Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

Domain: chemistry
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
The age-dependent transition of metastable, liquid-like protein condensates to amyloid fibrils is an emergent phenomenon in numerous neurodegeneration-linked protein systems. A key question is whether the thermodynamic driving forces underlying phase separation and maturation to amyloid fibrils are distinct and separable. Here, we address this question using an engineered version of microtubule-associated protein Tau, which forms biochemically-active condensates. These metastable protein condensates rapidly convert to amyloid fibrils under quiescent, cofactor-free conditions. In particular, the interfaces of condensates promote fibril nucleation, impairing condensate activity in recruiting tubulin and catalyzing microtubule assembly. Remarkably, a small molecule metabolite, L-arginine, selectively impedes age-dependent amyloid formation in a valence and chemistry-specific manner without perturbing phase separation. By enhancing condensate viscoelasticity, L-arginine counteracts the age-dependent decline in condensate activity. These results provide a proof-of-principle demonstration that small molecule metabolites can enhance the metastability of protein condensates and delay the formation of amyloid fibrils, thereby preserving biochemical function.

Introduction
Protein condensation via phase separation is facilitated by an interplay between chain solvation and multivalent sequence-encoded intermolecular interactions1–6. Typically, protein condensates possess physical properties akin to viscoelastic fluids7–9. In certain protein systems associated with neurodegenerative disorders (ND), including FUS, hnRNPA1, TDP-43, and Tau, phase separation is linked to age-dependent alterations in condensate dynamics, morphology, and structure—a process known as physical aging or maturation10–16. The aging of protein condensates is an emergent property of the system described in recent literature using the framework of a glass transition8, dynamical arrest of the dense phase9, and a fluid-to-amyloid fibril transition17. Importantly, several clinically relevant mutations in ND-linked proteins were found to accelerate the liquid-to-fibril transition of condensates11,12,16,18,19, indicating that protein phase separation may contribute significantly to disease-associated fibrillization20–23. However, an open question remains: Are the thermodynamic driving forces governing protein phase separation and fibrillization related or distinct?

Emerging experimental and computational results suggest that protein condensates are metastable relative to a globally stable solid state9,17,24. This may explain why stress granules hosting multiple aggregation-prone proteins become irreversible with prolonged persistence11,25. It is plausible that active cellular processes and molecular chaperones, previously shown to regulate protein phase behavior26–31, could counteract the age-dependent liquid-to-solid phase transitions of biomolecular condensates. Utilizing sequence engineering approaches for the prion-like low complexity domain of hnRNPA1, it was recently shown that the physical aging of protein condensates is sequence-encoded and partially distinct from the interactions that drive condensate formation24, leading to the postulate that distinct molecular grammars might exist for phase separation and physical aging. This indicates that macromolecules (such as proteins, RNAs, etc.) and/or small molecules that can selectively modulate sequence-specific interactions underlying amyloid formation could, in principle, buffer condensate liquid-to-solid transition independent of protein phase separation (Fig. 1a). Chemical targeting via small molecules has already been shown to modulate protein condensate formation and disassembly26,32–34. With the growing interest in targeting aberrant biomolecular condensates with small molecules35–39, a key focus is on using small-molecule approaches to disentangle the molecular driving forces of phase separation from the physical aging of condensates associated with ND-linked proteins.

Motivated by these open questions, we sought to establish a model protein condensate system guided by the following criteria: (i) the ability to form metastable fluid-like condensates that undergo age-dependent conversion to globally stable solids under quiescent conditions, and (ii) condensates possess a tractable biochemical activity, enabling direct assessment of how physical aging affects condensate function. Establishing this benchmark will help differentiate between selectively disrupting condensate aging and broadly perturbing condensate formation, which could impact their biochemical activity. ND-linked proteins such as α-synuclein, FUS, and hnRNPA1 form condensates that undergo aging to fibrillar solids12,15,17,40; however, the biochemical activities of condensates formed by these protein systems have not been established yet. Conversely, the microtubule-associated protein Tau, associated with a class of NDs called Tauopathies41–45, is known to form biochemically active condensates capable of microtubule (MT) assembly and stabilization implicated in intracellular trafficking in post-mitotic neurons46,47. Importantly, multiple studies have hypothesized a link between the pathological cascade of Tau and Tau condensation, as pathogenic mutations have been shown to accelerate Tau condensate maturation48–52. These important features provide a case for Tau as our model protein system. However, the aging of wild-type Tau condensates to amyloid fibrils is a kinetically sluggish process (Supplementary Fig. 1), hallmarked by the incredibly long timescale required for Tau aggregation and prion-like spread in vitro and in vivo53,54. Tau is also well known for being highly soluble and recalcitrant to aggregation in vitro, often attributed to its hydrophilic nature44,55. To enable Tau fibrillization within an experimentally accessible timescale, previous studies have used physicochemical perturbations, including negatively charged cofactors like heparin, shear stress, and modifications to the native protein, such as truncations or phosphorylation49,51,56,57. These strategies impose unintended effects on Tau condensation and their activity in MT assembly46,58, independent of condensate aging.

Here, we use SynTag-Tau, which comprises the full-length Tau protein (2N4R isoform) and a 40-amino-acid prionogenic N-terminal tag. Serendipitously, we found that this synthetic prion-like low-complexity sequence substantially reduces the barrier to nucleation of Tau condensates into fibrils. SynTag-Tau undergoes molecular crowding-dependent phase separation in vitro to form biochemically active liquid-like condensates, similar to the untagged wild-type Tau protein49. Importantly, unlike the untagged protein, SynTag-Tau condensates undergo physical aging to form mesoscale amyloid fibrils in the timescale of hours under quiescent conditions and in the absence of polyanionic cofactors, shear stress, or perturbations to the core microtubule-binding domain. Using the SynTag-Tau system, we uncovered that conditions that promote protein phase separation commensurately accelerate condensate aging to amyloid fibrils. SynTag-Tau condensate interface nucleates fibril formation, yielding a disorder-to-order transition with gradual changes in the condensate’s dynamical and structural properties. These age-dependent transitions in SynTag-Tau condensate physical properties accompany a progressive loss of biochemical activity in tubulin recruitment and catalyzing MT assembly. Remarkably, phase separation and physical aging in this system are decoupled by a naturally occurring small molecule metabolite abundantly present in the intracellular milieu, L-arginine (L-Arg). Our experiments show that the partitioning of L-Arg into SynTag-Tau condensates prevents age-dependent changes in the condensate microenvironment, leading to an inhibition of fibril formation. Instead, it facilitates intra-condensate percolation transitions that enhance condensate metastability. L-Arg treatment of SynTag-Tau condensates preserves their activity in MT assembly. Finally, we show that L-Arg counteracts the condensate-to-amyloid conversion of wild-type Tau similar to that of SynTag-Tau. Overall, this proof-of-principle study demonstrates that small molecule metabolites can stabilize protein condensates, preventing their transition from a liquid state to amyloid fibrils and thereby maintaining their function.

Results
Physical aging of SynTag-Tau condensates leads to the formation of ordered amyloid fibrils
Wild-type (WT) Tau form condensates in physiologically relevant buffer conditions in vitro. These condensates are biochemically active46, but they do not transition to amyloid fibrils under quiescent conditions, thereby limiting our investigations (Supplementary Fig. 1a,b). Recent reports employing a protein sequence engineering strategy have enabled probing the interplay between phase separation and amyloid formation on an experimentally accessible timescale and resolution that is otherwise difficult to probe59,60. These approaches exploit the principle that in many amyloid-forming protein systems, fibril formation is constrained by high nucleation barriers, which dictate the probability and kinetics of self-assembly61. Inspired by this phenomenon, we sought to design a sequence-engineered variant of Tau that selectively lowers the nucleation barrier for amyloid formation while preserving its intrinsic phase separation behavior and MT assembly function. Previous reports indicate that the fibrillar self-assembly of amyloidogenic proteins can be reinforced by fusing to a compact prionogenic low-complexity sequence62–64. Fusion to prion-like sequences flanking an amyloid-forming protein can act as soluble entropic bristles that favor the prion-like spread of protein aggregates without directly contributing to the primary nucleation mediated by the amyloid core65. In the current study, we adapted the strategy of adding a prion-like low-complexity sequence as an N-terminal tag to the Tau protein (Fig. 1b). We synthesized SynTag-Tau, which consists of the full-length Tau protein (2N4R isoform) with an amino-terminal tag containing a synthetic prionogenic sequence of 40 amino acid residues66,67 (Fig. 1b; Supplementary Fig. 2; Supplementary Table 1). The design of this specific tag is serendipitous. Alphafold368 predicted structure of SynTag-Tau indicates that this prion-like tag and most of the protein are intrinsically disordered, similar to that of the untagged Tau protein (Fig. 1c; Supplementary Fig. 3).

We first set out to characterize the formation and maturation of SynTag-Tau condensates utilizing a previously reported protocol of in vitro reconstitution in 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, 2 mM DTT, and a molecular crowder (10% PEG 8000)49,52. We tracked age-dependent morphological changes of SynTag-Tau condensates, doped with Atto488 (A488)-conjugated SynTag-Tau, by laser scanning confocal fluorescence microscopy (Fig. 1d). Within 2 to 3 hours (h or hr) after sample preparation, we observed fibrils from periphery of SynTag-Tau condensates growing in a heterogeneous manner—visible fibrils originated from some condensates but not all. The fibrils nucleated from one condensate grow into the dilute phase and attach to neighboring condensates, inducing further nucleation and propagation of fibrillar assemblies (Fig. 1d; Supplementary Video 1). This observation appears to be consistent with a recent fibril growth model in the presence of condensates24,69, where the growth of fibrils occurs in the dilute phase. Such a process engenders an efflux-generated gradient of protein molecules from nearby condensates, thereby creating a network of fibrils with condensates serving as nodes. Shuffling the primary sequence of the prionogenic tag without altering the overall sequence composition in a way that lowers the PLAAC score abrogates condensate aging to fibrils (Supplementary Fig. 4). Truncation of the tag sequence to limit it to the portion with the highest weighting in PLAAC score also renders the protein unable to trigger condensate-to-fibril transition (Supplementary Fig. 5). Thus, the specific composition and patterning of this synthetic prion-like tag sequence appear to be critical for lowering the nucleation barrier for the condensate-to-fibril conversion, assuming that the thermodynamic stability of fibrils remain unchanged (Fig. 1e). As a further validation, we sought to determine whether the SynTag could serve as the primary driver of SynTag-Tau fibril formation. ZipperDB70 analysis revealed that in comparison to the native Tau sequence, the putative steric zippers in SynTag are outweighed in absolute score (Rosetta energy expressed as kcal/mol) and frequency, i.e., number of steric zippers (Supplementary Fig. 6). This analysis suggests that the primary steric zippers capable of driving fibril nucleation likely originate from the native Tau sequence. However, without structural studies, we cannot rule out the possibility that the putative zipper motifs in SynTag act as auxiliary steric zippers that facilitate amyloid fibril formation.

We next employed in situ second-harmonic generation (SHG), a non-linear label-free optical phenomenon ideal for probing non-centrosymmetric structures with molecular ordering71. We observed that SynTag-Tau fibrils, but not SynTag-Tau condensates, contain spatially ordered structures (Fig. 1f). This bears a resemblance to the SHG signal originating from bona fide amyloid fibrils observed in the brain of a transgenic model of Alzheimer’s72. To evaluate whether these ordered fibrils nucleated by condensates are indeed enriched in cross-β-sheet structures, we employed an amyloid-sensitive dye, Thioflavin T (ThT). SynTag-Tau fibrils but not nascent SynTag-Tau condensates showed detectable ThT fluorescence (Fig. 1g). To probe the condensate-to-fibril transition in further detail, we measured the (a) timescale for droplet relaxation using a dual-trap optical tweezer, and (b) molecular mobility using fluorescence recovery after photobleaching (FRAP). We observed that the SynTag-Tau condensates undergo an age-dependent dynamical arrest, hallmarked by progressively slower condensate fusion and slower FRAP recovery, prior to the formation of visible amyloid fibrils in the mesoscale (Fig. 1h,i). At the molecular level, we hypothesized that a disorder-to-order conformational change of SynTag-Tau is likely to aid in dynamical arrest and concomitant fibrillization. To obtain an age-dependent structural fingerprint of SynTag-Tau molecules in the dense phase, we employed in situ spatially resolved broadband coherent anti-Stokes Raman scattering (BCARS)73, which is a label-free hyperspectral Raman imaging technique. Intra-condensate BCARS measurements revealed a progressive increase in the β-sheet content with condensate age, ultimately leading to amyloid fibrils that are predominantly enriched in β-sheet conformers (Fig. 1j; Supplementary Table 2).

So far, we have established that SynTag-Tau condensates convert from a fluid-like phase to fibrils, enriched in β-sheet structures, growing in the dilute phase. Next, we asked whether these two processes are correlated, meaning how perturbing SynTag-Tau condensate formation affects SynTag-Tau fibrillization. Previous literature suggests that electrostatic interactions play a dominant role in Tau phase separation56,74,75, which can be modulated by tuning the ionic strength of the sample buffer. We observed that increasing ionic strength diminishes SynTag-Tau phase separation (measured by condensate area fraction). Interestingly, the kinetics of SynTag-Tau fibrillization decrease in a correlated manner (Supplementary Fig. 7a,b). Further, we tested this interplay by titrating the molecular crowder concentration, wherein high [PEG 8000] favors protein phase separation propensity via depletion forces76. Upon increasing the concentration of the crowder, we find that SynTag-Tau phase separation is enhanced, judged by an increase in condensate area fraction, and concomitantly, the timescale for condensate-to-fibril conversion is reduced (Supplementary Fig. 7c). Taken together, these observations suggest that promoting SynTag-Tau condensation enhances fibrillization. Although the insights gained so far seemingly indicate an interplay between phase separation and fibril formation, a key question is how condensate physical aging impacts the function of SynTag-Tau condensates in tubulin recruitment and MT assembly.

Condensate physical aging impairs biochemical activity
We utilized an in vitro tubulin recruitment and MT assembly assay46 (Fig. 2a) to quantify the impact of SynTag-Tau condensate aging on biochemical activity. We first tested tubulin partitioning in the absence of GTP, both in nascent and aged SynTag-Tau condensates. We find that the nascent condensates can recruit tubulin (labeled with HiLyte647) with homogeneous distribution throughout dense phase (Fig. 2b; Supplementary Fig. 8). Contrastingly, tubulin partitioning was attenuated in aged SynTag-Tau condensates with tubulin molecules mostly concentrating close to the interface (Fig. 2c; Supplementary Fig. 8). Next, we tested the age-dependent activity of SynTag-Tau condensates using in vitro MT polymerization assay (experimental schematic in Fig. 2a). With increasing condensate age (time=Δt), we observed a gradual reduction in MT polymerization capacity (Fig. 2d,e; Supplementary Figs. 9–11). By the 16-h time point, the ability of SynTag-Tau condensates to drive MT polymerization ceases completely. Consistently, tubulin partitioning experiments conducted using similar assay conditions as the MT polymerization assay revealed increasingly heterogeneous tubulin partitioning at later time points, which corresponds to loss of activity (Supplementary Fig. 12; Fig. 2e). The ineffective partitioning of tubulin to condensates at later ages potentially indicates that either the characteristic mesh size progressively declines with physical aging or that the fibril formation occurring at the condensate interface blocks tubulin partitioning77,78. Nevertheless, it is worth pointing out that at the 8-h time point, despite observing a noticeable drop in MT assembly, we do not observe a dramatic blockade of tubulin partitioning via fluorescence microscopy, which might indicate that aging-induced changes in material state, as well as perturbed tubulin binding by conformationally altered SynTag-Tau within aged condensates, might underscore condensate loss-of-activity. Overall, the loss of activity of SynTag-Tau condensates with age demonstrates that the condensate material properties affect tubulin recruitment and MT assembly. Similar attenuation of SynTag-Tau condensate activity was observed (Supplementary Fig. 13) when we increased the molecular crowder concentration from 5% to either 7.5% or 10%, which accelerates SynTag-Tau condensate-to-fibril transition (Supplementary Fig. 7c) and was previously shown to modulate condensate material properties76.

