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Research article
Tau Conformers in FTLD-MAPT Undergo Liquid-liquid Phase Separation and Perturb the Nuclear Envelope
David Westaway
Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB Canada.
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1928-4616
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Germline mutations in the MAPT gene cause some forms of frontotemporal lobar degeneration (FTLD). Recent studies show that a single mutation in MAPT can promote alternative tau misfolding pathways engendering divergent tau conforms and representing clinical heterogeneity, and that under conditions of cell-free molecular crowding the repertoire of tau forms can include liquid-liquid phase separation (LLPS).
Methods: Neuronal nuclear morphologies in FTLD patients and TgTauP301L transgenic mice were analyzed by immunohistochemistry of nuclear lamina. Tau conformers associated with a common behavioral variant of frontotemporal dementia were cloned by endpoint dilution; the cells were assayed for viability and biochemical markers of cell death and were also assessed by video microscopy and photobleaching to determine dynamic aspects of aggregate formation.
Results: Analysis of post-mortem tissues from aged neurologically normal controls and other neurodegenerative syndromes indicated that microtubule-associated nuclear clefts were associated with chronological aging and disruptions of the nuclear envelope with FTLD-MAPT. Tau conformers present in FTLD cases and transduced into reporter cells had a high propensity to condense on the nuclear envelope and to disrupt nuclear-cytoplasmic transport. Nuclear envelope fluorescent tau signals and small fluorescent inclusions in a stable clonal line behaved as a demixed liquid state under live cell conditions; indicative of LLPS effects, these droplets exhibited spherical morphology, fusion events and recovery from photobleaching. While pathogenic mutations in some proteins can interfere with physiological functions of membrane-less organelles, a disease-causing MAPT mutation perturbed nuclear-cytoplasmic transport by gain-of-function formation of LLPS on the nuclear envelope, this acting as a molecular cue to trigger regulated cell death. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 µm2, inferring that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions.
Conclusions: Our findings indicate that within a spectrum of alternative conformers, tau undergoing LLPS is a notably toxic species; demixed droplets on the nuclear envelope hindering nuclear-cytoplasmic transport can serve to trigger cytotoxic pathways and may act as nurseries for the abundant fibrillar structures present at end-stage disease.
Keywords
tauopathy, focal tau pathology, liquid-solid phase transition, nuclear cytoplasmic transport, transgenic mouse
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryMovie11.mp4
Additional file 19: Supplementary Movie 11 Description: Live cell imaging of multinucleated cells containing nuclear envelope tau inclusions (ES1 cells). For FRAP analysis, 1 reference photograph was taken at the beginning and nuclear envelope tau inclusions were photobleached. Time-lapse movies were created by recording photographs for 30 min at one frame every 30 sec (1/30 frame/sec). Scale bar, 10 µm.
- SupplementaryMovie10.mp4
Additional file 18: Supplementary Movie 10 Description: Live cell imaging of nuclear envelope tau inclusions in ES1 cells. For FRAP analysis, 5 reference photographs were taken at the beginning and nuclear envelope tau inclusions were photobleached. Time-lapse movies were created by recording photographs for 30 min at one frame every 30 sec (1/30 frame/sec). Scale bar, 10 µm.
- SupplementaryMovie9.mp4
Additional file 17: Supplementary Movie 9 Description: Live cell imaging of ES1 cells transiently transfected with NCC reporter construct as per Supplementary Movie 8. Time-lapse movies were created by recording photographs for 6 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie8.mp4
Additional file 16: Supplementary Movie 8 Description: Live cell imaging of tau reporter cells (4RD-YFP P301L/V377M) transiently transfected with NCC reporter construct. For FRAP analysis, 5 reference photographs were taken at the beginning and RFP was photobleached. Time-lapse movies were created by recording photographs for 6 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie7.mp4
Additional file 15: Supplementary Movie 7 Description: Live cell imaging of tau reporter cells (4RD-YFP P301L/V377M) as per Supplementary Movie 1. Transiently seeded reporter cells containing nuclear envelope tau inclusions underwent apoptotic cell death. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie6.mp4
Additional file 14: Supplementary Movie 6 Description: Live cell imaging of tau reporter cells (Dox:GFP-0N4R P301L) as per Supplementary Movie 1. Nuclear envelope tau inclusions were transformed into amorphous shapes. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie5.mp4
Additional file 13: Supplementary Movie 5 Description: Live cell imaging of tau reporter cells (4RD-YFP P301L/V377M) as per Supplementary Movie 1. Nuclear envelope tau inclusions were transformed into speckle and then amorphous shapes. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie4.mp4
Additional file 12: Supplementary Movie 4 Description: Live cell imaging of tau reporter cells (4RD-YFP P301L/V377M) as per Supplementary Movie 1. Multinucleated cells emerged by a failure in cell division. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie3.mp4
Additional file 11: Supplementary Movie 3 Description: Live cell imaging of the seeded tau reporter cells (4RD-YFP P301L/V377M) as per Supplementary Movie 1. Tau inclusions were adsorbed by adjacent cells. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie2.mp4
Additional file 10: Supplementary Movie 2 Description: Live cell imaging of tau reporter cells (4RD-YFP P301L/V377M) seeded as per Supplementary Movie 1. Tau inclusions spread through cell division. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- SupplementaryMovie1.mp4
Additional file 9: Supplementary Movie 1 Description: Live cell imaging of the seeded tau reporter cells (4RD-YFP P301L/V377M) seeded with brain homogenate of clinically ill TgTauP301L with CSA Type 2 profiling. Cell-to-cell spread of tau inclusions through membrane nanotubes. Time-lapse movies were created at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- FigS8.tif
Additional file 8: Fig. S8 Condensation of tau-YFP into various inclusion morphologies. a. Transiently tau-seeded reporter cells (4RD-YFP P301L/V377M) as per Fig. 1q showed various inclusion morphologies. b and c. Plot profiling of the tau inclusions. Intensities of tau-YFP signals were measured from four different areas across the tau-positive and negative cells along with the arrows with a length of 100 µm. a.u., arbitrary units; ns, non-seeded cells. Scale bar, 40 µm and 20 µm in the boxed images.
