Abstract
Biomolecular condensates are membraneless compartments that impart spatial and temporal organization to cells. Condensates can undergo maturation, transitioning from dynamic liquid-like states into solid-like states associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease. Despite their important roles, many aspects of condensate biology remain incompletely understood, requiring tools for acutely manipulating condensate-relevant processes within cells. Here we used the BCL6 BTB domain and its ligands BI-3802 and BI-3812 to create a chemical genetic platform, BTBolig, allowing inducible condensate formation and dissolution. We also developed optogenetic and chemical methods for controlled induction of condensate maturation, where we surprisingly observed recruitment of chaperones into the condensate core and formation of dynamic biphasic condensates. Our work provides insights into the interaction of condensates with proteostasis pathways and introduces a suite of chemical–genetic approaches to probe the role of biomolecular condensates in health and disease.
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Data availability
All data analyzed during this study are included in the article and the Supplementary Information. Imaging data are provided as supplementary videos associated with this publication. Original TIFF formats of imaging data (not deposited in Source Data due to the large file sizes) are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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Acknowledgements
This work was funded by grants from the National Institutes of Health (R35GM136367 and R21MH134019) to C.L.T. C.N.H.-C. is a 2019 Latin American Fellow in the Biomedical Sciences, supported by the Pew Charitable Trusts. We thank Boehringer Ingelheim for generously providing BI-3802 and BI-3812 for use in studies. We also thank N. Fawzi, T. Inoue, D. Hodson, H. Kampinga, V. Timmerman, P. De Camilli, D. Picard and D. Altshuler for providing constructs (via Addgene) used in plasmid construction or studies, including MBP-FUS_FL_WT (Addgene, 98651)43; cyto-YFP-FKBPx5 (Addgene, 103777)13; MSCV-BCL6-t2A-BCL2 (Addgene, 135305)44; pcDNA5/FRT/TO GFP HSPA1L (Addgene, 19484), pcDNA5/FRT/TO GFP DNAJB1 (Addgene, 19495)45, pmScarlet-Hsp70 (HSPA1A) (Addgene, 163790)46, pEGFP.90beta (Addgene, 108221)47, mCherry-CRY2-5ptaseOCRL (Addgene, 66836) and iRFP-PH-PLCdelta1 (Addgene, 66841)33.
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C.L.T. and C.N.H.-C. conceived the project and wrote the paper. C.L.T., C.N.H.-C. and B.R.B. designed experiments, generated figures and edited the paper. C.N.H.-C. and B.R.B. performed and analyzed data for all experiments except Extended Data Figs. 5c, 6 and 7. E.H. performed experiments and analyzed data for Extended Data Figs. 5c, 6 and 7.
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Nature Chemical Biology thanks Eitan Lerner, Pilong Li and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Preclustering of BTBIDR-FRB prior to BI-3802 addition.
a) Quantification of % of BTBIDR-FRB in clusters in COS-7 cells pre and post 1 µm BI-3802 in nucleus and cytosol. Pre-clustering is predominantly nuclear. Mean and s.e.m., n = 34 cells from 5 independent experiments. b) Quantification of data from (a) from cells undergoing nuclear preclustering. A linear relationship between relative expression level (mean intensity/background) and nuclear preclustering (% of protein in cluster) is observed.
Extended Data Fig. 2 BTBIDR-FRB and BTB-EGFP can undergo multiple rounds of condensate induction and reversal without loss of efficacy.
Shown are representative images (top) and quantification of condensate fluorescence (bottom). a) COS-7 cells expressing BTBIDR-FRB and EGFP (as a cell-fill) were treated with 1 µM BI-3802 (filled arrowheads) for 10–15 min then 10 µM BI-3812 (open arrowheads) for 7–15 min. Between each cycle, the media was changed (wash). Data is representative of 2 independent experiments. b) COS-7 cells expressing BTB-EGFP were treated with 1 µM BI-3802 for 10–20 min (filled arrowheads), then incubated with 10 µM BI-3812 for 15–20 min (open arrowheads). Samples were washed in PBS between each treatment cycle. Data is representative of 2 independent experiments. Scale bars, 10 µm.