Recent reports suggest that the interface of protein condensates plays a critical role in nucleating the liquid-to-amyloid transition17,79,80. This is presumed to stem from the unique conformational ensemble of the protein molecules at the condensate interface, in contrast to that in the dilute or dense phases81. Therefore, we asked whether SynTag-Tau condensate interfaces play a similar role in fibril formation. To address this, we employed frequency-domain fluorescence lifetime imaging (FLIM) with Atto488-labeled SynTag-Tau. FLIM is a highly sensitive imaging technique that reports on the spatial heterogeneity in the molecular microenvironment of a fluorescent probe. In our experiment, we measured the lifetime of the Atto488-labeled SynTag-Tau to interrogate the local environment of protein molecules within the condensate as they undergo age-dependent morphological transitions. We find that there is a distinct lifetime signature of SynTag-Tau molecules on the interface of the condensates at an early age (Fig. 2f,g). The lifetimes of SynTag-Tau molecules at the condensate interface are longer than those of the interior, closer to the lifetimes of the fibrils (Fig. 2h). These data suggest that SynTag-Tau condensate interfaces nucleate amyloid formation. We infer that the altered tubulin partitioning in nascent versus aged protein condensates (Fig. 2b,c; Supplementary Fig. 12) is due to the physical aging of SynTag-Tau condensates that originates at the interface (Fig. 2i). Disabling and/or delaying the fluid-to-fibril transition of protein condensates could potentially protect against condensate loss-of-activity.

Naturally occurring small molecule metabolites modulate Tau condensate physical aging
Identifying small molecule inhibitors of protein fibrillization has been an active area of research for over two decades. In the context of phase separation, small molecules, such as 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) and adenosine triphosphate (ATP), were shown recently to modulate the phase behavior of ND-linked proteins26,32. These studies and mechanistic perspectives gleaned by quantitative frameworks such as polyphasic linkage82 and heterotypic buffering83,84 have postulated that tuning condensate physical properties may prevent their aberrant behavior. Motivated by this perspective, we set out to test whether SynTag-Tau condensate maturation timescale can be tuned independently of SynTag-Tau phase separation through small molecule treatment. We first tested generic chemical disruptors of protein-protein interactions and naturally occurring small molecule metabolites at low millimolar concentrations. Remarkably, we found that physiologically abundant cationic amino acids, L-arginine (L-Arg) and L-lysine (L-Lys), inhibit SynTag-Tau condensate-to-fibril transition without perturbing SynTag-Tau phase separation (Fig. 3a). The concentrations at which L-Arg showed such activity are similar to its physiologically relevant concentrations85,86. In the presence of anionic amino acids such as L-glutamic acid (L-Glu) and L-aspartic acid (L-Asp), however, the appearance of SynTag-Tau fibrils seemed to accelerate. The observed tunability of SynTag-Tau liquid-to-fibril transition by these small molecule metabolites is likely owed to their charge and the side chain chemistry as L-proline (L-Pro), which cannot participate in electrostatic interactions, failed to show any modulatory effect (Fig. 3a). The inhibitory effect of L-Arg and L-Lys was also not recapitulated by small molecules with pleiotropic effects on protein-protein interactions, such as those that disrupt protein backbone hydrogen bonding interactions, including guanidinium hydrochloride (GnHCl) and urea (Fig. 3a). The chemical chaperone trimethylamine N-oxide (TMAO) previously reported to selectively disfavor fibril conversion of TDP-43 condensates27 was unable to prevent SynTag-Tau phase separation coupled fibrillization (Fig. 3a). Conversely, ATP appears to hinder fibril formation, however, the condensate size and morphology are substantially affected (Fig. 3a).

We next turned our attention to L-Arg to determine its dose-dependent efficacy in perturbing SynTag-Tau fibrillization (Fig. 3b). At the lowest L-Arg concentration tested (1 mM), the partition coefficient of Atto488-labeled SynTag-Tau molecules in the dense phase is higher compared to the untreated condition. However, increasing the L-Arg concentration to 2 mM or 4 mM did not dramatically alter the partition coefficient relative to the untreated condition (Fig. 3b,c). This data suggests that SynTag-Tau phase separation is not fully inhibited by 1 mM to 4 mM L-Arg. Notably, at 2 mM L-Arg concentration, SynTag-Tau condensate size and morphology looked similar to those of the untreated condition, but the timescale of fluid-to-fibril transition shifted from 8 h to 7 days (Fig. 3b,c). At 4 mM L-Arg, however, we observed some differences in SynTag-Tau condensate size and surface wetting behavior compared to the untreated condition (Fig. 3b,c) despite the SynTag-Tau partition coefficient being largely similar. In order to ascertain whether the reduced condensate sizes stem from either altered condensate coarsening dynamics, inhibition of SynTag-Tau phase separation or both, we conducted a quantitative analysis of condensate area fraction using confocal fluorescence microscopy (Supplementary Fig. 14). We found that L-Arg concentrations of 1 mM and 2 mM yielded condensate sizes that are not significantly different than that of the untreated condition (0 mM). However, in the case of 4 mM L-Arg treatment, we observe a significantly reduced condensate area fraction, indicating that L-Arg at high concentrations (above 2 mM) inhibits SynTag-Tau phase separation and potentially affects condensate coarsening. The selective inhibition of fibrillization without abrogating condensate formation reveals a distinction in the molecular driving forces underlying these two processes, indicating that phase separation and fibrillization are biochemically separable. This decoupling phenomenon is distinct from global perturbation to condensate formation, as seen with the effect of modulating ionic strength or molecular crowding (Supplementary Fig. 7).

Next, we probed the partitioning behavior of L-Arg in SynTag-Tau condensates utilizing α-dansyl-L-arginine (dansyl-L-Arg), a fluorescent analog of L-Arg (Fig. 3d). Using dual-color fluorescence microscopy with SynTag-Tau condensates visualized with Alexa594-conjugated protein, we find favorable partitioning with a partition coefficient, k = ~4, of dansyl-L-Arg within SynTag-Tau condensates (Fig. 3e). This partition coefficient value is consistent with the known partition coefficients of similar small molecule metabolites in condensates39. The favorable partitioning of dansyl-L-Arg is not due to the dansyl group, as partitioning of dansyl chloride (dansyl-Cl) to SynTag-Tau condensates is markedly weaker (k = ~1.5) (Fig. 3d,e).

To investigate how the inhibitory effect of L-Arg on SynTag-Tau condensate aging is related to its chemical properties, we tested L-Arg ethyl ester (L-Arg EE) and observed no impact on SynTag-Tau condensate aging (Fig. 3f). Experiments with D-arginine (D-Arg) yielded similar results as L-Arg, revealing that the stereospecificity of arginine is not critical to its inhibitory activity (Fig. 3f). Next, we tested whether multivalent polymers of lysine or arginine as well as charged polyamines such as spermine and spermidine could impose an enhanced inhibitory potential to condensate-to-fibril transformation relative to small molecules (L-Arg/L-Lys). Intriguingly, treating condensates with multivalent cationic molecules did not offer any improvement in fibrillization timescale relative to the small molecule treatment (Supplementary Fig. 15; Fig. 3a). Given the observation that the positive hits in the small molecule screen are basic amino acids with chemical moieties of high pKa (>7), L-Lys (pKa of ε-amino group = 10.5), and L-Arg (pKa of guanidino group = 12.4), we wondered at the possibility that high pKa could be an important chemical property that confers protection against condensate-to-fibril transition. However, the other small molecules with basic chemical moieties failed to achieve a similar effect. For example, guanidinium hydrochloride (pKa of guanidinium group = 12.6), L-arginine ethyl ester (pKa of guanidino group = 12.4), as well as spermine and spermidine (pKa across multiple amine groups = ~10.7). Despite sharing highly basic, i.e., high pKa, chemical moieties, several of these compounds failed to inhibit SynTag-Tau fibrillization selectively (Fig. 3a; Supplementary Fig. 15), which suggests that strongly basic functional groups alone are a poor predictor of inhibitory potential. Therefore, we conclude that the observed inhibitory activity of L-Arg at its physiological concentration is chemistry-, valence-, and charge-specific but does not depend on chirality.

L-Arg prevents cross β-sheet formation and nucleation of fibrils at the condensate interface
Given that L-Arg can prevent age-dependent changes in SynTag-Tau condensate morphology (Fig. 3), we next sought to probe its effect on the structure and dynamics of SynTag-Tau condensates. Using the ThT fluorescence assay as a function of time, we observed that the age-dependent increase in the signal originating from amyloid fibrils is notably less pronounced for condensates in the presence of L-Arg (Fig. 4a). This observation suggests that L-Arg inhibits conformational conversion of disordered SynTag-Tau molecules to cross-β-rich assemblies, preventing nucleation of amyloid fibrils. One caveat of imaging condensates at a single plane using ThT fluorescence is the lack of spatial information in the z-axis, which would be critical for resolving whether fibrils are indeed nucleated at condensate interfaces or localize to condensate interiors. We tested this by capturing z-slices and measuring A594-labeled SynTag-Tau and ThT fluorescence in untreated SynTag-Tau condensates. We find that ThT fluorescence is preferentially enriched at condensate interfaces with amyloid fibrils growing into the dilute phase, but not condensate interiors (Fig. 4b). At a relatively higher z position (0.95 μm), i.e., approaching the topmost interface of condensates (with respect to the coverslip surface), the ThT signal originating from fibrils becomes more prominent, in line with interfacial nucleation of fibrils. This is consistent with the recently proposed framework that condensate interiors could act as sinks of amyloidogenic proteins; however, their interfaces present the risk of fibril nucleation, with fibril growth occurring outward into the dilute phase24,69. On the other hand, L-Arg-treated SynTag-Tau condensates fail to show any notable ThT fluorescence across z-sections (Fig. 4c).

For further examination, we employed BCARS hyperspectral imaging and observed that the amide I band of SynTag-Tau in nascent condensates is fairly similar in either absence or presence of L-Arg (Fig. 4d,e; Supplementary Table 2). However, the amide I band of aged SynTag-Tau condensates treated with 2 mM L-Arg appears distinct from that of untreated condensates. The most dramatic difference is the decrement in the mean population of β-sheet conformers in L-Arg-treated condensates (39.3%) versus the untreated condition (47.2%). We also note a moderate increase in non-β conformations in small molecule-treated condensates relative to untreated condensates, namely random coil (37.4% vs. 32.9%, respectively) and α-helix (23.3% vs. 19.9%, respectively) (Fig. 4e). This may suggest an exciting possibility that L-Arg might slow down the SynTag-Tau condensate liquid-to-fibril conversion by populating alternative conformational states of SynTag-Tau. Follow-up work is necessary to explore this possibility further.

To probe the spatially resolved SynTag-Tau condensate microenvironment with age in the presence of L-Arg, we performed FLIM measurements using an identical approach as outlined for Fig. 2f. When comparing with the SynTag-Tau condensates without L-Arg, we made two key observations. The first is that the lifetime of fluorescently labeled SynTag-Tau molecules does not change over the same timescale, and the interface of these condensates appears similar in terms of the fluorescence lifetime with respect to the SynTag-Tau molecules at the condensate core (Fig. 4f,g). These data collectively suggest that the mesoscale changes in the SynTag-Tau condensate morphology with age are inhibited by L-Arg. Finally, we tested the efficacy of L-Arg in the heparin-induced maturation of WT Tau condensates into amyloid fibrils. L-Arg proved effective in this in vitro model of condensate aging as well, indicating that the selective inhibitory potential of L-Arg against condensate-to-fibril transition does not stem from interactions between L-Arg and the synthetic prionogenic tag in SynTag-Tau but potentially via frustration of the interactions mediated by the zipper motifs in Tau (Supplementary Fig. 16). Contrarily, treatment with L-Glu resulted in progressively higher ThT fluorescence relative to the untreated condition, consistent with observations in the SynTag-Tau model system (Supplementary Fig. 16; Fig. 3a).

Small molecule-mediated enhancement in condensate viscoelasticity weakens fibrillization propensity
The effect of L-Arg in regulating condensate-to-fibril transition may stem from modulating the material properties of Tau condensates. To test this idea, we employed video particle tracking (VPT) nanorheology using 200 nm-sized probe particles passively embedded inside WT Tau condensates (Fig. 5a). From the mean-squared displacements (MSDs) of the fluorescent probes, we computed the frequency-dependent viscoelastic or dynamical moduli of Tau condensates. The timescale of the dominantly viscous versus dominantly elastic behavior of Tau condensates can be deduced by estimating the crossover frequency from the frequency-dependent viscoelastic moduli. In the absence of L-Arg, Tau condensates are highly viscoelastic, with probe particles exhibiting sub-diffusive motion (diffusivity coefficient, α < 1) within the experimental timescale (Fig. 5b,c). With aging, these condensates exhibit a progressive lowering of the crossover frequency and increase in the storage (G’) modulus, suggesting an enhancement in the network elasticity (Fig. 5d; Supplementary Fig. 17). Surprisingly, L-Arg-treated condensates at 2 h of age were found to be more viscoelastic relative to that of the untreated condition (Fig. 5b,c). At 4 h, these condensates exhibit a substantial increase in viscoelasticity (Fig. 5d; Supplementary Fig. 17). These data suggest a surprising role of L-Arg in strengthening intra-condensate networking interactions, otherwise referred to as percolation87,88. Optical tweezer-based active fusion measurements of SynTag-Tau condensates show a consensus with nanorheology measurements (Fig. 5b vs. Supplementary Fig. 18a, Fig. 1h) in that L-Arg leads to a slowdown in condensate fusion speed in a dose-dependent manner. In agreement with these results, FRAP measurements at longer timescales revealed progressively slower recovery traces for L-Arg-treated condensates (Supplementary Fig. 18b). We also tested the effect of L-Glu treatment on condensate viscoelasticity (Supplementary Fig. 19). We observed that it enhances condensate viscoelasticity similarly to L-Arg; however, it is worth noting that this enhancement might stem from faster fibril assembly (Supplementary Fig. 16) rather than percolation-driven increase in the network strength. Further systematic investigation will be required to clearly distinguish these mechanisms. In total, measurements of condensate material properties reveal that L-Arg suppresses condensate-to-fibril transition via enhancement of condensate viscoelasticity.

Selective inhibition of condensate-to-fibril transition preserves condensate biochemical activity
The protective effects of L-Arg against the condensate-to-fibril conversion of SynTag-Tau motivated us to test its role in MT assembly (Fig. 2a). We observe that, similar to untreated SynTag-Tau condensates, the L-Arg-treated condensates can assemble MTs with comparable efficiency up to 5 h of physical aging (Fig. 6a,b; Supplementary Fig. 20a). After the 5-h mark, these trends start to diverge. The untreated SynTag-Tau condensates progressively lose their MT assembly activity as seen by the reduced MT formation (Fig. 2d,e). In contrast, the L-Arg-treated SynTag-Tau condensates (1 mM and 2 mM L-Arg) show a steady trend in MT assembly activity on the same timescale without appreciable decline. Particularly, at the 16-h time point, the untreated SynTag-Tau condensates completely lose activity, whereas the L-Arg-treated condensates continue to retain their function (Fig. 6a,b; Supplementary Fig. 20a). Tubulin partitioning experiments conducted under similar assay conditions revealed that L-Arg treatment is associated with homogeneous tubulin partitioning into condensates at all time points tested, which is otherwise perturbed as a function of condensate age (Supplementary Fig. 12). Moreover, FRAP measurements reveal marginally slower mobility of tubulin with L-Arg treatment, relative to the untreated condition, likely owed to the small molecule-induced enhancement in condensate viscoelasticity (Supplementary Fig. 20b; Fig. 5). Nonetheless, the outcome of MT assembly does not appear to be dramatically affected by small molecule-mediated changes in condensate material state (Fig. 6b). With our overall observations, we posit that nucleation of amyloid assemblies at the SynTag-Tau condensate interface results in the accumulation of tubulin at the condensate interface, hindering MT assembly. In the presence of L-Arg, fibril nucleation at the condensate interface is inhibited, allowing favorable partitioning of tubulin into the condensate core and facilitating tubulin polymerization (Fig. 6c).

