SNAP-Cell TMR Star, red) before and after treatment with CuET. (f), (g) Pulse-chase experiments show that under proteotoxic stress, TDP-43 degrades slower when HSPA1A is overexpressed, and degrades faster when a family of HSP70 is inhibited. (f) In-gel fluorescence image of lysates from cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell TMR Star) upon treatment with CuET for different time periods. Left: control, middle: HSPA1A overexpression, right: HSP70 family inhibition. (g) Quantification of fluorescent band intensity from the pulse-chase experiment.

Proposed models for how RBPs behave under stress.

(a) Upon proteotoxic stress, TDP-43 may enter the PML nuclear bodies in its native state together with heat shock proteins, including HSPA1A and DNAJA1/2. As such, the PML nuclear bodies may play a protective role in preventing the degradation of TDP-43 under transient stress. However, the protective effect may have limited capacity that can be overwhelmed by prolonged stress, resulting in misfolding of TDP-43 within the PML nuclear bodies. (b) Upon inhibition of arginine methylation by AdOx, FUS and TAF15 may enter the nucleolus in their native states.

Synthetic scheme for SNAP-tag substrate P0.

Synthetic scheme for precursor 1.

Synthetic scheme for precursor 2.

Synthetic scheme for precursor 3 and 4, and SNAP-tag substrates P2 and P1.

Synthetic scheme for precursor 6 and SNAP-tag substrates P3.

Synthetic scheme for precursor 8 and 9.

Synthetic scheme for SNAP-tag substrate P4.

Immunofluorescence experiments show that TDP-43 does not enter other nuclear bodies, including nuclear speckles, nuclear stress bodies, nucleoli, and paraspeckles, upon treatment with CuET.

(a) Schematic illustration of the procedure for the immunofluorescence experiments with anti-nuclear body makers (red) for cells stably expressing SNAPf–TDP-43 (green). SNAPf–TDP-43 stable cells were treated with doxycycline (140 ng/mL) and SNAP-Cell Oregon Green (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were further treated with CuET (5 μM) for 3 hours, they were fixed, permeabilized, and stained with the primary antibodies for several different nuclear body markers, and the secondary antibody conjugated with red fluorophore. (b), (c) Immunofluorescence imaging with anti-SC35 (for nuclear speckles), anti-HSF1 (for nuclear stress bodies), anti-NPM1 (for nucleoli), or anti-SFPQ (for paraspeckles) for cells stably expressing SNAPf-TDP-43 (labeled by SNAP-Cell Oregon Green, green) (b) without CuET treatment, or (c) upon treatment with CuET for 3 hours. Scale bar = 10 µm.

Z-stack and Airyscan imaging experiments reveal that PML nuclear bodies reside in the core of the TDP-43 granular structures formed by CuET treatment.

Three-dimensional z-stack reconstruction of Airyscan imaging for the cell stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) upon treatment with CuET for 1 h. PML nuclear bodies were labeled with anti-PML (red) by immunofluorescence. (a) Merged image of blue (DAPI) and green (TDP-43). (b) Merged image of blue (DAPI) and red (PML). (c) Merged image of blue (DAPI), green (TDP-43) and red (PML). The unit of the grid is 3 µm.

Fluorescence recovery after photobleaching experiments indicate that TDP-43 does not exhibit dynamic properties in the granular structures formed by CuET treatment.

(a) Time-course of the fluorescence intensity profile at regions of interest from Oregon Green (TDP-43) channel in cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) upon treatment with CuET for 0.5 h. The error bar represents standard deviation from three independent measurements at each respective time point. (b) Representative time-lapse images of cells stably expressing SNAPf–TDP-43 (labeled by SNAP-Cell Oregon Green, green) with CuET treatment before and after photobleaching. The white arrow indicates the region of interest for the FRAP analysis. Scale bar = 5 µm.

Immunofluorescence experiments show that FUS does not enter other nuclear bodies, including nuclear speckles, nuclear stress bodies, PML nuclear bodies, and paraspeckles, upon pretreatment with AdOx.

(a) Schematic illustration of the procedure for the immunofluorescence experiments with anti-nuclear body makers (red) for cells stably expressing FUS–SNAPf (green). FUS–SNAPf stable cells were pretreated with AdOx (25 μM) or not 3 hours before they were further treated with doxycycline (140 ng/mL) and SNAP-Cell Oregon Green (0.5 μM) for 24 hours for the inducible expression and labeling of FUS–SNAPf, respectively. The cells were then fixed, permeabilized, and stained with the primary antibodies for several different nuclear body markers, and the secondary antibody conjugated with red fluorophore. (b), (c) Immunofluorescence imaging with anti-SC35 (for nuclear speckles), anti-HSF1 (for nuclear stress bodies), anti-PML (for the PML nuclear bodies), or anti-SFPQ (for paraspeckles) for cells stably expressing FUS–SNAPf (labeled by SNAP-Cell Oregon Green, green) (b) without AdOx pretreatment, or (c) with AdOx pretreatment. Scale bar = 10 µm.

