Title: Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates
Abstract: Article17 March 2022Open Access Transparent process Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates Janine Hochmair Janine Hochmair orcid.org/0000-0001-9384-9764 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Visualization, Writing - review & editing Search for more papers by this author Christian Exner Christian Exner orcid.org/0000-0001-9585-4743 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Maximilian Franck Maximilian Franck German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Data curation, Investigation Search for more papers by this author Alvaro Dominguez-Baquero Alvaro Dominguez-Baquero German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Investigation Search for more papers by this author Lisa Diez Lisa Diez orcid.org/0000-0003-3101-0191 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Hévila Brognaro Hévila Brognaro Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Investigation Search for more papers by this author Matthew L Kraushar Matthew L Kraushar orcid.org/0000-0002-7359-0318 Max Planck Institute for Molecular Genetics (MOLGEN), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Thorsten Mielke Thorsten Mielke Max Planck Institute for Molecular Genetics (MOLGEN), Berlin, Germany Contribution: Resources, Investigation, Methodology Search for more papers by this author Helena Radbruch Helena Radbruch Institute for Neuropathology, Charité Berlin, Berlin, Germany Contribution: Resources, Investigation, Visualization Search for more papers by this author Senthilvelrajan Kaniyappan Senthilvelrajan Kaniyappan orcid.org/0000-0002-0606-2769 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn, Bonn, Germany Correction added on 1 June 2022, after first online publication: The author's name has been corrected by providing the full first name. Contribution: Resources Search for more papers by this author Sven Falke Sven Falke orcid.org/0000-0003-3409-1791 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Data curation, Methodology Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow orcid.org/0000-0003-4655-4829 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn, Bonn, Germany Contribution: Conceptualization, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Christian Betzel Christian Betzel orcid.org/0000-0002-3879-5019 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Janine Hochmair Janine Hochmair orcid.org/0000-0001-9384-9764 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Data curation, Formal analysis, Investigation, Visualization, Writing - review & editing Search for more papers by this author Christian Exner Christian Exner orcid.org/0000-0001-9585-4743 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Data curation, Formal analysis, Investigation Search for more papers by this author Maximilian Franck Maximilian Franck German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Data curation, Investigation Search for more papers by this author Alvaro Dominguez-Baquero Alvaro Dominguez-Baquero German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Investigation Search for more papers by this author Lisa Diez Lisa Diez orcid.org/0000-0003-3101-0191 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Hévila Brognaro Hévila Brognaro Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Investigation Search for more papers by this author Matthew L Kraushar Matthew L Kraushar orcid.org/0000-0002-7359-0318 Max Planck Institute for Molecular Genetics (MOLGEN), Berlin, Germany Contribution: Investigation, Methodology Search for more papers by this author Thorsten Mielke Thorsten Mielke Max Planck Institute for Molecular Genetics (MOLGEN), Berlin, Germany Contribution: Resources, Investigation, Methodology Search for more papers by this author Helena Radbruch Helena Radbruch Institute for Neuropathology, Charité Berlin, Berlin, Germany Contribution: Resources, Investigation, Visualization Search for more papers by this author Senthilvelrajan Kaniyappan Senthilvelrajan Kaniyappan orcid.org/0000-0002-0606-2769 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn, Bonn, Germany Correction added on 1 June 2022, after first online publication: The author's name has been corrected by providing the full first name. Contribution: Resources Search for more papers by this author Sven Falke Sven Falke orcid.org/0000-0003-3409-1791 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Data curation, Methodology Search for more papers by this author Eckhard Mandelkow Eckhard Mandelkow orcid.org/0000-0003-4655-4829 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn, Bonn, Germany Contribution: Conceptualization, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Christian Betzel Christian Betzel orcid.org/0000-0002-3879-5019 Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - review & editing Search for more papers by this author Susanne Wegmann Corresponding Author Susanne Wegmann [email