Multiphase Complex Coacervate Droplets

Abstract

Liquid-liquid phase separation plays an important role in cellular organization. Many subcellular condensed bodies are hierarchically organized into multiple coexisting domains or layers. However, our molecular understanding of the assembly and internal organization of these multicomponent droplets is still incomplete, and rules for the coexistence of condensed phases are lacking. Here, we show that the formation of hierarchically organized multiphase droplets with up to three coexisting layers is a generic phenomenon in mixtures of complex coacervates, which serve as models of charge-driven liquid-liquid phase separated systems. We present simple theoretical guidelines to explain both the hierarchical arrangement and the demixing transition in multiphase droplets using the interfacial tensions and critical salt concentration as inputs. Multiple coacervates can coexist if they differ sufficiently in macromolecular density, and we show that the associated differences in critical salt concentration can be used to predict multiphase droplet formation. We also show that the coexisting coacervates present distinct chemical environments that can concentrate guest molecules to different extents. Our findings suggest that condensate immiscibility may be a very general feature in biological systems, which could be exploited to design self-organized synthetic compartments to control biomolecular processes.

Figure 1

Multiphase complex coacervate droplets formed by mixing different polymeric coacervates (structures are shown in Table S1, and fluorescently labeled polymers are underlined). (a,b) ssDNA/PLys(Me)₃ core coacervates in a ssDNA/GFP-K₇₂ outer coacervate phase, viewed in (a) bright-field and (b) confocal fluorescence microscopy with fluorescence from Alexa-647 labeled ssDNA (red channel) and GFP (green channel). (c) ATP/PAH cores ATP/PDDA outer phases, with fluorescence from rhodamine-labeled PAH. (d) PGlu/PAH cores in PGlu/PDDA outer coacervate phases, shown as overlay of bright-field and fluorescence microscope image with fluorescence from rhodamine-labeled PAH. (e) PAA/PLys(Me)₃ cores in PAA/GFP-K₇₂ outer coacervate phases, with fluorescence from GFP. (f) PSPMA/PAH cores in PSPMA/DEAE-Dex outer coacervate phases, with fluorescence from rhodamine-labeled PAH. (g) Dextran sulfate (S-Dex)/PLys(Me)₃ cores in S-Dex/GFP-K₇₂ outer coacervate phases, with fluorescence from GFP. (h) PSPMA/PAH cores in PSPMA/PDDA outer coacervate phases, with fluorescence from rhodamine-labeled PAH. (i) PSPMA/PDDA cores in PSPMA/Q-Dex outer coacervate phases, with fluorescence from fluorescein-labeled PSPMA. (j) ATP/PAH cores in PSPMA/PDDA outer coacervate phases, with fluorescence from fluorescein-labeled PSPMA (green channel) and rhodamine-labeled PAH (yellow channel).

Figure 2

Interfacial tension-governed arrangement and fusion in multiphase coacervate droplets. (a) Fusion of core PAA/PLys(Me)₃ coacervates inside a PAA/GFP-K₇₂ outer phase (cf. Figure 1e). (b) Fusion of PGlu/PDDA coacervates followed by fusion of their internal PGlu/PAH cores (cf. Figure 1d). (c) Engulfing of an ATP/PAH coacervate by a PSPMA/PDDA coacervate (cf. Figure 1j). (d) Schematic illustration of four scenarios of two coexisting liquid droplets. (e) Dual multiphase arrangement (1/2 and 2/1) in PSPMA/PLys/PLys(Me)₃.

Figure 3

Schematic phase diagram of complex coacervation at charge neutrality showing binodal curves for coacervates with increasing interaction strength.

Figure 4

Step-wise dissolution of PSPMA/PAH/PDDA multiphase droplets, shown by (a) confocal fluorescence and (b) bright-field microscopy.

Figure 5

Partitioning of guest molecules in PSPMA/PAH/PDDA (a–g,i,j) and PSPMA/Q-Dex/PDDA (h) multiphase droplets visualized by confocal fluorescence microscopy. Panels show partitioning of different fluorescent guest molecules: (a) porphyrin derivative tetrakis-carboxyphenylporphyrin (TCPP), (b) 5(6)-carboxyfluorescein, (c) eGFP, (d) Rhodamine B, (e) Thioflavin T (ThT), (f) Nile red, (g) 6-aminofluorescein, (h) PEG-difluorescein in PSPMA/Q-Dex/PDDA multiphase droplets, (i) SYBR Gold, and (j) Methyl blue. No fluorescently labeled polymers were used to form the coacervates.

Figure 6

Multiphase complex coacervate droplets with three coexisting condensed phases. (a,b) An ATP/PAH inner core, surrounded by a PSPMA/PDDA shell in a PAA/PDDA outer coacervate phase, visualized in bright-field (a) and confocal fluorescence microscopy (b) with fluorescence from fluorescein-labeled PSPMA. Note that (a) and (b) do not show the same position. (c,d) PSPMA/PAH inner core, surrounded by a PSPMA/PDDA shell in a PSPMA/DEAE-Dex coacervate phase, visualized at the same position in bright-field (c) and confocal fluorescence microscopy (d) with fluorescence from fluorescein-labeled PSPMA.