Methods
Protein purification
Codon-optimized constructs of wild-type Tau (2N4R isoform) and designed variants used in this study were synthesized and cloned into a pETHis6N10TEVLIC vector by GenScript. SynTag-Tau protein was expressed and purified under denaturing conditions from the E. coli BL21 strain (New England Biolabs). Initially, E. coli cells were grown to OD of 0.6–0.8 and subsequently induced with 0.1 mM IPTG at 37°C for 2h. After pelleting, the cells were resuspended in a lysis buffer composed of 50 mM phosphate buffer (pH 6.4), 1 M KCl, 10 mM imidazole, 2 mM DTT, 6 M urea, and a protease inhibitor cocktail (Thermo Fisher Scientific). The resuspended lysate solution was subject to ultrasonication followed by ultracentrifugation at 37,500×g for 30 mins at 30°C. The extracted supernatant from the previous step was introduced to a packed Ni-NTA column at 4°C (5 ml volume; Thermo Fisher Scientific) prewashed with the lysis buffer. After several washing steps, the proteins were eluted from the column using an elution buffer composed of 50 mM phosphate buffer (pH 6.4), 2 M KCl, 200 mM imidazole, 2 mM DTT, and 3 M urea. The elutes were pooled and introduced to a pre-equilibrated Zeba spin desalting column (Thermo Fisher Scientific) following the manufacturer’s instructions to get rid of residual imidazole and for storage in a buffer composed of 50 mM phosphate buffer (pH 6.4), 2 M NaCl, 2 mM DTT, and 3 M urea. This step was repeated twice to ensure efficient desalting. The resulting protein stock was tested for nucleic acid contamination, and the protein concentration was estimated by a NanoDrop 1C spectrophotometer.

In the case of wild-type (WT) Tau (2N4R isoform) with 6xHis tag, the protein was expressed and purified under native conditions from the E. coli BL21 strain (New England Biolabs). Initially, E. coli cells were grown to OD of 0.6–0.8 and subsequently induced with 0.1 mM IPTG at 37°C for 2.5h. After pelleting, the cells were resuspended in a lysis buffer composed of 50 mM MES (pH 6.0), 300 mM NaCl, 10 mM imidazole, 2 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich), and a protease inhibitor cocktail (Thermo Fisher Scientific). The resuspended lysate solution was subject to ultrasonication followed by ultracentrifugation at 37,500×g for 30 mins at 4°C. The extracted supernatant was then incubated with Ni-NTA resin (pre-equilibrated with the lysis buffer) overnight at 4°C (5 ml volume; Thermo Fisher Scientific). The protein was eluted from the Ni-column after several wash steps using an elution buffer composed of 50 mM MES (pH 6.0), 300 mM NaCl, 250 mM imidazole, 2 mM DTT, 1 mM EDTA, and 0.3 mM PMSF. Protein fractions containing 6xHis-tagged Tau protein were incubated with TurboTEV protease (Eton Bioscience) overnight at 4°C. The proteolysis reaction volume was loaded onto another Ni-column to remove the free 6xHis tag, non-cleaved protein, and TEV protease. The supernatant containing unbound Tau protein was diluted in a buffer containing 50 mM MES (pH 6.0), and 2 mM DTT and then loaded onto a Capto HiRes S 5/50 cation exchange column (Cytiva) for ion-exchange chromatography. Tau protein was eluted from the column by a step gradient of 0 to 1 M NaCl. Purified protein in storage buffer containing 50 mM MES (pH 6.0), 500 mM NaCl, and 2 mM DTT was tested for nucleic acid contamination, and the protein concentration was estimated by a NanoDrop 1C spectrophotometer.

Finally, SDS-PAGE analysis was conducted to test the quality of all the purified proteins prior to flash-freezing the protein aliquots and storage at −80°C. Site-specific fluorescence labeling of proteins was accomplished following the manufacturer’s instructions. For the sequence details of WT and other Tau variants used in this study, refer to Supplementary Table 1.

Preparation of protein condensate samples
After thawing an aliquot of purified Tau protein at room temperature, desalting was conducted using an equilibrated Zeba spin desalting column (Thermo Fisher Scientific), following the manufacturer’s instructions, using a buffer composed of 10 mM HEPES (pH 7.4), 200 mM NaCl, 0.1 mM EDTA, and 2 mM DTT. The choice of this buffer composition for a majority of the experiments reported in our study is based on the observation that it provides conditions for favorable protein phase separation into large condensate sizes that are amenable to quantitative experimental interrogation, and for clear demarcation of condensate interface from their interior (Supplementary Fig. 7). The previous step was repeated twice to ensure efficient desalting. The resulting protein was concentrated, and its concentration was measured using a NanoDrop 1C spectrophotometer. The final sample for experiments was then prepared by diluting the concentrated protein solution in the same buffer without NaCl. Next, PEG 8000 (Sigma Aldrich) was added at a desired concentration (specified in the figure legends and this Method section) to trigger phase separation. Whenever applicable, small molecules were introduced into the sample buffer during the protein dilution step. Small molecules, including L-arginine, D-arginine, L-arginine ethyl ester, L-lysine, L-proline, L-aspartic acid, L-glutamic acid, adenosine triphosphate, α-dansyl-L-arginine, dansyl chloride, GTP, and trimethylamine N-oxide, were procured from Sigma Aldrich; urea and guanidinium hydrochloride were procured from Thermo Fisher Scientific. Many of the small molecule stocks were prepared with water, except for dansyl chloride and α-dansyl-L-arginine, which were dissolved in water spiked with DMSO; the final concentration of DMSO in condensate samples is less than 1%. Multivalent polymers/polyamines such as penta-lysine, deca-lysine, and deca-arginine were procured from GenScript, whereas spermine and spermidine were procured from Sigma Aldrich. The stocks of these multivalent molecules were prepared in water and introduced into the sample buffer, similar to the small molecules. In Thioflavin T (ThT; Thermo Fisher Scientific) based experiments, ThT at a final concentration of 50 μM was introduced during the protein dilution step. Pipetting was carried out subsequently to ensure sufficient mixing of all components in the sample to form protein condensates. The final sample was sandwiched between a tween 20-coated coverslip and a glass slide, separated by three layers of double-sided tape. The remaining space was filled with mineral oil to seal the sample chamber and prevent evaporation. Fluorescence imaging was performed either using a Q2 laser scanning confocal microscope (ISS Inc., 63× objective), a Lumicks (60× objective) C-trap confocal microscope, or a Zeiss LSM710 (63× objective) confocal microscope. An Andor Dragonfly 600 confocal platform (Oxford Instruments), equipped with an Andor iXon Ultra EMCCD camera and a Leica HC PL APO 63x/1.40-0.60 oil objective, was used for generating z-stack fluorescence images.

The preparation of wild-type Tau condensates was carried out similarly to that of SynTag-Tau and related variants, with the exception of including 6.25 μM heparin (Santa Cruz Biotechnology) in the sample buffer (unless specified otherwise) prior to induction of phase separation by the addition of PEG 8000. The sample was then placed inside an incubator set at 37°C. These conditions were shown previously to enhance the physical aging of Tau condensates57.

The experimental conditions relevant to the data shown in Fig. 1 are as follows. For Fig. 1d, the concentration of SynTag-Tau used is 24 μM with 10% PEG 8000. For Fig. 1f,g,j, the concentration of SynTag-Tau used is 12 μM with 10% PEG 8000. For Fig. 1h,i, the concentration of SynTag-Tau used is 12 μM with 7.5% PEG 8000. The buffer composition in all samples here is 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT. Wherever applicable, the concentration of Atto488-labeled SynTag-Tau is 250 nM, and the concentration of ThT is 50 μM. The experimental conditions relevant to the data shown in Fig. 2 are as follows. The concentration of SynTag-Tau protein used in Fig. 2b,c is 24 μM with the following buffer composition, 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, along with 10% PEG 8000. The concentration of HiLyte647-labeled tubulin is 50 nM. In Fig. 2f–h, the concentration of SynTag-Tau protein is 12 μM with the same buffer composition as Fig. 2b,c. In Fig. 2d, the concentration of SynTag-Tau used is 12 μM with the following buffer composition, 80 mM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, and 2 mM DTT, along with 5% PEG 8000. In Fig. 2d, the tubulin concentration is 5 μM, and the concentration of GTP is 1 mM. Wherever applicable, the concentrations of Atto488-labeled SynTag-Tau and HiLyte647-labeled tubulin are 250 nM and 500 nM (unless specified otherwise), respectively. The experimental conditions relevant to the data shown in Fig. 3 are as follows. The composition of these samples is 12 μM SynTag-Tau in buffer containing 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT along with 7.5% PEG 8000. Small molecules were introduced prior to the induction of protein phase separation, and their concentrations used in these experiments are 2 mM unless specified otherwise. Wherever applicable, the concentration of Atto488-/Alexa594-labeled SynTag-Tau is 250 nM, and the concentration of dansyl-L-Arg/dansyl-Cl is 500 nM (doped either with or without L-Arg to make up a total small molecule concentration of 2 mM). The experimental conditions relevant to the data shown in Fig. 4 are as follows. The composition of these samples is 12 μM SynTag-Tau in buffer containing 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, along with 10% PEG 8000 crowder. The L-Arg concentration used in these experiments is 2 mM. Wherever applicable, the concentration of A488/A594 labeled SynTag-Tau is 250 nM, and the concentration of ThT is 50 μM. The experimental conditions relevant to the data shown in Fig. 5 are as follows. The composition of the samples used here is 40 μM Tau in buffer containing 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, along with 7.5% PEG 8000, and 6.25 μM heparin. Wherever applicable, the L-Arg concentration used in experiments is 2 mM. The experimental conditions relevant to the data shown in Fig. 6 are as follows. The composition of the samples used here is 12 μM SynTag-Tau protein in buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, and 2 mM DTT, along with 5% PEG 8000 crowder. The tubulin concentration used here is 5 μM, and the concentration of GTP is 1 mM. The concentrations of Atto488-labeled SynTag-Tau and HiLyte647-labeled tubulin are 250 nM and 500 nM, respectively. The L-Arg concentration used in the small molecule-treated conditions is either 1 or 2 mM (indicated in the figure legends). Other details of the samples can be found in the respective figure legends or in the Methods.

Fluorescence recovery after photobleaching (FRAP)
Protein condensate samples placed on a Tween 20-coated sample chamber were kept on a confocal microscope stage (Lumicks C-trap) and imaged with a 60× water immersion objective of 1.3 numerical aperture. Imaging steps of either Atto488-labeled SynTag-Tau or HiLyte647-labeled tubulin were accomplished using the respective excitation laser line. Selection of the region of interest (ROI) for reference, bleaching, and continuous imaging for monitoring fluorescence recovery was done using the BlueLake software (Lumicks). The bleaching ROI dimensions were kept consistent for all condensate samples. Each condensate sample was bleached for at least 5 frames at maximum laser power to achieve optimal bleach depth, with a dwell time of 150 ms per pixel. Recovery of fluorescence was recorded for a maximum of 300 seconds after the bleaching steps. The images from the FRAP experiments were analyzed using Fiji96 (version 1.54f) to quantify the fluorescence recovery as a function of time. The intensities were normalized and corrected as described previously97. Succinctly, the normalization accounts for a correction for background intensity after the bleaching step as well as the photo-fading that follows, using the following expression, wherein I_correct(t) is the intensity of the bleached ROI at time t corrected for photo-fading and I_correct(0) is the value of intensity before the start of the bleach step (reference ROI used for quantifying the bleaching depth):

I_normalized(t) = (I_correct(t) - min(I_correct)) / (I_correct(0) - min(I_correct))

In total, 4 to 6 fluorescence recovery traces representative of three independent replicates were gathered and averaged.

Second-harmonic generation (SHG) imaging
SHG measurements were made using a Nd:YAG microchip laser generating nanosecond pulses with a repetition rate of approximately 1 MHz at 1064 nm (Leukos Opera HP, Leukos). A 100×, 0.85 NA objective (LCPLN100XIR, Olympus) focused the beam to the sample plane, and the transmission SHG signal was collected with a 20×, 0.4 NA objective (M-20X, MKS Newport). The signal was measured with a spectrometer (IsoPlane 160, Teledyne Princeton Instruments) and a back-illuminated, deep-depletion CCD (Blaze 1340×400HS, Teledyne Princeton Instruments). The CCD integration time per pixel was 1000 ms. Samples were stage-scanned with a pixel spacing of 0.3 μm per pixel.

Optical tweezer-mediated coalescence assay
Optical trap-induced fusion of protein condensates was accomplished using a Lumicks C-trap optical tweezers system equipped with a dual trap and a laser scanning confocal fluorescence microscope, with a 60x water immersion objective of numerical aperture 1.3, following a protocol described previously98,99. In brief, for a typical condensate fusion experiment, two condensates were trapped using a 1064 nm laser with the minimum power setting (~100 μW) to minimize laser-induced heating effects. Typically, the 1064 nm laser used in the assay provides a power of 1 mW at the objective (measured at the exit of the laser light from the objective). For the experiments, a power of 10% of 1 mW, which corresponds to ~100 μW, was used. This power is sufficient to trap droplets using the dual-trap optical tweezers system and perform droplet fusion assays98. The trapping of condensates relies on the refractive index mismatch between the condensate dense phase and the surrounding dilute phase. After trapping, Trap 2 was kept fixed, and Trap 1 was moved towards Trap 2 at a constant velocity of 100 nm/s. The trap is set to maintain this velocity until the fusion process concludes, allowing the final droplet to relax to a spherical or equilibrium shape. A bright-field camera was used to take time-lapse images of the droplet fusion event at 15 frames per second.

Broadband coherent anti-Stokes Raman (BCARS) hyperspectral imaging
For structural analysis of SynTag-Tau conformers within condensates or fibrils, we used an in-house-built, broadband coherent anti-Stokes Raman scattering microscope. For excitation, a Nd:YAG microchip laser generates nanosecond pulses with a repetition rate of approximately 1 MHz at 1064 nm and a broadband supercontinuum that ranges from 1100–2400 nm (Leukos Opera HP, Leukos). The beams were focused on the sample plane via a 100×, 0.85 NA objective (LCPLN100XIR, Olympus). In a transmission configuration, the signal was collected with a 20×, 0.4 NA objective (M-20X, MKS Newport). The signal was measured with a spectrometer (IsoPlane 160, Teledyne Princeton Instruments) and a back-illuminated, deep-depletion CCD (Blaze 1340×400HS, Teledyne Princeton Instruments). Curve-fitting measurements were performed by scanning the sample with a pixel spacing of 0.4 μm. The integration time per pixel was 80-150 ms to maximize the signal without saturating the CCD.

BCARS data processing
As reported in previous studies, raw BCARS spectra were transformed into Raman-like spectra for quantitative analysis using a modified Kramers–Kronig transform for phase retrieval100,101. Then, a second-order Savitzky-Golay with a 151-point (approximately Δ492 cm-1) smoothing window was applied to the phase-retrieved spectra to produce the Raman-like spectra.

An average spectrum for phase-separated condensates was extracted using a 3×3 pixel mean. Fibril average spectra were calculated as the mean spectra within a drawn region of interest around the fibril. The average Raman-like spectra were subsequently normalized by the max spectral intensity102 arising from the CH3 vibration located at 2933.4 cm-1. BCARS post-processing is performed in Igor Pro 8.4 (WaveMetrics). For consistency, center wavelength dependencies of the spectrometer calibration required a spectral shift of less than or equal to 10 cm-1 before amide I curve-fitting. A Gaussian peak was fit to the CH3 peak to identify the center of the CH3 peak. Seven spectral points were linearly interpolated between each measured wavenumber, and the entire spectra shifted by the difference between the CH3 center and 2933.4 cm-1.

