Coacervate droplets as pH-regionalized protocells

Domain: neuroscience
Published: 2026-02-02
Editorial outcome: minor_revision

Abstract
The mechanisms governing pH regulation in membrane-less organelles (MLOs) are essentially different from those relying on membrane proteins, yet it remains poorly understood due to the difficulty in directly controlling the conditions across the MLOs interface. Here, we develop a coacervate-based, in vitro model to investigate how liquid-liquid phase separation (LLPS) could contribute to pH regulation in MLOs. We construct peptide-based coacervate droplets using microfluidics and find that charged polymers within the coacervates help create uneven H+/OH- distributions, resulting in pH-regionalized microenvironments similar to the nucleolus. More interestingly, such a pH difference weakens or even disappears following the destruction of LLPS, a phenomenon observed in both nucleolus and coacervate droplets. Based on these findings, we demonstrate the ability to finely tune the local pH of the coacervate droplets over a wide range by incorporating enzymes, which can drive and control cascade reactions, and perform basic molecular biology operations such as polymerase chain reaction (PCR) and in vitro transcription and translation reaction (IVTT). This study highlights the role of LLPS in pH modulation within MLOs and provides insights into the potential of coacervates as protocells for broader applications.

Introduction
Cellular functions rely on numerous enzyme-catalyzed reactions that occur spatially and temporally correctly under physiological conditions1–3. The maintenance of enzyme activity is essential for metabolism, energy production, and the synthesis of various substances1,3. A key factor that affects enzyme activity is pH, as each enzyme functions optimally within a specific pH range. Deviations from this range can impair the structure and function of enzymes, which can even lead to diseases or disorders. It has been well established that various membrane exchangers and transporters play significant roles in regulating intracellular pH4. In contrast, membrane-less organelles (MLOs), which host diverse biochemical reactions and play essential roles in cellular regulation, lack such membrane-based control mechanisms. Recent studies have revealed that MLOs can exhibit local pH variations, which may influence the kinetics and selectivity of enzymatic reactions5,6. However, given the complexity of living cells, it is challenging to directly investigate the mechanisms of pH regulation in vivo by controlling conditions across the MLOs interface7.

In this study, we found that a natural pH difference exists between the nucleolus and nucleoplasm in several tested cell lines, which can be modulated by drug treatments to alter their structure and properties (Fig. 1a). Since coacervates are liquid-liquid phase separation (LLPS) systems driven by noncovalent interactions, and they share similar properties to MLOs such as molecular organization and compartmentalization7–13, we then present peptide-based coacervate droplets as protocells to mimic the pH difference found in the nucleus. We use microfluidics to prepare these coacervate droplets to ensure their uniformity in size, thus allowing for the generation of a similar pH difference across the LLPS interface under precisely controlled conditions14–16. Through molecular dynamics simulations and quantitative experiments, we conclude that the dense phase of coacervates, rich in charged polymers, tends to attract a higher concentration of H+/OH- ions than the dilute phase. This results in an uneven apparent pH distribution between the interior and exterior of the coacervates. More importantly, such a pH difference weakens or even disappears following the destruction of LLPS, a phenomenon observed in both nucleolus and coacervate droplets. Based on these findings, we demonstrate that the pH-regionalized environment of the coacervate droplets can be customized by selectively accommodating enzymatic reactions (as shown in Fig. 1b), and such feature can be further employed to control cascade reactions and perform polymerase chain reaction (PCR) and in vitro transcription and translation reaction (IVTT), thereby recapitulating various cellular activities (as shown in Fig. 1c)17. Our findings suggest that coacervate-based protocells offer a valuable platform to explore how physicochemical compartmentalization influences biochemical reactions.

Results
Identification of the pH difference between the nucleolus and the nucleoplasm
To elucidate whether there is a pH difference across the MLOs interface in living cells, we chose the nucleolus as an example because it is the largest membrane-less condensate and is easily observable18. We assess the pH of nucleoli in different cells using SNARF-4F, a cell-permeable indicator that shows notable pH-dependent dual-emissions at 580 and 640 nm. By measuring the fluorescence intensity ratio at these wavelengths, we can determine the subcellular pH effectively.

The spectral shifts of SNARF-4F based on pH changes are described by Eq. (1)19, where Ka denotes the acid dissociation constant of the probe, R represents the fluorescence intensity ratio at two wavelengths λ1 (580 nm) and λ2 (640 nm) (E 580/E 640), and RA and RB represent the limiting values of R corresponding to the fully protonated and fully deprotonated states, respectively.

[H+] = Ka × (R - RB) / (RA - R) (1)

Using Eq. (1), we generate the standard curve for SNARF-4F (Supplementary Fig. 1) after adjusting intracellular pH with calibration buffers and obtain the functional expression given by Eq. (2) (detailed discussion can be found in the Supplementary Note).

pH = 6.1790 - lg [(R - 0.6741) / (1.8615 - R)] (2)

With this, we study the pH levels in the nucleolus and nucleoplasm under various physiological conditions, as shown in Fig. 2a. Based on the results, we find that the nucleolus is slightly more acidic than the surrounding nucleoplasm in various cell types, including 3T3, OC1, HELA, and AML12 Fig. 2b–f and 2k–n20.

We next examine whether the pH difference between the nucleolus and nucleoplasm is linked to the nucleolar functions and the LLPS status. To this end, we used CX-5461, a well-known inhibitor of RNA polymerase I, to suppress rRNA synthesis in the nucleolus. Previous studies have also shown that CX-5461 can disrupt the native organization of the nucleolar structure20,21. We find that inhibiting rRNA synthesis with CX-5461 deacidifies both the nucleolus and nucleoplasm, indicating a closer relationship between nucleolar transcriptional activity and its pH (Fig. 2a, 2b, and 2g–2n). Additionally, the pH difference between the nucleolus and nucleoplasm is smaller in CX-5461-treated cells than in untreated cells regardless of the cell type (Supplementary Fig. 2). Moreover, we treated HeLa cells with different concentrations of actinomycin D (Act D), an inhibitor of RNA and protein synthesis, and found that low concentrations of Act D can also induce nucleolar deacidification (Fig. 2o and 2p). Furthermore, after nucleolar disassembly in HeLa cells treated with high concentrations of Act D, no detectable pH difference was observed (Fig. 2o and 2p). Taken together, these observations clarify that a pH difference exists across the nucleolus interface and suggest that this difference is closely linked to the LLPS status and biological function of the nucleolus. These experiments imply that the transcriptional activity and structural integrity of the nucleolus might both contribute to maintaining this pH difference. However, as transcriptional inhibition and structural disruption may take place simultaneously under drug treatment, the available data are not sufficient to distinguish which of these processes predominantly drives the observed pH changes.

Peptide coacervate droplets recapitulating the pH difference in MLOs
For simplified modeling of MLOs in vitro, we utilize a coacervate composed of decapeptides arginine (R10) and aspartic acid (D10). These amino acids are representative sequences of phase-separating proteins found in cells and can form coacervates through ion-pairing interactions between cationic R10 and anionic D10 (Fig. 3a)22. We generate R10/D10 coacervate microdroplets using the direct mixing method. A state diagram is plotted to indicate the optimal concentration ranges of each component, within which coacervate droplets can be readily formed, albeit with polydispersity (Fig. 3b and Supplementary Fig. 3). Based on the phase diagram, we set 10 mM R10 and 10 mM D10 as the standard experimental conditions (the formed coacervate is denoted as 10 mM R10/D10 coacervate), unless otherwise specified. Additionally, to assess the stability of these coacervates under different pH conditions, we observed them using bright-field microscopy (Supplementary Fig. 4) and measured their turbidity (Fig. 3c) with a UV-Vis spectrophotometer across a pH range of 6.0–9.0. The results suggest that the coacervates remain stable in pH environments close to physiological conditions, making them suitable for modeling the environments of MLOs within cells.

The condensate and dilute phases of freshly prepared coacervates exhibited comparable pH values of approximately 8.0 (as shown in Fig. 3d, p>0.05). Then, to investigate whether a pH difference can be established across the coacervate interface in response to environmental variables, we introduce different concentrations of H+ or OH- into the R10/D10 coacervate system through the addition of HCl and NaOH. A micro-pH probe is used to separately measure the pH of the condensed and diluted phases obtained after centrifugation (Supplementary Fig. 5). We observe the establishment of a notable pH difference, with the condensate phase responding to pH changes: it becomes more acidic upon H+ addition and more alkaline upon OH- addition, compared with the dilute phase (Fig. 3d and Supplementary Fig. 6). In addition, we utilized the SNARF-4F dye to cross-compare the pH change trends of the condensate and dilute phases, further validating the same observations (Supplementary Fig. 7).

We then investigate whether varying the coacervate and salt concentrations affects the pH response behavior of the coacervates. The results indicate that decreasing the coacervate concentration reduces the tolerance of the system to added salts, making the pH difference (defined as the pH of the condensed phase minus that of the dilute phase) detectable within a narrower range of salt concentrations (Fig. 3e). We prepare coacervate solutions with different concentrations and measure their turbidity, as shown in Fig. 3f. The turbidity of the coacervate solution decreases as the concentration of R10 and D10 is reduced. Then, we measure the pH difference between the condensate phase and the dilute phase of coacervate after adding 1 μM HCl or NaOH to the coacervate system. We find that the pH difference between the two phases is larger at higher coacervate concentrations (Fig. 3g, Supplementary Fig. 8a and 8b). Additionally, adding NaCl into the 10 mM R10/D10 coacervate solution with increasing concentrations leads to decreased turbidity, suggesting that high salt concentrations can lead to disassembly of the coacervate due to the screening effect on the peptide charges (Fig. 3h). Accordingly, a smaller pH difference is found between the two phases (Fig. 3i, Supplementary Fig. 8c and 8d). These results suggest that the condensate and dilute phases of the coacervate can respond differently to external acid or base, and this effect becomes more pronounced when the stability of the coacervate is higher. To further validate the above findings, we prepared coacervates in Tris-HCl buffer and added varying concentrations of acid or base. We then measured the pH of both the condensate and dilute phases. The results showed that, compared with coacervates prepared in water at pH 8.0, those prepared in pH 8.0 Tris-HCl buffer exhibited a similar trend in pH difference between the two phases (Supplementary Fig. 9).

We reason that this phenomenon might be attributed to the difference in H+ or OH- distribution in the two phases of the coacervates, as illustrated in Fig. 3a. To support our hypothesis, we conduct molecular dynamics simulations using a coarse-grain (CG) model of the R10/D10 coacervate in 20×20×20 nm³ simulation boxes containing equal amounts of R10 and D10 molecules23,24. Meanwhile, 100 H3O+ (referred to as “H+” for simplicity below) or OH- are introduced into the simulation box, accompanied by Na+ or Cl- to maintain electrostatic balance. We first investigate the effect of coacervate concentration on H+ or OH- distribution. Starting with a random distribution of the decapeptides, we perform simulations with 100, 200, and 300 pairs of peptides. The energy curve of the system indicates that it has reached a relatively stable state at 5 μs (Supplementary Fig. 10a and Supplementary Fig. 11). Figure 3j shows snapshots of the simulation box at 5 μs, illustrating the status of coacervation of the system at increasing concentrations of R10 and D10. Supplementary Fig. 12 shows the density profiles of the decapeptides, water, H+, and OH-. We find that both H+ and OH- are enriched in the condensate phase (Supplementary Fig. 13). The peptide–ion (H+ or OH-) radial distribution functions (RDFs) at different coacervate concentrations are shown in Fig. 3k and Supplementary Fig. 14. The pronounced first peaks around 0.5 nm indicate direct contact between the peptide and ions. The contact numbers between H+ and OH- and peptides are also shown in Fig. 3l. It is evident that as the coacervate concentration increases, the contact number between the peptide and H+ or OH- also rises. To further analyze the movement of H+ or OH- within the coacervate solution, we assess the diffusive properties by calculating the mean square displacement (MSD) of H+ and OH- (Supplementary Fig. 15a and 15b). We find that the diffusion coefficients of H+ and OH- decrease significantly with increasing coacervate concentration (Supplementary Fig. 15c and 15d). It indicates reduced mobility of ions within the coacervate, likely due to stronger molecular interactions or limited space in the coacervate network. Notably, in the coacervate solution, the diffusion coefficient of H+ decreases more significantly than that of OH- when compared to their respective values in water. This finding aligns with the RDF results, which reveal greater recruitment of H+ to the peptide condensates and a higher contact number of H+ with peptides relative to OH-. Together, these results suggest that H+ may have an even more preferential partitioning affinity in the R10/D10 coacervate system over OH-.

