DOI: 10.5061/dryad.n8pk0p38v
title: Data from: Decoupling phase separation and fibrillization preserves activity of biomolecular condensates

description: Age-dependent conversion of metastable, liquid-like protein condensates
 into amyloid fibrils is observed across many protein systems linked to
 neurodegeneration. A critical question is whether the thermodynamic forces
 governing reversible phase separation can be decoupled from those driving
 irreversible amyloid formation. This study addresses this question using
 an engineered variant of the microtubule-associated protein Tau that forms
 biochemically active condensates. Under quiescent, cofactor-free
 conditions, these protein condensates undergo rapid aging into amyloid
 fibrils, with the condensate interface serving as a site for fibril
 nucleation. This accompanies loss of condensate activity in tubulin
 recruitment and microtubule assembly. The small-molecule metabolite
 L-arginine selectively suppresses the condensate-to-fibril transition
 without disrupting phase separation, acting in a valence- and
 chemistry-dependent manner. By increasing condensate viscoelasticity,
 L-arginine mitigates age-dependent functional decline of Tau condensates,
 demonstrating that small molecules can stabilize condensate metastability
 and preserve function.
description: # Data from: Decoupling phase separation and fibrillization preserves
 activity of biomolecular condensates DOI: doi.org/10.5061/dryad.n8pk0p38v
 This readme file was generated on 2026-02-06 by Tharun Selvam Mahendran ##
 DESCRIPTION: The dataset provided here contains the representative imaging
 data (figures/video) corresponding to the main text and supplementary
 information concerning the referenced publication. A description
 accompanying each figure is provided. Methodological details and
 additional acquisition parameters can be found in the relevant methods
 section of the publication. The images are organized into folders, broadly
 categorized into main text figures (Dryad_source_data_for_main_text.zip)
 and supplementary information figures/videos
 (Dryad_source_data_for_supplementary_information.zip). The main folders
 include subfolders specified for individual image panels if the concerned
 figure includes multiple panels of data. These subfolders are named in
 accordance with the corresponding panel letter, e.g., a, b, c, d, etc.
 Further, the series of images constituting a figure are categorized
 according to experimental parameters if multiple image data exists in a
 single figure panel: timepoint of imaging (mentioned in hours, minutes or
 seconds), fluorophore being imaged (e.g., Atto488 or A488, Alexa594 or
 A594, H647, ThT), and sample composition (e.g., SynTag-Tau, Tau, tubulin,
 L-Arg), etc. A description is provided along with each set of files to
 provide clarification on technical terms used. Additionally, a direct
 correspondence exists between the organized data in the repository and the
 data shown in the published article. Therefore, details of the sample and
 additional information about the sample or imaging conditions, if
 applicable, can be found in the publication, specifically, the relevant
 figure legend and/or method section. ## GENERAL INFORMATION: Title of
 Dataset: Decoupling Phase Separation and Fibrillization Preserves Activity
 of Biomolecular Condensates Author/Principal Investigator Information
 Name: Priya R. Banerjee ORCID:
 [https://orcid.org/0000-0002-6169-1461](https://orcid.org/0000-0002-6169-1461) Institution: The State University of New York at Buffalo Address: 229 Fronczak Hall Email: [prbanerj@buffalo.edu](mailto:prbanerj@buffalo.edu) Author Name: Tharun Selvam Mahendran ORCID: [https://orcid.org/0000-0002-7536-1236](https://orcid.org/0000-0002-7536-1236) Institution: The State University of New York at Buffalo Address: 206 Fronczak Hall Email: [tharunse@buffalo.edu](mailto:tharunse@buffalo.edu) Author/Alternate Contact Information Name: Anurag Singh ORCID: [https://orcid.org/0000-0002-8132-903X](https://orcid.org/0000-0002-8132-903X) Institution: The State University of New York at Buffalo Address: Email: [singh26@buffalo.edu](mailto:singh26@buffalo.edu) Date of data collection: between 2022-01-01 and 2025-11-01 