Amide I decomposition was performed with Igor Pro MultiPeak Fitting 2 functionality, which employs an iterative Levenberg–Marquardt algorithm. The parameters of the Lorentzian peaks for curve-fitting are found in Supplementary Table 2. Briefly, we used six peaks73,103,104 within the amide I band (1550 – 1725 cm-1): 1644 cm-1 for α-helices, 1660 cm-1 for random coils, 1673 cm-1 for β-sheets, a minor peak for β-turns at 1691 cm-1, and two minor peaks for tyrosine ring modes at 1600 cm-1 and 1612 cm-1.

Tubulin partitioning and microtubule assembly functional assay
Tubulin and HiLyte647-labeled tubulin were purchased from Cytoskeleton. Partitioning experiments, using HiLyte647-labeled tubulin, were conducted on condensate samples composed of SynTag-Tau protein at 24 μM protein concentration with a PEG 8000 concentration of 10% with the following buffer composition: 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT. The HiLyte647-labeled tubulin was introduced to SynTag-Tau condensates that were allowed to incubate within a microfuge tube for a defined period (as indicated in the text). The concentration of HiLyte647-labeled tubulin is specified in the text. In addition, the partitioning experiments were also performed under sample conditions corresponding to the microtubule polymerization assay (described below). The microtubule polymerization assay was carried out based on methods described previously46,58. For a schematic of the assay, refer to Fig. 2a. Briefly, SynTag-Tau protein solution was buffer-exchanged to general tubulin buffer (Cytoskeleton) consisting of 80 mM PIPES (pH 6.9), 2 mM MgCl2, and 0.5 mM EGTA, supplemented with 2 mM DTT. This buffer composition was specifically used for this assay, as it was shown to support robust and reproducible tubulin polymerization under near-physiological conditions52. The addition of PEG 8000 to the buffer-exchanged sample at a final concentration of 5% induced the formation of protein condensates. These condensates are allowed to incubate within a microfuge tube for a defined period, as mentioned in the text, before the addition of tubulin and GTP at final concentrations of 5 μM and 1 mM, respectively. 500 nM HiLyte647-labeled tubulin was doped along with unlabeled tubulin for MT polymerization assays. Finally, the sample was prepared for fluorescence microscopy as described in the ‘Preparation of protein condensate samples’ method section, with imaging conducted after a sample incubation period of 30 minutes.

Image analysis to estimate microtubule surface coverage
The analysis of microtubule surface coverage was performed using a custom-made Python-based image analysis program. Initially, raw images were loaded into the analysis program and subjected to median filtering to suppress noise, followed by Richardson-Lucy deconvolution to enhance the resolution and clarity. To further accentuate these structures, gray-scale dilation and erosion were applied alongside contrast stretching and Gabor filtering, which specifically highlighted the microtubular objects in the image. Following enhancement, the brightest objects, predominantly condensates, were isolated through an intensity threshold. These objects were then removed using binary morphological operations combined with region labeling, where the eccentricity of each region was calculated to differentiate spherical condensates from microtubule structures. After excluding the condensates, the processed images were subjected to another round of thresholding to selectively identify microtubules. This selection was refined by assessing the eccentricity of dimmer blobs, which were likely overlooked in the initial removal step. Such blobs were excluded to avoid falsely conflating condensates with the microtubule structures. Final mask refinement involved additional binary morphological operations to fine-tune the segmentation. The resulting microtubule mask was then measured quantitatively. The final mask was measured, and data, including an overlay image and a data frame of measurements, such as microtubular coverage, were exported for further analysis (Supplementary Fig. 11).

Frequency-domain fluorescence lifetime imaging (FD-FLIM)
FD-FLIM measurements of protein condensate samples were collected using a Q2 laser scanning confocal microscope (ISS Inc.) equipped with FastFLIM modules105. For calibration prior to FLIM measurements, Rhodamine 110 dye diluted in water, with a fluorescence lifetime of 4.0 ns, was used. The fitting of the phase delay and modulation ratio of the acquired FD-FLIM data of a given condensate sample at digital modulation frequencies ranging from 20 to 100 MHz was used to estimate the fluorescence lifetime distribution of Atto488-labeled SynTag-Tau. Subsequently, this was used to map the distribution of fluorescence lifetimes in the condensate images. Generation of the corresponding fluorescence lifetime histograms of condensate samples at different imaging time points, as well as thresholding, was carried out using the ISS VistaVision software. Gaussian fitting of the fluorescence lifetime histograms was accomplished using GraphPad Prism 10.

Image analysis for estimating partition coefficient
Condensate samples used for partition coefficient analysis were imaged either using a Lumicks C-trap confocal microscope (60×) or a Zeiss LSM710 confocal microscope (63×). For each set of experiments, the microscope used and the acquisition settings were kept consistent. The concentration of labeled proteins/molecules, as well as the settings used for the respective excitation laser lines, was kept consistent across samples. Fluorescence images collected for partition coefficient image analysis were analyzed using Fiji96 to segment individual condensates and estimate the mean intensity of each condensate (dense phase), referred to as I_dense. To estimate the background mean fluorescence intensity (dilute phase) referred to as I_dilute, three regions away from the condensates were selected at random. The partition coefficient (k) is finally estimated by dividing the dense phase mean intensity and the dilute phase mean intensity, k = I_dense / I_dilute.

Estimation of Thioflavin T (ThT) fluorescence intensity
Condensate samples were prepared following a protocol as outlined in the ‘Preparation of protein condensate samples’ method section. The samples were imaged either using a Lumicks C-Trap confocal microscope or an ISS Q2 laser scanning confocal microscope, wherein, for each set of experiments, the microscope used and the acquisition settings were kept consistent. The acquired fluorescence images were quantified using Fiji to estimate the mean ThT fluorescence intensity, with correction for the background ThT fluorescence intensity. These ThT measurements were performed either in the absence or presence of a small molecule to assess whether the small molecule treatment leads to enhanced or reduced fibril formation kinetics using ThT fluorescence intensity as a proxy.

Video particle tracking (VPT) based nanorheology
VPT-based nanorheology measurements were conducted on wild-type Tau condensates following the protocol outlined in a previous report84. In brief, we utilized 200 nanometer-sized carboxylate-modified polystyrene beads (yellow-green fluorescent tracers; FluoSpheres™, Invitrogen) introduced into the sample buffer (0.0005% solids) prior to induction of Tau phase separation, along with heparin. The remainder of the sample preparation was carried out as prescribed in the ‘Preparation of protein condensate samples’ method section. The protein concentration used for these measurements is higher (40 μM Tau) relative to the typical concentration used for other experiments, as reported in the main text, to allow for the formation of large condensate volumes, which is favorable for tracking a higher number of fluorescent tracers for nanorheology measurements. To determine the mean-squared displacements of the tracers, the Trackmate106 plugin of Fiji was used.

The acquired trajectories were subjected to correction for potential drift by subtracting from the center of mass (COM) trajectory, which was estimated using particle velocity as shown below:

X_COM(k) = X_0 + sum_{j=0 to k-1} (1/N_j) * sum_{i=1 to N_j} v_{i,j}

Here, k represents the frame number at which X_COM is calculated, j represents the individual frame, and N_j represents the number of particles in frame j. X_0 represents the center of the mass vector of the first frame. Subsequently, after the correction of the trajectories for drifting, the mean square displacement (MSD) is estimated as follows:

MSD(τ) = <R(t+τ) - R(t)>^2

Here, τ represents the lag time and R represents the position vector of the particle. Utilizing various lag times τ, the MSD was estimated based on the trajectories of the particles, with the use of in-house made Python scripts, which are publicly available (see ‘Code Availability’ section). Next, the ensemble-averaged MSD was estimated using measurements from approximately 20–100 individual probe particles. Fitting of the MSD7,98 was accomplished using:

MSD(τ) = 4Dτ^α + N

Here, D represents the diffusion coefficient of the particles and α represents the diffusivity exponent, which signifies the nature of the particle diffusion (normal or sub-diffusive) inside the condensates. With each sample, about 20 to 100 trajectories were acquired from 3 condensates across three independently prepared samples. In order to deduce the condensate aging dynamics either in the presence or absence of small molecules (L-Arg or L-Glu), VPT nanorheology was conducted at two distinct time points during physical aging.

Estimation of dynamical moduli from nanorheology measurements
The viscoelastic properties of Tau condensates were estimated from the MSD measurements, utilizing in-house developed Python scripts (refer to ‘Code Availability’ section). The protocol followed to estimate the dynamical moduli from MSDs is described in a previous report9. In brief, the compliance j(t) was calculated from the MSDs <Δr^2(τ)> estimated from VPT nanorheology measurements described in the previous section using the following equation:

j(t) = 3πa <Δr^2(τ)> / (n_d k_B T)

Here, a represents the probe particle radius, n_d = 2 represents the dimension of the probe particle trajectories based on which the MSDs were estimated, k_B represents the Boltzmann constant, and T represents the absolute temperature at which VPT nanorheology experiments were done.

The complex shear modulus can be estimated using a Fourier transform, which relates the complex shear modulus and the compliance via a convolution integral, as shown below:

integral_{0 to τ} G(t) j(τ-t) dt = τ

G*(ω) = iω / j*(ω)

Following the methodology proposed by Evans, R. et al.107, the complex shear modulus was estimated. Using discrete experimental data points t_i, j_i of the estimated compliance from Eq. (5), the frequency-space shear modulus G*(ω) is estimated following the equation below:

G*(ω) = iω [j(0) + (1 - e^(-iωt_1)) / (iωt_1) * j_1 + sum_{k=2 to N} (j_k - j_{k-1}) / (t_k - t_{k-1}) * (e^(-iωt_{k-1}) - e^(-iωt_k)) / (iω)]^(-1)

The MSD data were oversampled using a cubic spline. The remainder of the parameters required in Eq. (8) for estimating the shear modulus can be acquired as follows. j(0) is acquired through extrapolation of the experimental data for compliance t=0 using a linear fit of the initial four data points and η using a linear fit of the final ten data points following the relation, η = 1 / j_dot(t).

Estimation of dense phase area fraction
Confocal fluorescence images visualized with Atto488-labeled SynTag-Tau were used as input for dense phase area fraction estimation. The imaging was conducted after all condensates had settled on the coverslip. Random frames were imaged to ensure an accurate representation of the area fraction. For quantification, thresholding was performed using Fiji in order to isolate condensates from the background (dilute phase), yielding a binarized image highlighting the areas occupied by condensates. Using the ‘Analyze Particles’ function of Fiji, individual condensate sizes (μm^2) were determined. Subsequently, the fraction of cumulative area occupied by condensates with respect to the total area of image acquisition, set by image acquisition parameters, was estimated. The dense phase area fraction estimations were conducted at different conditions, including varying buffer ionic strength or small molecule concentrations, to assess their impact on phase separation.

Coverslip treatment for microscopy
The experiments of this study used Tween 20-coated coverslips. The coverslip cleaning procedure prior to surface coating involves treatment with 2% Hellmanex solution for 2 h, followed by rinsing with MilliQ water six times. Subsequently, coverslips were treated with 20% Tween 20 solution (v/v) for 30 min. Further, these coverslips were rinsed with MilliQ six times and dried with compressed air. Lastly, the coverslips were dried in an oven at 40°C overnight. The coverslips were later stored at room temperature for use in experiments.