Similarly, to analyze the influence of salt concentration on the pH compartmentalization behavior of the D10/R10 coacervates, we set up identical simulation boxes with 300 pairs of randomly distributed R10 and D10 and perform simulations with salt concentrations ranging from 0 to 1.27 M (Supplementary Fig. 10b). Among the snapshots at 5 μs with varying NaCl concentrations, coacervation behavior occurs only at lower salt concentrations (0 M and 0.26 M) (Fig. 3j and Supplementary Fig. 16). The peptide–ion (H+ or OH-) RDFs with different salt concentrations are presented in Fig. 3m and Supplementary Fig. 17. We also find that as the salt concentration increases, the contact numbers between the peptides and H+ or OH- are correspondingly decreased (Fig. 3n). Moreover, with increasing salt concentration, the diffusion coefficients of H+ and OH- are enhanced (Supplementary Fig. 18). As salt concentration increases, the electrostatic interactions between charged peptides are screened, weakening the coacervate formation and reducing pH compartmentalization. This suggests that Na+ and Cl- disrupt the attractive forces between oppositely charged peptides, allowing H+ and OH- ions to distribute more evenly between the condensate and dilute phases. In summary, coacervates can dynamically respond to external environments and create regionalized pH difference.

Construction of pH-regionalized protocell
The pH regionalization feature of the coacervate droplets makes them promising candidates as protocells by integrating pH-dependent biological reactions. Prior to this, we first examine two key points: whether enzymes can be accommodated within the coacervates and whether their activity can be sustained. To this end, we choose two common enzymes, glucose oxidase (GOx) and urease, for encapsulation in the R10/D10 coacervates. Similar to many biochemical reactions within cells, these two enzymes generate acidic or alkaline byproducts in the presence of their substrates. One of the key features of coacervates, and a significant factor in their potential role as protocells, is their ability to capture a wide range of guest molecules and concentrate them at high levels to facilitate reactions. Because the uniformity of the coacervate droplets might influence the reaction rate, we employ our previously reported microfluidic technology25 to prepare uniform R10/D10 coacervate droplets for subsequent experiments (Fig. 4a). Then, we assess the ability of the R10/D10, along with other types of coacervate droplets, poly(dimethyldiallylammonium chloride) (PDDA)/poly(sodium acrylate) (PAA) and DEAE-dextran/dsDNA to concentrate enzymes using fluorescence microscopy. As shown in Fig. 4b and Supplementary Fig. 19, both FITC and RBITC display a uniform distribution within three types of homogeneous coacervate droplets, indicating that both enzymes have been successfully encapsulated within the droplets. Next, we examined the turbidity of solutions after mixing coacervates with varying concentrations of enzymes to assess whether the enzymes affect coacervate stability. As shown in Supplementary Fig. 20, the results indicated that neither GOx nor urease had a significant impact on the stability of the coacervates.

To determine the partition coefficients of FITC-GOx and RBITC-urease, the R10/D10 coacervates were incubated with each fluorescently labeled enzyme, and the fluorescence intensities in the condensate and dilute phases were subsequently quantified (Supplementary Fig. 21). In addition, with the condensate volume fraction already determined (Supplementary Fig. 5b), these parameters enabled us to estimate the percentage of the total enzyme recruited into the condensate phase, yielding P_GOx = 80.45% and P_urease = 73.14%. To further elucidate the preferential partitioning of enzymes within the coacervates, we employ microscale thermophoresis (MST) to assess the interactions between GOx and the components of the coacervates, i.e., D10 and R10, respectively. As shown in Fig. 4c and 4d, R10 interacts directly with FITC-GOx at Kd = 737.07 ± 104.96 nM, whereas no detectable binding between D10 and FITC-GOx is observed. We hypothesize that the partitioning affinity of FITC-GOx into the D10/R10 coacervate is driven by the interactions between the positively charged R10 and the negatively charged FITC-GOx. Therefore, we test other coacervate systems, including DEAE-dextran/dsDNA and PDDA/PAA. Based on the results from MST, it is evident that similar to R10, there are interactions between GOx and the positively charged polyelectrolytes DEAE-dextran (Kd = 1377.90 ± 197.96 nM) and PDDA (Kd = 1938.61 ± 255.71 nM). However, no interactions were observed between GOx and the negatively charged dsDNA and PAA. These results suggest that the interaction between the charged polymer and the enzymes plays a crucial role in the affinity partitioning of enzymes in coacervates.

We further conduct molecular dynamics simulations of R10/D10 coacervate systems containing GOx or urease. Starting from random placements in the solution, GOx and urease gradually partition into the condensate phase and tend to move to the inside (Figs. 4e, 4h, Supplementary Fig. 22a, and 22c). Additionally, we separately performed molecular dynamics simulations for solutions containing R10 and GOx, as well as R10 and urease. Snapshot of the final configuration for the solution containing R10 and GOx is shown in Fig. 4f and Supplementary Fig. 22b, while that for the solution containing R10 and urease is presented in Fig. 4i and Supplementary Fig. 22d. Over time, R10 molecules interact with GOx or urease molecules, as evidenced by the peptide-protein RDF and the potential of mean force (PMF) obtained from the simulations (Figs. 4g and 4j). In both R10-GOx and R10-urease systems, the peak in the RDF around 0.5 nm suggests the presence of interaction zones between the peptides and the enzyme. The PMF provides insight into the energetic landscape of these interactions, implying that R10 contacts with both GOx and urease. For comparison, the interactions between D10 and the enzymes were also analyzed using molecular dynamics simulations. The results showed that, unlike the R10 peptide, D10 did not exhibit significant interactions with either GOx or urease (Fig. 4k–n). These observations align with the MST measurements.

Next, we use R10/D10 coacervate droplets as protocells accommodating enzyme reactions, as shown in Fig. 5a. Glucose or urea is added to the system, which acts as a substrate for GOx and urease, respectively. Glucose can be oxidized to produce gluconic acid, thus creating a continuous H+-supplying environment. Similarly, urease catalyzes the hydrolysis of urea to produce NH3, a basic substance in the solution, thereby establishing a continuous OH- supply. We measured the reaction rates of GOx and urease in both coacervate and bulk solutions. The reaction rate of GOx as a function of glucose concentration follows typical Michaelis–Menten kinetics in both bulk solution (pH 8.0) and R10/D10 coacervate solutions (Fig. 5b, 5c, Supplementary Figs. 23, and 24). Similarly, the reaction rate of urease as a function of urea concentration also follows typical Michaelis–Menten kinetics in both bulk solution and R10/D10 coacervate solutions (Fig. 5d, 5e, Supplementary Figs. 25, and 26). The Vmax and Km values, representing the maximum reaction rate and substrate binding affinity, respectively, were obtained by fitting the Michaelis–Menten plots (Fig. 5f and Supplementary Fig. 27). In the absence of additional H+ or OH- ions, the lower Vmax values observed for both enzymes in coacervates, compared with those in solution, could possibly arise from restricted enzyme mobility within the dense coacervate matrix, which might limit catalytic turnover. The decrease in Km indicates an enhanced apparent affinity for the substrate, which may arise from the microenvironmental effects inside the coacervates. Moreover, as shown in Fig. 5f, upon the addition of extra H+ ions, the Vmax values of GOx and urease (with optimal pH values of 5.5 and 7.4, respectively) in the condensate phase became more comparable to those in solution, likely because the preferential partitioning of ions toward the condensate phase resulted in a locally elevated concentration of H+26,27. In contrast, as OH- ions were added, the difference in Vmax between the coacervate and solution phases increased accordingly.

Adding 5 mM glucose to the GOx-containing coacervate solution causes both the condensate and dilute phases to become more acidic (Fig. 5g). Conversely, adding 5 mM urea to the urease-containing coacervate solution causes both phases to become more alkaline (Fig. 5g). The pH change triggered by adding glucose or urea are more prominent in the condensed phase than in the dilute phase. This is likely due to the partition of the enzymes and enrichment of H+/OH- in the condensate phase. To figure out whether this effect also applies to other coacervate systems, we test it in DEAE-dextran/dsDNA coacervate (Fig. 5h) and PDDA/PAA coacervate (Fig. 5i). In both cases, we observe similar pH regionalization behavior, confirming that the observed phenomenon is not limited to a single coacervate system.

We then measure the pH difference between the condensate and dilute phases of the coacervate systems after reacting with different concentrations of glucose and urea (Fig. 5j). We observed that, after reacting with glucose or urea, the enzyme-containing coacervates exhibited a pH difference between the condensate and the surrounding dilute phase, which is consistent with the results obtained from the direct addition of H+ or OH-. Additionally, the pH difference between the condensate and dilute phases of the coacervate is also affected by the concentration of the coacervate and salt (Fig. 5k, 5l, and Supplementary Fig. 28). Similar to the effect observed with direct H+ or OH- addition, when the stability of the coacervate droplets is reduced by decreasing the scaffold molecular concentration or increasing the salt concentration, the pH difference between the inside and outside of the coacervate droplets correspondingly decreases.

Controlling cascade reactions in the coacervate protocells
Since we have demonstrated that the D10/R10 coacervates can be used to accommodate biological enzymes and establish pH difference across the interface using enzymatic reactions, we aim to further exploit these properties to develop protocells capable of performing cascading enzymatic reactions (Fig. 6a). First, we examined the stability of R10/D10 coacervates upon mixing with various reagents using bright-field microscopy (as shown in Fig. 6b). Then, we determine the adjustable range of pH of the GOx and urease-loaded R10/D10 coacervate by reacting with the substrate (glucose or urea). We find that the pH of the condensate phase can be regulated within the range of 7-9 by adjusting the substrate type and concentration (Fig. 6c). The feasibility of this approach is also demonstrated using GOx and urease-loaded DEAE-dextran/dsDNA coacervate and PDDA/PAA coacervate (Supplementary Fig. 29). To exclude the potential influence of the added components on the microenvironmental pH of the condensate phase, we measured the pH of the condensate phase of R10/D10 coacervates without enzymes after mixing with different components, and found that the addition of the substrate alone did not significantly affect the internal pH of the coacervates (Supplementary Fig. 30). These results indicate that GOx- and urease-loaded coacervates can regulate the microenvironment’s pH within the normal physiological range, laying a solid foundation for constructing cascade protocells.

We then investigate the impact of the pH variation of the coacervate protocells caused by one enzymatic reaction on the activity of another enzyme. As shown in Fig. 6d, the activity of GOx (optimal pH 5.5) was significantly suppressed under alkaline conditions created by urease, while urease activity (optimal pH 7.4) was reduced under acidic conditions generated by GOx26,27. These results highlight how enzymatic cross-regulation within coacervates can modulate biochemical processes through pH-dependent mechanisms.

Based on this, we explore whether the coacervate protocells can be leveraged to recapitulate more complex physiological processes, such as gene expression and signal transduction. To investigate the ability of coacervates to conduct and regulate in vitro DNA replication, we use a qPCR kit with a green fluorescent dye to monitor the accumulation of PCR products. By pre-mixing the GOx- and urease-loaded coacervate solution with the qPCR kit and adjusting the pH microenvironment with varying concentrations (0, 2.5, and 5 mM) of glucose or urea, we create different experimental conditions. Following the complete reaction of the substrates, we analyze the PCR products to evaluate how the local pH influences the reaction efficiency. This approach allows us to better understand the role of coacervates in modulating gene expression under varying environmental conditions. After disassembling the coacervate, the PCR products are quantitatively analyzed using gel electrophoresis, and the fluorescence intensity of the generated DNA in the solution is measured using a microplate reader. As shown in Fig. 6e, PCR reactions are significantly inhibited when 2.5 or 5 mM glucose and urea are added to the solution. Because the optimal pH for PCR reactions is about 8.0, which is about the original pH of coacervate, this inhibitory effect may result from the suppression of PCR-related enzyme activity under acidic or alkaline conditions28.

Next, we perform IVTT experiments using the coacervate protocells. We utilize a cell-free transcription and translation kit combined with a GOx- and urease-loaded R10/D10 coacervate solution to create uniform droplets. The addition of 5 mM glucose or urea influences the reaction conditions within the coacervate environment. Following the incubation at 37°C, we observe the results under a fluorescence microscope. The successful transcription is evidenced by the presence of RNA exhibiting green fluorescence in the coacervate droplets, and successful translation is indicated by proteins displaying red fluorescence (Fig. 6fi). We then proceed with a quantitative analysis of the fluorescence intensity within the coacervate protocells, as shown in Fig. 6fii and 6fiii. The results reveal that the addition of urea decreases both RNA and protein production. In contrast, the introduction of glucose enhances the rates of transcription and translation within the coacervate droplets compared to the control group. This could be attributed to the fact that the optimal pH conditions for transcription and translation processes are typically neutral, while the initial pH of the coacervate is nearly 8.0. The addition of urea shifts the pH to a more alkaline level (about 9.6), while the addition of glucose brings the pH closer to neutral (about pH 6.8), facilitating the processes of transcription and translation. Furthermore, to evaluate the relative transcription and translation rates in the two phases of the coacervates, the mixtures containing coacervates and the IVTT kit were centrifuged to separate the condensate and dilute phases. Both phases were then individually incubated at 37°C for the IVTT reactions, and their fluorescence intensities were compared to determine the relative rates of transcription and translation. As shown in Fig. 6fiv, the results indicated that most transcription and translation reactions occurred within the condensate phase of the coacervates. These findings highlight the effective functioning of the coacervate protocells in accommodating and regulating transcription and translation, demonstrating its potential for applications in synthetic biology.

