Abstract
Membraneless organelles within cells have unique microenvironments that play a critical role in their functions. However, how microenvironments of biomolecular condensates affect their structure and function remains unknown. In this study, we investigated the micropolarity and microviscosity of model biomolecular condensates by fluorescence lifetime imaging coupling with environmentally sensitive fluorophores. Using both in vitro and in cellulo systems, we demonstrated that sufficient micropolarity difference is key to forming multilayered condensates, where the shells present more polar microenvironments than the cores. Furthermore, micropolarity changes were shown to be accompanied by conversions of the layered structures. Decreased micropolarities of the granular components, accompanied by the increased micropolarities of the dense fibrillar components, result in the relocation of different nucleolus subcompartments in transcription-stalled conditions. Our results demonstrate the central role of the previously overlooked micropolarity in the regulation of structures and functions of membraneless organelles.
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All data are available in the main text, supplementary materials and source data. Source data are provided with this paper.
Code availability
The computer code used in this study is available at https://github.com/ZhangGroup-MITChemistry/ELP.
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Acknowledgements
The authors thank the Instrumentation and Service Center for Molecular Sciences at Westlake University for assistance on fluorescence lifetime imaging microscopy. We thank P. S. Cremer for providing part of the ELP plasmids used in this work. X.Z. thanks support from the PEW Biomedical Scholars Program (00033066) and the Research Center for Industries of the Future at Westlake University. Y.L. thanks support from the National Natural Science Foundation of China (22222410). B.Z. and A.P.L. were supported by the National Institutes of Health (grant R35GM133580). A.P.L. acknowledges support from the National Science Foundation Graduate Research Fellowship Program (grant 1745302).
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S.Y. and X.Z. conceived the project. S.Y., Y.T. and C.-H.H. performed all in vitro and live cell experiments. A.P.L. performed the computational analysis. J.C. assisted with plasmids cloning. F.L. assisted with organic synthesis and characterization. Y.L., B.Z. and X.Z. supervised the project. S.Y. and X.Z. wrote the paper, with help from other co-authors.
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Extended data
Extended Data Fig. 1 Analyzation of the phase transition temperature and purity of ELP.
a-f, Phase transition processes of ELP were captured by the rapid increase of absorbance signal using a UV-Vis-NIR spectrometer. ELPs were mixed in buffers (50 mM HEPES, pH 7.0) with different molarity of NaCl (0.15 M, 0.5 M, 1.0 M, 1.5 M, 2.0 M) at a final peptide concentration of 70 µM on ice. An assay was designed to gradually increase the sample temperature from 1 ˚C to 50 ˚C at 0.5 ˚C/min rate, while the absorbance at 395 nm was recorded for every 0.5˚C of temperature increment. g, Summary of the phase transition temperature of ELPs at different NaCl concentrations. h, SDS-PAGE analysis of the purity of the ELP. The polyacrylamide gel was stained in 0.5 M CuCl2 solution to develop protein bands.
Extended Data Fig. 2 Fluorophore labelling does not alter the organization and partition of dual-component ELP condensates.
a, Swapping fluorophores on ELP preserved the organization of dual-component ELP condensates. Repeating dual colour imaging from Fig. 1c with swapped labelling fluorophores yields the dual component ELP condensates with identical organizations but inverted colours. b-c, Dual colour confocal images with varying concentrations of labelled ELPs yield similar core-shell structures with identical partition coefficients. b, Dual-color confocal images of V5A2G3-120/V-120 condensates with 0.5% (experimental condition), 1% and 1.5% fluorescent labels. c, Selected areas and values of partition coefficient calculated from the images in (b). Data represent mean and standard deviations from 3 distinct samples. Statistical significance was calculated using two-tailed t-tests. ns, non-substantial. p = 0.75 between 0.5% and 1.0% labelled ELPs, p = 0.42 between 1.0% and 1.5% labelled ELPs and p = 0.49 between 0.5% and 1.5% labelled ELPs. Experimental condition: 70 µM V5A2G3-120 and 70 µM of V-120 labelled with different percentages of fluorescein-NHS and AF 647-NHS were mixed in phase separation buffer (50 mM HEPES, pH 7.0, 2 M NaCl) and incubated at RT. Scale bar, 10 µm.