Software
Fiji96 (version 1.54f) was used for processing and analyzing microscopy images. A custom Python script was used to plot VPT nanorheology data and estimate dynamical moduli. The scripts are available publicly (see ‘Code Availability’ section). Trackmate (v7.14) was used for nanorheology-related analysis. For all other plots, GraphPad Prism 10 (v10.4.1) was used. MarvinSketch (ChemAxon) was used to visualize the chemical structures of small molecules. Adobe Illustrator (2024) was used for preparing figures and schematics. Igor Pro 8.4 was used for BCARS data processing. BlueLake (v1.6.11) was used for collecting fluorescence images as well as for performing FRAP and optical tweezer-induced condensate fusion measurements with the Lumicks C-Trap microscope. ZEN (SP5 2012 Black) was used to acquire fluorescence images with the LSM 710 laser-scanning confocal microscope. VistaVision (v4.2) was used to acquire fluorescence images and FLIM measurements with the ISS Q2 laser-scanning confocal microscope. Fusion Dragonfly software (v2.6) was used for acquiring fluorescence images with the Andor Dragonfly600 confocal platform. UCSF ChimeraX108 (v1.7) was used for visualizing AlphaFold368 predicted structures of Tau protein variants. ZipperDB was used for estimating fibrillization propensity scores70. DrugBank was used to generate predicted pKa values for small molecules109. PLAAC110 was used to generate prion scores of protein variants.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Mahendran et al. developed a model protein condensate system that allows to study the liquid-to-solid amyloid transition of\ntau condensates, as well as how this transition affects MT assembly. This system employs the fusion of a prion-like derived\ntag that accelerates the interface-mediated amyloid formation of tau. Using an extensive set of techniques (FLIM, SHG\nmicroscopy, and VPT nanorheology), alongside thorough controls, the authors demonstrate that the formation of tau fibrils at\nthe condensate interface compromises the biochemical activity of tau. They identify small molecules, such as arginine, as\nmodulators of this amyloid transition. These molecules can decouple phase separation from fibrillization, preserving the MT\nassembly activity of tau condensates. The study is well-designed, the conclusions are supported by robust data, and the\nmanuscript is clear and concise. Moreover, the identification of modulators that inhibit amyloid formation without affecting\nphase separation could inspire new strategies to inhibit aberrant phase transitions. I believe this study will be of great\ninterest to the protein condensate community and recommend its publication in Nature Communications after addressing the\nfollowing comments:\n1) Role of the prion-like tag: The authors show that different variants of the tag (truncation and shuffling) abolish amyloid\nformation. Please consider specifying what is the mechanistic role of this tag in tau aggregation. Is it the primary driver? The\ncontrol in Supplementary Figure 13 using wt tau is informative but does not fully exclude the contribution of the SynTag in\naccelerating tau aggregation.\n2) Supplementary Fig 5: please consider adding the PLAAC plot for this truncated variant.\n3) Effect of ionic strength on phase separation: the authors state that buffer ionic strength influences the phase separation\npropensity of the protein. Please consider quantifying more precisely, for example, by measuring the Csat of the protein at\ndifferent salt concentrations. While microscopy images provide qualitative insights, changes in salt concentration might\naffect PS by reducing condensate size but increasing droplet number.\n4) Effect of arginine on phase separation: the authors demonstrate that arginine increases condensate viscoelasticity by\nstrengthening intra-condensate interactions. However, higher arginine concentrations (4 mM) reduce condensate size. The\nauthors suggest this is due to altered coarsening dynamics, rather than condensate dissolution. If this is the case, one would\nexpect a higher number of smaller condensates at 4 mM compared to 2 mM, assuming a constant volume fraction. Please\nconsider quantifying both the number and size of condensates (or, ideally, the volume fraction of the dense phase) at 2 mM\nand 4 mM arginine to support this interpretation.\n5) MT assembly activity in the presence of arginine: given that arginine increases the strength of intra-condensate\ninteractions and reduces dynamics (as shown by FRAP in Supplementary Figure 15), could the diffusion of tubulin within the\ncondensates be affected by this percolated network? If so, can this reduced mobility affect MT assembly?\n6) Please consider comparing the tubulin uptake into the nontreated and L-arg treated condensates at different timepoints.\nSimilarly to the analysis in fig 2b,c, just at all the timepoints corresponding to the activity assay. This would allow to evaluate\nif the presence of arg enables a good partitioning of tubulin also at later time points compared to the non treated\ncondensates.\n7) Please consider adding some later timepoints in figure 2d and 6a, to better evaluate the impact of L-arg on aging. Same\nfor the aggregation data in Fig. 4a\n8) figures 2d and 6a: why does the tau signal dissapear in the tau channel after 16 hours? Shouldn't the aggregates still be\nvisible? (there has maybe been an intended switch of images between the tubulin and tau channels?) Please consider also\nassing images of tau condensates before adding gtp and tubulin.\n9) Please consider commenting on the selection of the buffers: a standard buffer is used for MT assambly, but not all the\nexperiments?", "reviewer_2": "In this article the authors developed an engineered-Tau protein, comprising the full length of the protein Tau and a 40 amino-\nacid priogenic tag, called SynTag-Tau. They showed that SynTag-Tau self organizes in protein condensates that with aging\ntransit irreversibly to fibrillar morphology, in a time scale of 2 to 3 hours. The aging in a wild-type Tau condensate is much\nslower than aging in SynTag-Tau condensates, what makes this engineered model suitable for investigations of the\nmechanisms related to the transition from condensate to fibrillar phases.\nThe fibrillar phase is in the origin of neurodegenerative diseases, like Alzheimer, and to get knowledge about the transition\nfrom condensate (liquid-like) to fibrillar (solid-like) is in my point of view of significant importance.\nThe authors also showed that the condensates of SynTag-Tau are biologically active in promoting the recruitment of tubulin\nin the formation of microtubules. Besides, they showed that L-Arg and L-Lys, abundant cationic amino acids, inhibit the\nliquid-fibrillar transition of SynTag-Tau condensates. They propose that these small proteins alter the metastable energy\nbarrier for the liquid-fibrillar phase transition, indicating a proof of principle of a mechanism that can be used to decrease\nfibrillar formation from Tau condensates, avoiding fibrils appearance, a characteristic of neurodegenerative diseases.\nThe authors employed a variety of biochemical and optical techniques to support their conclusions, including fluorescence\nrecovery after photobleaching, second harmonic generation imaging, Raman spectroscopy, optical tweezers and video\nparticle tracking for rheology experiments.\nThe data and the discussion are well presented, and the subject can be of high impact. Nevertheless, I have some concerns\nabout the presentation in the Supplementary Information regarding the optical tweezers and rheology results.\nThe coalescence assay performed with optical tweezers seemed detached from the main purpose of the article. The authors\nshould emphasize what the purpose of the coalescence assay is. I was expecting an analysis of the condensate mechanical\nproperties For example, surface tension, elastic modulus, and viscosity can be accessed with optical tweezers. Besides,\nthose physical properties may be related to the rheological storage and loss moduli, measured with the video particle\ntracking experiments. The authors claim that the aging effect on SynTag-Tau condensates is related to a liquid-solid\ntransition, what would alter the above-mentioned condensate physical properties. The authors should add this information to\nthe text.\nThe description of the optical tweezers assays lacks information about the system employed, such as what kind of objective\nlens is used and what is its numerical aperture. What is the maximum power employed? A laser power of 100 microW\nseems to be too small value to be used, even for a soft material like the protein condensate. Was the power measured at the\nentrance of the objective lens? The loss of power in the lens can reach 80% depending on the lens and laser employed in\nthe optical tweezers system, what leads to a power estimation of 30microW in the optical traps employed. A toy laser\npointing has more than ten times bigger power. It seems that the presented value is not the correct used in the experiments.\nWhat was the setup to build the double optical trap? Concerning the rheological experiments procedures described in the\nSupplementary Information, the Equation (6), line 314, is meaningless. I would\nsuggest a large revision of these two sections.\nIn summary, this article presents remarkably interesting and important results related to the transition of liquid-solid in protein\ncondensates. It brings proof of the principle of the use of small proteins to possibly prevent the liquid to fibrillar transition.\nIn my opinion, it is suitable for publication after clarification of the issues reported above and text alterations accordingly.", "reviewer_3": "In the manuscript, Tharun et al. investigated the process of biomolecular condensation and the subsequent formation of\namyloid fibrils. By engineering the terminal region of the Tau protein, the timescale of physical aging is significantly reduced\nto a few hours. Using fluorescence spectroscopy and nonlinear microscopy, the authors demonstrated that physical aging\noccurs through a disorder-to-order process. The β-sheet content is increased, ultimately leading to\namyloid fibril formation. It is noted that the fibril nucleation was observed to initiate at the condensate interface, which compromises the biochemical function of the condensates. The study further explored the effect of small molecules on protein fibrillation. Among the tested\ncompounds, L-arginine (L-Arg) was found to inhibit fibril formation, which was attributed to an increase in the viscoelasticity\nof the condensates. L-Arg selectively inhibited fibrillation without affecting the condensation process itself, suggesting that\ncondensation and fibrillation can be decoupled.\nOverall, the manuscript is well-written with great clarity. I would like to recommend considering\nthe publication of this manuscript because it tackles the important problems in the field of Biomolecular condensate.\nUnderstanding how the\nintrinsically disordered proteins in biomolecular condensates transform into irreversible fibrils is crucial for the ultimate cure\nof neurodegenerative disease pathology and identifying potential therapeutic strategies. The authors demonstrate that by\nutilizing the small molecules stabilizes protein condensates in a metastable state, thereby preserving their biochemical\nfunction. I only have several comments as follows:\nComments:\n1. In Figures 1(j) and 4(b), the authors present BCARS spectra obtained from hyperspectral imaging. However, the\ncorresponding BCARS hyperspectral imaging data are not included in the main text or the Supplementary Information. The\nBCARS hyperspectral imaging could significantly strengthen the manuscript. If the authors' hypothesis—that amyloid fibrils\nnucleate initially at the condensate interface—is true, one would expect to observe a heterogeneous spatial distribution of\nthe β-sheet signal intensity. Therefore, the hyperspectral imaging data and accompanying discussion are required.\n2. In Figures 1 (j) and 1(b), the BCARS spectra of nascent condensates shows higher background (~0.2 offset) than other\nspectra. Is it related to the physical aging?\n3. The authors report that L-arginine (L-Arg) inhibits fibril formation, which is due to an increase in condensate\nviscoelasticity. However, it remains unclear whether this represents a general mechanism that viscoelasticity governs the\nkinetics of fibril formation. Specifically, does the higher (lower) viscoelasticity correlate with slower (faster) fibril formation?\nTo address this, it would be benefit to examine the effects of other small molecules. For instance, if L-lysine (L-Lys),\nanother fibril-inhibiting molecule, also increases the condensate viscoelasticity. And if molecules such as L-glutamic acid (L-Glu) or\nL-aspartic acid (L-Asp), which will accelerate fibrillation, correspondingly reduce viscoelasticity. These results would support\nthe proposed mechanism. A more in-depth discussion is required.\n4. The fibril-inhibiting molecules, such as L-arginine and L-lysine, both are basic amino acids with pKa values greater than\n7. Is there a potential correlation between their pKa values and the mechanism of physical aging? Please comment on it.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Responses to reviewer comments\nWe thank the reviewers for their helpful comments and suggestions. We have revised our\nmanuscript extensively and tried our best to address all the points raised by the reviewers. Here,\nwe provide a detailed, point-by-point response to each of the comments made by each reviewer.\nFor ease of delineation hereon, the reviewers' comments are in black, while our responses are\nin blue. Major changes made in the manuscript are highlighted in yellow and reproduced here\nin the context of specific comments.\nReviewer #1 (Remarks to the Author):\nMahendran et al. developed a model protein condensate system that allows to study the liquid-\nto-solid amyloid transition of tau condensates, as well as how this transition affects MT assembly.\nThis system employs the fusion of a prion-like derived tag that accelerates the interface-\nmediated amyloid formation of tau. Using an extensive set of techniques (FLIM, SHG microscopy,\nand VPT nanorheology), alongside thorough controls, the authors demonstrate that the\nformation of tau fibrils at the condensate interface compromises the biochemical activity of tau.\nThey identify small molecules, such as arginine, as modulators of this amyloid transition. These\nmolecules can decouple phase separation from fibrillization, preserving the MT assembly activity\nof tau condensates. The study is well-designed, the conclusions are supported by robust data,\nand the manuscript is clear and concise. Moreover, the identification of modulators that inhibit\namyloid formation without affecting phase separation could inspire new strategies to inhibit\naberrant phase transitions. I believe this study will be of great interest to the protein condensate\ncommunity and recommend its publication in Nature Communications after addressing the\nfollowing comments:\nResponse: We are grateful for the reviewer’s encouraging and constructive feedback on our\nmanuscript.\n1) Role of the prion-like tag: The authors show that different variants of the tag (truncation and\nshuffling) abolish amyloid formation. Please consider specifying what is the mechanistic role of\nthis tag in tau aggregation. Is it the primary driver? The control in Supplementary Figure 13 using\nwt tau is informative but does not fully exclude the contribution of the SynTag in\naccelerating tau aggregation.\nResponse: We thank the reviewer for raising this important question. In the case of SynTag-Tau,\nwe propose that the N-terminal prionogenic tag lowers the nucleation barrier of Tau condensates\ntransitioning to amyloid fibrils. Many protein systems susceptible to forming amyloids are\nconstrained by nucleation barriers, which determine the probability of self-assembly (Khan et al.\n2018). Specifically, in aggregation-prone prion protein systems such as Sup35, a substantial\nnucleation barrier exists such that virtually all Sup35-containing yeast cells do not form amyloid\naggregates unless cross-seeding via heterologous prion aggregates is introduced (Derkatch et\nal. 2001). Similarly, in the case of Tau, the nucleation barrier to amyloid formation is considerably\n1\nhigher, in the context of both in vitro and in vivo conditions, which precludes it from forming\namyloid fibrils at experimentally-accessible timescales (Meisl, Knowles, and Klenerman 2022).\nPreviously, fusion tags have been shown to modulate the nucleation barrier of amyloid\nself-assembly (Goehler et al. 2010). However, this effect is only observed when the prionogenic\nfusion tag is introduced to amyloidogenic proteins and does not apply to non-amyloidogenic\nproteins that are innately incapable of amyloid fibril formation, suggesting that the tag itself may\nnot directly form the amyloid core (Chandramowlishwaran et al. 2018). We posit that the\nprionogenic SynTag in our current study operates via a similar mechanism. To include the\nessence of this discussion, the following text has been added to the revised manuscript:\nThese approaches exploit the principle that in many amyloid-forming protein systems, fibril\nformation is constrained by high nucleation barriers, which dictate the probability and kinetics of\nself-assembly61. Inspired by this phenomenon, we sought to design a sequence-engineered\nvariant of Tau that selectively lowers the nucleation barrier for amyloid formation while preserving\nits intrinsic phase separation behavior and MT assembly function.\nTo answer the reviewer’s question whether the synthetic prionogenic tag can directly\nserve as the primary steric zipper solely driving the formation of SynTag-Tau fibrils, we performed\nZipperDB (Goldschmidt et al. 2010) analysis and elucidated putative six-residue steric zipper\nmotifs across the entire SynTag-Tau protein sequence including the tag (see below;\nSupplementary Fig. 6). The analysis revealed a high frequency of steric zipper motifs with high\nscores (threshold set at Rosetta Energy of -23 kcal/mol) in the native Tau sequence. Only one\nout of the ten highest-scoring zipper motifs is found to be localized in the synthetic prionogenic\ntag (ranked #10 out of 10). Among the highest-scoring motifs are PHF6 and PHF6* motifs in the\nmicrotubule binding domain, as well as nearby overlapping motifs that are known to serve as the\namyloid core of Tau fibrils (Fitzpatrick et al. 2017). Overall, in comparison to the native Tau\nsequence, the putative steric zippers in SynTag are outweighed in absolute score and frequency.\nHowever, we cannot rule out the contribution of the putative zipper motifs in SynTag in serving\nas an auxiliary steric zipper aiding in amyloid fibril formation. Directly testing this possibility will\nrequire structural studies using cryo-EM or NMR, which represent an important direction for\nfuture investigation.