Live-cell visualization of tau aggregation in human neurons – Communications Biology

Plasmid generation

All protein expression plasmids generated in this work were constructed using restriction enzyme (NEB) digested backbone vectors and PCR-generated DNA fragments (Q5® High-Fidelity Polymerase, NEB) assembled with Gibson Assembly (HiFi DNA Assembly Master Mix, NEB). To generate DNA expression constructs that allow for the option of both transient and stable protein expression and doxycycline-inducible expression, a complete bi-directional, Tet-On® 3G sequence from (pCMV-Tet3G (Clontech) was assembled within 5’ and 3’ PiggyBac transposon-specific inverted terminal repeat sequences (ITR) of the PiggyBac(PB)-CAG-eGFP plasmid using EcoRV and SpeI restriction sites. All plasmids were constructed using the expression vector PB-pCMV-Tet3G (PB-Tet3G) by PCR fragment assembly using Ecor1 and Not1 restriction enzyme sites. An N-terminal truncated version of VVD (residues 37–186, 17.1 kDa) (Addgene #58689) was used for these studies. VVD mutations (I74V, I85V) were introduced by site-directed mutagenesis with Gibson Assembly to generate the VVD (fast) sequence. The addition of a V5 tag sequence to tau (2N4R) and VVD(fast) plasmids was constructed using PCR-generated fragments from 2N4R tau (Addgene #92204) and V5 (Addgene #107596) and inserted into the PB-pCMV-TET3G plasmid to generate tau (tau(2N4R))-V5, and VVD (I74V, I85V (fast))-V5. The following optogenetic plasmids: VVD-Tau(2N4R), Tau(2N4R)-VVD-V5, Cry2olig-Tau(2N4R), and Tau(2N4R)-Cry2olig, were constructed using PCR-generated fragments from the VVD plasmid and cryptochrome 2 (Cry2Olig) (Addgene #60032) plasmids and inserted into the described PB-Tet3G plasmid. Additional optoTAU isoforms (1N4R, 0N4R) were constructed using the original tau (2N4R)-VVD-V5 sequence and two-fragment Gibson assembly to remove one or both N-terminal tau inserts and insert fragments simultaneously into EcoR1 and Not1 sites of the PB-Tet3G plasmid. OptoTAU point mutations (^K280) and (^K280, I277P, I308P) were generated through site-directed mutagenesis with Gibson assembly and inserted into the PB-pCMV-TET3G plasmid. OptoTAU-Halo was generated using a pCMV-HaloTag® plasmid (Gift from Dr. Xin Zhang’s lab) to construct tau (2N4R)-VVD-V5-Halo into the PB-Tet3G plasmid. All plasmids were confirmed by Sanger sequencing (Genewiz).

HEK293 cell culture, transient transfection, and stable cell line generation

HEK293 cells were purchased from (ATCC). The cells were maintained in Dulbecco’s modified eagle medium (DMEM/F12) supplemented with 10% fetal bovine serum at 37 °C and 5% CO₂. On day 1, cells were seeded in six-well plates at 70–80% confluency. On day 2, at ≥90% confluency, cells were transfected with cDNA using X-tremeGENE HP DNA (Millipore Sigma) according to the manufacturer’s instructions. On day 3, the cell culture medium (1.5 ml for 6-well, and 500 µl for 24-well plates) was replaced with phenol-free DMEM/F12 containing 1 μg/ml doxycycline (Millipore Sigma) and placed in a dark incubator for 24 h. On day 4, the cells were incubated in the presence or absence of blue light exposure for the indicated times. For the generation of HEK293 cells that stably express Tau-VVD-V5-Halo, cells were seeded on six-well plates at 80% confluency. The following day, cells were co-transfected with PB-Tet3G-Tau-VVD-V5-Halo plasmid (2 μg DNA; transposon) and the PBase enzyme (transposase) expression plasmid (0.5 μg DNA) at a 4:1 transposon: transposase ratio using X-tremeGENE HP DNA. On the following day, fresh medium containing 5 µg/ml puromycin (Millipore Sigma) was added to the cell culture medium. Once confluent, the cells were trypsinized in 0.25% Trypsin-EDTA (ThermoFisher) and plated in a T75 flask in the presence of 5 μg/ml puromycin selection for up to 2 weeks.

