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Kinetic control of shape deformations and membrane phase separation inside giant vesicles

Nature Chemistry volume 16, pages 54–62 (2024)Cite this article

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Abstract

A variety of cellular processes use liquid–liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation—en route to the new equilibrium—is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane’s compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.

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Fig. 1: Segregative phase separation of PEG and dextran.
Fig. 2: Phase transition trajectory inside giant vesicles.
Fig. 3: Composition dependence of polymer phase separation inside giant vesicles.
Fig. 4: Coupling of bulk LLPS inside giant vesicles with membrane phase separation at the vesicle boundaries.

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Data availability

Data supporting the findings of this study are available within this article and its supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank the MCB Light Microscopy Imaging Facility, which is a UC-Davis Campus Core Research Facility, for the use of their spinning disk confocal fluorescence microscope. The 3i Marianas spinning disk confocal used in this study was purchased using National Institutes of Health Shared Instrumentation grant 1S10RR024543-01. W.-C.S., D.L.G. and A.N.P. acknowledge funding from the National Science Foundation (DMR-1810540). J.C.S.H. and A.N.P. acknowledge funding and support from the Singapore Centre for Environmental Life Sciences Engineering and the Institute for Digital Molecular Analytics and Science, Nanyang Technological University. C.D.K. and A.T.R. were supported by the US Department of Energy, Office of Science, Basic Energy Sciences under award no. DE-SC0008633.

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Authors and Affiliations

  1. Chemistry Graduate Program, University of California, Davis, CA, USA

    Wan-Chih Su & Atul N. Parikh

  2. Singapore Centre for Environmental Life Sciences Engineering and Institute for Digital Molecular Analytics and Science, Nanyang Technological University, Nanyang Technological University, Singapore, Singapore

    James C. S. Ho & Atul N. Parikh

  3. Chemical Engineering Graduate Program, University of California, Davis, CA, USA

    Douglas L. Gettel & Atul N. Parikh

  4. Department of Chemistry, The Pennsylvania State University, University Park, PA, USA

    Andrew T. Rowland & Christine D. Keating

  5. Biomedical Engineering Graduate Programs, University of California, Davis, CA, USA

    Atul N. Parikh

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  1. Wan-Chih Su
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Contributions

W.-C.S., C.D.K. and A.N.P. conceived and designed the study. W.-C.S., J.C.S.H. and D.L.G. designed and executed the experimental protocols for the preparation of the vesicle system and carried out the fluorescence microscopy measurements. A.T.R. performed the polarimetry and refractometry measurements of the phase compositions. W.-C.S., J.C.S.H., C.D.K. and A.N.P. co-wrote the paper, with contributions from all authors.

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Correspondence to Christine D. Keating or Atul N. Parikh.

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Extended data

Extended Data Fig. 1 Fluorescence intensity measurements of solution encapsulated in an LLPS GUV to determine the partition coefficient (K) of dextran in PEG-rich and dextran-rich phases.

(a, d) Normalized fluorescent intensity profile of dextran (Alexa flour® 488 dextran) in PEG-rich and dextran-rich phases after phase separation. Photobleaching effect was taken into account in the calculation (Supplementary Fig. S2). Data are presented as mean values ± 5% error, (n = 3). For the number of dextran-rich droplets measured, refer to Supplementary Data. (b, e) The ratio of fluorescence intensity of dextran-rich phase versus the PEG-rich phase. (c, f) Measurement of partition coefficient (K) of dextran in LLPS GUV. GUVs contain 96.8 mol% POPC, 2.2 mol% DOPE-mPEG, and 1 mol% Rho-DOPE encapsulate a mixture of 6 wt% PEG 8 kDa and 6.4 wt% dextran 10 kDa upon immersion in 143 mM sucrose solution.

Source data

Extended Data Fig. 2 Ostwald ripening and fusion trajectories.

Trajectory of individual droplets during coarsening in GUVs encapsulating a mixture of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa doped with 0.001% AlexaFluor® 488-Dextran upon immersion in 143 mM sucrose solution. (a) Purely Ostwald ripening. (b) Fusion events. Arrows indicate coarsening droplets. All scale bar, 10 µm.