Z-stack and Airyscan imaging experiments reveal that FUS resides within the nucleolus when pretreated with AdOx.

Three-dimensional z-stack reconstruction of Airyscan imaging for the cell stably expressing FUS–SNAPf (labeled by SNAP-Cell Oregon Green, green) upon AdOx treatment. Nucleoli were labeled with anti-NPM1 (red) by immunofluorescence. (a) Merged image of blue (DAPI) and green (FUS). (b) Merged image of blue (DAPI) and red (NPM1). (c) Merged image of blue (DAPI), green (FUS) and red (NPM1). The unit of the grid is 3 µm.

Fluorescence recovery after photobleaching experiments indicate that FUS remains diffuse in the nucleolus when pretreated with AdOx.

(a) Time-course of the fluorescence intensity profiles at regions of interest from EGFP (NPM1) and TMR (FUS) channels in cells co-expressing EGFP–NPM1 (green) and FUS– SNAPf (labeled by SNAP-Cell TMR Star, red) with AdOx pretreatment. The error bar represents standard deviation from three independent measurements at each respective time point. (b) Representative time-lapse images of live cells co-expressing EGFP–NPM1 (green) and FUS–SNAPf (labeled by SNAP-Cell TMR Star, red) with AdOx pretreatment before and after photobleaching. The blue arrow indicates the region of interest for the FRAP analysis. Scale bar = 5 µm.

Immunofluorescence experiments show that TAF15 does not enter other nuclear bodies, including nuclear speckles, nuclear stress bodies, PML nuclear bodies, and paraspeckles, upon pretreatment with AdOx.

(a) Schematic illustration of the general experimental procedure for the immunofluorescence imaging with anti-nuclear body makers (red) for cells stably expressing SNAPf–TAF15 (green). SNAPf–TAF15 stable cells were pretreated with AdOx (25 μM) or not 3 hours before they were further treated with doxycycline (140 ng/mL) and SNAP-Cell Oregon Green (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TAF15, respectively. The cells were then fixed, permeabilized, and stained with the primary antibodies for several different nuclear body markers, and the secondary antibody conjugated with red fluorophore. (b), (c) Immunofluorescence imaging with anti-SC35 (for nuclear speckles), anti-HSF1 (for nuclear stress bodies), anti-PML (for the PML nuclear bodies), or anti-SFPQ (for the paraspeckles) for cells stably expressing SNAPf–TAF15 (labeled by SNAP-Cell Oregon Green) (b) without AdOx pretreatment, or (c) with AdOx pretreatment. Scale bar = 10 µm.

Z-stack and Airyscan imaging experiments reveal that TAF15 resides within the nucleolus when pretreated AdOx.

Three-dimensional z-stack reconstruction of Airyscan imaging for the cell stably expressing SNAPf–TAF15 (labeled by SNAP-Cell Oregon Green, green) upon AdOx treatment. The nucleoli were labeled with anti-NPM1 (red) by immunofluorescence. (a) Merged image of blue (DAPI) and green (TAF15). (b) Merged image of blue (DAPI) and red (NPM1). (c) Merged image of blue (DAPI), green (TAF15) and red (NPM1). The unit of the grid is 3 µm.

Photophysical characterization of P1 and P2 shows that they are spectrally orthogonal one another and have distinct viscosity sensitivity.

(a) Structure of P1. (b) Fluorescence excitation spectrum of P1 (5 μM) in glycerol. (c) Fluorescence emission spectrum of P1 (5 μM) in glycerol. (d) The viscosity sensitivity of P1 was determined to be 0.20 from the slope of the linear plot of the logarithm of the fluorescence emission intensity of P1 at 633 nm as a function of logarithm of viscosity. (e) Structure of P2. (f) Fluorescence excitation spectrum of P2 (5 μM) in glycerol. (g) Fluorescence emission spectrum of P2 (5 μM) in glycerol. (h) The viscosity sensitivity of P2 was determined to be 0.54 from the slope of the linear plot of the logarithm of the fluorescence emission intensity of P2 at 524 nm as a function of logarithm of viscosity. All fluorescence was measured by using a Tecan infinite M1000Pro fluorescence microplate reader at room temperature.

Average fluorescence lifetime (τAvInt) of P3 increases as the dielectric constant (ε) of solvent tested decreases.

The fluorescence lifetime images (left panel), lifetime histograms (center panel), and lifetime decay curves (right panel) of P3 in various polar protic solvents with different dielectric constant values. (a) in ethylene glycol, (b) in methanol, (c) in ethanol, (d) isopropanol, (e) n-butanol, (f) tert-butanol.

FLIM measurements of P3 with live cells stably expressing SNAPf–TDP-43 without CuET treatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment 30 minutes.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 30 minutes at 37 °C under CO2 (5%) and subsequently the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 1 hour.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 1 hour at 37 °C under CO2 (5%) and subsequently the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 3 hours.

The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 3 hours at 37 °C under CO2 (5%) and subsequently the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 8 hours.