protected] orcid.org/0000-0002-5388-2479 German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Janine Hochmair1,†, Christian Exner2,†, Maximilian Franck1, Alvaro Dominguez-Baquero1, Lisa Diez1, Hévila Brognaro2, Matthew L Kraushar3, Thorsten Mielke3, Helena Radbruch4, Senthilvelrajan Kaniyappan5,6, Sven Falke2, Eckhard Mandelkow5,6, Christian Betzel2 and Susanne Wegmann *,1 1German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany 2Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, Hamburg, Germany 3Max Planck Institute for Molecular Genetics (MOLGEN), Berlin, Germany 4Institute for Neuropathology, Charité Berlin, Berlin, Germany 5German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany 6Department of Neurodegenerative Diseases and Geriatric Psychiatry, University of Bonn, Bonn, Germany † These authors contributed equally to this work *Corresponding author. Tel: +49 30450539834; E-mail: [email protected] The EMBO Journal (2022)41:e108882https://doi.org/10.15252/embj.2021108882 See also: VI Wiersma et al (June 2022) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations. SYNOPSIS Condensation of the microtubule-associated protein Tau is induced by the presence of polyanions like RNA or by molecular crowding. Here we show that these conditions have synergistic effects on Tau condensation and Tau-dependent microtubule bundling in vitro, and on the seeding of Tau aggregates in cells. Synergy between molecular crowding and RNA enables Tau condensation at physiological buffer conditions. Phosphorylation and RNA content change Tau-condensate surface wetting, and formation and organization of microtubule bundles. Tau condensates develop seeding potential with time regardless of their physicochemical origin, and even in the absence of percolation Aged Tau condensates seed different cellular Tau accumulations, including condensate-like Tau at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Introduction The intrinsically disorded protein Tau is a neuronal microtubule (MT) binding protein that helps to stabilize axons in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, Tau accumulates intraneuronally and aggregates as neurofibrillary tangles (NFTs) into highly ordered β-structured Tau aggregates, which are a hallmark of the disease and are associated with progressive neuronal death (Braak & Braak, 1991). Tau aggregation and spreading in the brain are thought to involve the seeding of Tau aggregation by small amounts of misfolded Tau (Frost & Diamond, 2010; Dujardin & Hyman, 2019), a mechanism described as “templated misfolding” or “seeded aggregation”. Interestingly, the morphology and molecular content of intraneuronal Tau aggregates in the brain is variable and disease-specific (Ferrer et al, 2014), and also, more subtle forms of Tau accumulations, for example around the nucleus, can occur (Eftekharzadeh et al, 2018; Cornelison et al, 2019). Tau accumulating at the nuclear envelope was shown to inhibit neuronal nucleocytoplasmic transport of proteins (Eftekharzadeh et al, 2018) and RNA (Cornelison et al, 2019), induce changes in nuclear morphology (Sheffield et al, 2006) and lamin folds (Frost et al, 2016), and lead to MTs invading the nucleoplasm (Paonessa et al, 2019). The molecular assembly state of Tau at the nuclear envelope is not known, but it appears that they differ from amyloid-like large cytosolic aggregates since they have never been reported by investigations of Tau pathology using amyloid dyes. Cell models of seeded Tau aggregation can in part recapitulate different types of Tau accumulations (Holmes et al, 2014), whereby the morphology and subcelluar localization of the accumulations is governed by the Tau material used as seeds (Kaufman et al, 2016), and the molecular packing of Tau in various described accumulations seems to differ (Kaniyappan et al, 2020). Tau can have different assembly forms that all display unique biophysical and biochemical characteristics and have different cellular (mis)functions. Monomers and dimers are widely thought to resemble the soluble “native” form of Tau in the cytosol. Tau oligomers seem to encode neurotoxicity (Ward et al, 2012) and are discussed to seed aggregation and mediate Tau pathology spreading between neurons (Gerson & Kayed, 2013). Tau aggregates rich in β-structure are the stable end product of Tau aggregation processes and are deposited in long-lasting neuronal inclusions in the brain (Brion, 1998). In addition, liquid-like protein condensates of Tau have been suggested to be involved in different Tau functions, including the binding and polymerization of MTs (Hernández-Vega et al, 2017; Siahaan et al, 2019; Tan et al, 2019; Zhang et al, 2020) as well as the de novo formation of seeding competent Tau oligomers (Wegmann et al, 2018; Boyko et al, 2020; Kanaan et al, 2020). Liquid-like condensates of Tau form via liquid–liquid phase separation (LLPS), a process that is used by cells to assemble membrane-less organelles of various functions (Hyman et al, 2014; Mitrea & Kriwacki, 2016). From studies in vitro, two different modes of Tau condensation have been identified. Tau LLPS can be initiated through the addition of macromolecular crowding agents like polyethylene glycol (PEG) or dextrane to a dilute Tau protein solution (Ambadipudi et al, 2017; Wegmann et al, 2018). The crowding-induced colloid osmotic pressure (Mitchison, 2019), together with attractive forces between Tau molecules, triggers the de-mixing of Tau into liquid-dense condensates. Crowding-induced biomolecular Tau condensates have been suggested to harbor pathological seeding potential since they can convert—at least in vitro—into oligomeric Tau species with properties similar to in vitro generated Tau aggregates (Wegmann et al, 2018; Boyko et al, 2020; Kanaan et al, 2020). Tau LLPS can also be induced through complex coacervation, which is the spontaneous organization of oppositely charged (parts of) biomolecules into higher order assemblies, for example, liquid-like condensates. This process is driven by electrostatic interactions of the engaging molecules. In the case of Tau, polyanionic RNA (or heparin; Lin et al, 2020) co-condensate with the positively charged Tau microtubule-assembly domain into liquid-like droplets (Zhang et al, 2017; Lin et al, 2019). It is not known whether Tau:RNA coacervates can trigger the formation of pathological Tau aggregates similar to crowding-induced Tau condensates. Notably, high concentrations of RNA do not induce Tau LLPS but can trigger Tau aggregation (Kampers et al, 1996) without the formation of liquid-condensed intermediates (Lin et al, 2020). In cells, Tau coacervation may, for example, be important for the co-condensation of Tau with RNA-containing ribonucleoparticles such as stress granules (Vanderweyde et al, 2016; Ash et al, 2021), as well as for the binding of Tau to the anionic MT surface (Mukrasch et al, 2007; Kadavath et al, 2018). It has been suggested that the binding of Tau to MTs involves both single Tau molecules (Janning et al, 2014) as well as liquid-condensed forms of Tau (Hernández-Vega et al, 2017; Tan et al, 2019; reviewed in Mitchison, 2020). In the following, we will use “condensation” to describe all LLPS processes including those induced by crowding, polyanions, or both. The term “coacervation” refers solely to the condensation of Tau with polyanionic co-factors, in the absence of crowding. In the neuronal cytoplasm, Tau encounters RNA, microtubules, and other polyanions and thus may undergo crowding- and polyanion-induced condensation at the same time. To mimic this scenario, we characterized Tau LLPS in model systems that consider the contribution of both crowding and polyanions. We find that, in fact, molecular crowding is necessary to enable Tau and phosphorylated Tau coacervation with RNA at intracellular ion compositions. The Tau binding partners RNA and tubulin seem to compete for co-condensation with Tau, and the presence of RNA during MT polymerization out of Tau condensates enables pronounced MT bundling and a unique geometric bundle arrangement, likely a result of the high binding affinity (wetting) to negatively charged MT surfaces combined with strong cohesive forces inside of Tau:PEG:RNA condensates. The generation of seeding-competent Tau is similar for Tau condensates with PEG, with RNA, or with both, and all aged Tau condensates convert into similar types of cytosolic and nuclear Tau accumulations, including Tau at the nuclear envelope. Combining FRAP and FLIM of Tau-CFP, we find that different types of Tau accumulations display modes of molecular packing reminiscent of cytosolic and nuclear aggregates versus less densely packed condensates at the nuclear envelope. These accumulations further enhance Tau-induced nucleocytoplasmic transport deficits present in Tau-expressing cells. Interestingly, binary Tau:RNA coacervates promote Tau seed formation in the absence of condensate liquid-to-gel transition, which deviates from the current model of progressive percolation formulated for RNA-binding protein condensates. Collectively, these observations provide a framework to explain the formation, interactions, functions, and pathological activities of different biomolecular Tau condensates and thereby improve the understanding of how condensation contributes to Tau biology and disease-related cellular Tau accumulations. Results RNA and crowding synergize during Tau condensation Tau condensation in vitro can be induced by molecular crowding agents or by polyanions like RNA. The cell has a generally crowded environment, and RNA may be a potent inducer of Tau LLPS in the cytosol. Cellular Tau condensation may thus be influenced by both factors at the same time. To test whether Tau condensates could form in an environment where both RNA and molecular crowding are present, we mixed human full-length wild-type Tau (5 µM Tau, 2N4R isoform, 441 aa, net charge at pH 7.4 = +1.4 (unphosphorylated); Fig 1A) with polyA RNA at charge