Methods
Materials
Arginine (R10) and aspartic acid (D10) polypeptides are obtained from Anhui Guoping Pharmaceuticals. The OC1 (FH0777), 3T3 (FH0380), HELA (FH0314), and AML12 (FH0338) cell lines are sourced from Fuheng Biology. DMEM, fetal bovine serum, PBS, penicillin/streptomycin, SNARF-4F, 0.25% trypsin-ethylenediaminetetraacetic acid, intracellular pH calibration buffer kit, live cell imaging solution, urease, and urea are purchased from Thermo Fisher Scientific. Actinomycin D (ActD), and CX-5461 are obtained from MedChemExpress. Diethylaminoethyl dextran hydrochloride (DEAE-dextran), low molecular weight deoxyribonucleic acid (derived from salmon sperm), and poly (dimethyldiallylammonium chloride) (PDDA, average Mw 200,000-350,000) are sourced from Sigma-Aldrich. Poly (sodium acrylate) (PAA, average Mw 5000) is obtained from J&K Scientific. Glucose oxidase is ordered from Sangon Biotech. Cell-free gene expression kits and qPCR kits are ordered from MiniPCR Bio. The Glucose Oxidase Activity Assay Kit is ordered from Solarsbio. Urease Activity Assay Kit is ordered from Boxbio. FITC-GOx and RBITC-urease are ordered from AGOMA.

Nucleolar pH quantification using SNARF-4F
SNARF-4F is dissolved in high-grade DMSO to prepare a 5 mM stock solution. For cell co-culture, the concentration of SNARF-4F is adjusted to 7.5 μM. Intracellular pH calibration is performed using a calibration buffer kit following the manufacturer’s instructions. After staining with SNARF-4F, fluorescence images are captured using confocal microscopy with excitation at 488 nm and emissions detected at 580 nm and 640 nm. The intracellular pH is determined based on the emission intensity ratio of at 580 nm and 640 nm (Em580/Em640). The detailed calculation method for the standard curve is provided in the Supplementary Note.

CX-5461 and Actinomycin D inhibitors treatment
For CX-5461-treated cells, 1 mM CX-5461 in high-grade DMSO is added to the culture media and incubated with living cells at a concentration of 1 μg/mL for 1 hour. For low concentration ActD-treated cells, ActD stock solution in DMSO is diluted to 0.063 μg/mL and applied to the cells for 1 hour. For high concentration ActD-treated cells, an ActD stock solution in high-grade DMSO is diluted to 5 μg/mL and applied to the cells for 2 hours. After these treatments, the cells are stained with SNARF-4F, and the intracellular pH is measured with confocal fluorescence.

Coacervate generation
D10/R10 coacervates are produced by directly mixing a 10 mM stock solution of R10 (the pH is adjusted to 8.0 using 1 M NaOH or HCl, and other solutions are adjusted similarly unless otherwise specified) and D10 (pH 8.0) in water at a 1:1 volume ratio. The coacervate samples are freshly prepared for characterization and equilibrated at room temperature for 15 minutes before analysis.

For the GOx-loaded coacervate protocells, 50 nM GOx is added to the 10 mM R10/D10 coacervate solution. Similarly, urease-loaded coacervate protocells are prepared by adding 50 nM urease to the 10 mM R10/D10 coacervate solution.

To create DEAE-dextran/dsDNA coacervates, 10 mg/mL DEAE-dextran (pH 8.0) stock solution and 10 mg/mL dsDNA (pH 8.0) stock solution are mixed directly at a volume ratio of 1:1. To prepare the PDDA/PAA coacervates, 10 mM PDDA (pH 8.0) and 10 mM PAA (pH 8.0) stock solutions are mixed at a volume ratio of 1:4.

Measuring the turbidity of the coacervate solution
To evaluate the stability of the coacervate solution across different pH values, 10 mM R10 and 10 mM D10 solutions are first adjusted to target pH levels ranging from 6.0 to 9.0 with 1 M HCl or 1 M NaOH. The R10 and D10 solutions were then thoroughly mixed at the corresponding pH values and sit for 15 minutes to equilibrate. Next, the turbidity of the coacervate solutions was measured at 600 nm (λ600) using UV-vis spectroscopy, and the turbidity (T) is calculated using the following equation:

T = [1 - 10^(-λ600)] × 100%

Determining the pH value of coacervate in condensate phase and dilute phase
Varying volumes (0, 0.4, 0.8, 1.2, 1.6, and 2.0 μL) of 1 M NaOH or 1 M HCl are mixed with 1 mL of 10 mM R10 solution and 1 mL of 10 mM D10 solution. After mixing, the coacervate solutions are left to stand for 15 minutes. Then, the solutions are centrifugated to separate the condensate phase. The pH values of the condensate and dilute phases are measured using a micro-pH meter.

To investigate the effect of coacervate concentration on the pH difference between the two phases, 1 μM HCl or NaOH is mixed with R10 and D10 at different concentrations (1, 2, 4, 6, 8, and 10 mM), respectively. After 15 minutes, the condensate and dilute phases of the coacervate solution are separated by centrifugation at 2.4g for 20 minutes, and the pH of each phase is measured using a micro-pH meter.

To examine the impact of salt concentration on the pH difference between the two phases, varying concentrations (0, 10, 20, 30, 40, and 50 mM) of NaCl are added to a 10 mM R10 and D10 solution mixed with 1 μM HCl or NaOH. The pH of both phases is then measured using the method described above.

When measuring the pH of the coacervate solution using SNARF-4F, 10 mM coacervates were mixed with different concentrations of NaOH or HCl and thoroughly vortexed. Subsequently, 7.5 μM SNARF-4F was added to the solution. Fluorescence images were then captured using confocal microscopy with excitation at 488 nm and emissions collected at 580 nm and 640 nm. The pH was determined based on the emission intensity ratio at 580 nm to 640 nm (Em580/Em640).

Molecular dynamics simulation of R10/D10 coacervate formation and ion distribution
CG molecular dynamics simulations are conducted with the Martini 3.0 coarse-grained force field, generating R10 and D10 CG peptides using the martinize.py script in Martini 3.023,29. In all simulations, equal numbers of peptides are randomly distributed in a 20×20×20 nm³ simulation box and solvated with Martini water beads, where one bead represents four water molecules. Subsequently, 100 H3O+ or OH- beads are introduced into the simulation box, replacing some water beads. Specifically, H3O+ was modeled as a TQ5 bead with a net charge of +1e, and OH- as a TQ5 bead with a net charge of -1e. All other parameters were set according to the default MARTINI 3.0 force field30. Na+ and Cl- ions are also added for electrostatic balance.

To investigate the effect of coacervate concentration on H+ and OH- distribution, simulations are carried out with 100, 200, and 300 pairs of R10 and D10 CG polypeptides. Following steepest descent minimization, the systems are equilibrated for 10 ns with a 10 fs time step, followed by a 5 μs production run at the same time step. To explore the impact of salt concentration on ion distribution, 0, 1000, and 4000 pairs of Na+ and Cl- are added to the simulation box with 300 pairs of R10 and D10. All other parameters are maintained consistently throughout the experiments.

Post-simulation snapshots of the simulation box are captured using the VMD software. For data analysis, normalized RDFs are calculated using the GROMACS tool gmx rdf. The contact number is calculated using gmx mindist with a cut-off of 0.6 nm. The density profile is computed using gmx energy, and the PMF is determined through umbrella sampling (US) simulations.

Microfluidic generation of uniform coacervate droplets
Uniform coacervate droplets are generated using a previously developed microfluidic device integrated with mechanical vibration25. The pre-formed R10/D10, DEAE-dextran/dsDNA, and PDDA/PAA coacervate solutions are centrifuged at 2.4g for 20 minutes. The precipitated coacervate phase, serving as the dispersed phase, is then injected into the microfluidic device via a syringe, while the dilute phase flows into the collection tube as the continuous phase. The typical flow rates are 20 μL/h for the discrete phase and 5 mL/h for the continuous phase. A sinusoidal perturbation with a 500 Hz actuation frequency is applied to the dispersed phase using a mechanical vibrator to facilitate active droplet generation. The microfluidic process for preparing uniform coacervates is consistent throughout the study.

Microscale thermophoresis analysis
The binding affinity between FITC-GOx and DEAE-dextran/PDDA/R10/dsDNA/PAA/D10 is measured using MST methods31,32. Binding reactions are conducted in water. Ligands (DEAE-dextran/PDDA/R10/dsDNA/PAA/D10) are initially prepared at a concentration of 20 μM and dilution series at 0.6104, 1.2207, 2.4414, 4.8828, 9.7656, 19.5313, 39.0625, 78.125, 156.25, 312.5, 625, 1250, 2500, 5000, 10000, and 20000 nM, respectively, as prepared. The resulting ligand solutions are mixed with 200 nM FITC-GOx in a 1:1 volume ratio. To prevent non-specific adsorption, the samples are loaded into NanoTemper monolithic capillaries immediately after preparation.

Molecular dynamics simulation of protein partitioning affinity into R10/D10 coacervate
To investigate the partitioning affinity of GOx and urease for the coacervates, the files of GOx (1GAL) and urease (4G7E) are downloaded from the Protein Data Bank, and their CG models are generated using the martinize.py script. For the partitioning affinity simulations, 300 pairs of polypeptides and 2 GOx or urease molecules are randomly distributed in a 20×20×20 nm³ simulation box and individually solvated with Martini water beads. Na+ and Cl- ions are introduced to maintain electrostatic balance. After minimization, the systems are equilibrated for 10 ns with a 10 fs time step, followed by a 5 μs production run with the same time step.

In simulations examining the interaction between R10/D10 and either GOx or urease, 100 R10/D10 molecules and 1 GOx or urease molecule are randomly distributed in a 20×20×20 nm³ simulation box and solvated with Martini water beads. Na+ and Cl- ions are also added for electrostatic balance. After the steepest descent minimization, the systems are equilibrated for 10 ns with a 10 fs time step, then a 5 μs production run is performed using the same time step. The RDFs are calculated using the gmx RDF tool, and the PMF is determined through umbrella sampling simulations.

Detection of enzymatic activities in coacervates
To assess the catalytic activity of GOx within coacervates, a 50 nM GOx-loaded R10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM GOx were each mixed with glucose solutions at various concentrations (0, 1, 2, 3, 4, 5, 10, 15, and 20 mM). After different reaction times (5 min, 10 min, 15 min, 30 min, 45 min, and 60 min), to minimize turbidity interference from the protocell-containing samples, the mixtures were centrifuged, and the supernatants were collected for GOx activity measurement using a Glucose Oxidase Activity Assay Kit. The initial reaction rate at each substrate concentration was determined by analyzing the linear region of the product formation curve over a 60-minute period (Supplementary Figs. 23 and 24).

Similarly, to evaluate urease activity in coacervates, a 50 nM urease-loaded R10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM urease are mixed with urea solutions at concentrations of 0, 1, 2, 3, 4, 5, 10, 15, and 20 mM. After different reaction times, to minimize turbidity interference from the protocell-containing samples, the mixtures were centrifuged and the supernatants collected for urease activity measurement using a Urease Activity Assay Kit. The initial reaction rate at each substrate concentration was determined by analyzing the linear region of the product formation curve over a 60-minute period (Supplementary Fig. 25 and 26).

Enzymatic reaction-induced pH difference across the two phases of coacervate protocells
To generate a pH difference across the two phases of R10/D10 coacervate, glucose and urea at concentrations ranging from 0 to 5 mM are added to a 50 nM GOx- or urease-loaded 10 mM R10/D10 coacervate solution. After incubating for 1 hour, the condensate and dilute phases are separated by centrifugation at 2.4g for 20 minutes, and the pH of each phase is measured using a micro-pH meter.

To examine the effect of coacervate concentration on the pH difference between the two phases, glucose or urea (5 mM) is added to R10/D10 coacervates at concentrations of 1, 2, 4, 6, 8, and 10 mM, each containing 50 nM GOx or urease. After 1 h of incubation, the two phases were separated by centrifugation, and their pH values were measured using a micro-pH meter.

To explore the impact of salt concentration on the pH difference across the phases, salts at concentrations of 0, 10, 20, 30, 40, and 50 mM are added to a 10 mM R10/D10 coacervate solution containing 50 nM GOx or urease, together with 5 mM substrates. After 1 h of incubation, the two phases were separated by centrifugation, and their pH values were measured using a micro-pH meter.

The pH difference between the two phases of DEAE-dextran/dsDNA and PDDA/PAA coacervates is generated by adding 5 mM substrates (glucose or urea) to coacervate solutions containing 50 nM GOx or urease and incubating for 1 h. The condensate and dilute phases are then separated by centrifugation, and their pH values are measured using a micro-pH meter.

Regulating enzymatic activities of GOx and urease in coacervate protocells
To establish acidic conditions, 10 mM glucose is added to a 10 mM R10/D10 coacervate solution co-loaded with 50 nM GOx and 50 nM urease. After the glucose reaction for 1 hour, 1 mM urea is introduced to evaluate urease activity. Following a 15-minute reaction, the solution is centrifuged, and the supernatant is collected for urease activity measurement using the urease activity assay kit.