Extended Data Fig. 3 Dual component ELP droplets prepared by mixing two already phase-separated ELP samples resulted in identical core-shell structures compared to the co-phase separation of binary ELP solution mixtures.
Two different ELPs (labelled with fluorescein or AF 647) were allowed to phase separate individually in phase separation buffer (50 mM HEPES, pH 7.0, 2 M NaCl) at a final concentration of 140 µM at room temperature. The two phase-separated samples were then mixed with an equal amount and allowed to settle for 15 min on glass coverslips for confocal imaging. Scale bars, 10 µm.
Extended Data Fig. 4 Chemical structure and photophysical property of SBD.
a, The chemical structure of free SBD-NHS probe and labelled SBD structure on the N-terminus or lysine residuals of ELP. b, The chemical structure of SBD-methyl ester. SBD-methyl ester resembled the chemical structure of labelled SBD on proteins, hence was used for photophysical property measurements. c, Fast FLIM images of SBD-methyl ester in solvent mixtures of methanol and 1,4-dioxane. Scale bars, 10 µm. d, SBD lifetime-dielectric constant calibration curve calculated from measured SBD fluorescence lifetime in methanol and 1,4-dioxane solvent mixtures with known dielectric constant. The dielectric constant values of methanol-1,4-dioxane mixtures were calculated by the weighted average of the mixture components by assuming a simple additive effect. e, Fluorescence lifetime decay curves of SBD in samples described in (c-d). f, Fluorescence lifetime decay curves of SBD in ethylene glycol-glycerol solvent mixtures demonstrated minimal fluorescence lifetime changes in comparison with SBD in methanol-1,4-dioxane mixtures. Ethylene glycol-glycerol mixtures are commonly used solvent standards bearing similar polarity but contrasting viscosities. Therefore, SBD fluorescence lifetime is insensitive to viscosity changes. g, Excitation and emission spectra of SBD-NHS in pure methanol. h, Excitation and emission spectra of SBD-NHS in 1,4-dioxane. The dielectric constant values of methanol-1,4-dioxane mixtures and the viscosity values of ethylene glycol-glycerol mixtures were available in Supplementary Tables 3 and 4, respectively.
Extended Data Fig. 5 Hydrophobicity of the ELP condensates microenvironment is inversely correlated with the micropolarity.
a, Chemical structure of solvatochromic fluorophore, Nile Red. b, Lambda scan imaging to quantify the hydrophobicity of the Nile Red-stained ELP condensates. The false colour of the images is the weighted average from 14 individual images within a wavelength range from 570 nm to 694 nm, with each image measuring the emission intensity of an 8.9 nm interval. c, Fluorescence emission spectra of Nile Red calculated from the lambda scan images. d, Summary of the Nile Red emission maximum wavelength from c as a reflection of the hydrophobicity in ELP condensates. e, Chemical structure of bis-ANS. f. Fluorescence emission spectra of bis-ANS in phase-separated ELP condensates. g. Summary of the bis-ANS peak emission wavelength and fluorescence intensity at peak wavelength in f. 70 μM ELP were mixed in phase separation buffer (50 mM HEPES, pH 7.0, 2 M NaCl) to allow LLPS with the presence of 5 µM Nile Red or bis-ANS.
Extended Data Fig. 6 Chemical structure and photophysical property of BODIPY.
a, The structure of BODIPY-NHS probe and labelled BODIPY structure on the N-terminus or lysine residuals of ELP. b, Chemical structure of BODIPY-Halo. The chromophore core part of BODIPY-Halo resembles the chemical structure of labelled BODIPY, whereas the HaloTag reactive warhead does not affect the photophysical property of BODIPY. BODIPY-Halo was used for photophysical property measurements. c, Fast FLIM images of BODIPY-Halo in glycerol at different temperatures. The viscosity of glycerol is known to change dramatically in response to temperature changes, hence was used for the calibration of the BODIPY viscosity response. Scale bars, 10 µm. d, BODIPY lifetime-viscosity calibration curve calculated from measured BODIPY fluorescence lifetime in glycerol at different temperatures. e, Fluorescence lifetime decay curve of BODIPY-Halo in samples described in (c-d). f, Fluorescence lifetime decay curve of BODIPY-Halo in non-viscous solvents with different polarity. BODIPY displayed minimal fluorescence lifetime changes in response to different polarities. g, Excitation and emission spectra of BODIPY-NHS in pure methanol. h, Excitation and emission spectra of BODIPY-NHS in pure glycerol. The viscosity values of glycerol at different temperatures are available in Supplementary Table 4.