matching concentrations, and with the molecular crowding agent polyethylene glycol (PEG8000; 5% (w/vol)). Light microscopy confirmed the previously reported formation of liquid-like Tau phases (observed as droplets) in the presence of RNA (Tau:RNA) or PEG (Tau:PEG), as well as in tertiary systems containing both polyA and PEG (Tau:PEG:RNA; Fig 1B). Using fluorescently labeled Tau-Dylight488, polyA-Cy3, and PEG-Dylight647, we observed that Tau and RNA become enriched inside the condensates, whereas the crowding agent PEG is excluded (Fig 1C). Figure 1. Kinetics of Tau condensation and mesoscopic cluster formation Domain structure of the longest human Tau isoform (2N4R) used in this study: the positively charged C-terminal half of Tau contains four repeats (R1-R4) and two proline-rich regions (P1, P2), and comprises the microtubule binding region of Tau. This region binds to polyanionic macromolecules such as RNA through electrostatic interactions. The N-terminal ~200 amino acids (aa) project from the microtubule surface and contain two negatively charged N-terminal inserts (N1, N2). The homogenous charge distribution (sliding window of 10 aa) along its aa sequence assigns Tau an amphiphilic character. Addition of polyA RNA, PEG (PEG8000), or both induces the de-mixing of Tau at physiological concentrations (5 μM Tau in 25 mM HEPES, 1 mM DTT, pH 7.4). Scale bars = 10 μm. Confocal fluorescence microscopy shows co-condensation of polyA-Cy3 (10:1, polyA:polyA-Cy3) with Tau (5 µM Tau with 1% Tau-DyLight488). PEG8000-Dylight647 is excluded from Tau condensates. Scale bar = 10 μm. Representative size distribution of molecular Tau assemblies during Tau LLPS measured by time-resolved DLS (trDLS). At 25 μM Tau in low salt (10 mM NaCl) buffer, trDLS detects Tau monomers (radii ~10 nm) and mesoscopic Tau clusters (radii ~100–200 nm). Addition of PEG, polyA, or both after ~15 min induced the formation of Tau condensates with a radius of >103 nm, at the cost of monomers and mesoscopic clusters in the solution. Data point bubble sizes correspond to DLS amplitudes, which are proportional to the intensity of scattered light of the respective particles. Data shown are the same as in Fig 2D for 0 <t<45 min. Kinetics of Tau:PEG, Tau:RNA, and Tau:PEG:RNA condensate growth assessed through power law, R = a*tb, fitting to trDLS data starting at the time of LLPS inducer addition. Kinetics of cluster growth in low salt (25 mM HEPES, 10 mM NaCl) at 30 μM and 50 μM Tau using the same fitting model as in (E). trDLS of Tau:PEG condensation at increasing NaCl concentrations. Radius of Tau:PEG condensates at different NaCl concentrations. N = 3 experiments, 10 last data points per experiments shown, data shown as mean ± SEM. Radius of Tau clusters at different NaCl concentrations. N = 2–3 experiments, 3 last data points per experiments shown, data shown as mean ± SEM. Radius of Tau monomers at different NaCl concentrations. N = 2–3 experiments, 3 last data points per experiments shown, data shown as mean ± SEM. Model of Tau condensation: Tau monomers (grey circles) and clusters coacervate with RNA (pink) to form condensates that grow through droplet fusion (Oswald ripening). Molecular crowding agents, like PEG (blue), are excluded from the dense phase. Inset: excluded volume effects exerted by PEG force individual Tau molecules to interact, thereby trigger LLPS and stabilize Tau:RNA coacervates against the electrostatic shielding of molecular interactions inside the dense phase. As a result, Tau:PEG:RNA condensates can form at cytosol-like ion concentrations. R0 = radius of hydration. Data information: Data in H, I, J have been compared by one-way ANOVA with Tukey test for multiple comparison. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint To describe the effects of RNA and crowding on the Tau condensation process, we performed time-resolved dynamic light scattering (trDLS), which allows following the evolution and size distribution of particles in a particle radius (R0, radius of hydration) range between 100 and 105 nm in solution over time (Schubert et al, 2017; Falke et al, 2019; Van Lindt et al, 2021). During the first ~15 min of measuring the particle radii in a Tau solution (25 µM for Tau:polyA, 50 µM for Tau:PEG and Tau:PEG:polyA), we observed Tau monomers (RMono~10 nm) and spontaneously formed “mesoscopic” Tau clusters (RClust~100–200 nm; Figs 1D and EV1A and B). Addition of polyA (50 µg/ml) or PEG (5%) induced an immediate increase in particle radius, which could be attributed to the formation of “microscopic” Tau condensates (RCond~1,000 nm), as judged by light microscopy. Simultaneous addition of polyA and PEG induced a similar increase of particle radii in the solution. Notably, the trDLS measurements were recorded in low salt buffer (25 mM HEPES, pH7.4, 10 mM NaCl) to enable Tau:RNA coacervation. In the following, we use the descriptions monomers, clusters, and condensates to refer to the degree of Tau assembling. Click here to expand