For alkaline conditions, 10 mM urea is added to a 10 mM R10/D10 coacervate solution co-loaded with 50 nM urease and 50 nM GOx. After the urea reaction for 1 hour, 1 mM glucose is introduced to assess the relative activity of GOx. Similarly, the solution undergoes a 15-minute reaction, centrifugation, and collection of the supernatant, followed by GOx activity measurement using the glucose oxidase activity assay kit.

Regulating PCR efficiency in coacervate protocells
GOx- or urease-loaded R10/D10 coacervate protocells are prepared as previously described. The resulting coacervate solutions are then mixed with a commercial PCR reagent solution at a 10:1 volume ratio. After reacting with glucose or urea at concentrations of 0, 2.5, or 5 mM, the PCR process is performed using a PCR machine (Arhat 96-Deep Well MP60401). The specific reaction parameters are set as per the manufacturer’s instructions. After the reaction, the products are collected and treated with a 1 M NaCl solution with a volume equal to that of the mixture for further study.

For quantitative analysis through nucleic acid gel electrophoresis, 15 μL of the product solution is mixed with 4 μL of loading dye. The samples are loaded onto a gel and subjected to electrophoresis for approximately 15 minutes. Finally, the results are analyzed using a gel imaging system. For quantitative analysis through UV-vis spectroscopy, this solution is added to a 96-well plate. Then, the fluorescence intensity of the solution is quantitatively analyzed using a microplate reader.

Regulating IVTT efficiency in coacervate protocells
Cell-free transcription and translation reaction kits are mixed with 50 nM GOx- or urease-loaded R10/D10 coacervate protocells (10 mM) at a 10:1 volume ratio, followed by incubation with 5 mM urea, water, or 5 mM glucose. After 1 hour of reaction with glucose or urea, the coacervate solution is centrifuged at 2.4g for 20 minutes to separate the condensate phase. The condensate is then injected into a microfluidic device, as previously described, to generate uniform coacervate microdroplets. These uniform coacervate droplets are subsequently incubated at 37°C for 30 minutes to initiate transcription and translation reactions. Green fluorescence signals (indicating RNA synthesis) and red fluorescence signals (indicating protein synthesis) are observed in the uniform coacervate microdroplets. The gray values of the droplets are analyzed and recorded.