\nThe following sentences have been added to the revised text:\nAs a further validation, we sought to test whether the SynTag could serve as the primary driver\nof SynTag-Tau fibril formation. ZipperDB70 analysis revealed that in comparison to the native Tau\nsequence, the putative steric zippers in SynTag are outweighed in absolute score (Rosetta\nEnergy expressed as kcal/mol) and frequency, i.e., number of steric zippers (Supplementary\nFig. 6). This suggests that the primary steric zippers capable of driving fibril nucleation likely\noriginate from the native Tau sequence. However, without structural studies, we cannot rule out\nthe possibility that the putative zipper motifs in SynTag act as auxiliary steric zippers that facilitate\namyloid fibril formation.\nsequence generated using zipperDB23, with the Rosetta energy threshold set at -23 kcal/mol. The putative\nsteric zippers in SynTag and the remainder of the full-length Tau sequence are colored green and orange,\nrespectively. (b) Rank order of zipperDB predicted putative steric zippers in SynTag-Tau, corresponding\nto the analysis done in (a). The row shaded in green indicates a steric zipper confined to the N-terminally\nattached SynTag, whereas those shaded in orange are confined to the C-terminus of wild type Tau.\n2) Supplementary Fig 5: please consider adding the PLAAC plot for this truncated variant.\nWe thank the reviewer for pointing out this inconsistency. We have now amended\nSupplementary Fig. 5 to include the PLAAC plot corresponding to the truncated SynTag-Tau\nvariant.\nSupplementary Figure 5. Truncating the prionogenic tag sequence in SynTag-Tau to the Asn(N)-rich\nsegment, which corresponds to the major weighting in the PLAAC score (top), is insufficient to accelerate\nthe condensate-to-fibril transition (below). Refer to Supplementary Table 1 for sequence details of the\n3\ntruncated SynTag-Tau variant used here. The composition of the sample is 12 μM truncated SynTag-Tau\nin buffer containing 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT, along with 7.5%\nPEG 8000 crowder. The concentration of Atto488-labeled truncated SynTag-475 Tau is 250 nM. This\nexperiment was independently repeated three times.\n3) Effect of ionic strength on phase separation: the authors state that buffer ionic strength\ninfluences the phase separation propensity of the protein. Please consider quantifying more\nprecisely, for example, by measuring the Csat of the protein at different salt concentrations.\nWhile microscopy images provide qualitative insights, changes in salt concentration might affect\nPS by reducing condensate size but increasing droplet number.\nResponse: We thank the reviewer for raising this important question. In order to quantitatively\nassess the impact of buffer ionic strength on SynTag-Tau condensates, we estimated the\ncondensate area fraction (expressed in percentages with respect to the total image acquisition\narea) utilizing confocal fluorescence microscopy. The methodological details of the image\nanalysis and related quantification are now included in the method section (see section on\n‘Estimation of dense phase area fraction’). If higher ionic strengths are indeed suppressing the\ndriving forces of SynTag-Tau phase separation, the condensate area fraction would trend lower\nand vice versa. Consistent with this expectation, our measurements reveal a substantial\nreduction in total condensate area fraction commensurate with increasing [NaCl], suggesting\nthat SynTag-Tau condensates are stabilized by electrostatic interactions. We have now\nincorporated this data as part of Supplementary Fig. 7, a part of which is reproduced below:\n25\n20\n15\n10\n5\n0\n50 mM 75 mM 100 mM\n[NaCl]\nSupplementary Figure 7. (b) Estimated SynTag-Tau condensate area fraction (expressed in percentages with\nrespect to the total image acquisition area) at different ionic strengths, measured 2 hours after sample preparation.\nThe dashed line represents the mean, and the error bars represent the standard error of the mean (S.E.M.) based\non three independent replicates. Individual points represent data from independent experiments. Statistical\n4\nnoitcarf\naera\netasnednoc\n%\nP = 0.0006\nP = 0.0020\nsignificance was determined using the unpaired two-sided Student’s t-test. The respective P values are\nindicated in the plot.\n4) Effect of arginine on phase separation: the authors demonstrate that arginine increases\ncondensate viscoelasticity by strengthening intra-condensate interactions. However, higher\narginine concentrations (4 mM) reduce condensate size. The authors suggest this is due to\naltered coarsening dynamics, rather than condensate dissolution. If this is the case, one would\nexpect a higher number of smaller condensates at 4 mM compared to 2 mM, assuming a\nconstant volume fraction. Please consider quantifying both the number and size of condensates\n(or, ideally, the volume fraction of the dense phase) at 2 mM and 4 mM arginine to support this\ninterpretation.\nWe thank the reviewer for posing this important question. As described in point # 3 above, we\nconducted a similar quantitative analysis of condensate area fraction (expressed in percentages)\nat all L-Arg concentrations tested, corresponding to conditions reported in Fig. 3b, utilizing\nconfocal fluorescence microscopy. We found that L-Arg concentrations of 1 mM and 2 mM\nyielded condensate sizes that are not significantly different than that of the untreated condition\n(0 mM). However, in the case of 4 mM L-Arg treatment, we find a significantly reduced\ncondensate area fraction, suggesting that L-Arg at high concentrations (greater than 2 mM)\npartially inhibits SynTag-Tau phase separation. In addition, based on previous condensate\ncoalescence measurements using optical tweezers (Supplementary Fig. 18a), we deduced that\nL-Arg treatment slows down condensate coarsening dynamics. In light of this finding, we have\nrevised the following statement in the main text.\nIn order to ascertain whether the reduced condensate sizes stem from either altered condensate\ncoarsening dynamics, inhibition of SynTag-Tau phase separation or both, we conducted a\nquantitative analysis of condensate area fraction using confocal fluorescence microscopy\n(Supplementary Fig. 14). We found that L-Arg concentrations of 1 mM and 2 mM yielded\ncondensate sizes that are not significantly different than that of the untreated condition (0 mM).\nHowever, in the case of 4 mM L-Arg treatment, we observed a significantly diminished\ncondensate area fraction, indicating that L-Arg at high concentrations (greater than 2 mM)\ninhibits SynTag-Tau phase separation and potentially affects condensate coarsening.\nWe have also incorporated this data as Supplementary Fig. 14, which is reproduced below:\n5\n40\n30\n20\n10\n0\n0 mM 1 mM 2 mM 4 mM\n[L-Arg]\nSupplementary Figure 14. Estimated SynTag-Tau condensate area fraction (expressed in percentages with\nrespect to the total image acquisition area) at different concentrations of L-Arg, measured 2 hours after sample\npreparation. The dashed line represents the mean, and the error bars represent the standard error of the mean\n(S.E.M.) based on three independent replicates. Individual points represent data from independent experiments.\nStatistical significance was determined using the unpaired two-sided Student’s t-test. The respective P values are\nindicated in the plot. This experiment was independently repeated three times.\n5) MT assembly activity in the presence of arginine: given that arginine increases the strength\nof intra-condensate interactions and reduces dynamics (as shown by FRAP in Supplementary\nFigure 15), could the diffusion of tubulin within the condensates be affected by this percolated\nnetwork? If so, can this reduced mobility affect MT assembly?\nWe thank the reviewer for raising this important question. To address the first part of the question,\nwe conducted FRAP measurements of tubulin (labeled with HiLyte647) partitioned within\nSynTag-Tau condensates either in the presence or absence of L-Arg. For condensates probed\n1 hour after sample preparation, we observe a marginal decrease in tubulin diffusion within the\ndense phase in the presence of L-Arg, relative to the absence of L-Arg. This is possibly due to\nL-Arg-induced enhancement in condensate viscoelasticity.\nIn response to the second part of the question, we know from earlier measurements that the MT\nassembly kinetics are similar between the untreated and L-Arg treated condensates at the same\ntime point (1 hour) after preparation, before the onset of aging-induced loss-of-activity of\nuntreated condensates. This observation indicates that although L-Arg treatment alters tubulin\ndiffusion to a certain degree within condensates, MT assembly, as estimated from the surface\ncoverage of microtubules, is similar in both untreated and L-Arg treated conditions (Fig. 6b). We\nnote that our observations described above are similar to a recent study that showed that a small\n6\nnoitcarf\naera\netasnednoc\n%\nP = 0.0006\nP = 0.2004\nP = 0.0993\nmolecule, methylene blue, promotes Tau condensate dynamical arrest without substantially\naltering the rate of Tau condensate mediated MT assembly (Huang et al. 2023).\nWe have now incorporated the following text in the revised manuscript:\nMoreover, FRAP measurements reveal marginally slower mobility of tubulin with L-Arg treatment,\nrelative to the untreated condition, likely owed to the small molecule-induced enhancement in\ncondensate viscoelasticity (Supplementary Fig. 20b; Fig. 5). Nonetheless, the outcome of\ntubulin assembly does not appear to be dramatically affected by small molecule-mediated\nchanges in condensate material state (Fig. 6b).\nWe have now incorporated the newly conducted FRAP measurements as part of\nSupplementary Fig. 20b, which is reproduced below:\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0\n0 5 10 15 20 25 30\nTime (s)\nSupplementary Figure 20. (b) FRAP profiles of HiLyte647-labeled tubulin in SynTag-Tau condensates either\nwithout L-Arg (untreated) or with L-Arg (1 mM). Measurements were taken 1 hour after sample preparation. Intensity\nerror bars were plotted based on the standard error of the mean (S.E.M.) at each interval and are represented as\nthe shaded regions. Each of these experiments was independently repeated three times.\n6) Please consider comparing the tubulin uptake into the nontreated and L-arg treated\ncondensates at different timepoints. Similarly to the analysis in fig 2b,c, just at all the timepoints\ncorresponding to the activity assay. This would allow to evaluate if the presence of arg enables\na good partitioning of tubulin also at later time points compared to the non-treated condensates.\nWe thank the reviewer for raising this important comment. We have now examined the\npartitioning of tubulin (labeled with HiLyte647) in SynTag-Tau condensates prepared either\nwithout L-Arg (untreated, UT) or with L-Arg. We observe that in untreated condensates at time\npoints corresponding to significant loss of activity, there is a concomitant decline in\nhomogeneous tubulin partitioning, similar to that reported in Fig. 2b, c. Contrastingly, at the\nsame time points, in L-Arg-treated condensates, tubulin partitioning is homogeneous. It is worth\nnoting that in the 8 hr timepoint, despite observing a significant drop in MT assembly in untreated\n7\nyrevoceR\ndezilamroN\nUntreated L-Arg\nconditions versus L-Arg treated conditions, we do not observe a notable blockade of tubulin\npartitioning in the untreated condition via microscopy. We speculate that a combination of factors,\nnamely, physical aging–induced changes in the condensate material state and perturbed tubulin\nbinding by conformationally altered Tau within aged condensates, likely contributes to the\ninhibition of efficient tubulin polymerization within the condensate microenvironment, as\nsupported by FLIM and nanorheology measurements. We have included this discussion in the\nrevised text and incorporated the corresponding data as Supplementary Fig. 12.\nThe following paragraphs have been incorporated into the appropriate Results and Discussion\nsection:\nResults & Discussion Section: Condensate physical aging impairs biochemical activity\nConsistently, tubulin partitioning experiments conducted using similar assay conditions as the\nMT polymerization assay revealed increasingly heterogeneous tubulin partitioning at later time\npoints, which correspond to loss of activity (Supplementary Fig. 12; Fig. 2e). The ineffective\npartitioning of tubulin to condensates at later ages suggests that the characteristic mesh size\nmight progressively decline with physical aging77, 78. Nevertheless, it is worth pointing out that in\nthe 8 hr time point, despite observing a noticeable drop in MT assembly, we do not observe a\ndramatic blockade of tubulin partitioning via fluorescence microscopy, which might indicate that\naging-induced changes in material state, as well as perturbed tubulin binding by conformationally\naltered SynTag-Tau within aged condensates, might underscore condensate loss-of-activity.\nResults & Discussion Section: Selective inhibition of condensate-to-fibril transition preserves\ncondensate biochemical activity\nTubulin partitioning experiments conducted under similar assay conditions revealed that L-Arg\ntreatment is associated with homogeneous tubulin partitioning into condensates at all time points,\nwhich is otherwise perturbed as a function of condensate age (Supplementary Fig. 12).\nvarious time points after sample preparation, after the addition of 50 nM HiLyte647-labeled tubulin but not GTP (b)\nshows the line profiles corresponding to 12 hr and 16 hr time points, as indicated in (a). The composition of the\nsample is 12 μM SynTag-Tau in a buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl , 0.5 mM EGTA, and 2 mM\n7) Please consider adding some later timepoints in figure 2d and 6a, to better evaluate the impact\nof L-arg on aging. Same for the aggregation data in Fig. 4a\nWe believe that the data presented in the manuscript provide clear evidence that L-Arg\nsignificantly delays condensate aging and, in doing so, protects condensate activity from loss-\nof-function. The 16-hour time point in Fig. 2d was chosen because the activity of untreated\ncondensates is completely abolished by that stage. Consequently, comparisons between treated\nand untreated samples beyond this point would be uninformative and would not further illustrate\nthe efficacy of L-Arg treatment. For these reasons, we believe it is reasonable to retain the figure\nas currently presented.\nof untreated condensates was approximately tenfold higher than that of small molecule–treated\nsamples. Furthermore, the data in Fig. 3b provide complementary evidence showing that L-Arg–\ntreated condensates remain free of fibrils for several days in the presence of 2 mM L-Arg,\nwhereas untreated condensates form fibrils within just a few hours. Therefore, we believe it is\nreasonable to retain the figure in its current form.\nNevertheless, in response to a comment from another reviewer, we conducted additional\nexperiments comparing untreated and L-Arg–treated condensates using ThT fluorescence and\nA594-labeled SynTag-Tau at a much later time point (48 hours). These results continue to show\na striking difference in ThT intensity (figure reproduced below).\nCollectively, these lines of evidence reinforce the conclusion that L-Arg exerts a robust protective\neffect against condensate aging and the associated loss of activity.\nFigure 4. (b) Individual Z-slices of untreated SynTag-Tau condensates visualized using ThT (green) as well as\nA594-labeled SynTag-Tau (red). The indicated z-position is relative to the leftmost Z-slice in the panel. The inset\nshown represents the area demarcated by the white-dashed box. Sample age is 48 hours. (c) Maximum intensity\nprojection (MIP) image of 2 mM L-Arg treated SynTag-Tau condensates with A594-labeled SynTag-Tau and ThT.\nSample age is 48 hours. Notably, the ThT fluorescence intensity is substantially lower relative to the A594-labeled\nSynTag-Tau fluorescence for these condensates, in contrast to that of the untreated SynTag-Tau condensates,\nsuggesting a lack of fibrils with L-Arg treatment.\n8) figures 2d and 6a: why does the tau signal dissapear in the tau channel after 16 hours?\nShouldn't the aggregates still be visible? (there has maybe been an intended switch of images\nbetween the tubulin and tau channels?) Please consider also assing images of tau condensates\nbefore adding gtp and tubulin.\nThe images reported at the 16-hour time point have not been switched. The purpose of these\ntwo-color images is to report microtubule coverage (via fluorescence signal of HiLyte647-labeled\ntubulin) with the fluorescence signal of Atto488-labeled SynTag-Tau showing condensates\nwetting the microtubules, consistent with previously reported observations (Hernández-Vega et\nal. 2017). For the untreated sample, by the 16-hour time point, only a few amorphous clusters,\n10\nbut not long MTs, are observable, with SynTag-Tau fluorescence signal overlapping with aberrant\ntubulin-rich structures.\nIn response to the reviewer’s comment, we recognize that the 16-hour timepoint fluorescence\nimage corresponding to the SynTag-Tau channel was not the ideal representative image in terms\nof Z-focus and contrast. As such, it did not accurately reflect the absence of microtubules (MTs)\nas quantified in our raw data analysis (Fig. 2e). We have therefore replaced the 16-hour images\nin Fig. 2d with another set from the same experimental dataset. The revised images more clearly\nconvey the lack of MTs at the 16-hour time point, consistent with Fig. 2e, and better depict the\nmorphology of amorphous clusters composed of tubulin and SynTag-Tau. The updated Fig. 2d\nis reproduced below.\nRegarding the reviewer’s note on Fig. 6a concerning the apparent loss of Tau signal, we\nemphasize that the SynTag-Tau fluorescence channel in these images colocalizes with the MT\nsignal. This observation demonstrates that L-Arg treatment preserves condensate activity, in\nagreement with the quantitative analysis presented in Fig. 6b. Therefore, Fig. 6a remains\nunchanged.\nFigure 2. (d) Condensate age-dependent microtubule polymerization assay in SynTag-Tau condensates.\nIn response to the second part of the question, we reported the requested control data\nearlier as Supplementary Figs. 8, 9 in the initial version of the manuscript, which are now\nrenumbered as Supplementary Fig. 9, 10. We have reproduced the figures below:\nto the addition of tubulin and GTP in MT polymerization assays. The composition of the sample is 12 μM SynTag-\nTau in a buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl , 0.5 mM EGTA, and 2 mM DTT with 5% PEG 8000.\ntimes.