Extended Data Fig. 3 FRAP of BTBIDR-FRB and BTBIDR at later timepoints reveals a condensate maturation process for BTBIDR-FRB.
a) Normalized FRAP analysis of BTBIDR-FRB condensates (in COS-7 cells incubated with BI-3802 for 1–1.5 h). Graph shows mean and s.e.m., n = 26 condensates, from 2 independent experiments. Inset shows representative condensates pre and post photobleach. Scale, 1 µm. b) Normalized FRAP analysis of BTBIDR-FRB condensates in COS-7 cells incubated with BI-3802 for 3–6 h. Graph shows mean and s.e.m., n = 12 condensates from a single experiment. Inset shows representative condensate pre and post photobleach. Scale, 1 µm. c) Normalized FRAP analysis of BTBIDR condensates 4 h post BI-3802. Graph shows mean and s.e.m., n = 11 cells from 2 independent experiments. Inset shows representative condensate pre and post photobleach. Scale, 1 µm. d) BTBIDR condensates in COS-7 cell treated with BI-3802 for 19 h (added 24 h post transfection). Representative of 3 independent experiments. Scale, 10 µm. e) Normalized FRAP analysis of BTBIDR condensates 19 h post BI-3802. Graph shows mean and s.e.m., n = 10 cells from a single experiment. Inset, representative condensates pre and post photobleach. Scale, 1 µm.
Extended Data Fig. 4 Rapamycin-induced CoSMo.
a) Schematic of CoSMo approach with rapamycin (rap). b) COS-7 cells expressing BTBIDR-FRB and mCh-FKBP2x were treated with 1 µM BI-3802 for <30 min. Condensate circularity was quantified pre or post 333 nM rapamycin. Graph shows box plot (defined in Methods), n = 140 (-rap) or 325 (+rap) condensates from one experiment. **, p < .001, unpaired two-tailed t-test. c) FRAP analysis of BTBIDR-FRB condensates treated as in (b), pre or 10 min post rapamycin. Mean and s.e.m., n = 7 (pre) or 10 (post) condensates from 2 independent experiments. d, e) COS-7 cells expressing BTBIDR-FRB and mEGFP-FKBP2x treated with 1 µM BI-3802 for <30 min, before or 10 min post 333 nM rapamycin. Representative images in (d) (different cells, with higher magnification images at right), representative of 2 independent experiments. Scale, 10 µm. FRAP analysis in (e), mean and s.e.m., n = 12 (pre) or n = 13 condensates (post) from 2 independent experiments. f) FRAP analysis of condensates in cells expressing BTBIDR-FRB and mEGFP-FKBP2x, 30–60 min after 1 µM BI-3802 and 15–60 min after adding 10, 60, or 333 nM rapamycin. Mean and s.e.m., n = 10 (10 nM), 25 (60 nM), or 13 (333 nM) condensates from 2 independent experiments. g) COS-7 cells expressing BTBIDR-FRB and miRFP-FKBP5x < 30 min after 1 µM BI-3802, before and 10 min after 333 nM rapamycin. Images representative of 3 independent experiments. Scale, 10 µm. Higher magnification region at bottom. h) Condensate circularity with miRFP-FKBP5x recruitment as in (g), pre or 10 min post rapamycin. Data shows box plot (defined in Methods), n = 394 (pre) or 359 (post) condensates from 2 experiments. ****, p = 6.6 × 10–13, two-tailed unpaired t-test.