Geographic location of data collection: Buffalo, New York, United States of America, and Austin, Texas, United States of America. Information about funding sources that supported the collection of the data: This work was supported by the US National Institutes of Health (NIH) through grants R03 AG070510 (to P.R.B.), R35 GM138186 (to P.R.B.), and the St. Jude Children’s Research Collaborative on the Biology and Biophysics of RNP Granules (to P.R.B.). We are grateful for financial support from the Welch Foundation F-2008-20220331, the Chan Zuckerberg Initiative #2021-236087, and the National Science Foundation (NSF) #2146549 to S.H.P., and C.M.J. is grateful for support from NSF (DGE-1610403) and NIH T32 EB007507. ## SHARING/ACCESS INFORMATION Licenses/restrictions placed on the data: CC Zero Links to other publicly accessible locations of the data: Links/relationships to ancillary data sets: [https://github.com/BanerjeeLab-repertoire/Decoupling-Phase-Separation-and-Fibrillization-Preserves-Condensate-Biochemical-Activity](https://github.com/BanerjeeLab-repertoire/Decoupling-Phase-Separation-and-Fibrillization-Preserves-Condensate-Biochemical-Activity) Was data derived from another source? NO If yes, list source(s): Recommended citation for this dataset: T. S. Mahendran et al., Decoupling Phase Separation and Fibrillization Preserves Activity of Biomolecular Condensates. bioRxiv, 2025.2003.2018.643977 (2025). Usage notes: File types are either in .tif or .png formats. Either file type can be accessed via Fiji/ImageJ, a freely available software. ## DATA & FILE OVERVIEW File List: Source data for the main text (contains all the microscopy data shown in the main text)  ### Dryad_source_data_for_main_text.zip #### Figure 1 \- d \- 1 hr.tif \- 2 hr.tif \- 2.5 hr.tif \- 2.7 hr.tif \- 2.8 hr.tif \- 3 hr.tif \- 3.7 hr.tif Description: Time-lapse imaging of Atto488-labeled SynTag-Tau condensates. These image files correspond to different time points. hr indicates hours. \- f \- SHG of condensate.tif \- SHG of fibrils.tif Description: Second harmonic generation (SHG) microscopy shows the absence of molecular ordering in nascent SynTag-Tau condensates (indicated by dashed circles; sample age = 2 hours) and the presence of spatial ordering in fibrils (sample age = 10 hours). These image files correspond to second harmonic generation (SHG) of either condensates or fibrils. \- g -condensate.tif -fibrils.tif Description: Representative Thioflavin T (ThT) fluorescence line profiles from a nascent SynTag-Tau condensate (indicated by a dashed circle; sample age = 2 hours) and fibrils (sample age = 10 hours). The contrast was adjusted independently in order to better visualize the nascent condensate that is very dim. These image files correspond to either condensate or fibrils. \- h -0 hr 0s.png -0 hr 2.5s.png -0 hr 4.9s.png -2 hr 0s.png -2 hr 5.3s.png -2 hr 300s.png Description: Optical tweezer-controlled fusion of nascent SynTag-Tau condensates (sample age = 30 mins, indicated as the 0-hour time point) and condensates at 2 hours of age. These image files correspond to different time points. hr indicates hours and s indicates seconds. #### Figure 2 -b -Merge.tif -A488 Tau.tif -H647 tubulin.tif Description: Nascent SynTag-Tau condensates (age = 2 hours) in the presence of 50 nM HiLyte647-labeled tubulin show enrichment of tubulin in the dense phase. These image files correspond to different fluorescence channels. -c -Merge.tif -A488 Tau.tif -H647 tubulin.tif Description: Aged SynTag-Tau condensates (age = 4 hours) with the addition of 50 nM HiLyte647-labeled tubulin show altered tubulin partitioning to the condensate interface. These image files correspond to different fluorescence channels. -d -A488 1 hr.png -A488 2.5 hr.png -A488 5 hr.png -A488 8 hr.png -A488 12 hr.png -A488 16 hr.png -H647 1 hr.png -H647 2.5 hr.png -H647 5 hr.png -H647 8 hr.png -H647 12 hr.png -H647 16 hr.png -Merge 1 hr.png -Merge 2.5 hr.png -Merge 5 hr.png -Merge 8 hr.png -Merge 12 hr.png -Merge 16 hr.png Description: Condensate age-dependent MT polymerization assay in SynTag-Tau condensates. The naming organization described here is in reference to a series of images, corresponding to different time points (hr or hours) and