Self-programmed enzyme phase separation and multiphase coacervate droplet organization

Abstract

Membraneless organelles are phase-separated droplets that are dynamically assembled and dissolved in response to biochemical reactions in cells. Complex coacervate droplets produced by associative liquid-liquid phase separation offer a promising approach to mimic such dynamic compartmentalization. Here, we present a model for membraneless organelles based on enzyme/polyelectrolyte complex coacervates able to induce their own condensation and dissolution. We show that glucose oxidase forms coacervate droplets with a cationic polysaccharide on a narrow pH range, so that enzyme-driven monotonic pH changes regulate the emergence, growth, decay and dissolution of the droplets depending on the substrate concentration. Significantly, we demonstrate that time-programmed coacervate assembly and dissolution can be achieved in a single-enzyme system. We further exploit this self-driven enzyme phase separation to produce multiphase droplets via dynamic polyion self-sorting in the presence of a secondary coacervate phase. Taken together, our results open perspectives for the realization of programmable synthetic membraneless organelles based on self-regulated enzyme/polyelectrolyte complex coacervation.

Fig. 1. (a) Scheme of the complex coacervation process between glucose oxidase (GOx) and diethylaminoethyl (DEAE)-dextran. (b) Plot of the absorbance at 700 nm of solutions of GOx (0.25 mg mL⁻¹) in the presence of varying concentrations of DEAE-dextran in phosphate buffer (2.5 mM, pH 7.4). The maximum turbidity (red dotted line) corresponds to the optimal ratio for coacervation. (c) Plot of the absorbance at 700 nm of a solution of GOx (0.25 mg mL⁻¹) and DEAE-dextran (0.04 mg mL⁻¹) as a function of the pH. The maximum turbidity (red dotted line) corresponds to the optimal pH for coacervation. On (b) and (c), the DEAE-dextran (positive) : GOx (negative) molar charge ratio is also reported (see ESI Note 1†). Error bars represent the standard deviation of three independent repeats. (d) Optical microscopy images of GOx/DEAE-dextran mixtures ([GOx] = 0.25 mg mL⁻¹, [DEAE-dextran] = 0.04 mg mL⁻¹) prepared at different pH, as indicated, and corresponding schematic representations of the charge conditions on both polyions. Scale bars, 10 μm.

Fig. 2. (a) Schematic representation of enzyme-mediated self-regulated complex coacervation of GOx with DEAE-dextran in the presence of glucose. GOx catalyzes the oxidation of glucose into gluconolactone that spontaneously hydrolyses into gluconic acid, producing a pH decrease that can drive coacervate formation and dissolution. (b) Time-dependent evolution of the absorbance at 700 nm of a solution of GOx (0.25 mg mL⁻¹) and DEAE-dextran (0.04 mg mL⁻¹) produced at pH 10.2 after the sequential addition of glucose (0.6 mM at each addition). The colored area represents error as the standard deviation of three independent repeats. (c) Time-dependent evolution of the absorbance at 700 nm of a solution of GOx (0.25 mg mL⁻¹) and DEAE-dextran (0.04 mg mL⁻¹) produced at pH 10.2 after the single-step addition of varying final glucose concentrations, as indicated. Above a certain glucose concentration, a bell-shape is observed, attributed to the nucleation, grow, decay and dissolution of coacervate droplets. In such conditions, τ_1/2 denotes the full width at half maximum turbidity (here shown on the example of 1.4 mM glucose). The colored area represents error as the standard deviation of three independent repeats. (d and e) Optical microscopy snapshots of GOx/DEAE-dextran mixtures ([GOx] = 0.4 mg mL⁻¹, [DEAE-dextran] = 0.064 mg mL⁻¹) prepared at pH 10.2 at different times after addition of 0.5 mM (d) or 25 mM (e) glucose, showing the formation of stable or transient coacervate droplets, respectively. Scale bars, 20 μm. (f) Evolution of τ_1/2 as defined in c as a function of the final glucose concentration. The red line represents a mono-exponential fit of the data. Error bars represent standard deviations of three independent repeats. (g) Time-dependent evolution of the absorbance at 700 nm of a solution of GOx (0.25 mg mL⁻¹) and DEAE-dextran (0.04 mg mL⁻¹) produced at pH 10.2 after the single-step addition of 5 mM glucose and the repeated additions of 10 mM NaOH (black arrows). The dilution factor after the last NaOH addition was ∼1.05, so the final concentrations of components did not appreciably change.

Fig. 3. (a–c) Optical (a) and confocal fluorescence (b and c) microscopy images of multiphase ATP/pLL-in-GOx/DEAE-dextran coacervate micro-droplets doped with RITC-GOx (b, red fluorescence) and FITC-DEAE-dextran (c, green fluorescence) in phosphate buffer (2.5 mM, pH 7.4). False coloring to magenta and cyan was used, respectively. Scale bars, 20 μm. (d) Schematic representation of GOx-mediated dynamic formation of multiphase coacervate droplets. At low glucose concentration, stable multiphase droplets are formed as the pH stabilizes to physiological values while higher glucose turnover gives rise to a transient multiphase droplet organization. (e and f) Optical microscopy snapshots of ATP/pLL/GOx/DEAE-dextran mixtures produced at pH 10.2 at different times after addition of 25 mM (e) or 100 mM (f) glucose, showing the formation of stable or transient multiphase coacervate droplets, respectively. Scale bars, 20 μm. Insets show zoomed areas (white box). Scale bars, 5 μm.