Extended Data Fig. 5 Additional quantification of biphasic condensates induced by light using CoSMo approach.
a) Circularity of CIBN-BTBIDR-FRB condensates coexpressed with CRY2olig-mEGFP, 30 min post BI-3802, before and after light (488 nm, 5% laser power, 7x50ms pulses, every 30 s). Post light separated into monophasic or biphasic. Box plot (defined in Methods), n = 271 (pre-light), 103 (post, monophasic) or 62 (post, biphasic) condensates from 2 independent experiments. ns, not significant (p = .21 and .12), unpaired two-tailed t-test. b) Quantification of size of CIBN-BTBIDR-FRB condensates remaining monophasic or transitioning to biphasic, for COS-7 cells also expressing CRY2olig-mEGFP, treated with 1 µM BI-3802 then light. Size quantified before light treatment. Data represents individual points and median, n = 59 condensates from 2 independent experiments. **, p = .000004, unpaired two-tailed t-test. c) COS-7 cells expressing CIBN-BTBIDR-FRB, CRY2olig-miRFP, and GFP-tagged HSP90b, DNAJB1, or EGFP alone, treated with BI-3802 and light to induce biphasic condensates. Yellow boxes denote a higher magnification image of the boxed region. Scale, 10 µm. Images representative of 2 independent experiments. d) Normalized FRAP analysis of EGFP-HSPA1L within the core of biphasic CIBN-BTBIDR-FRB + CRY2olig-miRFP condensates (treated with BI-3802 and blue light). Mean and s.e.m., n = 5 condensates from one experiment. e) Condensates undergoing fusion in cells expressing CIBN-BTBIDR-FRB (labeled as ‘BTB’), CRY2olig-miRFP, and EGFP-HSPA1L (Hsp70), treated with 1 µm BI-3802 and exposed to blue light. Scale, 2 µm. Images representative of 3 independent experiments.
Extended Data Fig. 6 Tracking biphasic condensates generated through light-induced CoSMo with extended light treatment.
a–c) COS-7 cells expressing CIBN-BTBIDR-FRB, CRY2olig-miRFP, and HSPA1L-EGFP were treated with BI-3802 then blue light (465 nm, 1.1 mW/cm2, 2 s pulse every 2 min) for 2 h. Images representative of 3 independent experiments. a) Representative image of cell after 2 h blue light. Condensates remain biphasic and circular and are distributed throughout the cell periphery. Scale, 10 µm. b) Timelapse of 2 h light-treated condensates that continue to merge and coalesce suggestive of a liquid-like state. Scale, 1 µm. c) Timelapse of 2 h light-treated condensates showing popping/release behavior with Hsp70. Scale, 1 µm. d-f) 24 h light treatment. COS-7 cells expressing CIBN-BTBIDR-FRB (mCh), CRY2olig-miRFP, and HSPA1L-EGFP were treated with 1 µM BI-3802 then blue light for 24 h (465 nm, 1.1 mW/cm2, 2 s pulse every 2 min). Images representative of 2 independent experiments. d) Cells show no aggresomal accumulation of CIBN-BTBIDR-FRB after 24 h light. Scale, 10 µm. e) Timelapse of 24 h light-treated biphasic condensates merging and coalescing. Scale, 1 µm. f) Timelapse of biphasic condensate showing popping and releasing behavior with HSPA1L-EGFP after 24 h of light. Scale, 1 µm.
Extended Data Fig. 7 Removal of light after light-induced CoSMo does not result in transition of condensates to monophasic state.