Statistics and reproducibility
All confocal and other microscopy images represent experiments independently repeated at least 3 times, yielding similar result. For all graphs and statistical analyses, the number of independent replicates (n) is provided. Statistical tests are specified in the legends or on the figure panels, and exact P values are provided whenever possible.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al. developed a strategy to modulate the local pH of peptide-based coacervates by incorporating different enzymatic reactions. Using this system, the authors claim they can manipulate the pH within coacervates to enable reactions such as PCR and in vitro transcription and translation (IVTT). Currently, the experimental evidence does not completely support the conclusions. Many additional major/minor experiments and clarifications are needed before considering the potential of this manuscript for publication.\n1) Line 67: The sentence “coacervate-based protocells are highly potential in revealing basic life mysteries” should be rephrased. Consider more accurate terminology.\n2) Figure S1: Why did the authors calibrate the intracellular pH for the standard curve of SNARF-4F? Why not simply use in vitro calibration with buffer solutions at defined pH values? Please justify.\n3) Line 122-128: The authors claim that CX-5461 disrupts nucleolar structure and also that deacidification of the nucleolus in its presence is due to transcriptional inhibition. Could the structural disruption also (or possibly alone) contribute to the observed pH changes? Please clarify whether the deacidification is exclusively attributed to transcriptional inhibition or if structural effects also play a role.\n4) Figure S2: The differences between the pH with and without the treatment are negligible. Are they significant? Consider adding error bars and statistics.\n5) Line 143: “in which coacervate droplets can be facilely generated, albeit with polydispersity” What does “facilely” mean?\n6) Figure 3C: Consider including representative bright-field microscopy images of the coacervates formed at different pHs. This can help to confirm the turbidity data, and check the shape and size of the coacervates. It is important to rule out the possibility that turbidity arises from non-liquid aggregates.\n7) Line 179-180: “The pH of the condensate and dilute phase of freshly prepared coacervates is slightly different, at 8.01 and 7.98, respectively.” Is this pH-difference significant? What is the variability across independent replicates, including biological replicates?\n8) Figure S5: There is a typo with the concentration of HCl and NaOH. It should be “µM” instead of “mM”.\n9) Figure 3G: It is surprising that only 50 µM NaCl disrupts electrostatic interactions in a 10 mM peptide solution. This concentration of NaCl is relatively low. Please comment on this observation and reconsider the interpretation.\n10) Figure 3L: The concentration of NaCl where coacervation occurs (from 0 to 260 mM) overlaps with the one used to dissolve the coacervates in Figure 3G (50 µM). Can you explain the discrepancy between these two results?\n11) Major point: what percentage of the total enzyme concentration is inside the coacervates? And what is the volume fraction of these coacervates? These values are critical, as the majority of enzymatic activity may be occurring in the dilute phase rather than inside the coacervates. This clarification is key for interpreting the conclusions.\n12) Moreover, the volume fraction of the R10/D10 coacervates would give an indication on the accuracy of the micro pH probe method in distinguishing the two phases. An image of the centrifuged sample would also provide additional validity to the method. Based on Figure 3d there is no difference between the dense and dilute phase as no statistics are provided. It is recommend to use the SNARF-4F dye previously employed to cross-validate the results from Figure 3d, e and f.\n13) It is suggested that the authors repeat the experiments of Figure 3 where they note a difference in the dense and dilute phases in a system which is actually buffered (as per the Method section coacervates are prepared in water without an additional buffering agent). Without a buffering agent, the observations of basification or acidification of the dense phase are merely a partitioning of charged guest species as opposed to a true pH modulatory effect. Indeed the comparison made between free enzymes and enzymes+coacervates the enzyme only solution is buffered in PBS. It is unclear if the coacervate containing samples are also in PBS. If not, this needs to be performed to make the Michaelis Menten Kinetics truly comparable.\n14) Can the strength of electrostatic interactions (KD in the nM range) between enzymes and R10, decrease the phase separation propensity of R10/D10 peptides? This can be analysed by comparing the volume fraction or turbidity of the coacervates in the presence and absence of the enzymes.\n15) How did the authors measure the reaction rates of GOx and urease within the coacervates? According to the methods, the activity was not measured over time, just at one point of the reaction (activity in the supernatant after removing the coacervates). Reaction rates in µM/min are typically obtained from the linear section of the curves (substrate consumption or product formation over time).\n16) Regarding the Vmax and KM values reported, please explain the observed changes in these parameters in the presence of coacervates. Why does vmax of both enzymatic reactions decrease in the presence of condensates, while Km increases in the presence of condensates? This should be indicated in the main text. Also, include standard deviations for these values.\n17) Since gluconic acid and ammonia (reaction products) are commercially available, what is the advantage of using enzymatic reactions to modulate the pH of coacervates, instead of adding these compounds directly? Please justify the use of this strategy over simpler alternatives.\n18) Figure 6: Is the formation of coacervates compatible with the buffer composition of the qPCR kit? Please, show microscopy images of coacervates in presence of this kit. How the components of this kit influence the pH inside the coacervates?\n19) Figure 6A: Replace “activited” and “inactivited” by “activated” and “inactivated”.\n20) Line 432: Replace “urease” by “urea”.\n21) Please perform a control experiment to assess the activity of the PCR kit in the presence of urea and glucose alone (without coacervates or enzymes). This will help discard any direct effects of these metabolites on PCR efficiency.\n22) Figure 6F-H: Please quantify how much of the reaction is really happening within the coacervates. It can be possible that the reaction is mainly happening in the dilute phase, with the products partitioning into the coacervates.\n23) For both enzymatic systems (PCR and IVTT), please measure the pH inside the coacervates after the addition of different concentrations of glucose and urea.\n24) Figure 1 shows various in vivo and in vitro processes related to a pH shift in coacervates or MLOs. These are however not specifically discussed in the introduction or elsewhere in the text. Could the authors please address each of the three panels?\n25) Please indicate what Ka, RB and RA are referring to in Equation 1.\n26) The information provided in lines 367 – 373 about the influence of substrate concentration, salt and scaffold material on the pH gradient is not commented or explained. Can the authors please elaborate on these results and their implications.\n27) The discussion of the data in Figure 6b in the main text comes after the discussion of 6d. The order of Figure panels should be congruent with the appearance in the text.\n28) The authors indicate the optimal activity of GOx to be at pH 5.5, of Urase to be at pH 7.4 and of PCR reactions pH 7.0. As they do not test this themselves, a reference should be given for each for this information.", "reviewer_2": "In this current study, Wang et al studies the differential pH between the dense and dilute phases of complex coacervates across the condensate interface. They first show (confirm) previously reported results that such a difference in pH exists between nucleoplasm and nucleolus in live cells. Then they use a simple model system, complex coacervates formed by R10+D10, and study the differential pH across the interface and how that changes by the addition of salt and H+, and OH- ions. Although the results are interesting, by no means, these coacervates formed by polyelectrolyte mixtures close to the cellular structures. Very interestingly, from their cellular experiments, they observed that inhibition of rRNA production by CX5461 disrupts the pH difference between the nucleolus and nucleoplasm, hinting at the role of active processes in creating such a proton gradient. Such processes are absent in R10+D10 coacervates. Finally, the authors employ a few common enzymes, such as Gox and urease, that can produce H+ or OH- ions as byproducts in these coacervate droplets. They also attempted PCR reaction and IVTT in these droplets.\nThe reported studies are highly qualitative in nature. There are at least a few recent reports(Ausserwöger et al. 2024; King et al. 2024; Dai et al. 2024) that established that the concentration of H+ ions and other types of ions across the condensate interface can be unequal, leading to an apparent difference in pH between the dense and dilute phases. However, it is important to note that there is no “gradient” per se, since that would mean that the system is out of equilibrium. The concentrations of the ions and/or protons are different across the condensate interface, similar to the concentration of macromolecules that make up the condensate. This is also supported by the data showing CX5461 disrupts the pH difference between the nucleolus and nucleoplasm.\nThe model system (R10+D10) is interesting, but the characterization of such systems by the addition of salt and pH change is not new. It is conceivable that as the system moves closer to the single phase regime by the addition of salt, the differences in the H+ or OH- concentrations decrease between the dense and dilute phase since the density of the macromolecules that make up the dense phase decreases as well. It will be good to establish quantitative phase diagrams and how they relate to the differences in the pH between the dense and dilute phases.\nThe addition of enzymes to these condensates is also interesting, but similar studies have been done at a much greater depth(Jambon-Puillet et al. 2024; Testa et al. 2021).\nFinally, it will be great if the authors can focus on understanding the enzyme kinetics in condensates and how they are impacted via differences in the intra-condensate pH. Such active systems may show emergent properties due to phase separation-driven changes in the bulk pH, but to decipher that, more rigorous analysis is needed.\nMinor comments: the MD simulations and the use of microfluidics to generate coacervates are not really needed for this study, are they? Some claims are not supported by the data, such as “bioreactors,” “protocells,” etc.; such terms should not be used loosely. Finally, schematics like Fig. 1 are not helpful to occupy one figure in an article; rather, they may be better reserved for SI. Describing how the enzyme reaction rates were measured at the section where the results were described would be useful for the readers.\nReferences:\nAusserwöger, Hannes, Rob Scrutton, Charlotte M. Fischer, Tomas Sneideris, Daoyuan Qian, Ella de Csilléry, Ieva Baronaite, et al. 2024. “Biomolecular Condensates Sustain PH Gradients at Equilibrium through Charge Neutralisation.” BioRxiv. https://doi.org/10.1101/2024.05.23.595321.\nDai, Yifan, Zhengqing Zhou, Wen Yu, Yuefeng Ma, Kyeri Kim, Nelson Rivera, Javid Mohammed, et al. 2024. “Biomolecular Condensates Regulate Cellular Electrochemical Equilibria.” Cell 187 (21): 5951-5966.e18.\nJambon-Puillet, Etienne, Andrea Testa, Charlotta Lorenz, Robert W. Style, Aleksander A. Rebane, and Eric R. Dufresne. 2024. “Phase-Separated Droplets Swim to Their Dissolution.” Nature Communications 15 (1): 3919.\nKing, Matthew R., Kiersten M. Ruff, Andrew Z. Lin, Avnika Pant, Mina Farag, Jared M. Lalmansingh, Tingting Wu, et al. 2024. “Macromolecular Condensation Organizes Nucleolar Sub-Phases to Set up a PH Gradient.” Cell 187 (8): 1889-1906.e24.\nTesta, Andrea, Mirco Dindo, Aleksander A. Rebane, Babak Nasouri, Robert W. Style, Ramin Golestanian, Eric R. Dufresne, and Paola Laurino. 2021. “Sustained Enzymatic Activity and Flow in Crowded Protein Droplets.” Nature Communications 12 (1): 6293.", "reviewer_3": "The manuscript \"Coacervate Droplets as pH-Regionalized Protocells\" aims to explore the potential mechanisms that govern pH regulation in membrane-less organelles (MLOs). To this end, the authors first investigate differences in the condensed phase nucleolus and nucleoplasm pH using a fluorescent indicator. The nucleoli report lower pH compared to the nucleoplasm, with a less pronounced difference when RNA Pol I activity is inhibited or complete loss of a pH gradient when they are disassembled upon treatment with actinomycin D. To capture a similar LLPS dependent pH gradient in vitro, the investigators design a peptide based complex coacervate formed by electrostatic complexation of decapeptides R10 and D10. LLPS in this system is investigated as a function of pH, peptide concentration and presence of salt with this system behaving in line with previously reported coacervates of similar nature. A small intrinsic pH gradient is noted with the condensate phase being slightly more acidic, and coarse-grained molecular dynamics simulations are used to explain the preferential partitioning of the hydronium (H 0+) ions inside the peptide-rich droplets. Subsequently, enzymes are encapsulated in these coacervates to build minimalistic protocell bioreactors. The enzymatic reactions of GOx and Urease release products that influence the pH and therefore the substrate concentration serves as a handle to regulate enzyme kinetics.\nThe authors argue that this work provides potential insights into the mechanism for pH regulation in MLOs, but this is not entirely evident as no similar examples are provided for sequestration of pH regulating enzymes in biological MLOs. Nonetheless, the work provides an interesting demonstration of pH gradients in minimalistic peptide condensates with the ability to regulate enzyme activity by tuning substrate concentration of the orthogonal pH reaction. The following concerns should be addressed and clarifications provided prior to being considered for publication -\n1) In Figure 3 j-k and m-n, the RDFs indicate that with increasing peptide concentration or at lower salt-concentration, a higher number of hydronium ion beads are recruited to the peptide-condensates. Similar RDFs for hydroxide (OH-) ions are substantially lower (Figure S10) at g(r) ~ 7 vs. ~27 for H30+. The number of contacts measured from the simulations between the peptides and ions does not appear to reflect this trend however with both H+ and OH- showing ~ 350 contacts within 0.6 nm with the peptides. This would require some clarification in the manuscript to explain the pH difference and preferential partitioning of H+ into the condensates.\n2) Figure S5 (a-d): The caption reads 1 μM HCl/NaOH however the figure panels say 1 mM and should be corrected to be consistent.\n3) Using the CG-MD simulations, the authors argue that the diffusion coefficients of the H+/OH- ions are reduced by interaction with the coacervate network. In Figure S11, the H+ diffusion as quantified by the MSD always appears lower than the OH- diffusion even at low coacervate concentration. H+ is known to have higher diffusion compared to OH- due to the Grotthus effect and other mechanisms. Due the authors attribute this lower diffusion to preferential incorporation of the H+ beads in the peptide aggregates? A control simulation in the absence of peptides but presence of H+/OH- ions in similar box volumes would be required to validate the MARTINI models' ability to capture the intrinsic differences in diffusivity of these ions.\n4) The partitioning of enzymes was demonstrated to show electrostatic selectivity, with no significant binding and recruitment noted between the D10 peptides and the FITC-GOx or other negatively charged systems (dsDNA/PAA). Although the corresponding MD simulations for the favorable interaction are presented (GOx/Urease-R10), the manuscript/supplementary information would benefit from a similar simulation with D10 indicating exclusion/poor interaction. Furthermore, this would also rule out peptide-enzyme interactions as arising from excluded volume effects.\n5) The Michaelis-Menten plots in Figure 5 b-c show slower reaction rates inside the coacervates than in PBS. Condensates that strongly partition the substrates and enzymes typically improve such reaction rates but the opposite effect can be noted here. Is this due to lower diffusion inside the condensates or do some of the enzymes lose activity upon being bound by peptides?\n6) The introduction and conclusions would generally benefit from a rewrite that emphasizes the pH differences, the ability to regulate enzymatic reactions and consequently control IVVT or cascade reactions rather than the direct connection to pH regulation in biological MLOs. The mechanisms of this are not entirely revealed in this manuscript, as the fundamental reason of H+ enrichment in coacervates is not completely deciphered or explained but rather simply noted, observed and exploited."