ReNcell culture, differentiation, and stable cell line generation

The ReNcell® VM (Millipore) neural progenitor cell (NPC) line was maintained and differentiated as previously described⁴⁰. Briefly, cells were maintained on Matrigel^TM-coated flasks in proliferation medium (DMEM/F12 supplemented with 1× B27 (ThermoFisher), 2 μg/ml heparin (Millipore Sigma), 20 ng/ml bFGF (Millipore Sigma), and 20 ng/ml hEGF (Millipore Sigma) and filtered through a 0.2-μm PES filter (Fisher Scientific)). For stable expression of PB-Tet3G-Tau-VVD-V5-Halo, and PB-Tet3G-VVD-V5, cells were seeded on six-well Matrigel^TM-coated plates at 80% confluency in proliferation medium. On day 2, cells were co-transfected with appropriate PB-Tet3G plasmid (2 μg DNA) and the PBase (500 μg DNA) using the jetOPTIMUS® (Polyplus transfection) transfection reagent according to the manufacturer’s instructions. On day 3, fresh proliferation medium supplemented with 1 μg/ml puromycin was added to the cell culture medium. When the cells reached confluency, they were treated with Dispase (1U/ml, StemCell Technologies), scraped, and transferred to T75 flasks in proliferation medium supplemented with 1 μg/ml puromycin for 2 weeks.

Differentiation

ReNcell® VM neural progenitor cells were seeded on Matrigel^TM coated 6-well plates, 24-well coverslips, or 24-well glass imaging plates in differentiation medium (DMEM/F12 supplemented with 1× B27 and 2 μg/ml heparin and filtered through a 0.2-μm filter). Cells were allowed to differentiate for 25 days with medium changes every 3 days.

Peptide inhibitor preparation

The previously described W-MINK inhibitor³¹ with the following amino acid sequence (DVWMINKKRK) was synthesized by Genscript with a minimum purity of 90%. The stock peptide was dissolved in sterile DPBS to a final stock concentration of 5 mM, aliquoted for single use, and stored at −20 °C. For each experiment, W-MINK was diluted to 10 μM in phenol red-free medium.

Blue light stimulation

Chronic blue light exposure was performed in 24- or six-well plates using a custom-built LED stage (5 μM, 465 nm) housed in a cell culture incubator (37 °C and 5% CO₂). For multiday Confocal imaging experiments, light-exposed cells were transferred from the cell culture incubator to a preheated (37 °C and 5% CO₂) microscope stage top incubator and allowed to equilibrate for 10 min prior to imaging. For the 16 h time-lapse imaging experiments, a custom-built LED stage (5 μM, 465 nm) designed for the Confocal stage top incubator allowed simultaneous light exposure and imaging. A dark cell culture incubator was used for the control dark condition.

LDH cytotoxicity assay in HEK293 and ReN cells

Cell culture medium was collected from six-well plates after the respective blue light exposure experiment. For each experimental condition, 50 μl of the cell culture medium was added to 3 wells of a 96-well plate to perform the lactate dehydrogenase (LDH) release assay using the CyQUANT™ LDH Cytotoxicity assay kit (ThermoScientific) according to the manufacturer’s instructions. Briefly, 50 μl of the Reaction Mixture was added to each sample. The plate was protected from light and incubated for 30 min at room temperature (RT). In total, 50 μl of Stop Solution was then added to each well. The absorbance was measured at 490 nm and 680 nm using a Synergy microplate reader (Biotek). To determine LDH activity, the 680 nm absorbance value (background signal) was subtracted from the 490 nm absorbance value (LDH signal).