fluorescence channels (A488, H647, or Merge). -f -1 hr.tif -3 hr.tif -5 hr.tif Description: Frequency-domain (FD) FLIM map of SynTag-Tau condensates at various time points. These image files correspond to different time points. -h -image.tif Description: FD-FLIM map of aged SynTag-Tau condensates with emergent amyloid fibrils (sample age = 8 hours). #### Figure 3 -a -ATP 2 hr.png -ATP 24 hr.png -Control 2 hr.png -Control 24 hr.png -GnHCl 2 hr.png -GnHCl 24 hr.png -L-Arg 2 hr.png -L-Arg 24 hr.png -L-Asp 2 hr.png -L-Asp 24 hr.png -L-Glu 2 hr.png -L-Glu 24 hr.png -L-Lys 2 hr.png -L-Lys 24 hr.png -L-Pro 2 hr.png -L-Pro 24 hr.png -TMAO 2 hr.png -TMAO 24 hr.png -Urea 2 hr.png -Urea 24 hr.png Description: Effect of naturally occurring small molecule metabolites, chaotropic compounds, and small molecule modulators of protein-protein interactions on phase separation and fibrillization of SynTag-Tau. These image files correspond to different small molecule treatment groups at two different time points. -b -0 mM 2 hr.png -0 mM 4 hr.png -0 mM 7 d.png -0 mM 8 hr.png -0 mM 12 hr.png -0 mM 24 hr.png -0 mM 48 hr.png -1 mM 2 hr.png -1 mM 4 hr.png -1 mM 7 d.png -1 mM 8 hr.png -1 mM 12 hr.png -1 mM 24 hr.png -1 mM 48 hr.png -2 mM 2 hr.png -2 mM 4 hr.png -2 mM 7 d.png -2 mM 8 hr.png -2 mM 12 hr.png -2 mM 24 hr.png -2 mM 48 hr.png -4 mM 2 hr.png -4 mM 4 hr.png -4 mM 7 d.png -4 mM 8 hr.png -4 mM 12 hr.png -4 mM 24 hr.png -4 mM 48 hr.png Description: These image files correspond to different small molecule conditions at two different time points (e.g., hr, d). -d -dansyl-L-Arg -Merge.tif -A594.tif -dansyl.tif Description: These image files correspond to different fluorescence channels. -dansyl Cl -Merge.tif -A594.tif -dansyl.tif Description: these image files correspond to different fluorescence channels. Description: Partitioning of either dansyl chloride (dansyl-Cl) or dansyl-L-Arg doped along with L-Arg in SynTag-Tau condensates -f -D-Arg 24 hr.png -D-Arg 2 hr.png -L-Arg EE 24 hr.png -L-Arg EE 2 hr.png Description: L-Arg ethyl ester (L-Arg EE), a derivative of L-Arg that lacks a carboxyl group, failed to prevent condensate aging to fibrils. D-Arginine (D-Arg) treated condensates do not transition to fibrils. These image files correspond to different small molecule conditions at two different time points (e.g., hr). #### Figure 4 -a -2 mM L-Arg 4 hr.tif -2 mM L-Arg 3 hr.tif -2 mM L-Arg 1 hr.tif -2 mM L-Arg 2 hr.tif -2 mM L-Arg 5 hr.tif -UT 2 hr.tif -UT 1 hr.tif -UT 3 hr.tif -UT 4 hr.tif -UT 5 hr.tif Description: ThT fluorescence measurements at different sample ages, either without (untreated) or with 2 mM L-Arg. These image files correspond to different small molecule conditions at two different time points (e.g., hr). -b -untreated_condensates.tif Description: Individual Z-slices of untreated SynTag-Tau condensates visualized using ThT (green) as well as A594-labeled SynTag-Tau (red). These image files correspond to condensates of untreated condition. -c -MIP Arg treated condensates Composite.tif Description: Maximum intensity projection (MIP) image of 2 mM L-Arg treated SynTag-Tau condensates visualized as a merge of A594-labeled SynTag-Tau and ThT fluorescence. These image files correspond to maximum intensity projection (MIP) image of Arg-treated condensates. -f -1 hr.tif -3 hr.tif -5 hr.tif Description: FD-FLIM map of L-Arg treated condensates at various time points. These image files correspond to different time points. #### Figure 6 -a -Merge 1 hr.png -Merge 2.5 hr.png -Merge 5 hr.png -Merge 8 hr.png -Merge 12 hr.png -Merge 16 hr.png -Tau channel 1 hr.png -Tau channel 2.5 hr.png -Tau channel 5 hr.png -Tau channel 8 hr.png -Tau channel 12 hr.png -Tau channel 16 hr.png -Tubulin channel 1 hr.png -Tubulin channel 2.5 hr.png -Tubulin channel 5 hr.png -Tubulin channel 8 hr.png -Tubulin channel 12 hr.png -Tubulin channel 16 hr.png Description: MT polymerization assay of SynTag-Tau condensates treated with 1 mM L-Arg at various time points. The naming organization described here is in reference to a series of images, corresponding to different time points (hr or hours) and fluorescence channels (A488, H647, or Merge). --- &gt; Source data for supplementary text (contains all the microscopy data shown