}, "author_response": "Response to reviewers\nDear Editor:\nWe hereby submit the revised manuscript (NCOMMS-25-41534-T), entitled\n“Coacervate Droplets as pH-Regionalized Protocells” by Chong Wang, Zhonglin Fang,\nLinyi Zhang, Yuanjin Zhao, and Luoran Shang, for your consideration as a Research\nArticle in Nature Communications. We sincerely appreciate your careful reading of our\nmanuscript and the valuable suggestions provided by you and the reviewers. In response\nto the reviewers’ comments, we have made the following revisions:\nReviewer 1#\nComment: Wang et al. developed a strategy to modulate the local pH of peptide-based\ncoacervates by incorporating different enzymatic reactions. Using this system, the\nauthors claim they can manipulate the pH within coacervates to enable reactions such\nas PCR and in vitro transcription and translation (IVTT). Currently, the experimental\nevidence does not completely support the conclusions. Many additional major/minor\nexperiments and clarifications are needed before considering the potential of this\nmanuscript for publication.\nResponse: We appreciate the reviewer’s recognition of this work. Accordingly, we have\nmade these revisions as follows:\nComment 1: Line 67: The sentence “coacervate-based protocells are highly potential\nin revealing basic life mysteries” should be rephrased. Consider more accurate\nterminology.\nResponse: As suggested by the reviewer, this sentence has been rephrased using more\naccurate terminology in the revised manuscript (Revised Introduction, Paragraph 2).\n“Our findings suggest that coacervate-based protocells offer a valuable platform to\nexplore how physicochemical compartmentalization influences biochemical reactions.”\nComment 2: Figure S1: Why did the authors calibrate the intracellular pH for the\nstandard curve of SNARF-4F? Why not simply use in vitro calibration with buffer\nsolutions at defined pH values? Please justify.\nResponse: We appreciate the reviewer’s insightful question. The intracellular pH\ncalibration for SNARF-4F was performed according to the method reported in\nMolecular Cell (2023, “Nucleolus activity-dependent recruitment and biomolecular\ncondensation by pH sensing”) and the manufacturer’s instructions for the Intracellular\npH Calibration Buffer Kit (Thermo Fisher, P35379). The corresponding reference has\nbeen cited in the manuscript.\nComment 3: Line 122-128: The authors claim that CX-5461 disrupts nucleolar\nstructure and also that deacidification of the nucleolus in its presence is due to\ntranscriptional inhibition. Could the structural disruption also (or possibly alone)\ncontribute to the observed pH changes? Please clarify whether the deacidification is\nexclusively attributed to transcriptional inhibition or if structural effects also play a role.\nResponse: We appreciate the reviewer’s insightful comment and acknowledge that our\nprevious description was not sufficiently precise. CX-5461 is generally recognized as\nan inhibitor of RNA polymerase I; however, some studies have also reported that CX-\n5461 can disrupt the natural organization of the nucleolus (e.g., The chemotherapeutic\nagent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release\nto cause nucleolar disruption, DNA damage and cell inviability. NAR Cancer, 2020).\nSimilarly, high concentrations of actinomycin D (Act D) are known to induce nucleolar\ndisassembly, whereas low concentrations can also cause nucleolar deacidification (e.g.,\nNucleolus activity-dependent recruitment and biomolecular condensation by pH\nsensing. Molecular Cell, 2023). The corresponding reference has been cited in the\nmanuscript. To further support these observations, we have performed additional\nexperiments and included the relevant data in the revised manuscript (Revised in\nFigure 2, and Identification of the pH difference between nucleolus and\nnucleoplasm, Paragraph 4). Based on these results, we consider that transcriptional\ninhibition and structural disruption are not mutually exclusive processes but rather\ninterrelated phenomena.\na. Confocal fluorescence images of HELA cells stained with SNARF-4F after treatment\nwith different concentrations of Act D. b. Profile of the 580/640 nm emission signal for\nthe nucleus in HELA cells treated with different concentrations of Act D. The white\nlines in (a) denote the region where the data points are taken.\nComment 4: Figure S2: The differences between the pH with and without the treatment\nare negligible. Are they significant? Consider adding error bars and statistics.\nResponse: As the reviewer suggested, we have added error bars and statistical analysis\nin the revised manuscript (Revised in Figure S2).\npH differences between the nucleolus and nucleoplasm (the pH of the nucleolus minus\nthe pH of the nucleoplasm) in various cells under different treatments. Each dot\nrepresents the pH difference for an individual cell (n = 10). Data are presented as mean\n± standard deviation (SD). Statistical significance is determined using a Student’s t-test,\nwith asterisks indicating levels of significance: p < 0.05 (*), p < 0.01 (**), and p < 0.001\n(***).\nComment 5: Line 143: “in which coacervate droplets can be facilely generated, albeit\nwith polydispersity” What does “facilely” mean?\nResponse: We sincerely thank the reviewer for pointing this out. In this context, it\nindicates that coacervate droplets can be conveniently generated by directly mixing the\ncomponents, although the resulting droplets are polydisperse. To improve clarity, we\nhave revised the sentence in the revised manuscript. (Revised in Peptide coacervate\ndroplets recapitulating the pH difference in MLOs, Paragraph 1).\n“A state diagram is plotted to indicate the optimal concentration ranges of each\ncomponent, within which coacervate droplets can be readily formed, albeit with\npolydispersity (Figures 3 and S3).”\nComment 6: Figure 3C: Consider including representative bright-field microscopy\nimages of the coacervates formed at different pHs. This can help to confirm the turbidity\ndata, and check the shape and size of the coacervates. It is important to rule out the\npossibility that turbidity arises from non-liquid aggregates.\nResponse: We thank the reviewer for the valuable suggestion. In response, we have\nadded representative bright-field microscopy images of the coacervates formed at\ndifferent pH values in the Supporting Information to complement the turbidity data and\nconfirm the shape and size of the coacervates (Revised in Figure S4).\nRepresentative bright-field microscopy images of R10/D10 coacervates formed at\ndifferent pHs.\nComment 7: Line 179-180: “The pH of the condensate and dilute phase of freshly\nprepared coacervates is slightly different, at 8.01 and 7.98, respectively.” Is this pH-\ndifference significant? What is the variability across independent replicates, including\nbiological replicates?\nResponse: We appreciate the reviewer’s comment. The observed pH difference\nbetween the condensate and dilute phases of freshly prepared coacervates is very small.\nWe have assessed the variability across independent replicates and performed statistical\nanalysis. The results indicate that this difference is not statistically significant (p>0.05).\nThese details, including replicate numbers and statistical results, have been added to\nthe revised manuscript (Revised in Figure 3).\nThe pH changes in the condensate phase and dilute phase upon the addition of different\namounts of H+ or OH-. n=10. Data are presented as mean ± SD. Statistical significance\nis determined using a Student’s t-test.\nIn addition, we have modified the relevant descriptions in the manuscript\naccordingly (Revised in Peptide coacervate droplets recapitulating the pH\ndifference in MLOs, Paragraph 2).\n“The condensate and dilute phases of freshly prepared coacervates exhibited\ncomparable pH values of approximately 8.0 (as shown in Figure 3d, p>0.05).”\nComment 8: Figure S5: There is a typo with the concentration of HCl and NaOH. It\nshould be “µM” instead of “mM”.\nResponse: We sincerely thank the reviewer for pointing this out. The typo has been\ncorrected in the revised manuscript (Revised in Figure S8).\n(a,b) pH values of the condensate and dilute phases of R10/D10 coacervate at different\ncoacervate concentrations after adding 1 μM HCl (a) and NaOH (b). (c,d) pH values of\nthe condensate phase and dilute phases at different salt concentrations are measured\nfollowing the addition of 1 μM HCl (c) and NaOH (d). n=30.\nComment 9: Figure 3G: It is surprising that only 50 µM NaCl disrupts electrostatic\ninteractions in a 10 mM peptide solution. This concentration of NaCl is relatively low.\nPlease comment on this observation and reconsider the interpretation.\nResponse: We thank the reviewer for pointing this out. The concentration of NaCl\nshould indeed be expressed in “mM”. We apologize for the oversight in the original\nmanuscript. This has been corrected in Figure 3i in the revised manuscript. In addition,\nwe have carefully checked the entire manuscript and made the corresponding\ncorrections in all relevant sections.\npH difference between the condensate phase and the dilute phase of coacervate after\nmixing 2 μmol H+ or OH- with 2 mL R10/D10 coacervate solution, at different salt\nconcentrations. n=9.\nComment 10: Figure 3L: The concentration of NaCl where coacervation occurs (from\n0 to 260 mM) overlaps with the one used to dissolve the coacervates in Figure 3G (50\nµM). Can you explain the discrepancy between these two results?\nResponse: We thank the reviewer for this insightful question. In Figure 3h, at a NaCl\nconcentration around 50 mM, the turbidity of the coacervate is approximately 20%,\nindicating that coacervates are not completely dissolved at this salt concentration.\nTherefore, the observed overlap with the concentration range in Figure 3j reflects partial\ndisruption rather than complete dissolution.\nComment 11: Major point: what percentage of the total enzyme concentration is inside\nthe coacervates? And what is the volume fraction of these coacervates? These values\nare critical, as the majority of enzymatic activity may be occurring in the dilute\nphase rather than inside the coacervates. This clarification is key for interpreting the\nconclusions.\nResponse: We appreciate the reviewer’s suggestion and have supplemented the\nmanuscript with measurements of the enzyme partition coefficient (Revised in Figure\nS21) and the volume fraction of the coacervates (Revised in Figure S5) to provide a\nclearer understanding of the proportion of the total enzyme concentration contained\nwithin the coacervates and to better interpret the distribution of enzymatic activity.\na. Concentration of FITC-GOx in the condensate and dilute phases, representing its\npartition coefficient between the two phases. n=5. b. Concentration of RBITC-urease\nin the condensate and dilute phases, representing its partition coefficient between the\ntwo phases. n=5. Data are presented as mean ± SD.\nThe centrifuged R10/D10 coacervate solution.\nComment 12: Moreover, the volume fraction of the R10/D10 coacervates would give\nan indication on the accuracy of the micro pH probe method in distinguishing the two\nphases. An image of the centrifuged sample would also provide additional validity to\nthe method. Based on Figure 3d there is no difference between the dense and dilute\nphase as no statistics are provided. It is recommend to use the SNARF-4F dye\npreviously employed to cross-validate the results from Figure 3d, e and f.\nResponse: We thank the reviewer for these valuable suggestions. We have added an\nimage of the centrifuged coacervate solution, along with schematic illustrations and\nrepresentative images showing the use of the micro pH probe for measurements\n(Revised in Figure S5). An Orion™ PerpHecT™ ROSS™ Micro pH electrode was\nused for pH measurements. The electrode requires a minimum sample volume of 15 µL.\nNotably, the volume of the centrifuged condensate phase exceeded this requirement,\nensuring reliable and accurate pH measurements. A comparable measurement method\nwas reported in Physicochemical Characterization of Polymer-Stabilized Coacervate\nProtocells (ChemBioChem, 2019), which further validates the reliability of our pH\ndetermination approach.\na. A schematic illustration showing the measurement of the pH of the R10/D10\ncondensate phase using a micro-pH probe. b. The centrifuged R10/D10 coacervate\nsolution. c. Measurement of the pH of the condensate phase using a micro-pH meter.\nIn addition, Figure 3d has been redrawn to include the statistical analysis of the pH\ndifferences between the dense and dilute phases (Revised in Figure 3).\nThe pH changes in the condensate phase and dilute phase upon the addition of different\namounts of H+ or OH-. n=10. Data are presented as mean ± SD. Statistical significance\nis determined using a Student’s t-test.\nMoreover, as the reviewer suggested, we have also used the SNARF-4F dye to\ncross-validate these results in Figure 3 (Revised in Figure S7).\npH changes in the condensate and dilute phases upon the addition of different amounts\nof H⁺ or OH⁻, measured using SNARF-4F. n=50. Data are presented as mean ± SD.\nStatistical significance is determined using a Student’s t-test.\nThe results indicate that the addition of exogenous H⁺ or OH⁻ leads to a\ncorresponding decrease or increase in the pH of the condensate phase. However, the pH\nvalues determined by SNARF staining differ noticeably from those measured directly\nusing a micro-pH meter. We attribute this discrepancy to two main factors. First, the\nelevated local ion concentrations within the condensate phase may interfere with the\naccuracy of SNARF-based pH measurements, consistent with observations reported in\nrecent studies (Catalytic peptide-based coacervates for enhanced function through\nstructural organization and substrate specificity. Nature Communications, 2023; Effect\nof Polypeptide Complex Coacervate Microenvironment on Protonation of a Guest\nMolecule. J. Phys. Chem. B, 2023). Second, fluorescence-based detection methods\ninherently introduce a certain degree of measurement error. Considering that the micro-\npH electrode provides a more reliable measurement under these conditions, we believe\nthat the pH values obtained using the micro-pH meter better reflect the actual pH of the\ncondensate phase.\nComment 13: It is suggested that the authors repeat the experiments of Figure 3 where\nthey note a difference in the dense and dilute phases in a system which is actually\nbuffered (as per the Method section coacervates are prepared in water without an\nadditional buffering agent). Without a buffering agent, the observations of basification\nor acidification of the dense phase are merely a partitioning of charged guest species as\nopposed to a true pH modulatory effect. Indeed the comparison made between free\nenzymes and enzymes+coacervates the enzyme only solution is buffered in PBS. It is\nunclear if the coacervate containing samples are also in PBS. If not, this needs to be\nperformed to make the Michaelis Menten Kinetics truly comparable.\nResponse: As suggested by the reviewer, we have repeated the relevant experiments\nusing coacervates prepared in Tris-HCl buffer (pH 8.0) (Revised in Figure S9).\n000\na. The pH changes in the condensate phase and dilute phase upon the addition of\ndifferent amounts of H+ or OH-. n=10. Data are presented as mean ± SD. Statistical\nsignificance is determined using a Student’s t-test. b. The turbidity of the coacervate\nsystem prepared by mixing 1, 2, 4, 6, 8, and 10 mM R10 and D10 solutions (1:1). n=3.\nc. pH difference between the condensate phase and the dilute phase of coacervate upon\nmixing 2 μmol H+ or OH- with 2 mL R10/D10 coacervate solution, at various\ncoacervate concentrations. n=30. d. The turbidity of the R10/D10 coacervate solution\nat various salt concentrations. n=3. e. pH difference between the condensate phase and\nthe dilute phase of coacervate after mixing 2 μmol H+ or OH- with 2 mL R10/D10\ncoacervate solution, at different salt concentrations. n=30.\nTo ensure that the reaction conditions were consistent, the coacervates were\nprepared in water adjusted to pH 8.0, and the corresponding enzyme-only reactions\nwere also conducted in the same pH 8.0 solution (Revised in Figure 5).\na. Michaelis−Menten plots showing GOx activity in bulk solution with different\nconcentrations of H+/OH-. n=5. b. Michaelis−Menten plots showing GOx activity in\nR10/D10 coacervates with different concentrations of H+/OH-. n=5. c.\nMichaelis−Menten plots showing urease activity in bulk solution with different\nconcentrations of H+/OH-. n=5. d. Michaelis−Menten plots showing urease activity in\nR10/D10 coacervates with different concentrations of H+/OH-. n=5.\nComment 14: Can the strength of electrostatic interactions (KD in the nM range)\nbetween enzymes and R10, decrease the phase separation propensity of R10/D10\npeptides? This can be analysed by comparing the volume fraction or turbidity of the\ncoacervates in the presence and absence of the enzymes.\nResponse: According to the reviewer’s suggestion, we have added turbidity\nmeasurements of the coacervate solutions after mixing them with varying\nconcentrations of enzymes in the revised manuscript (Revised in Figure S20).\na. Turbidity measurements of 10 mM R10/D10 coacervate solutions after mixing with\nvarious concentrations of GOx. n=3. b. Turbidity measurements of 10 mM R10/D10\ncoacervate solutions after mixing with various concentrations of urease. n=3.\nComment 15: How did the authors measure the reaction rates of GOx and urease within\nthe coacervates? According to the methods, the activity was not measured over time,\njust at one point of the reaction (activity in the supernatant after removing the\ncoacervates). Reaction rates in µM/min are typically obtained from the linear section\nof the curves (substrate consumption or product formation over time).\nResponse: We thank the reviewer for this valuable comment. In this study, the\nenzymatic activities of GOx and urease inside and outside the coacervates were\nevaluated by quantifying the reaction products at multiple fixed time points rather than\nby continuous monitoring. The time-dependent product formation profiles under\ndifferent conditions are provided in the Supporting Information (Figure S23-S26), and\nthe reaction rates were determined from the linear regions of these curves.