Detergent solubility assay (SarkoSpin)

All detergent solubility assays were performed from HEK293 and ReN cells plated on 6-well dishes. Detergent solubility assays were performed according to the SarkoSpin²⁸ method with minor modifications. For the stable optoTau-Halo HEK293 cell line and transient transfection experiments, cells were washed with RT DPBS following removal of the cell culture medium. The cells were then scraped in 170 µl of RT 1× homogenization-solubilization (HS) buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA,1 mM dithiothreitol, complete EDTA-free protease inhibitors (Roche) and phosphatase inhibitors (Millipore Sigma), and 0.5% Sarkosyl), followed by the addition of a mixture of 2 mM MgCl₂ and 25U (per well) Benzonase (Millipore Sigma) before cell scraping. For sample solubilization, 52 μl 1× HS buffer (no Sarkosyl) and 178 μl 2× HS buffer with 4% Sarkosyl were added to aliquots of the 170 μl lysate for a final concentration of 2% sarkosyl in 400 μl of lysate. Alternatively, for ReNcell cultures, three wells from a six-well plate were combined in a total of 400 μl of lysis buffer. After a 3 min of incubation at RT with vortexing every 10 min, the samples were diluted by adding 200 µl of 1× HS buffer. For fractionation, the lysate was centrifuged at 21,200 × g on a benchtop centrifuge (Eppendorf) for 45 min at RT. Supernatants (soluble fractions) were collected in a fresh tube. The insoluble pellet was washed with 250 µl of wash buffer (1× HS buffer, 1.5% Sarkosyl). The pellet was briefly vortexed and then centrifuged at 21,200 × g for 30 min at RT. The wash buffer was completely removed prior to freezing the insoluble pellet. Both the soluble fraction and insoluble pellet were frozen (−20 °C) prior to western blot analysis.

Western blot analysis

For gel loading, the soluble fractions were mixed with loading sample buffer (4× Bolt™ LDS Sample buffer (ThermoFisher) containing 4% β-mercaptoethanol (BioRad). Insoluble pellets were resuspended in 30 μl of 1× HS buffer containing 0.5% Sarkosyl and 10 μl of loading sample buffer (4× Bolt™ LDS Sample buffer (ThermoScientific). The samples were then heated for 10 min at 70 °C. Soluble and insoluble (20 µl, 50% of total pellet) samples were loaded onto NuPAGE™ 3–8% Tris-Acetate,1.0 mm gels (ThermoFisher). The samples were run at 50 V for 1 h followed by 100 V for 1 h using Novex NuPAGE Tris-Acetate SDS Running Buffer (ThermoFisher). Gels were equilibrated in fresh Towbin transfer buffer (25 mM Tris-base, 192 mM glycine, 20% (v/v) ethanol, 0.05% SDS (pH 8.3)) for 10 min. Protein was then transferred to 0.45 µm nitrocellulose membranes (BioRad) using a wet transfer method (Invitrogen™ Mini Blot Module) at 10 V for 90 min. After the transfer, the membranes were rinsed for 5 min in distilled water, stained with Ponceau S (0.1% (w/v) in 5% acetic acid; Sigma Aldrich) for 5 min, and rinsed with distilled water to remove excess Ponceau S. For total protein measurement, Ponceau S-stained membranes were imaged on an Amersham ImageQuant 800 imager (GE Healthcare). Membranes were then rinsed for 10 min and blocked in 1× TBS (Tris-buffered Saline) + 5% non-fat milk powder for 30 min at RT, and then briefly rinsed with 1× TBS-T (1× TBS + 0.1% Tween 20) and incubated with primary antibodies diluted in 1× TBS-T + 5% milk overnight at 4 °C on a shaker. Membranes were washed with 1× TBS-T (3× 10 min) and then incubated with a species-specific IgG (H + L) HRP-conjugated secondary antibody (BioRad) in 1× TBS-T, 5% milk for 1 h at RT. Membranes are then washed with 1X TBS-T (3× 10 min) and 1× TBS (3× 5 min). Western blots were developed using Western Lightning Plus-ECL (PerkinElmer) or SuperSignal™ West Femto substrate (Fisher Scientific) and imaged on an Amersham ImageQuant 800 imager. Quantification was performed using ImageLab software 6 (BioRad). For both soluble and insoluble fractions, background-adjusted band densities were obtained for the total tau monomer (75 kDa) and the total HMW tau signal (>100 kDa). For soluble fractions, tau protein was normalized to total protein (Ponceau S), and a HMW/Monomer tau ratio was calculated from a single non-overexposed image. All HEK293 data presented was from at least n = 3 independent biological experiments using two technical replicates per experimental condition. All ReN data presented is from at least n = 3 independent biological experiments with three technical replicates/wells pooled together during cell processing.

Immunocytochemical analysis

Subcellular distribution and colocalization of optoTau(2N4R)-V5-Halo with tau antibodies and amyloid dyes were investigated by multi-labeling immunocytochemical analysis of fixed optoTau-V5-Halo expressing HEK293 cells or ReN cells grown on Matrigel^TM-coated coverslips. Following the respective experimental protocol, cells were rinsed with 1× DPBS at RT and then fixed with freshly made 4% paraformaldehyde (PFA) for 15 min at RT. Cells were washed with 1× DPBS (3× 5 min) before incubation with a permeabilization/blocking solution (0.1%Triton X-100, 0.1% Tween 20, 5% normal goat serum in 1× DPBS) for 30 min (3× 10 min) at RT. Cells were incubated with primary antibodies overnight at 4 °C, washed with 1× DPBS (3× 10 min) at RT, followed by incubation of Alexa Fluor-conjugated secondary antibodies for 1 h at RT in the dark. All antibodies were diluted in a permeabilization/blocking solution. After primary and secondary antibody incubations, cells were stained with the amyloid Amytracker 680 dye (1:1000; Ebba Biotech) for 30 min at RT followed by sequential washes with 50% ethanol/1× DPBS (2× 3 min), 30% ethanol/1× DPBS (2× 3 min), 10% ethanol/1× DPBS (3× 1 min), and 1× DPBS (3× 5 min). 4”6’-diamino-2-final-indol (DAPI), diluted in 1× DPBS, was then added to the cells for nuclei counterstaining followed by 1× DPBS washes (3× 5 min). The coverslips were mounted with ProLong® Diamond Antifade mounting medium (ThermoFisher) and stored in the dark. For fixed-cell HaloTag fluorescence imaging, the HaloTag-specific dye, Oregon Green (Promega), was added at 1 µM to phenol-free DMEM/F12 containing 1 μg/ml doxycycline for 24 h before light stimulation followed by cell fixation, antibody/nuclei staining, and mounting as described above.

AggFluor live-cell imaging

OptoTau-V5-Halo expressing HEK293 cells were seeded on Matrigel™–coated 24-well glass-bottom plates at 80% confluency. For the 16 h time-lapse imaging experiments, on the day after plating the cells, the culture medium was removed, and the cells were incubated with DMEM/F12 supplemented with 1 μg/ml Hoechst dye (ThermoFisher) for 10 min. OptoTAU protein expression was then induced by refreshing the cell culture medium with DMEM/F12 containing the following components: 1 μg/ml doxycycline to induce protein expression, 10 µM cytosine β-D-arabinofuranoside (AraC; Millipore Sigma) to mitotically arrest cells, and both Aggfluor probes 1 μM P1h (red) and 1 µM P18h (green). For cells treated with W-MINK inhibitor, an aliquot of the above dye solutions was combined with a 10 μM W-MINK solution and added to cells. Cells were then placed in a dark incubator for 4 h to allow protein expression and optoTau-V5-Halo labeling with Aggfluor probes. Cells were then allowed to equilibrate on the preheated (37 °C and 5% CO₂) stage top incubator for 10 min prior to imaging. Two fields of view were followed for both untreated and treated cells. After the initial imaging of a pre-light timepoint (time = 0 h), cells were exposed to light and imaged every two hours over a 16-h experiment. For multiday imaging, protein expression was induced by replacement with fresh DMEM/F12 containing the following components: 1 μg/ml doxycycline, 10 μM AraC, and both Aggfluor probes 1 μM P1h and P18h or 0.5 μM coumarin (Blue) HaloTag ligand (Promega). For cells treated with the W-MINK inhibitor, an aliquot of the above dye solutions was combined with a 10 μM W-MINK solution and added to cells. Cells were then placed in a dark incubator for 24 h to allow protein expression and optoTau(2N4R)-V5-Halo labeling with Halo-tag ligands. The culture medium was replaced with fresh DMEM/F12 to remove excess probes and placed in a dark incubator for 5 min. Medium was removed, and cells were incubated with DMEM/F12 supplemented with 1 μg/ml Hoechst for 10 min in a dark incubator. Then, the medium was replaced with fresh phenol-free DMEM/F12 containing 1 μg/ml doxycycline and 10 μM AraC and incubated in the dark for 1 h before live-cell imaging to capture pre-blue light conditions. After imaging, cells were treated with light for 24 h after which cells were imaged to capture post-light conditions. After imaging, cells were placed under dark conditions for 24 h and then imaged to capture a post 24 h light/post 24 h dark (aggregate persistence) condition. For stable optoTau-V5-Halo expressing ReN 25-day, differentiated cells had protein expression induced by replacement with fresh differentiation medium containing the following components: 1 μg/ml doxycycline and both Aggfluor probes P1h and P18h or coumarin HaloTag. For cells treated with the W-MINK inhibitor, an aliquot of the above dye solutions was combined with a 10 μM W-MINK solution and added to cells. Thereafter, both ReN and HEK293 cells were treated identically except for the use of the appropriate cell culture medium.

Image acquisition

Confocal images were obtained with a Nikon A1R HD25 confocal microscope using a Galvano scanner. For fixed-cell imaging: n = 6 fields of view per coverslip from at least n = 3 independent biological experiments were taken from cells under either a 40× or 60× oil-immersion objective (either 512 × 512 or 1024 × 1024 frame size; 1.39× zoom; 1.2 Airy units pinhole dimension; unidirectional scanning; Offset unchanged) with appropriate negative controls (dox-free conditions with appropriate combinations either primary/secondary antibodies and Halo dyes). For every image, we performed Z-stacks with a step size of 0.15 μm allowing imaging of an entire layer of cells. Acquisition settings (laser intensity, gain, and offset) were kept constant for all images within a staining group. For live-cell imaging: All live-cell imaging experiments were performed on a Nikon A1R HD25 laser-scanning confocal microscope outfitted with a Tokai HIT stage top incubator using a resonant scanner. Cells were allowed to equilibrate on the preheated (37 °C and 5% CO₂) stage top incubator for 10 min before imaging. For 16 h time-lapse imaging: n = 2 fields of view from n = 4 independent biological experiments were taken from cells under 60× oil-immersion objective (512 × 512 frame size; 1.39× zoom; 1.2 Airy units pinhole dimension; unidirectional scanning; Offset unchanged). For every image, we performed Z-stacks with a step size of 0.3 μm allowing imaging of an entire layer of cells. Acquisition settings (laser intensity, gain, and offset) were kept constant throughout the experiment. For multiday imaging: n = 6–10 fields of view per well (two wells per condition) were taken from cells under the 60× oil-immersion objective. (512 × 512 frame size; 1.39× zoom; 1.4 Airy units pinhole dimension; unidirectional scanning; Offset unchanged). For every image, we performed Z-stacks with a step size of 0.3 μm, allowing imaging of an entire layer of cells. Timing and order of image acquisition were alternated across experiments between experimental groups. Acquisition settings (laser intensity, gain, and offset) were kept constant for all time points during a multiday imaging experiment. For both fixed- and live-cell imaging, representative images of optoTau-V5-Halo expressing HEK293 or ReN cells were acquired with higher resolution settings (Nyquist sampling) using a 60× oil-immersion objective.

Image analysis

Confocal images were post-processed using Nikon 3-D deconvolution, and MaxIP images were further processed and analyzed using Nikon’s NIS-Elements software. Intensity-based analysis was conducted using the General Analysis 3 (GA3) plugin (NIS-Elements). Before any quantification was assessed, all the channels collected within an image were corrected for background subtraction and potential uneven illumination using the “rolling ball” method. The general analysis included the counting of nuclei number (DAPI) per field of view and the total summed intensity (per cell or field of view) for the specific channels being imaged (FITC, TRITC, and Cy5).

Statistical analysis

Statistical significance was calculated with GraphPad Prism software (Version 9.3) and resulting p-values less than or equal to 0.05 were considered significant. No statistical methods were used to predetermine sample sizes. The normality of the datasets was verified with the Shapiro-Wilk normality test and equality of variances with an F test. Otherwise, data distribution was assumed to be normal. Unpaired Student t tests were used to determine statistical significance in datasets comparing two variables. One-way or two-way ANOVA followed by Tukey’s multiple comparison test was used for multiple variables. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For details of statistical analysis, see respective figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s42003-024-06840-z