\nSupplementary Figure 10. (a) Lack of visible microtubules (MTs) in the absence of SynTag-Tau condensates. The\nsample was imaged using HiLyte647-labeled tubulin. It contains 5 μM tubulin and 1 mM GTP in a buffer containing\n80 mM PIPES (pH 6.9), 2 mM MgCl , 0.5 mM EGTA, and 2 mM DTT with 5% PEG 8000. (b) SynTag-Tau\n2\ncondensates, as visualized using Atto488-labeled SynTag-Tau, in the absence of tubulin and GTP (1 hour since\nsample preparation) in a buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl , 0.5 mM EGTA, and 2 mM DTT with\n2\n5% PEG 8000. Wherever applicable, the concentrations of Atto488-labeled SynTag-Tau and HiLyte647-labeled\ntubulin are 250 nM and 500 nM, respectively. Each experiment was independently repeated three times.\n9) Please consider commenting on the selection of the buffers: a standard buffer is used for MT\nassambly, but not all the experiments?\nWe thank the reviewer for this thoughtful comment. The use of different buffers for the\nmicrotubule (MT) assembly experiments versus the other assays was intentional and based on\nthe distinct physicochemical requirements of each experimental objective.\nPIPES, pH 6.9; 2 mM MgCl₂; 0.5 mM EGTA; 2 mM DTT; 5% PEG8000), which is a well-\nestablished composition that supports robust and reproducible tubulin polymerization under\nnear-physiological conditions (Hochmair et al. 2022). EGTA chelates Ca²⁺ ions that otherwise\ndestabilize MTs, Mg²⁺ stabilizes polymerized tubulin, and GTP provides the nucleotide cofactor\nfor polymerization. PIPES serves a buffering role similar to that of HEPES, used in other\nexperiments, as both are Good’s buffers.\nIn contrast, for tubulin partitioning experiments (Fig. 2b, c) and biophysical assays of\ncondensate aging (e.g., confocal imaging, FLIM, FRAP, nanorheology, and coalescence), we\nused a low-ionic-strength HEPES-based buffer (10 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, 2\nmM DTT). This buffer promotes the formation of larger condensates, which is critical for resolving\nthe condensate interior versus interface and for monitoring condensate-to-fibril transitions with\nhigher spatial and temporal resolution (compare Supplementary Figs. 7a and 9). The lower ionic\nstrength also maintains the fibrillization window within an experimentally tractable timescale\n(Supplementary Fig. 7a).\nAdditionally, achieving these larger condensates required a crowder concentration of 7.5–10%,\nwhich, while optimal for condensate characterization, inhibits MT polymerization (Supplementary\nFig. 13). Therefore, the MT assembly experiments necessarily used lower crowder levels to allow\nmicrotubule formation.\nTo further validate consistency, we have now repeated the tubulin partitioning experiments\nunder the same conditions as the MT polymerization buffer (Supplementary Fig. 12) and\nobserved the same overall trends.\nWe have added clarifying statements to the Methods sections — “Preparation of Protein\nCondensate Samples” and “Tubulin Partitioning and Microtubule Assembly Functional Assay” —\nexplaining the rationale for these buffer choices and their alignment with the specific\nexperimental goals.\nReviewer #2 (Remarks to the Author):\nIn this article the authors developed an engineered-Tau protein, comprising the full length of the\nprotein Tau and a 40 amino-acid priogenic tag, called SynTag-Tau. They showed that SynTag-\nTau self organizes in protein condensates that with aging transit irreversibly to fibrillar\nmorphology, in a time scale of 2 to 3 hours. The aging in a wild-type Tau condensate is much\nslower than aging in SynTag-Tau condensates, what makes this engineered model suitable for\ninvestigations of the mechanisms related to the transition from condensate to fibrillar phases.\nThe fibrillar phase is in the origin of neurodegenerative diseases, like Alzheimer, and to get\nknowledge about the transition from condensate (liquid-like) to fibrillar (solid-like) is in my point\nof view of significant importance.\npromoting the recruitment of tubulin in the formation of microtubules. Besides, they showed that\nL-Arg and L-Lys, abundant cationic amino acids, inhibit the liquid-fibrillar transition of SynTag-\nTau condensates. They propose that these small proteins alter the metastable energy barrier for\nthe liquid-fibrillar phase transition, indicating a proof of principle of a mechanism that can be\nused to decrease fibrillar formation from Tau condensates, avoiding fibrils appearance, a\ncharacteristic of neurodegenerative diseases.\nThe authors employed a variety of biochemical and optical techniques to support their\nconclusions, including fluorescence recovery after photobleaching, second harmonic generation\nimaging, Raman spectroscopy, optical tweezers and video particle tracking for rheology\nexperiments.\nThe data and the discussion are well presented, and the subject can be of high impact.\nNevertheless, I have some concerns about the presentation in the Supplementary Information\nregarding the optical tweezers and rheology results.\nResponse: We thank the reviewer for their positive and insightful comments, which have helped\nus further strengthen the manuscript.\nThe coalescence assay performed with optical tweezers seemed detached from the main\npurpose of the article. The authors should emphasize what the purpose of the coalescence\nassay is. I was expecting an analysis of the condensate mechanical properties For example,\nsurface tension, elastic modulus, and viscosity can be accessed with optical tweezers. Besides,\nthose physical properties may be related to the rheological storage and loss moduli, measured\nwith the video particle tracking experiments. The authors claim that the aging effect on SynTag-\nTau condensates is related to a liquid-solid transition, what would alter the above-mentioned\ncondensate physical properties. The authors should add this information to the text.\nResponse: We thank the reviewer for this thoughtful comment and the opportunity to clarify the\npurpose of the coalescence assay. The droplet fusion (coalescence) experiments were designed\nto qualitatively demonstrate the progressive aging of SynTag-Tau condensates (Fig. 1h) and to\nassess, on a relative scale, how L-Arg modulates their material properties. This qualitative\nassessment was complemented by quantitative nanorheology measurements using video\nparticle tracking (VPT) in other experimental systems (Fig. 5d).\nWe agree that optical tweezer–based assays can, in principle, provide quantitative access to\ncondensate physical properties such as surface tension, viscosity, and elastic modulus. Indeed,\nour laboratory has previously established such methods for characterizing biomolecular\ncondensates (Alshareedah et al. 2021; Alshareedah, Kaur, and Banerjee 2021; Alshareedah,\nSingh, et al. 2024; Alshareedah, Borcherds, et al. 2024). However, in the present study, the non-\nequilibrium nature of the SynTag-Tau condensates precluded such quantitative rheological\ncharacterization. Specifically, inverse capillary velocity (fusion time) could only be measured at\nthe initial time point (~30 minutes post-assembly; response Fig. 1). Beyond one hour, the\n14\ndroplets exhibited arrested fusion, beginning to coalesce but failing to fully relax into spherical\nshapes, consistent with rapid aging and increasing viscoelasticity.\nFurther, ThT fluorescence imaging confirmed that these condensates undergo amyloid formation\nwithin 3–4 hours, highlighting a rapid transition from fluid-like to solid-like states. Such\naccelerated kinetics render standard optical tweezer rheology impractical within the\nexperimental window. Thus, the coalescence assay served as a qualitative indicator of\ncondensate aging and as evidence that L-Arg treatment delays this process, enhancing\nviscoelasticity even at concentrations as low as 0.25–0.5 mM (Supplementary Fig. 18).\nFor quantitative measurements, we employed VPT nanorheology using a more experimentally\nstable WT Tau–heparin system, which does not undergo rapid fibrillization. These data (Fig. 5d)\ndemonstrate that L-Arg enhances condensate viscoelasticity and promotes intra-\ncondensate percolation, providing a mechanistic context consistent with the qualitative trends\nobserved in the coalescence assay.\nTogether, the optical tweezer and VPT experiments complement one another—the former\ncapturing qualitative signatures of aging and the latter providing quantitative rheological\nvalidation in a closely related, slower-aging model system.\n!\"#\n#\"A\n#\"%\n#\"&\n#\"'\n#\"#\nResponse Figure 1. Laser signal for SynTag-Tau droplet fusion event measured 30 min after sample preparation.\nThe laser signal was fitted with the fusion relaxation model (Alshareedah, Kaur, and Banerjee 2021), F(t)=aexp(-\nt/τ)+bt+c, where τ is the relaxation time of the droplets and a, b, and c are the fitting parameters, to estimate the\nfusion relaxation timescale of the droplets. The fitting revealed the droplet relaxation time, τ=143 ± 0.33 ms, from\nthe droplet fusion event.\nThe description of the optical tweezers assays lacks information about the system employed,\nsuch as what kind of objective lens is used and what is its numerical aperture. What is the\n15\n6\"R\"F4-3-21F0L.*-,+*F(\n:;<+,-=#-9.0\n-(F*+,-*.L0F1-\n->+1F?F<.@0-9@A+1-;.<- \n#\"# #\"T !\"# !\"T '\"# '\"T\n8.9+-4*6\nmaximum power employed? A laser power of 100 microW seems to be too small value to be\nused, even for a soft material like the protein condensate. Was the power measured at the\nentrance of the objective lens? The loss of power in the lens can reach 80% depending on the\nlens and laser employed in the optical tweezers system, what leads to a power estimation of\n30microW in the optical traps employed. A toy laser pointing has more than ten times bigger\npower. It seems that the presented value is not the correct used in the experiments.\nWhat was the setup to build the double optical trap? Concerning the rheological experiments procedures\ndescribed in the Supplementary Information, the Equation (6), line 314, is meaningless. I would\nsuggest a large revision of these two sections.\nResponse: We appreciate the reviewer’s detailed feedback and the opportunity to clarify the\noptical tweezers methodology. There appears to be some misunderstanding regarding the laser\npower requirements for trapping and manipulating biomolecular condensates. Over the past five\nyears, our laboratory has pioneered and standardized optical tweezer–based microrheology to\nstudy the material properties and aging dynamics of protein and RNA condensates, establishing\nvalidated protocols that have been reported across multiple peer-reviewed publications\n(Alshareedah et al. 2021; Alshareedah, Kaur, and Banerjee 2021; Alshareedah, Singh, et al.\n2024; Alshareedah, Borcherds, et al. 2024).\nFor the droplet coalescence experiments, we used a Lumicks C-Trap instrument integrating\ndual optical traps with a confocal fluorescence microscope, equipped with a 60× water-\nimmersion objective (NA 1.3). A 1064 nm infrared laser provides 1 mW power at the\nobjective (measured at the objective exit). For condensate fusion assays, we employed 10%\nof this maximum power (~100 µW)—a level sufficient for stable trapping of protein and RNA\ncondensates while minimizing laser-induced heating. This power range has been repeatedly\nvalidated in our prior work for both droplet trapping and bead-based microrheology\n(Alshareedah, Thurston, and Banerjee 2021; Mahendran et al. 2025; Alshareedah, Borcherds,\net al. 2024). The trapping mechanism relies on the refractive-index contrast between the\ncondensate dense phase and the surrounding dilute phase, which requires substantially lower\noptical power than that typically used for rigid dielectric beads.\nWe emphasize that the reported laser power was empirically measured at the objective, not\nestimated from the source output, and is consistent with published optical-tweezer assays of\nsimilarly soft, refractive droplets. Hence, the concern that 100 µW would be insufficient does not\napply to condensate systems, whose low interfacial tension and density contrast permit efficient\ntrapping at this power level (Alshareedah et al. 2021).\nWe have revised the method section outlining the details of ‘Optical tweezer-mediated\ncoalescence assay’, which has been reproduced below:\nOptical trap-induced fusion of protein condensates was accomplished using a Lumicks C-trap\noptical tweezer system equipped with a dual trap and a laser scanning confocal fluorescence\nmicroscope, with a 60x water immersion objective of numerical aperture 1.3, following a protocol\n16\ndescribed previously4, 5. In brief, for a typical condensate fusion experiment, two condensates\nwere trapped using a 1064 nm laser with the minimum power setting (~ 100 µW) to minimize\nlaser-induced heating effects. Typically, the 1064 nm laser used in the assay provides a power\nof 1 mW at the objective (measured at the exit of the laser light from the objective). For the\nexperiments, a power of 10% of 1 mW, which corresponds to ~100 µW, was used. This power\nis sufficient to trap droplets using the dual-trap optical tweezer system and perform droplet fusion\nassays4. The trapping of condensates relies on the refractive index mismatch between the\ncondensate dense phase and the surrounding dilute phase. After trapping, Trap 2 was kept fixed,\nand Trap 1 was moved towards Trap 2 at a constant velocity of 100 nm/s. The trap is set to\nmaintain this velocity until the fusion process concludes, allowing the final droplet to relax to a\nspherical or equilibrium shape. A bright-field camera was used to take time-lapse images of the\ndroplet fusion event at 15 frames per second.\nRegarding the reviewer’s comment on Equation (6) in the section “Estimation of\ndynamical moduli from nanorheology measurements,” we note that this equation defines the\nrelationship between the complex shear modulus (G) and the compliance (j) in the time\ndomain, forming the theoretical basis for estimating viscoelastic moduli from VPT nanorheology\ndata using the Evans method (Evans et al. 2009). This approach has been successfully applied\nin our prior publications (Alshareedah, Borcherds, et al. 2024), and we therefore believe it is both\nappropriate and important to retain Equation (6) as part of the methodological description.\nOverall, the optical tweezer parameters reported are accurate, experimentally verified,\nand consistent with established standards for condensate trapping. The Methods section has\nbeen updated to explicitly include all instrumental and experimental details.\nIn summary, this article presents remarkably interesting and important results related to the\ntransition of liquid-solid in protein condensates. It brings proof of the principle of the use of small\nproteins to possibly prevent the liquid to fibrillar transition. In my opinion, it is suitable for\npublication after clarification of the issues reported above and text alterations accordingly.\nResponse: We sincerely thank the reviewer for the highly positive and encouraging feedback.\nWe appreciate their recognition of the significance of our findings and have tried our best to\naddress all the points raised above through clarifications and revisions to the text as suggested.\nWe hope the reviewer will be persuaded that the revised manuscript satisfactorily addresses all\nconcerns.\nReviewer #3 (Remarks to the Author):\nIn the manuscript, Tharun et al. investigated the process of biomolecular condensation and the\nsubsequent formation of amyloid fibrils. By engineering the terminal region of the Tau protein,\nthe timescale of physical aging is significantly reduced to a few hours. Using fluorescence\nspectroscopy and nonlinear microscopy, the authors demonstrated that physical aging occurs\n17\nthrough a disorder-to-order process. The β-sheet content is increased, ultimately leading to\namyloid fibril formation. It is noted that the fibril nucleation was observed to initiate at the\ncondensate interface, which compromises the biochemical function of the condensates. The\nstudy further explored the effect of small molecules on protein fibrillation. Among the tested\ncompounds, L-arginine (L-Arg) was found to inhibit fibril formation, which was attributed to an\nincrease in the viscoelasticity of the condensates. L-Arg selectively inhibited fibrillation without\naffecting the condensation process itself, suggesting that condensation and fibrillation can be\ndecoupled.\nOverall, the manuscript is well-written with great clarity. I would like to recommend considering\nthe publication of this manuscript because it tackles the important problems in the field of\nBiomolecular condensate. Understanding how the intrinsically disordered proteins in\nbiomolecular condensates transform into irreversible fibrils is crucial for the ultimate cure of\nneurodegenerative disease pathology and identifying potential therapeutic strategies. The\nauthors demonstrate that by utilizing the small molecules stabilizes protein condensates in a\nmetastable state, thereby preserving their biochemical function. I only have several comments\nas follows:\nResponse: We sincerely appreciate the reviewer’s thoughtful and positive evaluation of our\nwork.\nComments:\n1. In Figures 1(j) and 4(b), the authors present BCARS spectra obtained from hyperspectral\nimaging. However, the corresponding BCARS hyperspectral imaging data are not included in\nthe main text or the Supplementary Information. The BCARS hyperspectral imaging could\nsignificantly strengthen the manuscript. If the authors' hypothesis—that amyloid fibrils\nnucleate initially at the condensate interface—is true, one would expect to observe a heterogeneous\nspatial distribution of the β-sheet signal intensity. Therefore, the hyperspectral imaging data and\naccompanying discussion are required.\nWe thank the reviewer for raising this important point. We fully agree that, if our hypothesis—\nthat amyloid fibrils nucleate preferentially at the condensate interface—is correct, BCARS\nhyperspectral imaging should ideally reveal a heterogeneous spatial distribution of the β-sheet\nsignal.\nHowever, the pixel-wise signal-to-noise ratio (SNR) of the condensate BCARS scans limited our\nability to resolve β-sheet content with sufficient spatial fidelity on a per-pixel basis. To ensure\nquantitative robustness, our analysis in the submitted manuscript therefore focused on spectra\naveraged over multiple condensates for each treatment condition and time point, thereby\nimproving the effective SNR and reliability of the spectral fits.\nTo directly address the reviewer’s request, we have now performed additional analyses of the\nBCARS hyperspectral dataset to assess potential β-sheet enrichment at the condensate\n18\ninterface. Specifically, we averaged spectra collected from the periphery versus the interior of\naged (6 h) SynTag-Tau condensates, with and without L-arginine treatment, and fitted the\nAmide I band to compare β-sheet composition between these regions. The resulting multi-peak fits\nreveal minimal differences in β-sheet content between the periphery and interior under both\nconditions (see Response Fig. 2).