\nTime-dependent product formation curves of GOx in solution catalyzing glucose at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of GOx in coacervate catalyzing glucose at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of urease in solution catalyzing urea at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of urease in coacervate catalyzing urea at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nWe have also clarified this measurement process in the revised manuscript\n(Revised in the Detection of enzymatic activities in coacervates).\n“To assess the catalytic activity of GOx within coacervates, a 50 nM GOx-loaded\nR10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM\nGOx were each mixed with glucose solutions at various concentrations (0, 1, 2, 3, 4, 5,\n10, 15, and 20 mM). After different reaction times (5 min, 10 min, 15 min, 30 min, 45\nmin, and 60 min), to minimize turbidity interference from the protocell-containing\nsamples, the mixtures were centrifuged and the supernatants were collected for GOx\nactivity measurement using a Glucose Oxidase Activity Assay Kit. The initial reaction\nrate at each substrate concentration was determined by analyzing the linear region of\nthe product formation curve over a 60-minute period (Figure S23 and Figure S24).\nSimilarly, to evaluate urease activity in coacervates, a 50 nM urease-loaded\nR10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM\nurease are mixed with urea solutions at concentrations of 0, 1, 2, 3, 4, 5, 10, 15, and 20\nmM. After different reaction times, to minimize turbidity interference from the\nprotocell-containing samples, the mixtures were centrifuged and the supernatants\ncollected for urease activity measurement using a Urease Activity Assay Kit. The initial\nreaction rate at each substrate concentration was determined by analyzing the linear\nregion of the product formation curve over a 60-minute period (Figure S25 and Figure\nS26).”\nComment 16: Regarding the V and K values reported, please explain the observed\nmax M\nchanges in these parameters in the presence of coacervates. Why does V of both\nmax\nenzymatic reactions decrease in the presence of condensates, while K decreases in the\nm\npresence of condensates? This should be indicated in the main text. Also, include\nstandard deviations for these values.\nResponse: We thank the reviewer for this important comment. The observed decrease\nin V in the presence of coacervates may result from restricted enzyme mobility or\nmax\nsubstrate accessibility within the dense coacervate phase, which could limit the\nmaximum catalytic turnover. The decrease in K indicates an enhanced apparent\nm\naffinity for the substrate, which may arise from the microenvironmental effects inside\nthe coacervates. A similar trend in the changes of enzyme V and K of within\nmax m\ncoacervates has also been observed by Jan C. M. van Hest and colleagues\n(Physicochemical Characterization of Polymer-Stabilized Coacervate Protocells.\nChemBioChem, 2019). As the reviewer suggested, we have added corresponding\nexplanations in the revised manuscript to clarify the observed changes in V and K\nmax m\nvalues in the presence of coacervates (Revised in Construction of pH-regionalized\nprotocell protocells, Paragraph 5).\n“We measured the reaction rates of GOx and urease in both coacervate and bulk\nsolutions. The reaction rate of GOx as a function of glucose concentration follows\ntypical Michaelis–Menten kinetics in both bulk PBS solution and R10/D10 coacervate\nsolutions (Figures 5b, 5c, S23, and S24). Similarly, the reaction rate of urease as a\nfunction of urea concentration also follows typical Michaelis–Menten kinetics in both\nbulk solution and R10/D10 coacervate solutions (Figures 5d, 5e, S25, and S26). The\nV and K values, representing the maximum reaction rate and substrate binding\nmax m\naffinity, respectively, were obtained by fitting the Michaelis–Menten plots (Figure 5f\nand Figure S27). In the absence of additional H⁺ or OH⁻ ions, the lower Vₘₐₓ values\nobserved for both enzymes in coacervates, compared with those in solution, could\npossibly arise from restricted enzyme mobility within the dense coacervate matrix,\nwhich might limit catalytic turnover. The decrease in K indicates an enhanced apparent\nm\naffinity for the substrate, which may arise from the microenvironmental effects inside\nthe coacervates.”\nAdditionally, we have included supplementary figures presenting the standard\ndeviations of these parameters to provide a clearer representation of the variability in\nthe kinetic data (Revised in Figure 5 and Figure S27).\na. V of GOx and urease in solution and within R10/D10 coacervates upon the\nmax\naddition of different concentrations of H+ or OH-. n=5. b. K of GOx and urease in\nm\nsolution and within R10/D10 coacervates upon the addition of different concentrations\nof H+ or OH-. n=5. Data are presented as mean ± SD.\nComment 17: Since gluconic acid and ammonia (reaction products) are commercially\navailable, what is the advantage of using enzymatic reactions to modulate the pH of\ncoacervates, instead of adding these compounds directly? Please justify the use of this\nstrategy over simpler alternatives.\nResponse: We appreciate the reviewer’s insightful question. From the perspective of\nprotocell construction and biomimetic design, our aim was not merely to modulate pH,\nbut to recreate a biologically relevant mechanism by which enzymatic reactions\ngenerate and regulate microenvironmental heterogeneity within membrane-less\ncompartments. In living cells, enzymatic reactions occurring inside organelles or\ncondensates may give rise to localized pH gradients, which could in turn influence\nbiochemical reactions, molecular assembly, and functional compartmentalization. By\nemploying enzyme-catalyzed reactions within coacervates, we sought to mimic this\nendogenous regulation, enabling the coacervate protocells to autonomously modulate\ntheir internal pH in response to metabolic-like processes. Direct addition of gluconic\nacid or ammonia could, of course, induce a rapid and homogeneous pH shift; however,\nthis approach fails to capture the spatiotemporal precision and dynamic cross-regulation\ncharacteristic of enzymatic reactions in living systems. Therefore, using enzymes\nprovides a more physiologically relevant and dynamic model for studying how\nbiochemical reactions within condensates influence their physicochemical state and\npotentially affect other coupled reactions.\nComment 18: Figure 6: Is the formation of coacervates compatible with the buffer\ncomposition of the qPCR kit? Please, show microscopy images of coacervates in\npresence of this kit. How the components of this kit influence the pH inside the\ncoacervates?\nResponse: As suggested by the reviewer, we have added microscopy images illustrating\nthe morphology of coacervates after mixing with different components in the revised\nmanuscript (Revised in Figure 6).\nBright-field microscopy images demonstrating the stability of R10/D10 coacervates\nupon mixing with different reagents.\nFurthermore, we measured the internal pH of the coacervates in the presence of\nthese components to assess how they influence the internal microenvironment (Revised\nin Figure S30).\npH of the condensate phase of R10/D10 coacervates without enzymes after mixing with\ndifferent components. n=10. Data are presented as mean ± standard SD. Statistical\nsignificance is determined using a Student’s t-test.\nComment 19: Figure 6A: Replace “activited” and “inactivited” by “activated” and\n“inactivated”.\nResponse: We appreciate the reviewer for catching this typographical error. It has been\ncorrected in Figure 6a in the revised manuscript (Revised in Figure 6).\nSchematic illustrating the working mechanism of the coacervate-based protocells: by\nregulating the pH environment through enzymatic reactions, the downstream enzymatic\nreaction activity can be further controlled.\nComment 20: Line 432: Replace “urease” by “urea”.\nResponse: We thank the reviewer for pointing out this typographical error. It has been\ncorrected to “urea” in the revised manuscript. (Revised in Controlling cascade\nreactions in the coacervate protocells, Paragraph 3)\n“As shown in Figure 6e, PCR reactions are significantly inhibited when 2.5 or 5\nmM glucose and urea are added to the solution.”\nComment 21: Please perform a control experiment to assess the activity of the PCR kit\nin the presence of urea and glucose alone (without coacervates or enzymes). This will\nhelp discard any direct effects of these metabolites on PCR efficiency.\nResponse: As suggested by the reviewer, we conducted control experiments with urea\nand glucose alone, and the results have been added to the revised manuscript. (Revised\nin Figure 6)\nGel electrophoresis of PCR products from coacervate protocells at different glucose\nand urea concentrations. The “No enzyme” group represents reactions lacking both\nenzymes and coacervates.\nComment 22: Figure 6F-H: Please quantify how much of the reaction is really\nhappening within the coacervates. It can be possible that the reaction is mainly\nhappening in the dilute phase, with the products partitioning into the coacervates.\nResponse: To address the reviewer’s concern, we performed control experiments to\ndetermine the extent to which the reaction occurred within the coacervates. Specifically,\nafter thoroughly mixing the PCR kit with the coacervate solution, the mixture was\ncentrifuged to separate the condensate and dilute phases, which were then reacted\nseparately. After disrupting the coacervates, the fluorescence intensity of each phase\n(corresponding to the amounts of transcription and translation products) was measured\n(Revised in Figure 6). The results indicated that the vast majority of the reaction took\nplace within the coacervate phase.\nComparison of fluorescence intensity between the condensate and dilute phases after\nIVTT reactions, representing the relative transcription and translation rates in the two\nphases. n=5. Data are presented as mean ± SD.\nComment 23: For both enzymatic systems (PCR and IVTT), please measure the pH\ninside the coacervates after the addition of different concentrations of glucose and urea.\nResponse: As suggested by the reviewer, we have measured the pH inside the\ncoacervates after the addition of different concentrations of glucose and urea, and the\ncorresponding data have been added to the revised manuscript. (Revised in Figure S30)\npH of the condensate phase of R10/D10 coacervates without enzymes after mixing with\ndifferent components. n=10. Data are presented as mean ± standard SD. Statistical\nsignificance is determined using a Student’s t-test.\nComment 24: Figure 1 shows various in vivo and in vitro processes related to a pH\nshift in coacervates or MLOs. These are however not specifically discussed in the\nintroduction or elsewhere in the text. Could the authors please address each of the three\npanels?\nResponse: We thank the reviewer for the valuable suggestion. In response, we have\nrevised the Introduction section to specifically address and discuss the processes\nillustrated in each of the three panels in Figure 1, providing clearer context and more\ndirectly linking these illustrations to the objectives of our study. (Revised in\nIntroduction, Paragraph 2)\n“In this study, we found that a natural pH difference exists between the nucleolus\nand nucleoplasm in several tested cell lines, which can be modulated by drug treatments\nto alter their structure and properties (Figure 1a). Since coacervates are liquid-liquid\nphase separation (LLPS) systems driven by noncovalent interactions, and they share\nsimilar properties to MLOs such as molecular organization and compartmentalization,\nwe then present peptide-based coacervate droplets as protocells to mimic the pH\ndifference found in the nucleus. We use microfluidics to prepare these coacervate\ndroplets to ensure their uniformity in size, thus allowing for the generation of a similar\npH difference across the LLPS interface under precisely controlled conditions. Through\nmolecular dynamics simulations and quantitative experiments, we conclude that the\ndense phase of coacervates, rich in charged polymers, tends to attract a higher\nconcentration of H+/OH- ions than the dilute phase. This results in an uneven apparent\npH distribution between the interior and exterior of the coacervates. More importantly,\nsuch pH difference weakens or even disappears following destruction of LLPS, a\nphenomenon observed in both nucleolus and coacervate droplets. Based on these\nfindings, we demonstrate that the pH-regionalized environment of the coacervate\ndroplets can be customized by selectively accommodating enzymatic reactions (as\nshown in Figure 1b), and such feature can be further employed to control cascade\nreactions and perform polymerase chain reaction (PCR) and in vitro transcription and\ntranslation reaction (IVTT), thereby recapitulating various cellular activities (as shown\nin Figure 1c).”\nComment 25: Please indicate what Ka, RB and RA are referring to in Equation 1.\nResponse: We appreciate the reviewer’s comment on this point. In Equation 1, Kₐ\ndenotes the acid dissociation constant of the probe; R represents the fluorescence\nintensity ratio at two wavelengths, λ₁ (580 nm) and λ₂ (640 nm); and R and R\nA B\nrepresent the limiting values of R corresponding to the fully protonated (acidic) and\nfully deprotonated (basic) states, respectively. These definitions have been added to the\nrevised manuscript for clarity. (Revised in Identification of the pH difference\nbetween nucleolus and nucleoplasm, Paragraph 1)\n“The spectral shifts of SNARF-4F based on pH changes are described by Equation\n(1), where Kₐ denotes the acid dissociation constant of the probe, R represents the\nfluorescence intensity ratio at two wavelengths λ1 (580 nm) and λ2 (640 nm)\n(E 580/E 640), and R and R represent the limiting values of R corresponding to the\nm m A B\nfully protonated and fully deprotonated states, respectively.”\nComment 26: The information provided in lines 367 – 373 about the influence of\nsubstrate concentration, salt and scaffold material on the pH gradient is not commented\nor explained. Can the authors please elaborate on these results and their implications.\nResponse: We appreciate the reviewer’s comment and have incorporated corresponding\nexplanations in the manuscript. In response, we have added detailed explanations in the\nmanuscript regarding the influence of substrate concentration, salt, and scaffold\nmaterial on the pH gradient, and discussed the implications of these results accordingly.\n(Revised in Construction of pH-regionalized protocell protocells, Paragraph 7)\n“We observed that, after reacting with glucose or urea, the enzyme-containing\ncoacervates exhibited a pH difference between the condensate and the surrounding\ndilute phase, which is consistent with the results obtained from the direct addition of\nH+ or OH-. Additionally, the pH difference between the condensate and dilute phases of\nthe coacervate is also affected by the concentration of the coacervate and salt (Figures\n5k, 5l, and S28). Similar to the effect observed with direct H+ or OH- addition, when\nthe stability of the coacervate droplets is reduced by decreasing the scaffold molecular\nconcentration or increasing the salt concentration, the pH difference between the\ninside and outside of the coacervate droplets correspondingly decreases.”\nComment 27: The discussion of the data in Figure 6b in the main text comes after the\ndiscussion of 6d. The order of Figure panels should be congruent with the appearance\nin the text.\nResponse: We appreciate the reviewer’s suggestion and have revised Figure 6 so that\nthe panel order now corresponds to their discussion sequence in the main text. (Revised\nin Figure 6)\nFigure 6. R10/D10 coacervate protocells for cascade enzymatic reactions.\nComment 28: The authors indicate the optimal activity of GOx to be at pH 5.5, of\nUrase to be at pH 7.4 and of PCR reactions pH 7.0. As they do not test this themselves,\na reference should be given for each for this information.\nResponse: As the reviewer suggested, the optimal pH values of different enzymes and\nreactions have now been supported with appropriate references. Specifically, the\noptimal pH of GOx was referenced from Kinetics and Thermodynamic Properties of\nGlucose Oxidase Obtained from Aspergillus fumigatus ASF4, Tropical Journal of\nNatural Product Research, 2022; that of urease from Kinetic properties of Helicobacter\npylori urease compared with jack bean urease, FEMS Microbiology Letters, 1992; and\nthat of PCR reactions from Quantitative analysis of temperature, salinity and pH on\nWSSV proliferation in Chinese shrimp Fenneropenaeus chinensis by real-time PCR,\nAquaculture, 2011. These relevant references have been added in the revised\nmanuscript (Revised in Controlling cascade reactions in the coacervate protocells,\nParagraph 2, and Controlling cascade reactions in the coacervate protocells,\nParagraph 3).\nReviewer 2#\nComment: In this current study, Wang et al studies the differential pH between the\ndense and dilute phases of complex coacervates across the condensate interface. They\nfirst show (confirm) previously reported results that such a difference in pH exists\nbetween nucleoplasm and nucleolus in live cells. Then they use a simple model system,\ncomplex coacervates formed by R10+D10, and study the differential pH across the\ninterface and how that changes by the addition of salt and H+, and OH- ions. Although\nthe results are interesting, by no means, these coacervates formed by polyelectrolyte\nmixtures close to the cellular structures. Very interestingly, from their cellular\nexperiments, they observed that inhibition of rRNA production by CX5461 disrupts the\npH difference between the nucleolus and nucleoplasm, hinting at the role of active\nprocesses in creating such a proton gradient. Such processes are absent in R10+D10\ncoacervates. Finally, the authors employ a few common enzymes, such as Gox and\n and\nurease, that can produce H+ or OH- ions as byproducts in these coacervate droplets.