The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 8 hours at 37 °C under CO2 (5%) and subsequently the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

Confocal and FLIM imaging experiments with insoluble aggregates of P3•SNAPf–TDP-43 conjugate.

(a) DIC and FL imaging, (b) lifetime imaging, (c) lifetime histogram, (d) lifetime decay fitting with insoluble aggregates of P3•SNAPf–TDP-43 conjugate. Scale bar = 10 µm. The insoluble aggregates of P3•SNAPf–TDP-43 conjugate were generated from recombinantly purified SNAPf–TDP-43–TEV–Halo fusion protein. The protein was constructed and purified from E. Coli according to the previously reported procedures.73 For labeling, the fusion protein (10 µM) was incubated with P3 (5 µM) at 37 °C for 30 minutes in HEPES buffer (20 mM, pH 7.5, 140 mM NaCl) containing DTT (1 mM). After labeling, the protein conjugate solution was treated with TEV protease (0.75 µM) and PEG3350 (5%) at room temperature for 1 hour to induce the insoluble aggregates.

Average fluorescence lifetime (τAvInt) of P4 increases as the viscosity (η) of solvent mixtures tested increases.

The fluorescence lifetime images (left panel), lifetime histograms (center panel), and lifetime decay curves (right panel) of P4 in mixtures of glycerol and ethylene glycol with different volume fractions (v/v, %). (a) in 60% glycerol and 40% ethylene glycol, (b) in 70% glycerol and 30% ethylene glycol, (c) in 80% glycerol and 20% ethylene glycol, (d) in 90% glycerol and 10% ethylene glycol, (e) in 100% glycerol.

FLIM measurements of P4 with live cells stably expressing SNAPf–TDP-43 without CuET treatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements, (c), (f), (i) lifetime histograms from the images b, e, and h, respectively, (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 30 minutes.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 30 minutes at 37 °C under CO2 (5%) and subsequently the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 1 hour.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 1 hour at 37 °C under CO2 (5%) and subsequently the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 3 hours.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 3 hours at 37 °C under CO2 (5%) and subsequently the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g),(j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TDP-43 upon CuET treatment for 8 hours.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TDP-43, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the cells were treated with CuET for 8 hours at 37 °C under CO2 (5%) and subsequently the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

Confocal and FLIM imaging experiments with insoluble aggregates of P4•SNAPf–TDP-43 conjugate.

(a) DIC and FL imaging, (b) lifetime imaging, (c) lifetime histogram, (d) lifetime decay fitting with insoluble aggregates of P4•SNAPf–TDP-43 conjugate. Scale bar = 10 µm. The insoluble aggregates of P4•SNAPf–TDP-43 conjugate were generated from recombinantly purified SNAPf–TDP-43–TEV–Halo fusion protein. The protein was constructed and purified from E. Coli according to the previously reported procedures.73 For labeling, the fusion protein (10 µM) was incubated with P4 (5 µM) at 37 °C for 30 minutes in HEPES buffer (20 mM, pH 7.5, 140 mM NaCl) containing DTT (1 mM). After labeling, the protein conjugate solution was treated with TEV protease (0.75 µM) and PEG3350 (5%) at room temperature for 1 hour to induce the insoluble aggregates.

FLIM measurements of P3 with live cells stably expressing FUS–SNAPf without AdOx pretreatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of FUS–SNAPf, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing FUS–SNAPf with AdOx pretreatment.

(a) The stable cells were pretreated with AdOx (25 μM) 3 hours before they were further treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of FUS– SNAPf, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing FUS–SNAPf without AdOx pretreatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of FUS–SNAPf, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing FUS–SNAPf with AdOx pretreatment.

(a) The stable cells were pretreated with AdOx (25 μM) 3 hours before they were further treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of FUS– SNAPf, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TAF15 without AdOx pretreatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TAF15, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P3 with live cells stably expressing SNAPf–TAF15 with AdOx pretreatment.

(a) The stable cells were pretreated with AdOx (25 μM) 3 hours before they were further treated with doxycycline (140 ng/mL) and P3 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf– TAF15, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P3 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TAF15 without AdOx pretreatment.

(a) The stable cells were treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf–TAF15, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.

FLIM measurements of P4 with live cells stably expressing SNAPf–TAF15 with AdOx pretreatment.

(a) The stable cells were pretreated with AdOx (25 μM) 3 hours before they were further treated with doxycycline (140 ng/mL) and P4 (0.5 μM) for 24 hours for the inducible expression and labeling of SNAPf– TAF15, respectively. After the cells were washed with fresh fluorobriteTM DMEM media supplemented with fetal bovine serum (10%) to remove excess probes, the lifetime of P4 was measured using Zeiss LSM 880 microscope with a PicoQuant-FLIM LSM upgrade KIT. (b), (e), (h) lifetime images from three individual measurements. (c), (f), (i) lifetime histograms from the images b, e, and h, respectively. (d), (g), (j) lifetime decay fitting from the images b, e, and h, respectively. Scale bar = 10 µm.