\nBecause the pixel-wise SNR of individual scans remains inherently limited, our primary\nhyperspectral analysis continues to rely on ensemble-averaged spectra. Nevertheless, to\nillustrate this point visually, we generated representative BCARS hyperspectral intensity\nprojections of the secondary-structure components within the Amide I band (Response Fig. 3).\nThese projections do not show a clear or systematic enrichment of β-sheet signal at the\ncondensate interface relative to the interior at 6 h. At earlier (2 h) time points, condensates were\nweakly adhered to the substrate and subject to optical trapping by the excitation beam,\nprecluding reliable mapping of their periphery.\nTo further resolve the spatial localization of fibrils, we conducted confocal fluorescence z-\nstack imaging using ThT fluorescence together with A594-labeled SynTag-Tau. Unlike\nearlier single-plane imaging (Fig. 4a), this approach allowed us to visualize fibril distribution\nacross the condensate volume. The resulting images show that fibrils predominantly localize at\nthe condensate interface and in the surrounding dilute phase, but not within condensate interiors\n(Fig. 4b). When the z-focus is shifted toward the upper interface of the condensate, fibrils\nbecome even more apparent—supporting our model that fibril nucleation occurs at the interface.\nUnder equivalent conditions, L-Arg–treated condensates show no detectable ThT fluorescence\nacross z-sections (Fig. 4c), consistent with their protection against amyloid formation.\nTogether, these additional analyses and imaging data reinforce our central conclusion that fibril\nnucleation occurs at condensate interfaces and that L-Arg effectively suppresses this process.\nTo this end, we have included below an excerpt of the revised main text explaining these\nobservations:\nA caveat of imaging condensates at a single plane using ThT fluorescence is the lack of spatial\ninformation in the z-axis that would be critical to resolve whether fibrils are indeed nucleated at\ncondensate interfaces or if they localize to condensate interiors. We tested this by capturing z-\nslices, utilizing A594-labeled SynTag-Tau and ThT fluorescence, in samples of untreated\nSynTag-Tau condensates. We find that ThT fluorescence is preferentially enriched at\ncondensate interfaces with amyloid fibrils growing into the dilute phase, but not condensate\ninteriors (Fig. 4b). At a relatively higher z position (0.95 μm), i.e., approaching the topmost\ninterface of condensates (with respect to the coverslip surface), the ThT signal originating from\nfibrils becomes more prominent, in line with interfacial nucleation of fibrils. This is consistent with\nthe recently proposed framework that condensate interiors could act as sinks of amyloidogenic\nproteins; however, their interfaces present the risk of fibril nucleation, with fibril growth occurring\noutward into the dilute phase24. On the other hand, L-Arg-treated SynTag-Tau condensates fail\nto show any notable ThT fluorescence across z-sections (Fig. 4c).\nsubmission (Fig. 4f, g)—together with the newly added ThT-based z-stack confocal\nfluorescence microscopy (Fig. 4b, c), provides more sensitive and spatially resolved\napproaches than BCARS hyperspectral imaging for visualizing the localization of early amyloid\nfibril formation at condensate interfaces.\nResponse Fig. 2: Amide I band fitting of BCARS spectra averaged from aged (6 hours) SynTag-Tau condensates,\nwith and without L-Arg treatment, acquired at the condensate periphery and interior. The fits show minimal\ndifferences in β-sheet signal intensity between the edge and center regions for both treatment conditions. The fitting\nprocedure is identical to that described in the original submission.\nSynTag-Tau condensates at two time points (2 hours and 6 hours). The first column shows the Amide I band\nintensity projection (1642–1675 cm⁻¹), summed at each pixel following 3-point spectral smoothing. The second\nthrough fourth columns display maps of the α-helix (1642–1662 cm⁻¹), random coil (1660–1675 cm⁻¹), and β-sheet\n(1671–1693 cm⁻¹) regions, respectively. For each map, the summed peak intensity within the specified range is\nnormalized pixel-wise by dividing by the cumulative Amide I band intensity at the same pixel. To suppress\nbackground contributions, a dynamic threshold was applied: pixels with a cumulative Amide I intensity below the\nimage mean were excluded in the 2-hour projections, and pixels below the mean plus one standard deviation were\nexcluded in the 6-hour projections. The 2-hour scans do not exhibit clear structural features, as the condensates\nwere optically trapped by the excitation beams during measurement.\nA594-labeled SynTag-Tau (red). The indicated z-position is relative to the leftmost z-slice in the panel. The inset\nshown represents the area demarcated by the white-dashed box. Sample age is 48 hours. (c) Maximum intensity\nprojection (MIP) image of 2 mM L-Arg treated SynTag-Tau condensates with A594-labeled SynTag-Tau and ThT.\nSample age is 48 hours. Notably, the ThT fluorescence intensity is substantially lower relative to the A594-labeled\nSynTag-Tau fluorescence for these condensates, in contrast to that of the untreated SynTag-Tau condensates,\nsuggesting a lack of fibrils with L-Arg treatment.\n2. In Figures 1 (j) and 1(b), the BCARS spectra of nascent condensates shows higher\nbackground (~0.2 offset) than other spectra. Is it related to the physical aging?\nWe believe that the apparent offset observed in the BCARS spectra of nascent condensates can\nresult from the BCARS post-processing procedure, similar to any background subtraction in\nspontaneous Raman, rather than being indicative of condensate physical aging. Specifically, this\noffset may arise from the phase-retrieval procedure used to convert the raw BCARS signal at\neach pixel into a Raman-like spectrum that more closely resembles a spontaneous Raman\nspectrum (Camp, Lee, and Cicerone 2016). Phase-retrieved BCARS spectra can exhibit\nbaseline fluctuations due to the nature of the Kramers–Kronig transformation and the finite\nspectral acquisition window. Truncation of the spectral range introduces edge effects and\nconstrains the integral used for phase retrieval, which can produce residual baseline offsets in\nthe reconstructed spectra.\n3. The authors report that L-arginine (L-Arg) inhibits fibril formation, which is due to an increase\nin condensate viscoelasticity. However, it remains unclear whether this represents a general\nmechanism that viscoelasticity governs the kinetics of fibril formation. Specifically, does the\nhigher (lower) viscoelasticity correlate with slower (faster) fibril formation?\nTo address this, it would be benefit to examine the effects of other small molecules. For instance, if L-lysine (L-\nLys), another fibril-inhibiting molecule, also increases the condensate viscoelasticity. And if\nmolecules such as L-glutamic acid (L-Glu) or L-aspartic acid (L-Asp), which will accelerate\nfibrillation, correspondingly reduce viscoelasticity. These results would support the proposed\nmechanism. A more in-depth discussion is required.\nWe thank the reviewer for raising this important point regarding whether alterations in\ncondensate viscoelasticity correlate with the kinetics of fibril formation. In a recent collaborative\n22\nstudy with Dr. Tanja Mittag’s group on the prion-like low complexity domain of hnRNPA1, we\nreported that protein condensates of higher viscoelasticity, such as those formed by sequence-\nengineered variants with higher sticker strength (e.g., tyrosine (Y) to tryptophan (W)), lead to\nslower fibril formation via constraining the protein efflux-driven mechanism of amyloid fibril\ngrowth in the dilute phase (Das et al. 2025). In the context of whether small molecules may\ncontribute to a similar mechanism, it is worth noting that methylene blue—a clinically studied\ncompound for Tauopathies—was recently shown to promote Tau condensate gelation or\npercolation. Interestingly, it simultaneously suppressed Tau condensate fibrillization (Huang et\nal. 2023). This observation is reminiscent of the effect of L-Arg on Tau condensate viscoelasticity\nand amyloid formation kinetics, as quantitatively reported in our study. These parallels further\nsupport the notion that modulation of condensate material properties can influence fibrillization\npathways. However, counterexamples of small molecules that promote condensate fluidity while\napparently reducing Tau aggregation also exist, such as myricetin (Dai et al. 2021). These\ncontrasting reports suggest that small molecules with inhibitory potential against Tau condensate\nto amyloid transition may act via distinct mechanisms having contrasting effects on condensate\nmaterial properties.\nAs per the reviewer’s suggestion of testing the correlation between small molecule-induced\nchanges to SynTag-Tau condensate viscoelasticity and the kinetics of fibril formation, we set out\nto determine whether L-Glu treatment, which we observed to enhance the formation of fibrils,\nleads to reduced condensate viscoelasticity. Employing VPT-based nanorheology, we observed\nthat upon L-Glu treatment, condensate viscoelasticity appears to be increased relative to\nthe untreated condition, but remains similar to that of the L-Arg-treated condition. We have\nnow incorporated the newly acquired data as Supplementary Fig. 19, which is also reproduced\nbelow:\nwith small molecule treatment [L-arginine, L-Arg or L-glutamic acid, L-Glu] or without small molecule treatment\n(untreated). Dynamical moduli of Tau condensates in the absence (b) or presence of L-Arg (c) or L-Glu (d). These\nmeasurements were conducted 2 hours after sample preparation. The ‘untreated’ and ‘L-Arg’ data are replotted\nhere based on data reproduced from Fig. 5b, c. The dashed line in (b-d) represents the crossover frequency. The\ncomposition of the samples used here is 40 μM Tau in buffer containing 10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1\nmM EDTA, and 2 mM DTT, along with 7.5% PEG 8000 crowder, and 6.25 μΜ heparin. Wherever applicable, the\nsmall molecule concentration used in these experiments is 2 mM. Each of these experiments was independently\nrepeated three times.\nWe wondered whether the observed increase in L-Glu–treated Tau condensate viscoelasticity\nresults from enhanced fibrillization. ThT measurements revealed that indeed L-Glu−treated Tau\ncondensates display higher ThT signals (Supplementary Fig. 16), when compared to the\nuntreated (UT) condition, consistent with observations in the SynTag-Tau system (Fig. 3a).\nHence, fibril-induced apparent enhancements in L-Glu–treated Tau condensate\nviscoelasticity make it difficult to determine whether changes in viscoelasticity directly\ncorrelate with fibril formation kinetics. A systematic investigation of this relationship will\nbe necessary in future work. We have appended the new data to the already existing plot in\nthe Supplementary Information to show the normalized ThT intensities of UT and small molecule-\ntreated conditions, which is now labeled as Supplementary Fig. 16. A note on the normalized\nThT fluorescence intensity estimation is now included in the relevant method section (see\n‘Estimation of Thioflavin T (ThT) fluorescence intensity’).\n24\n1000\n100\n10\nh\nA\nr\nr g\nh\nG\nr\nl u\n2\nhr\nU T\nd\nAr g\nd\nGl u\n1\nd\nU T\nd\nAr g\nd\nGl u\n3\nd\nSupplementary Figure 16. Wild-type Tau condensate samples (UT: untreated condition) show an increased\nThioflavin T (ThT) fluorescence signal upon aging, which is diminished in the presence of 2 mM L-Arg (L-Arg) and\nis enhanced in the presence of 2 mM L-Glu (L-Glutamic acid). The time points are indicated in the figure (hr- hours;\nd- days). The center line in the box plot represents the median, and the whiskers represent min to max. Statistical\nsignificance was determined by performing an unpaired two-sided Student’s t-test (**** means p<0.0001) between\nthe ThT fluorescence intensities of either UT and L-Arg-treated condensates or UT and L-Glu-treated condensates\nat different time points. The composition of the sample is 25 μM Tau in buffer containing 10 mM HEPES (pH 7.5),\n50 mM NaCl, 0.1 mM EDTA, and 2 mM DTT with 7.5% PEG 8000 and 6.25 μM heparin. Each experiment was\nindependently repeated three times.\nOverall, these newly appended experimental data and the cited literature reports discussed\nabove suggest that small molecule-induced selective inhibition of Tau condensate physical aging\nis likely due to a number of factors and may not be restricted to condensate viscoelastic\nproperties. We uncovered, through FLIM-based measurements of condensate\nmicroenvironments (Fig. 2f, Fig. 4f), CARS-based structural measurements (Fig. 4e), as well\nas ThT fluorescence z-stack imaging (Fig. 4b, c), that L-Arg treatment tunes fibril nucleation at\nthe condensate interface, thereby attenuating structural changes associated with aging. To glean\nthe underlying mechanism of L-Glu in promoting fibrillization, a similar comprehensive approach\nand high-resolution structural studies would be required, which would be an exciting direction\nfor future studies.\nThe following paragraph has been incorporated in the Results and Discussion section:\nWe also tested the effect of L-Glu treatment on condensate viscoelasticity (Supplementary Fig.\n19). We observed that it enhances condensate viscoelasticity similarly to L-Arg; however, it is\nworth noting that this enhancement might stem from faster fibril assembly (Supplementary Fig.\n16) rather than elevated condensate metastability. Further systematic investigation will be\nrequired to clearly distinguish these mechanisms.\n4. The fibril-inhibiting molecules, such as L-arginine and L-lysine, both are basic amino acids\n25\n).u.a(\nytisnetni\nThT\ndezilamroN\n****\n****\n****\n****\n****\n****\nwith pKa values greater than 7. Is there a potential correlation between their pKa values and the\nmechanism of physical aging? Please comment on it.\nWe appreciate the reviewer’s question regarding a potential link between the pKa of candidate\nsmall molecules and their ability to inhibit the condensate-to-amyloid transition. In the current\nstudy, we tested a panel of small molecules with chemical moieties possessing strongly basic\npKa (values exceeding 7), including L-arginine (pKa of guanidino group = 12.4), L-lysine (pKa of\nε-amino group = 10.5), guanidinium hydrochloride (pKa of guanidinium group = 12.6), L-arginine\nethyl ester (pKa of guanidino group = 12.4), spermine, and spermidine (pKa across multiple\namine groups = ~10.7). Despite sharing highly basic (i.e., high pKa) chemical moieties, several\nof these compounds, such as spermine and spermidine, failed to inhibit SynTag-Tau fibrillization\nselectively (Fig. 3a; Supplementary Fig. 15), which suggests that elevated pKa alone may not\nbe a good predictor of inhibitory potential in the current case. Rather, selective inhibition of\ncondensate aging may rely on a number of physicochemical properties, including but not limited\nto the following: valence (number of interaction sites), specificity of chemical moieties, and pKa.\nA combination of these properties confers the ability to interfere with the maturation of\ncondensates to irreversible amyloid fibrils, without disrupting functional phase separation.\nWe have included a brief excerpt of the discussion above in the Results and Discussion, which\nhas been reproduced below:\nGiven the observation that the positive hits in the small molecule screen are basic amino acids\nwith chemical moieties of high pKa (>7), L-Lys (pKa of ε-amino group = 10.5), and L-Arg (pKa\nof guanidino group = 12.4), we wondered at the possibility that high pKa could be an important\nchemical property that confers protection against condensate-to-fibril transition. However, the\nremaining small molecules with basic moieties failed to achieve a similar effect. For example,\nguanidinium hydrochloride (pKa of guanidinium group = 12.6), L-arginine ethyl ester (pKa of\nguanidino group = 12.4), and spermine and spermidine (pKa across multiple amine groups =\n~10.7). Despite sharing highly basic, i.e., high pKa chemical moieties, several of these\ncompounds failed to inhibit SynTag-Tau fibrillization selectively (Fig. 3a; Supplementary Fig.\n15), which suggests that strongly basic (high pKa) functional groups alone are a poor predictor\nof inhibitory potential.\nPlease note that the pKa values were obtained using computational cheminformatics tools of\nDrugBank (Knox et al. 2024), which has been noted in the method section, accompanied by a\nreference.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. 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Klenerman. 2022. \"Mechanistic Models of Protein\nAggregation Across Length-Scales and Time-Scales: From the Test Tube to\nNeurodegenerative Disease.\" Front Neurosci 16:909861. doi: 10.3389/fnins.2022.909861.\n28"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done an outstanding job in addressing all my comments. I recommend this excellent manuscript for\npublication.", "reviewer_2": "In this article the authors developed an engineered-Tau protein, comprising the full length of the protein Tau and a 40 amino-\nacid priogenic tag, called SynTag-Tau. They showed that SynTag-Tau self organizes in protein condensates that with aging\ntransit irreversibly to fibrillar morphology, in a time scale of 2 to 3 hours. The aging in a wild-type Tau condensate is much\nslower than aging in SynTag-Tau condensates, what makes this engineered model suitable for investigations of the\nmechanisms related to the transition from condensate to fibrillar phases.\nThe fibrillar phase is in the origin of neurodegenerative diseases, like Alzheimer, and to get knowledge about the transition\nfrom condensate (liquid-like) to fibrillar (solid-like) is in my point of view of significant importance.\nThe authors also showed that the condensates of SynTag-Tau are biologically active in promoting the recruitment of tubulin\nin the formation of microtubules. Besides, they showed that L-Arg and L-Lys, abundant cationic amino acids, inhibit the\nliquid-fibrillar transition of SynTag-Tau condensates. They propose that these small proteins alter the metastable energy\nbarrier for the liquid-fibrillar phase transition, indicating a proof of principle of a mechanism that can be used to decrease\nfibrillar formation from Tau condensates, avoiding fibrils appearance, a characteristic of neurodegenerative diseases.\nThe authors employed a variety of biochemical and optical techniques to support their conclusions, including fluorescence\nrecovery after photobleaching, second harmonic generation imaging, Raman spectroscopy, optical tweezers and video\nparticle tracking for rheology experiments.\nThe data and the discussion are well presented, and the subject can be of high impact. All the questions I raised in the first\nreview were answered. I recommend publication in Nature Communications.", "reviewer_3": "I recommend the work to be published as is.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter