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A Proline-Rich-Domain-Binding Single Domain Antibody Selectively Inhibits RNA-Induced Phase Separation of Tau
Open accessAbstract
Phase separation mediates the formation of biomolecular condensates, which organize cellular processes such as synaptic plasticity in neurons. The neuronal protein tau undergoes phase separation under specific conditions, regulating synaptic vesicle clustering and microtubule dynamics. In vitro, tau phase separation is induced by cofactors such as polyethylene glycol (PEG) or RNA, mainly via weak multivalent electrostatic interactions. However, the molecular mechanisms governing the formation of tau phase separation, including domain specific contribution, remain unclear. In this study, we used eight single-domain antibodies (VHHs), targeting six distinct short sequences of tau, to explore the mechanisms of tau phase separation in vitro. By combining several biophysical methods, we evaluated the effect of each anti-tau VHH on tau phase separation. With PEG as an inducer, VHHs A31 and Z70 targeting the tau microtubule binding domain enhanced tau phase separation by increasing the droplet size. With RNA as an inducer, the effect of the VHHs was mixed: VHH F8-2 targeting the C-terminal domain promoted condensation, while VHH B1-1, which binds the proline-rich domain (PRD), abolished droplet formation. Nuclear magnetic resonance and surface plasmon resonance spectroscopies confirmed 1 to 1 binding of VHH B1-1 to the PRD, and competition assays with a PRD peptide restored phase separation, demonstrating mechanistic specificity. Our findings provide domain-resolved insights into the regulation of tau phase separation and introduce VHHs as selective probes to modulate biomolecular condensates in physiological and pathological contexts.
Overview
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TL;DR
Eight anti-tau single-domain antibodies (VHHs) were used to probe domain-specific regulation of tau phase separation in vitro. VHH B1-1, binding the tau proline-rich domain, selectively abolished RNA-induced tau droplet formation, while other VHHs enhanced PEG- or RNA-induced condensation.
Key findings
- Under PEG crowding, all anti-tau VHHs increased tau turbidity; VHH Z70, F8-2, and E2-2 increased OD400 more than 1.5-fold (OD400 = 1.84 ± 0.38, 1.73 ± 0.25, and 1.76 ± 0.25, respectively). VHHs A31 and Z70 increased total labeled droplet surface area (37.6 and 40.4 μm²) compared with tau PEG and anti-V5 controls (18.7 and 30.1 μm²).
- Under RNA coacervation, VHH F8-2 enhanced tau droplet formation by 30% (OD400 = 1.30 ± 0.13), whereas VHH B1-1 inhibited it (OD400 = 0.13 ± 0.18). DLS showed RNA-induced tau droplets at Rh ~450 nm, while B1-1 disrupted them to smaller particles (Rh = 24.4 ± 1.9 nm).
- VHH B1-1 bound the tau PRD epitope 221REPKKVAVVRTPPKSPSSAKSRLQT245; SPR with peptide 221REPKKVAVVRTP232 gave KD = 4.3 ± 1.3 μM and stoichiometric ratio SR = 0.7, confirming 1:1 binding. Competition with PRD peptide restored RNA-induced phase separation with EC50 = 4 μM.
- NMR and SPR indicated RNA and VHH B1-1 affected overlapping tau regions in the PRD and R2-R3 repeats, but B1-1 did not directly bind the isolated tau MTBD.
Methods
- Eight VHHs targeting six distinct tau sequences were tested against recombinant tau2N4R. Phase separation was induced by 7% PEG (10 μM tau) or 50 μg/mL RNA polyA (20 μM tau), with VHHs at 1:1 molar ratio unless dose effects were assessed.
- Biophysical assays included OD400 turbidity, phase diagrams, dynamic light scattering, confocal fluorescence microscopy, FRAP, NMR spectroscopy, surface plasmon resonance, yeast two-hybrid epitope mapping, and a heparin-induced tau aggregation assay.
- Main analyses normalized OD400 to tau phase-separation controls, quantified total labeled droplet surface, droplet number and size, and correlated turbidity with microscopy using Pearson correlation (r = 0.85 for PEG; r = 0.79 for RNA).
Limitations
- The paper states that the work is in vitro and that the cellular environment, with complex binding partners, post-translational modifications, and crowding agents, may affect tau phase separation in ways not captured. It also states that whether regulating tau phase separation is a viable therapeutic strategy remains an open question.
- Evident from the text: RNA polyA is a model inducer rather than a specific cellular RNA; the VHH panel covers only six tau regions; RNA-induced microscopy showed more variability than PEG experiments; and the heparin-induced aggregation assay with VHH B1-1 was n = 1 performed in triplicate.
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In the paper
“Detailed quantification analysis revealed that VHHs A31 and mainly Z70 increase the droplet size, rather than their number (Figure S6).”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Anti-tau VHHs A31 and Z70 increase the size of tau-PEG droplets.
plainstates a cause- Asserted: passes — The authors put it forward as true.
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In the paper
“Nuclear magnetic resonance and surface plasmon resonance spectroscopies confirmed 1 to 1 binding of VHH B1-1 to the PRD”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Abstract
plainstates an association- Asserted: passes — The authors put it forward as true.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Adding 300 mM of NaCl inhibited the formation of tau droplets as no larger particles were detected.”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Anti-tau VHHs increase turbidity associated to tau condensation in molecular crowding conditions.
plainstates a cause- Asserted: passes — The authors put it forward as true.
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- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“competition assays with a PRD peptide restored phase separation”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Abstract
plainstates a cause- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Under these conditions, the signal began to increase at 4% PEG and reached a plateau at 12% (Figure 2a).”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Anti-tau VHHs increase turbidity associated to tau condensation in molecular crowding conditions.
plainstates an association- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Production and purification of tau2N4R, tauF3 and tauMTBD fragment
“tauF3 fragment (residues 208-324 of tau2N4R)”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > 221-REPKKVAVVRTPPKSPSSAKSRLQT-245
“free tauF3 fragment, residues 208-334”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > 221-REPKKVAVVRTPPKSPSSAKSRLQT-245
“The Pearson plot shows the total labeled area of the tau droplets versus the normalized optical density values obtained in Figure 2b.”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > 221-REPKKVAVVRTPPKSPSSAKSRLQT-245
“c.The OD400 value corresponding to tau phase separation in the presence of 10 μM tau2N4R and 7% PEG (tau PEG) was normalized to 1.Under the same conditions, tau2N4R was pre-incubated with either 10 μM of an anti-V5 VHH (anti-V5), 10% hexanediol (Hexanediol), 300 mM NaCl (NaCl), or 10 μM of each anti-tau VHH.”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > The binding of anti-tau VHH B1-1 to its proline-rich domain epitope inhibits tau phase separation in complex coacervation conditions.
“comprising 116 amino acid residues instead of 441”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > The binding of anti-tau VHH B1-1 to its proline-rich domain epitope inhibits tau phase separation in complex coacervation conditions.
“residues from 208 to 324 of tau”
Rated minor: it would not touch a result — a value no result rests on, a p-value that stays on the same side of the significance level, or a conflict that is clerical, resolved elsewhere in the paper, or outside its own findings.
In the paper
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > The binding of anti-tau VHH B1-1 to its proline-rich domain epitope inhibits tau phase separation in complex coacervation conditions.
“MCK consists of ten cycles, with each cycle corresponding to the injection of an increasing peptide concentration”
§ A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced phase separation of tau > Characterization of the interaction of VHH B1-1 with tau epitope-derived peptide
“Nine consecutive and increasing half-log concentrations of the tau peptide 221REPKKVAVVRTP232 were sequentially injected at a flow rate of 30 µL/min in a multi-cycle process.”
Citations
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Matplotlib: a 2D graphics environment
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Liquid phase condensation in cell physiology and disease
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Naturally occurring antibodies devoid of light chains
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High mobility of proteins in the mammalian cell nucleus
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Tau protein liquid–liquid phase separation can initiate tau aggregation
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Global Hairpin Folding of Tau in Solution
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Cited by
Caruso, A. C., Kavanagh, T., Waardenberg, A. J., Graham, M. E., Drummond, E., & Nisbet, R. M. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.08.13.665777
Metadata
- Authors
- Thiou, S., Martin, L., Manousaki, E., Nguyen, M., Mortelecque, J., Heidsieck, L., Cantrelle, F.-X., Blum, D., Buée-Scherrer, V., Buée, L., Landrieu, I., Dupré, E., & Danis, C.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Univ. Lille, CNRS, Institut Pasteur de Lille, UMR9031 - BSI - Integrative Structural Biology
- Inserm (FR)
- Univ. Lille, Inserm, CHU-Lille, U1172 - LilNCog - Lille Neuroscience & Cognition
- Corresponding author
- Danis, Clément
- ORCID
- Venue
- ACS Chemical Neuroscience
- Publisher
- American Chemical Society (ACS)
- Published
- 2026-05-08
- Volume
- 17
- Issue
- 10
- Pages
- 1955-1970
- ISSN
- 1948-7193 (ISSN-L)
- OpenAlex
- W7160604819
- Cited by
- 1
- References
- 35
- FWCI
- 0.00
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- ACS Chemical Neuroscience (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (submittedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion, cc-by-nc-nd)
- Funders
- Agence Nationale de la Recherche (ANR-11LABX-01, ANR-18-CE44-0016, ANR-22CE92-0061)
- Alzheimer's Association (T-PEP-23-969176)
- Rainwater Charitable Foundation (T-PEP-23-969176)
- HORIZON EUROPE Marie Sklodowska-Curie Actions (GA101119596)
- Fondation pour la Recherche M?dicale (SMC202405019)
- Association France Alzheimer (6433)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Health Sciences › Medicine › Clinical Neurology › Amyotrophic Lateral Sclerosis Research
- Keywords
- tau protein, phase separation, biomolecular condensates, nanobodies, VHH, proline-rich region, microtubule-binding domain, C-terminal domain, droplet formation, electrostatic interactions, protein-protein binding, neurodegeneration
- MeSH terms
- Protein Domains, Phase Separation, Animals, Humans, Proline, Protein Binding, RNA, tau Proteins, Single-Domain Antibodies
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