in the supplementary text, including videos) ### Dryad_source_data_for_supplementary_information.zip #### Supplementary Figure 1 -a -2 hr.png -3 d.png -5 d.png -24 hr.png Description: Wild-type Tau forms phase-separated condensates that do not age to form mesoscale fibrils under quiescent cofactor-free conditions. The files correspond to images collected at different time points. -b -2 hr.png -3 d.png -5 d.png -24 hr.png Description: Tau condensates show an absence of Thioflavin T (ThT) staining as a function of time. The files correspond to images collected at different time points. For example, 0 hr refers to 0 hours. #### Supplementary Figure 4 -2 hr.png -5 d.png Description: Sequence perturbations that reduce the PLAAC score of the prionogenic tag in SynTag-Tau without changing its overall amino acid composition prevent the physical aging of condensates into mesoscale fibrillar solids. The files correspond to images collected at different time points. #### Supplementary Figure 5 -2 hr.png -5 d.png Description: Truncating the prionogenic tag sequence in SynTag-Tau to the Asn(N)-rich segment, which corresponds to the major weighting in the PLAAC score (top), is insufficient to accelerate the condensate-to-fibril transition. The files correspond to images collected at different time points. #### Supplementary Figure 7 -a -50 mM NaCl -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png -75 mM NaCl -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png -100 mM NaCl -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png Description: Effect of modulation of ionic strength through NaCl titration in sample buffer on SynTag-Tau condensate formation and transition to fibrils. The files correspond to images collected at different time points. -c -5% PEG 8000 -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png -7.5% PEG 8000 -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png -10% PEG 8000 -2 hr.png -7 d.png -8 hr.png -14 d.png -24 hr.png -48 hr.png Description: Effect of modulation of molecular crowding through PEG8000 titration in sample buffer on SynTag-Tau condensate formation and transition to fibrils. The files correspond to images collected at different time points. #### Supplementary Figure 8 -nascent condensates -250 nM Merge.png -250 nM A488-Tau.png -250 nM Tubulin.png -100 nM Merge.png -100 nM A488-Tau.png -100 nM Tubulin.png -aged condensates -250 nM Merge.png -250 nM A488-Tau.png -250 nM Tubulin.png -100 nM Merge.png -100 nM A488-Tau.png -100 nM Tubulin.png Description: Partitioning of tubulin (HiLyte647-labeled) in nascent (1 hour since sample preparation) versus aged (4 hours since sample preparation) SynTag-Tau condensates. Here, the files are organized in terms of labeled tubulin concentration and fluorescence channel, in either nascent or aged condensates. #### Supplementary Figure 9 -2 mM 16 hr.png -2 mM 8 hr.png -2 mM 5 hr.png -2 mM 2.5 hr.png -2 mM 1 hr.png -1 mM 16 hr.png -1 mM 8 hr.png -1 mM 5 hr.png -1 mM 2.5 hr.png -1 mM 1 hr.png -UT 16 hr.png -UT 8 hr.png -UT 5 hr.png -UT 2.5 hr.png -UT 1 hr.png Description: SynTag-Tau condensates imaged at various time points after sample preparation prior to the addition of tubulin and GTP in MT polymerization assays. Here, the files are organized in terms of sample conditions (UT or Arg concentration) and time points. #### Supplementary Figure 10 \- a -image.png \- b -image.png Description: (a) Lack of visible microtubules (MTs) in the absence of SynTag-Tau condensates. (b) SynTag-Tau condensates, as visualized using Atto488-labeled SynTag-Tau, in the absence of tubulin and GTP (1 hour since sample preparation). #### Supplementary Figure 11 -raw_image_and_masked_image.png Description: (left) A fluorescence image of HiLyte647-labeled microtubule (MT) structures formed in the presence of SynTag-Tau condensates. (right) Image analysis allows selective segmentation of microtubules while ignoring spherical puncta, yielding an estimation of MT surface coverage area. This refers to a pair of images, the left is the actual fluorescence image, and the right one is its masked version made via image analysis. #### Supplementary Figure 12 -1HR arg.tif -1HR UT.tif -2.5hr arg.tif -2.5HR UT.tif -5 HR UT.tif -5HR arg.tif -8HR arg.tif -8HR UT.tif -12HR ARG.tif -12HR ut.tif -16HR arg.tif -16HR UT.tif Description: SynTag-Tau condensates, either untreated (UT) or L-Arg treated (1 mM), imaged at various time points after sample preparation, after the addition of 50 nM HiLyte647-labeled tubulin but not GTP. Here, the files are organized in terms of sample conditions (UT or Arg) and time points. #### Supplementary Figure 13 -a -10% PEG 8000 Merge channel.png -10% PEG 8000 Tau channel.png -10% PEG 8000 Tubulin channel.png -7.5% PEG 8000 Merge channel.png -7.5% PEG 8000 Tau channel.png -7.5% PEG 8000 Tubulin channel.png -5% PEG 8000 Merge channel.png -5% PEG 8000 Tau channel.png -5% PEG 8000 Tubulin channel.png Description: Representative fluorescence images of microtubule (MT) formation in the presence of SynTag-Tau condensates. These condensates were prepared at variable concentrations of PEG 8000 (w/v). Here, the files are organized in terms of sample conditions (PEG 8000 %) and fluorescence channel (e.g., Tau, Tubulin, Merge) #### Supplementary Figure 15 -Spermidine 24 hr.tif -Spermidine 1 hr.tif -Spermine 24 hr.tif -Spermine 1 hr.tif -R10 24 hr.tif -R10 1 hr.tif -K10 24 hr.tif -K5 1 hr.tif -K10 1 hr.tif -K5 24 hr.tif Description: Effect of multivalent cationic molecules on SynTag-Tau condensate formation and transition to amyloid fibrils. Here the files are organized in terms of the polymer and time points. #### Supplementary Figure 18 -a -0.25 mM L-Arg after.png -0.25 mM L-Arg before.png -0.5 mM L-Arg after.png -0.5 mM L-Arg before.png -2 mM L-Arg after.png -2 mM L-Arg before.png Description: Optical tweezer-mediated fusion of SynTag-Tau condensates treated with L-Arg at the specified concentrations as indicated. Here the files are organized in terms of the small molecule (L-Arg) concentration and either before or after the fusion event. #### Supplementary Figure 20 -a -Merge 1 hr.png -Merge 2.5 hr.png -Merge 5 hr.png -Merge 8 hr.png -Merge 12 hr.png -Merge 16 hr.png -Tau channel 1 hr.png -Tau channel 2.5 hr.png -Tau channel 5 hr.png -Tau channel 8 hr.png -Tau channel 12 hr.png -Tau channel 16 hr.png -Tubulin channel 1 hr.png -Tubulin channel 2.5 hr.png -Tubulin channel 5 hr.png -Tubulin channel 8 hr.png -Tubulin channel 12 hr.png -Tubulin channel 16 hr.png Description: Microtubule polymerization assay shows that SynTag-Tau condensates treated with 2 mM L-Arg remain active in catalyzing microtubule assembly as compared to untreated condensates. The naming organization described here is in reference to a series of images, corresponding to different time points (hr or hours) and fluorescence channels (A488, H647, or Merge). -Supplementary Video 1 -video.tif Description: The time-dependent conversion of SynTag-Tau condensates to fibrils as visualized by Atto488-labeled SynTag-Tau. Note: A series of images compiled as an image stack. Relationship between files, if important: Additional related data collected that was not included in the current data package: Are there multiple versions of the dataset? If yes, name of file(s) that was updated: Why was the file updated? When was the file updated? METHODOLOGICAL INFORMATION Description of methods used for collection/generation of data: for methodological details, please see [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) Methods for processing the data: for methodological details, please see [https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2](https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2) Instrument- or software-specific information needed to interpret the data: Files are available in standard formats that are accessible using Fiji. For further details, please see [https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2](https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2) Environmental/experimental conditions: for methodological details, please see [https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2](https://www.biorxiv.org/content/10.1101/2025.03.18.643977v2) People involved with sample collection, processing, analysis, and/or submission: Tharun Selvam Mahendran, Anurag Singh, Sukanya Srinivasan, Christian M. Jennings, Christian Neureuter, Bhargavi H. Gindra, Sapun H. Parekh, and Priya R. Banerjee.