this figure. Figure EV1. trDLS data of Tau coacervation Averaged experimental autocorrelation function (black circles) and data fit applying CONTIN analysis (red line) of example trDLS data. Similar data was recorded and fitted for each 20 s interval of the trDLS measurements. Distribution of hydrodynamic radii derived from autocorrelation data example in (A). Representative trDLS of Tau condensation upon addition of heparin, tRNA, and polyU RNA. Data point bubble sizes correspond to DLS amplitudes, which are proportional to the intensity of scattered light of the respective particles. Kinetics of condensate growth are assessed through power law, R = a*tb, fitting to trDLS data starting at the time of LLPS inducer addition. Data shown as mean ± SD. A representative single experiment is show. Download figure Download PowerPoint Liquid-like protein condensates grow through the recruitment of monomers from the dilute phase and through droplet coalescence (Ostwald ripening; Lifshitz & Slyozov, 1961; Siggia, 1979; Lee et al, 2021), which can be described by the power law R = a*tb, with R being the condensate radius, t the time after LLPS initiation, and b the coarseningexponent describing the increase in particle sizes (Berry et al, 2018); b ~ 1/3 describes the growth by Ostwald ripening of most biomolecular condensates. The growth kinetics of Tau:PEG:RNA condensates (b = 0.44; R25min: 1167 ± 103 nm) indicated a major contribution of Ostwald ripening during their formation. Binary Tau:RNA and Tau:PEG condensates showed fast initial growth, which then slowed down (polyA: b = 0.15; R25min: 1460 ± 78 nm; PEG: b = 0.18; R25min: 886 ± 78 nm), indicating less contribution of Ostwald ripening compared to the triple LLPS system (Fig 1E). Tau:PEG systems produced the smallest condensates. We also induced Tau coacervation with other polyanions (heparin, tRNA, and polyU RNA) that were previously used to induce Tau LLPS and found that all conditions resulted in similar Tau condensation responses (Fig EV1C). In summary, we found that polyanions and crowding synergize during Tau condensation producing Tau condensates that exhibit growth kinetics indicative of Ostwald ripening (= growth through fusion of condensates) as previously reported for other biomolecular condensates (Van Lindt et al, 2021). In cells, this growth behavior would enable progressive Tau condensation limited by the availability of Tau and/or RNA; this type of Tau condensation could be used as a dynamic response to changes in RNA or protein content or concentration. In contrast, slow condensate growth with limited condensate fusion, for example, as observed for crowding-induced Tau condensates, would rapidly produce Tau condensates of limited and distinct size; this type of condensation could enable a less responsive on/off switch for Tau condensation. Mesoscopic Tau clusters below csat for microscopic condensation The spontaneous formation of microscopic liquid condensates, that is, protein droplets large enough to be identified as such by light microscopy, occurs when the protein concentration exceeds the critical saturation concentration, csat (Shin & Brangwynne, 2017). For Tau, spontaneous microscopic condensation, in the absence of cofactors, was previously observed by light microscopy at concentrations of ~50–100 μM Tau (Ambadipudi et al, 2017; Wegmann et al, 2018). In our trDLS measurements, before the addition of RNA and PEG, we observed the formation of small spontaneous Tau clusters with a radius of ~150–250 nm already at 25–50 µM Tau (Fig 1D). Size and formation kinetics of these clusters depended on the Tau concentration and followed kinetics similar to Tau coacervates, reminiscent of Ostwald ripening (30 µM Tau: R12min = 173.0 ± 10.7 nm, b = 0.30; 50 µM Tau: R12min: 230.8 ± 22.7 nm, b = 0.45; Fig 1F). To test if Tau cluster formation depended on electrostatic interactions between and within Tau molecules, we performed trDLS of Tau condensation at different NaCl concentrations (Fig 1G). The size of Tau:PEG condensates (Fig 1H) and spontaneous Tau clusters (Fig 1I) both decreased with increasing salt concentration. Only few Tau clusters formed at 100 mM NaCl and none at 150 mM NaCl, indicating that spontaneously formed Tau clusters below csat depended on electrostatic interactions between Tau molecules. Notably, monomeric Tau had a radius of 9.6 ± 1.0 nm (mean ± SD) at 30 µM Tau and 10 mM NaCl, consistent with previous reports (Mylonas et al, 2008), which was rather insensitive to increasing ion concentrations (Fig 1J). Tau clusters coexisted with Tau monomers and disappeared upon addition of RNA or PEG to induce microscopic LLPS (Fig 1D and G). We suggest that the amphiphilic character of Tau (Fig 1A) enables the formation of small (=mesoscopic) Tau clusters at c < csat, therefore preceding microscopic LLPS (Fig 1K). In summary, we identified mesoscopic Tau clusters that form spontaneously in solution based on electrostatic interactions and coexist with monomeric T