- FigS7.tif
Additional file 7: Fig. S7 Disruption of nuclear-cytoplasmic transport. a and b. Tau reporter cells (a) and ES1 cells (b) were transiently transfected with NCC reporter construct and imaged after 24 hours. Scale bar, 20 µm and 10 µm in the boxed images. c and d. Intensities of green and red fluorescence signals (colored in magenta) in the reporter cells (a) and ES1 cells (b) were measured along with the arrows with a length of 60 µm. e. Tau reporter cells (Ctrl) and ES1 cells transfected with NCC reporter construct were imaged after 24 hours. Cells showing normal nuclear-cytoplasmic compartmentalization were subjected to fluorescence recovery after photobleaching (FRAP) analysis. Scale bar, 20 µm and 10 µm in the boxed images.
- FigS6.tif
Additional file 6: Fig. S6 TEM image analysis of disrupted nuclear envelope. TEM analysis revealed disruption of the double membrane architecture of the nuclear envelope was found in ES1 clonal cells in comparison with control cells (Ctrl). Arrowheads indicate the nuclear envelope. C, cytoplasm; N, nucleoplasm; NR, nucleoplasmic reticulum. Scale bar, 500 nm and 250 nm in the boxed images.
- FigS5.tif
Additional file 5: Fig. S5 Mis-localization of NUP98 with tau inclusions. Tau reporter cells (4RD-YFP P301L/V377M) were seeded with tau and imaged as per Fig. 1q and Fig. 4, respectively. Nucleoporin 98 (NUP98) is one of the most abundant nucleoporins and contains Phe and Gly-rich repeats. In comparison with control cells seeded with non-Tg brain homogenate (a), the mis-localization of NUP98 signals, which surrounded tau inclusions, were observed in the seeded cells (b). Tau in green; NUP98 in magenta; nuclei were counterstained with DAPI (blue). Scale bar, 20 µm and 10 µm in the boxed images.
- FigS4.tif
Additional file 4: Fig. S4 Increase in the level of cleaved Cas-3 with tau inclusions. Tau reporter cells (4RD-YFP P301L/V377M) were transiently seeded tau as per Fig. 1q and performed immunocytochemistry for cleaved Caspase-3 (cCas-3) and Fig. 4. In comparison with control cells seeded with non-Tg brain homogenate (a), the level of cleaved Cas-3 was increased in the tau seeded cells and adjacent cells (b). Tau in green; cleaved Cas-3 in magenta; nuclei were counterstained with DAPI (blue). Scale bar, 20 µm and 10 µm in the boxed images.
- FigS3.tif
Additional file 3: Fig. S3 Heterogeneous tau inclusion morphology and limited proteolytic digestions. a. ES1 cells were re-subcloned by limiting dilution to obtain monoclonal cell populations representing each tau inclusion morphology. All six subclones (1 to 6) showed a heterogeneous phenotype the same as ES1 parental cells seen in Fig. 2a. Scale bar, 10 µm. b to e. To differentiate the protected fibrillar cores of tau in individual cells, the cell lysates (b) were digested using pronase E (c), proteinase K (d), and thermolysin (e), and analyzed by western blot using anti-tau mAbs, ET3 and RD4. The limited proteolytic digestions revealed resistant core peptides in each subline (1 to 6) ranging from 10 to 25 kDa in size, while tau species in the reporter controls (Ctrl, 4RD-YFP P301L/V377M) were completely cleaved. The 10 kDa protease-resistant core appeared in all digestion conditions, and one or two bands between 15 to 20 kDa were shown depending on the enzymes tested. The patterns of the fragmented resistant cores were identical to each other, indicating that ES1 cells as well as the subclones reflect phenotypically similar monoclonal cell populations. f. Morphological changes in tau inclusions. Live-cell imaging revealed that in the seeded reporter cells (4RD-YFP P301L/V377M) as per Fig. S2, tau inclusions underwent morphological changes. Nuclear envelope morphology (TI-2) was turned into speckles (TI-3) and then amorphous (TI-1). The cells were imaged every 10 min for 16 hours. Scale bar, 10 µm. Related to Fig. 3.
- FigS2.tif
Additional file 2: Fig. S2 Heterogeneous morphology of tau inclusions and live-cell imaging analysis. Live cell imaging analysis of the seeded reporter cells (4RD-YFP P301L/V377M). The cells were seeded with tau as per Fig. 1q. a. Cell-to-cell spread of tau inclusions through tunneling nanotube-like protrusion of plasma membrane (both red and yellow arrowheads). b. Tau inclusion-positive cells underwent cell division and produced two daughter cells containing inclusions (yellow cycles). c. Tau inclusions within cellular debris were absorbed by adjacent cells and combined with others, resulting in a bigger inclusion (yellow cycles). d. Multinucleated cells emerged through a failure in cell division (yellow arrowheads). Time-lapse images were collected at 6 days post seeding by recording photographs for 16 hours at one frame every 10 min (1/10 frame/min). Scale bar, 10 µm.
- FigS1.tif
Additional file 1: Fig. S1 Tau accumulation on neuronal nuclear envelopes in vivo and heterogeneous morphology of inclusions in vitro. a. Immunofluorescent staining of phosphorylated tau (magenta, AT8) and lamin B1 (green) revealed that areas of the discontinued nuclear membrane were overlapped with tau deposits (yellow arrowheads) in TgTauP301L. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm and 10 µm in the boxed images. b. Tau reporter cells (Dox:GFP-0N4R P301L) were seeded with brain homogenate of aged TgTauP301L including CSA Type 2 tau conformers and imaged at 6 days post seeding. Diverse tau inclusion (TI) morphologies were observed; a large mass of aggregated tau with no specific pattern (amorphous, TI-1), juxtanuclear and nuclear membrane inclusions (nuclear envelope, TI-2), and threads (TI-4). Multinucleated giant cells (MNGCs) were characterized by thread shaped tau inclusions. Scale bar, 10 µm. c. The 4RD-YFP tau reporter cells (P301L/V377M) were seeded with tau derived from aged TgTauP301L mice as per Fig. 1q. Tau inclusions were visualized using the YFP fusion tag (green) and nuclei were counterstained with DAPI (blue). Cytoplasmic and nuclear localization of various tau inclusion morphologies were confirmed with the nuclear staining. Scale bar, 20 µm and 10 µm in the boxed images.
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Research article
Tau Conformers in FTLD-MAPT Undergo Liquid-liquid Phase Separation and Perturb the Nuclear Envelope
David Westaway
Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB Canada.
Corresponding Author
ORCiD: https://orcid.org/0000-0003-1928-4616
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 28 Sep, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Germline mutations in the MAPT gene cause some forms of frontotemporal lobar degeneration (FTLD). Recent studies show that a single mutation in MAPT can promote alternative tau misfolding pathways engendering divergent tau conforms and representing clinical heterogeneity, and that under conditions of cell-free molecular crowding the repertoire of tau forms can include liquid-liquid phase separation (LLPS).
Methods: Neuronal nuclear morphologies in FTLD patients and TgTauP301L transgenic mice were analyzed by immunohistochemistry of nuclear lamina. Tau conformers associated with a common behavioral variant of frontotemporal dementia were cloned by endpoint dilution; the cells were assayed for viability and biochemical markers of cell death and were also assessed by video microscopy and photobleaching to determine dynamic aspects of aggregate formation.
Results: Analysis of post-mortem tissues from aged neurologically normal controls and other neurodegenerative syndromes indicated that microtubule-associated nuclear clefts were associated with chronological aging and disruptions of the nuclear envelope with FTLD-MAPT. Tau conformers present in FTLD cases and transduced into reporter cells had a high propensity to condense on the nuclear envelope and to disrupt nuclear-cytoplasmic transport. Nuclear envelope fluorescent tau signals and small fluorescent inclusions in a stable clonal line behaved as a demixed liquid state under live cell conditions; indicative of LLPS effects, these droplets exhibited spherical morphology, fusion events and recovery from photobleaching. While pathogenic mutations in some proteins can interfere with physiological functions of membrane-less organelles, a disease-causing MAPT mutation perturbed nuclear-cytoplasmic transport by gain-of-function formation of LLPS on the nuclear envelope, this acting as a molecular cue to trigger regulated cell death. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 µm2, inferring that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions.
Conclusions: Our findings indicate that within a spectrum of alternative conformers, tau undergoing LLPS is a notably toxic species; demixed droplets on the nuclear envelope hindering nuclear-cytoplasmic transport can serve to trigger cytotoxic pathways and may act as nurseries for the abundant fibrillar structures present at end-stage disease.
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