Nonspherical Coacervate Shapes in an Enzyme-Driven Active System

Abstract

Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient nonspherical shapes, and we systematically study how PNPase concentration, UDP concentration, and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of nonspherical coacervate shapes may have broader implications for the relationship between (bio)chemical activity and coacervate biology.

Figure 1

Poly(U)/spermine coacervates can be generated and activated by the enzyme polynucleotide phosphorylase (PNPase). (a) Schematic of PNPase RNA polymerization. PNPase (green circle) polymerizes short RNA seeds (blue lines) by adding UMP monomers from UDP (blue dots), giving rise to long homopolymeric polyuridylic RNA (poly(U)). (b) Schematic description of PNPase induced coacervation. Poly(U)₂₀ does not phase separate within the presence of the polycation spermine, but once elongated, phase separation is initiated. (c) Coacervation induced by PNPase polymerization of poly(U) can be visualized macroscopically through solution turbidity. (d) Turbidity measurements of the solutions in panel c. Turbidity values were calculated from absorbance measurements at 500 nm wavelength (n = 9). (e) Time-lapse micrographs of the experiment shown schematically in panel b. The reaction contained 4 μM PNPase, 40 mM UDP, 1.0 wt % spermine, and 5 μM Cy5-U20 and was carried out at 30 °C. Images are false-colored and the scale bar indicates 10 μm.

Figure 2

Quantification of nonequilibrium coacervate shapes by average circularity of cross sections. (a) Schematic depiction of PNPase reaction in the presence of poly(U)/spermine coacervates in equilibrium, supplemented with poly(U) seeds, UDP, and PNPase. Initially, the coacervates become active but as the reaction reaches a dynamic equilibrium (t → ∞), they assume their spherical equilibrium shape. (b,c) PNPase reaction in the presence of preformed coacervates with 1 μM (b) and 4 μM (c) PNPase. There is a significant qualitative difference in terms of the coacervate shapes prior to assuming a spherical equilibrium shape. Images are false-colored and the scale bar indicates 10 μm. (d) Plot of the average circularity of coacervate cross sections for PNPase concentrations of 4 μM (black), 2 μM (red), and 1 μM (blue) PNPase. The control (green) contained passive poly(U)_∞/spermine coacervates, which were produced by incubation of PNPase, poly(U)₂₀, and UDP (see Materials and Methods for details). Dashed lines indicate the minimum observed average circularity per condition. In all images, PNPase was added 5 min before the start of image acquisition. (e) Average circularity profiles for 20 mM (black), 5 mM (red), and 2 mM (blue) UDP. (f) Average circularity profiles for temperatures of 30 °C (black), 34 °C (red), and 26 °C (blue). In panels d–f, the error bars indicate standard error from the mean.

Figure 3

Analysis of the circularity recovery after merging (CRAM) provides the time scale of coacervate merging. (a) Micrograph images of a merging event. The first image shows two coacervates prior to the merging process. Time zero is defined as the moment at which the coacervates start merging. (b) Circularity profile corresponding to the merging event of panel a. Prior to merging (t < 0), two spherical coacervates are observed. As they merge, the circularity of the resulting coacervate drops sharply, and recovers to a stationary value as the coacervates regains spherical shape. The red line indicates the best fit of a single exponential (see Materials and Methods) (c) Scatterplot of the time scale of merging plotted against the radius of the resulting droplet. The inverse capillary velocity was found to be 0.67 s/μm by linear regression (R² = 0.53, n = 53), indicated by the black dashed line. (d) Merging of passive coacervates containing Cy3- and Cy5-poly(U) (yellow and red, respectively, images false-colored) to demonstrate the mobility of the poly(U) within the coacervate phase. Scale bars of panels a and d indicate 5 μm.

Figure 4

Localization of reaction components in active poly(U)/spermine coacervates. (a,b) PNPase reaction with 5′-end Cy5-labeled poly(U) (panel a) and FITC-labeled PNPase (panel b). To illustrate the difference, images were false-colored with Cy5-poly(U) in yellow and FITC-PNPase in blue. Scale bar indicates 10 μm. (c) Fluorescence intensities of FITC-PNPase in the coacervate phase (CP), solvent phase (SP), and at the CP–SP interface. This implies that the PNPase concentration inside the coacervate phase is 3.9-fold higher than in the surrounding solvent phase (see Materials and Methods). Error bars indicate the standard deviation. (d) Fluorescence recovery after photobleaching (FRAP) of FITC-PNPase fluorescence indicates recovery of FITC-PNPase within 2.5 min. This indicates that there is continuous exchange of PNPase with the surrounding solvent phase and that the PNPase is redistributed throughout the coacervate phase.

Figure 5

PNPase degrades coacervates in high-phosphate buffer. (a) Provided with phosphate instead of nucleotides, PNPase has 3′-to-5′ exoribonuclease activity, through which it forms UDP from poly(U) and free phosphate. (b) Implementation of PNPase-degradation of coacervates inside high-phosphate buffer. (c) Micrograph images of the experiment shown in panel b. Poly(U)_∞/spermine coacervates were prepared in a buffer containing 10 mM disodium phosphate and 4 μM PNPase. After 30 min, significant degradation of coacervate was observed. Images are false-colored and the scale bar indicates 10 μm.