Extended Data Fig. 3 Membrane tubule formation.

(a-c) Selected frames from a time-lapse movie of wide-field fluorescence microscopy images of GUVs encapsulating a mixture of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa doped with 0.001% AlexaFluor 488-Dextran upon immersion in 143 mM sucrose solution. (d) The plot refers to the line intensity measurement (ii) of the distance covered by the dash arrow on the adjacent image (i).

Extended Data Fig. 4 Examples of uncoated droplets at the membrane of GUVs.

Selected frames of confocal microscopy images of GUVs encapsulating a mixture of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa doped with 0.001% AlexaFluor® 488-Dextran upon immersion in 143 mM sucrose solution. Trajectories are obtained from Supplementary Video S1. Arrows indicate examples of uncoated droplets at the membrane. All scale bar, 10 µm.

Extended Data Fig. 5 Droplet growth analysis for GUVs of different sizes.

(a) The plot shows exponent as a function of the starting GUV radius (top left, n = 10, where n refers to the number of GUV analyzed). Each data point refers to individual GUV. The droplet growth rate plots for individual GUV are also shown (b). Data are presented as mean values ± SD, (n = 10). For the number of dextran-rich droplets measured, refer to Source Data.

Source data

Extended Data Fig. 6 Long-term (>24 h) follow-up of bud morphological transition.

Selected frames of confocal fluorescence microscopy images of GUVs encapsulating a mixture of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa doped with 0.001% AlexaFluor® 488-Dextran upon immersion in 143 mM sucrose solution. GUVs contain 96.8 mol% POPC, 2.2 mol% DOPE-mPEG, 1 mol% Rho-DOPE. Scale bar, 10 µm.

Extended Data Fig. 7 Reversibility of osmotic deflation driven liquid-liquid phase separation.

(a) Confocal fluorescence microscopy image of a single GUV consists of a mixture containing 96.8 mol% POPC, 2.2 mol% DOPE-mPEG, 1 mol% Rho-DOPE encapsulating 6.0 % (w/w) PEG 8 kDa and 6.4 % (w/w) dextran 10 kDa aqueous phase subjected to pure water (till exterior sucrose concentration equal to 62 mM) after hypertonic trigger deformation in 143 mM sucrose solution. Scale bar, 10 µm. (b) Box plot shows fluorescence intensity of individual dextran-rich droplets and PEG-rich phase as a function of time for the reversal kinetic analysis. Each data point refers to an individual measurement. The intensity values of PEG-rich phase are sampled at random spots within the GUV (n = 1). Mean values are indicated by the red dots. For the number of data points shown in the plot for each time point, refer to Source Data. The inset shows the ratio of fluorescence intensity of dextran-rich phase verses the PEG-rich phase.

Source data

Extended Data Fig. 8 Time-lapse confocal fluorescence microscopy image of GUVs encapsulating different LLPS mixtures.

(a) 5:1 (namely 5P1D), (b) 3:1 (namely 3P1D), (c) 1:2 (namely 1P2D) and (d) 1:6 (namely 1P6D) ratios of PEG 8 kDa and dextran 10 kDa. All GUVs imaged consist of a mixture containing 96.8 mol% POPC, 2.2 mol% DOPE-mPEG, 1 mol% Rho-DOPE encapsulating PEG and dextran mixtures doped with 0.001 wt% AlexaFluor® 488 dextran upon immersion in (a) 143, (b) 120, (c) 263, and (d) 298 mM sucrose solution. All scale bar, 10 µm.

Extended Data Fig. 9 Control GUVs without labelled dextran.

The POPC: Egg-SM: Cholesterol (2:2:1) GUV, doped with 2.2% DOPE-mPEG, 1% Rho-DOPE and 3% NBD-PE with encapsulation of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa is immersed in 143 mM sucrose solution. Scale bar: 10 µm.

Extended Data Fig. 10 Osmotic deflation driven LLPS in PBD-PEO GUV.

(a) Selected frames from a time-lapse video of 99 % PBD-PEO GUV doped with 1% Rhodamine-DOPE, encapsulating a mixture of 6.0% (w/w) PEG 8 kDa and 6.4% (w/w) dextran 10 kDa doped with 0.001% AlexaFluor® 488-Dextran upon immersion in 143 mM sucrose. Confocal section (b) and 3D projection (c) of the final morphology are shown. All scale bar, 10 µm.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–5 and captions for Videos 1–12.

Supplementary Video 1

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 6.0% (wt/wt) PEG 8 kDa and 6.4% (wt/wt) dextran 10 kDa doped with 0.001% AlexaFluor 488-dextran.

Supplementary Video 2

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 6.0% (wt/wt) PEG 8 kDa and 6.4% (wt/wt) dextran 10 kDa doped with 0.001% AlexaFluor 488-dextran, recorded on a wide-field fluorescence microscope.

Supplementary Video 3

Reversibility of osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 6.0% (wt/wt) PEG 8 kDa and 6.4% (wt/wt) dextran 10 kDa doped with 0.001% AlexaFluor 488-dextran.

Supplementary Video 4

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 10 wt% PEG (8 kDa) and 2 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran (5P1D).

Supplementary Video 5

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 9.5 wt% PEG (8 kDa) and 3.1 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran (3P1D).

Supplementary Video 6

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 4 wt% PEG (8 kDa) and 8.3 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran (1P2D).

Supplementary Video 7

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 2.5 wt% PEG (8 kDa) and 15 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran (1P6D).

Supplementary Video 8

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs encapsulating a mixture of 4.5 wt% PEG (8 kDa) and 2.02 wt% dextran (450 kDa) doped with 0.001% AlexaFluor 488-dextran.

Supplementary Video 9

Osmotic deflation driven liquid–liquid phase separation in POPC GUVs with 4 mol% GM1 in membrane, encapsulating a mixture of 6 wt% PEG (8 kDa) and 6.4 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran.

Supplementary Video 10

Osmotic deflation driven liquid–liquid phase separation of 6 wt% PEG (8 kDa) and 6.4 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 561-dextran in GUVs with ternary lipid mixtures (POPC:Egg-SM:cholesterol in a ratio of 2:2:1), doped with 1% Rho-DOPE and 3% NBD-PE.

Supplementary Video 11

Control experiment of GUVs encapsulated 6 wt% PEG (8 kDa) and 6.4 wt% dextran (10 kDa) with ternary lipid mixtures, doped with 1% Rho-DOPE and 3% NBD-PE immersed in isotonic sucrose solution.

Supplementary Video 12

Osmotic deflation driven liquid–liquid phase separation of 6 wt% PEG (8 kDa) and 6.4 wt% dextran (10 kDa) doped with 0.001% AlexaFluor 488-dextran in GUVs consisting of 99% polybutadiene1200-b-polyethyleoxide600 (PBD-PEO; the unit for the number in subscript is Dalton) GUV, doped with 1% Rho-DOPE.

Supplementary Data 1

Source data for Supplementary Fig. 1

Supplementary Data 2

Source data for Supplementary Fig. 2

Supplementary Data 3

Source data for Supplementary Fig. 3

Supplementary Data 4

Source data for Supplementary Fig. 5

Source data

Source Data Fig. 1

Raw numerical data for Fig. 1b–c.

Source Data Fig. 2

Raw numerical data for Fig. 2f–i.

Source Data Fig. 3

Raw numerical data for Fig. 3a.

Source Data Fig. 4

Raw numerical data for Fig. 4d.

Extended Data Fig. 1

Source data for Extended Data Fig. 1.

Extended Data Fig. 5

Source data for Extended Data Fig. 5.

Extended Data Fig. 7

Source data for Extended Data Fig. 7.

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Su, WC., Ho, J.C.S., Gettel, D.L. et al. Kinetic control of shape deformations and membrane phase separation inside giant vesicles. Nat. Chem. 16, 54–62 (2024). https://doi.org/10.1038/s41557-023-01267-1

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