a) COS-7 cells expressing HSPA1L-EGFP, CRY2olig-miRFP, and CIBN-BTBIDR-FRB (with a K70N non-fluorescent mCherry, to allow quantification of CRY2olig-miRFP signal without overlap with mCherry) were incubated 40 min with 1 µM BI-3802, then 488 nm light (10% laser power, 12 × 100 ms pulses, every 2 min) for 30 min, followed by 60 min with no 488 nm light to allow dissociation of CRY2olig, then a second 488 nm light treatment. Graphs show quantification of normalized EGFP and CRY2olig signal in clusters during experiment. Data normalized to the amount at the end of the initial light period, set to 1. Graphs show mean and s.e.m, n = 5 cells (EGFP) or 4 cells (CRY2olig), of a single experiment, independently performed 2 times with similar results. b) Individual cell data for experiment in (a) showing % of clustered CRY2olig-miRFP at each timepoint. n = 4 cells. c) A parallel experiment with identical design as in (a), but using wildtype mCherry to allow visualization of CIBN-BTBIDR-FRB signal. BTBIDR-FRB signal in condensates remains constant. Data normalized to the amount at the end of the initial light period, set to 1. Graph shows mean and s.e.m., n = 5 cells, of a single experiment, independently performed 2 times with similar results. d,e) Images of BTBIDR-FRB (mCh) and HSPA1L-EGFP (d) or CRY2olig-miRFP and HSPA1L-EGFP (coexpressed with K70N CIBN-BTBIDR-FRB) (e) signal in condensates 30 min after initial 488 nm light, or after 60 min without 488 nm light. Condensates do not revert to monophasic. Scale, 2 µm. Representative of 2 independent repeats.
Extended Data Fig. 8 HSPA1L does not colocalize with initially formed rigid or liquid-like condensates.
Shown are representative images of COS-7 cells expressing EGFP-HSPA1L (or mScarlet-HSPA1L) and either BTB2x-mCh, CRY2olig-mCh, BTBIDR, or BTB-EGFP, 5 min to 2 h post inducing condensates (1 µM BI-3802 or light, as appropriate). Scale, 10 µm. Data representative of 2 independent experiments.
Extended Data Fig. 9 Model of biphasic condensate behavior.
Light-induced CoSMo results in condensates that retain liquid-like behavior, allowing dynamic interaction with chaperones. Rapamycin-induced CoSMo results in initial recruitment of Hsp70 and rearrangement to a shell/core structure. Subsequent gelation locks the shell structure in place but allows additional Hsp70 recruitment.
Supplementary information
Supplementary Information
Supplementary Figs. 1–8 and Supplementary Video legends (Videos 1–10).
Supplementary Table 1
Plasmid sequences and oligos. Sequences of plasmids created during this study and oligos used for cloning.
Supplementary Video 1
Induction and dissolution of BTB2x-mCh condensates.
Supplementary Video 2
Induction and dissolution of BTBIDR condensates.
Supplementary Video 3
Induction and dissolution of BTBIDR-FRB condensates.
Supplementary Video 4
Induction and dissolution of BTB-EGFP condensates.
Supplementary Video 5
Cells undergoing multiple rounds of formation and then dissolution of BTB-EGFP condensates.
Supplementary Video 6
BI-3812-dependent dissolution of BTB-EYFP fiber-like structures.
Supplementary Video 7
Light-induced formation of biphasic condensates.
Supplementary Video 8
Dynamics of light-induced BTB/chaperone biphasic condensates.
Supplementary Video 9
Magnified view of 'popping and chaperone release' behavior.
Supplementary Video 10
CoSMo-induced transition of BTBIDR-FRB condensates, resulting in recruitment of chaperones and transition to a biphasic state.
Source data
Source Data Fig. 1
Source data for Fig. 1d.
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Source data for Fig. 2a–c,e.
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Source data for Fig. 3e.
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Source data for Fig. 4c,f,i.
Source Data Fig. 5
Source data for Fig. 5c,e,g.
Source Data Fig. 6
Source data for Fig. 6a,c,e,f,h–j,l.
Source Data Extended Data Fig. 1
Source data for Extended Data Fig. 1.
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Source data for Extended Data Fig. 2.
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Source data for Extended Data Fig. 3.
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Source data for Extended Data Fig. 4.
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Source data for Extended Data Fig. 5.
Source Data Extended Data Fig. 7
Source data for Extended Data Fig. 7.
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Hernandez-Candia, C.N., Brady, B.R., Harrison, E. et al. A platform to induce and mature biomolecular condensates using chemicals and light. Nat Chem Biol 20, 452–462 (2024). https://doi.org/10.1038/s41589-023-01520-1
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DOI: https://doi.org/10.1038/s41589-023-01520-1