Extended Data Fig. 7 The impact of microviscosity on the organization and partition of the multi-component ELP protein condensates.
a, Pseudo-colour fast FLIM images and histogram plots of dual BODIPY labelled binary ELP condensates. Scale bars, 10 µm. b, Relationship between the partition coefficient of binary ELP condensates and the viscosity differences between individually formed component ELP condensates. Data represent mean and SD from 3 independent samples. Data were fitted to an exponential mathematical model.
Extended Data Fig. 8 Ratiometric imaging using Di-4-ANEPPS revealed minimal electric potential across the core-shell interfaces in multilayer condensates.
a, Fluorescence intensity images of Di-4-ANEPPS stained multilayer condensates in the channel of 535-545 nm, 610-640 nm and bright field images generated from KV6-112/V-120, V5A5-120/V120 and V5A2G3-120/V120 condensates, respectively. Scale bar, 10 µm. b, Selected areas for fluorescence intensity ratio calculation from the images in (a). c, Di-4-ANEPPS intensity integration ratio of shells and cores in a. Experimental condition: 70 µM of each ELP were mixed in phase separation buffer (50 mM HEPES, pH 7.0, 2 M NaCl) in the presence of 1 µM Di-4-ANEPPS. Data calculated from the ratio of the mean intensity in the channel of 535-545 nm versus the mean intensity in the channel of 610-640 nm from (a). Error bars represent propagated errors calculated from the s.d. from both channels.
Extended Data Fig. 9 Interfacial tensions of ELP condensates are reversely correlated with their micropolarity.
a, Schematic and example of the contact angle measurements of ELP condensates on glass surfaces. Scale bar, 5 µm. b, Measured contact angles of ELP condensates. 70 µM ELP labelled with AF 647 were mixed in phase separation buffer (50 mM HEPES, pH 7.0, 2 M NaCl) to allow contact angle measurements. Data points and labels represent mean and SD for n = 10 for V2I7E-40; n = 6 for V-120; n = 8 for QV6-112, n = 7 for KV6-112, n = 6 for V5A5-120 and n = 7 for V5A2G3-120.
Extended Data Fig. 10 Photophysical property of S-SBD-Halo.
a, Fast FLIM images of S-SBD-Halo in solvent mixtures of methanol and 1,4-dioxane. Scale bars, 10 µm. b, SBD lifetime-dielectric constant calibration curve calculated from measured SBD fluorescence lifetime in methanol and 1,4-dioxane solvent mixtures with known dielectric constant. The dielectric constant values of methanol-1,4-dioxane mixtures were calculated by the weighted average of the mixture components by assuming a simple additive effect. The dielectric constant values of methanol-1,4-dioxane mixtures was available in Supplementary Table 3.
Supplementary information
Supplementary Information
Supplementary Figs. 1–16, Tables 1–4, Notes 1 and 2, caption for Supplementary Video 1 and references.
Supplementary Video 1
Sequential phase separation of AF 647-labeled V-120 and fluorescein-labeled V5A2G3-120 from well-mixed solution as the solution temperature surpasses their Tph. Buffer condition: 70 μM AF 647-labeled V-120 (magenta) and 70 μM fluorescein-labeled V5A2G3-120 (green) mixed in 20 mM HEPES, pH 7.0, and 1 M NaCl. Imaging was conducted with a 1-frame-per-2-minute rate. The temperature was increased from 20 °C to 40 °C with a 2-°C-per-minute rate using a built-on heat plate on confocal microscopy.
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Ye, S., Latham, A.P., Tang, Y. et al. Micropolarity governs the structural organization of biomolecular condensates. Nat Chem Biol 20, 443–451 (2024). https://doi.org/10.1038/s41589-023-01477-1
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DOI: https://doi.org/10.1038/s41589-023-01477-1
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