\nThey also attempted PCR reaction and IVTT in these droplets.\nResponse: We appreciate the reviewer’s recognition of our work and their constructive\nsummary of the study. In response, we have carefully revised the manuscript to clarify\nthe scope and significance of our work.\nComment 1: The reported studies are highly qualitative in nature. There are at least a\nfew recent reports (Ausserwöger et al. 2024; King et al. 2024; Dai et al. 2024) that\nestablished that the concentration of H+ ions and other types of ions across the\ncondensate interface can be unequal, leading to an apparent difference in pH between\nthe dense and dilute phases. However, it is important to note that there is no “gradient”\nper se, since that would mean that the system is out of equilibrium. The concentrations\nof the ions and/or protons are different across the condensate interface, similar to the\nconcentration of macromolecules that make up the condensate. This is also supported\nby the data showing CX5461 disrupts the pH difference between the nucleolus and\nnucleoplasm.\nResponse: We appreciate the reviewer’s valuable suggestion and the helpful references.\nIn accordance with this clarification, the term “gradient” has been replaced with\n“difference” throughout the manuscript. We have also cited the relevant recent studies\nin the revised manuscript to support our findings.\nComment 2: The model system (R10+D10) is interesting, but the characterization of\nsuch systems by the addition of salt and pH change is not new. It is conceivable that as\nthe system moves closer to the single phase regime by the addition of salt, the\ndifferences in the H+ or OH- concentrations decrease between the dense and dilute phase\nsince the density of the macromolecules that make up the dense phase decreases as well.\nIt will be good to establish quantitative phase diagrams and how they relate to the\ndifferences in the pH between the dense and dilute phases.\nResponse: We sincerely appreciate the reviewer’s insightful comment. According to\nthe reviewer’s advice, we have supplemented our study with a state diagram to illustrate\nhow the phase behavior correlates with the pH differences between the dense and dilute\nphases. The corresponding results and discussion have been added in the revised\nmanuscript (Revised in Figure 3).\nState diagram showing the effects of salt and coacervate concentrations on the pH\ndifference between dense and dilute phases upon addition of 1 μM H⁺ or OH⁻.\nComment 3: The addition of enzymes to these condensates is also interesting, but\nsimilar studies have been done at a much greater depth (Jambon-Puillet et al. 2024;\nTesta et al. 2021).\nResponse: We appreciate the reviewer’s helpful suggestion. In this study, our main\nfocus was to utilize the catalytic reactions of GOx and urease to generate H⁺ and OH⁻\nions, thereby forming a pH difference across the coacervate interface and demonstrating\nits influence on other biochemical reactions. This approach aims to model the possible\nlocal pH regulation within membrane-less organelles in vitro. As the reviewer suggested,\nthe relevant references have been cited in the revised manuscript.\nComment 4: Finally, it will be great if the authors can focus on understanding the\nenzyme kinetics in condensates and how they are impacted via differences in the intra-\ncondensate pH. Such active systems may show emergent properties due to phase\nseparation-driven changes in the bulk pH, but to decipher that, more rigorous analysis\nis needed.\nResponse: We appreciate the reviewer’s valuable suggestion. Following the reviewer’s\nadvice, we have examined the enzyme kinetics of the catalytic reactions of GOx and\nurase both in bulk solution and within the coacervates under identical H+/OH⁻ ion\nconditions. These results have been included in the revised manuscript to further\nillustrate how the pH differences influence enzymatic activity. Our analysis indicates\nthat upon the addition of extra H⁺ ions, the Vₘₐₓ values of GOx and urease (with optimal\npH values of 5.5 and 7.4, respectively) in the condensate phase became more\ncomparable to those in solution, likely because the preferential partitioning of ions\ntoward the condensate phase resulted in a locally elevated concentration of H⁺. In\ncontrast, as OH⁻ ions were added, the difference in Vₘₐₓ between the coacervate and\nsolution phases increased accordingly. Taken together, these results demonstrate that\nthe altered local ion environment within the coacervates significantly influences the\ncatalytic behavior of the encapsulated enzymes.\nComment 5: The MD simulations and the use of microfluidics to generate coacervates\nare not really needed for this study, are they?\nResponse: We appreciate the reviewer’s comment. The molecular dynamics\nsimulations were included to provide a deeper understanding of the ion distribution\ninside and outside the coacervates. The microfluidic approach was employed to enable\nmore precise quantitative analysis, as coacervate droplets generated by microfluidics\nexhibit superior uniformity and controllable size. Compared with coacervates formed\nby simple mixing, this method allows for more consistent reaction conditions within\nthe droplets, thereby facilitating more reliable quantitative measurements.\nComment 6: Some claims are not supported by the data, such as “bioreactors,”\n“protocells,” etc.; such terms should not be used loosely.\nResponse: In this study, we define coacervates composed solely of R10/D10 peptides\nas “coacervate droplets” and those incorporating enzymes as “protocells.” In response\nto the reviewer’s suggestion, we have systematically reviewed the manuscript and\nrefined the terminology, replacing inconsistent or loosely used terms such as\n“bioreactors” with “protocells” where appropriate. This revision aims to ensure\nconceptual precision and improve clarity for the reader.\nComment 7: Finally, schematics like Fig. 1 are not helpful to occupy one figure in an\narticle; rather, they may be better reserved for SI.\nResponse: We appreciate the reviewer’s comment. In response, we have expanded the\nIntroduction to provide a more detailed explanation of Figure 1, emphasizing its role\nin illustrating the overall conceptual framework of the study. This integration enhances\nthe reader’s understanding of the rationale and design of our work, thereby justifying\nthe inclusion of Figure 1 in the main text. (Revised in Introduction, Paragraph 2)\n“In this study, we found that a natural pH difference exists between the nucleolus\nand nucleoplasm in several tested cell lines, which can be modulated by drug treatments\nto alter their structure and properties (Figure 1a). Since coacervates are liquid-liquid\nphase separation (LLPS) systems driven by noncovalent interactions, and they share\nsimilar properties to MLOs such as molecular organization and compartmentalization,\nwe then present peptide-based coacervate droplets as protocells to mimic the pH\ndifference found in the nucleus. We use microfluidics to prepare these coacervate\ndroplets to ensure their uniformity in size, thus allowing for the generation of a similar\npH difference across the LLPS interface under precisely controlled conditions. Through\nmolecular dynamics simulations and quantitative experiments, we conclude that the\ndense phase of coacervates, rich in charged polymers, tends to attract a higher\nconcentration of H+/OH- ions than the dilute phase. This results in an uneven apparent\npH distribution between the interior and exterior of the coacervates. More importantly,\nsuch pH difference weakens or even disappears following destruction of LLPS, a\nphenomenon observed in both nucleolus and coacervate droplets. Based on these\nfindings, we demonstrate that the pH-regionalized environment of the coacervate\ndroplets can be customized by selectively accommodating enzymatic reactions (as\nshown in Figure 1b), and such feature can be further employed to control cascade\nreactions and perform polymerase chain reaction (PCR) and in vitro transcription and\ntranslation reaction (IVTT), thereby recapitulating various cellular activities (as shown\nin Figure 1c).”\nComment 8: Describing how the enzyme reaction rates were measured at the section\nwhere the results were described would be useful for the readers.\nResponse: We appreciate the reviewer’s suggestion. In response, we have added a\ndetailed description of the enzyme reaction rate measurements in the Materials and\nMethods section of the manuscript to enhance clarity for the readers (Revised in\nDetection of enzymatic activities in coacervates).\n“To assess the catalytic activity of GOx within coacervates, a 50 nM GOx-loaded\nR10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM\nGOx were each mixed with glucose solutions at various concentrations (0, 1, 2, 3, 4, 5,\n10, 15, and 20 mM). After different reaction times (5 min, 10 min, 15 min, 30 min, 45\nmin, and 60 min), to minimize turbidity interference from the protocell-containing\nsamples, the mixtures were centrifuged and the supernatants were collected for GOx\nactivity measurement using a Glucose Oxidase Activity Assay Kit. The initial reaction\nrate at each substrate concentration was determined by analyzing the linear region of\nthe product formation curve over a 60-minute period (Figure S23 and Figure S24).\nSimilarly, to evaluate urease activity in coacervates, a 50 nM urease-loaded\nR10/D10 coacervate solution and a bulk aqueous solution (pH 8.0) containing 50 nM\nurease are mixed with urea solutions at concentrations of 0, 1, 2, 3, 4, 5, 10, 15, and 20\nmM. After different reaction times, to minimize turbidity interference from the\nprotocell-containing samples, the mixtures were centrifuged and the supernatants\ncollected for urease activity measurement using a Urease Activity Assay Kit. The initial\nreaction rate at each substrate concentration was determined by analyzing the linear\nregion of the product formation curve over a 60-minute period (Figure S25 and Figure\nS26).”\nFurthermore, the original reaction curves over 60 minutes under different ion and\nsubstrate concentrations have been included as supplementary figures in the Supporting\nInformation (Revised in Figure S23-S26).\nTime-dependent product formation curves of GOx in solution catalyzing glucose at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of GOx in coacervate catalyzing glucose at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of urease in solution catalyzing urea at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nTime-dependent product formation curves of urease in coacervate catalyzing urea at\ndifferent concentrations under varying H+/OH- conditions. n=5.\nReviewer 3#\nComment: The manuscript \"Coacervate Droplets as pH-Regionalized Protocells\" aims\nto explore the potential mechanisms that govern pH regulation in membrane-less\norganelles (MLOs). To this end, the authors first investigate differences in the\ncondensed phase nucleolus and nucleoplasm pH using a fluorescent indicator. The\nnucleoli report lower pH compared to the nucleoplasm, with a less pronounced\ndifference when RNA Pol I activity is inhibited or complete loss of a pH gradient when\nthey are disassembled upon treatment with actinomycin D. To capture a similar LLPS\ndependent pH gradient in vitro, the investigators design a peptide based complex\ncoacervate formed by electrostatic complexation of decapeptides R10 and D10. LLPS\nin this system is investigated as a function of pH, peptide concentration and presence\nof salt with this system behaving in line with previously reported coacervates of similar\nnature. A small intrinsic pH gradient is noted with the condensate phase being slightly\nmore acidic, and coarse-grained molecular dynamics simulations are used to explain\nthe preferential partitioning of the hydronium (H 0+) ions inside the peptide-rich\ndroplets. Subsequently, enzymes are encapsulated in these coacervates to build\nminimalistic protocell bioreactors. The enzymatic reactions of GOx and Urease\nrelease products that influence the pH and therefore the substrate concentration serves as a handle to regulate enzyme kinetics.\nThe authors argue that this work provides potential insights into the mechanism for\npH regulation in MLOs, but this is not entirely evident as no similar examples are\nprovided for sequestration of pH regulating enzymes in biological MLOs. Nonetheless,\nthe work provides an interesting demonstration of pH gradients in minimalistic peptide\ncondensates with the ability to regulate enzyme activity by tuning substrate\nconcentration of the orthogonal pH reaction. The following concerns should be\naddressed and clarifications provided prior to being considered for publication.\nResponse: We thank the reviewer for their thoughtful and constructive comments on\nour manuscript. In response, we have carefully considered the concerns raised and\nprovided clarifications and corresponding revisions in the manuscript to address these\npoints.\nComment 1: In Figure 3 j-k and m-n, the RDFs indicate that with increasing peptide\nconcentration or at lower salt-concentration, a higher number of hydronium ion beads\nare recruited to the peptide-condensates. Similar RDFs for hydroxide (OH-) ions are\nsubstantially lower (Figure S10) at g(r) ~ 7 vs. ~27 for H 0+.\nThe number of contacts\n3\nmeasured from the simulations between the peptides and ions does not appear to reflect\nthis trend however with both H+ and OH- showing ~ 350 contacts within 0.6 nm with\nthe peptides. This would require some clarification in the manuscript to explain the pH\ndifference and preferential partitioning of H+ into the condensates.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion. To more clearly\nillustrate the variation in contact numbers of H⁺ and OH⁻ under different conditions, we\nperformed repeated simulations and computed the averaged contact numbers. The\nresults show that, under identical conditions, H⁺ exhibits a slightly higher average\ncontact number with peptides compared to OH⁻, which may explain why the differences\nappeared less pronounced in the previous figure. This clarification has been added to\nthe revised manuscript (Revised in Figures 3).\na. Simulated contact number between peptides and H+ and OH- at various coacervate\nconcentrations. n=10. Data are presented as mean ± SD. Statistical significance is\ndetermined using a Student’s t-test. b. Simulated contact number between peptides and\nH+ and OH- at different salt concentrations. n=10. Data are presented as mean ± SD.\nStatistical significance is determined using a Student’s t-test.\nComment 2: Figure S5 (a-d): The caption reads 1 μM HCl/NaOH however the figure\npanels say 1 mM and should be corrected to be consistent.\nResponse: We thank the reviewer for pointing out this inconsistency. The error has\nbeen corrected in the revised manuscript to ensure consistency between the figure\npanels and the caption (Revised in Figure S8).\n(a,b) pH values of the condensate and dilute phases of R10/D10 coacervate at different\ncoacervate concentrations after adding 1 μM HCl (a) and NaOH (b). (c,d) pH values of\nthe condensate phase and dilute phases at different salt concentrations are measured\nfollowing the addition of 1 μM HCl (c) and NaOH (d). n=30.\nComment 3: Using the CG-MD simulations, the authors argue that the diffusion\ncoefficients of the H+/OH- ions are reduced by interaction with the coacervate network.\nIn Figure S11, the H+ diffusion as quantified by the MSD always appears lower than\nthe OH- diffusion even at low coacervate concentration. H+ is known to have higher\ndiffusion compared to OH- due to the Grotthus effect and other mechanisms. Due the\nauthors attribute this lower diffusion to preferential incorporation of the H+ beads in\nthe peptide aggregates? A control simulation in the absence of peptides but presence of\nH+/OH- ions in similar box volumes would be required to validate the MARTINI\nmodels' ability to capture the intrinsic differences in diffusivity of these ions.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion. To address this\npoint, we have performed a control simulation in the absence of peptides but in the\npresence of H⁺/OH⁻ ions under similar box volumes (Revised in Figure S15 and Figure\nS18).\na. Simulated MSD of H+ at different concentrations of R10/D10 coacervate. b.\nSimulated MSD of OH- at different concentrations of R10/D10 coacervate. c. Diffusion\ncoefficient of H+ at different concentrations of R10/D10 coacervate based on the\nsimulation results. n=10. d. Diffusion coefficient of OH- at different concentrations of\nR10/D10 coacervate based on the simulation results. n=10. Data are presented as mean\n± SD.\na. Simulated MSD of H+ at different NaCl concentrations. b. Simulated MSD of OH-\nat different NaCl concentrations. c. Diffusion coefficient of H+ at different NaCl\nconcentrations based on the simulation results. n=10. d. Diffusion coefficient of OH- at\ndifferent NaCl concentrations based on the simulation results. n=10. Data are presented\nas mean ± SD.\nThe results indicate that, compared to OH⁻, H⁺ diffusion is indeed more strongly\ninfluenced by the coacervate system. This observation is consistent with the RDF\nresults showing a higher number of H⁺ beads recruited to the peptide condensates, as\nwell as the higher contact number of H⁺ with peptides compared to OH⁻. Taken together,\nthese findings imply that within the R10/D10 coacervate system, H⁺ may interact more\nstrongly with the condensate phase, which could reflect a slight preferential partitioning\naffinity of the coacervate for H⁺ over OH⁻. In response, we have also included a related\ndiscussion in the revised manuscript to address this observation. (Revised in Peptide\ncoacervate droplets recapitulating the pH difference in MLOs, Paragraph 5)\n“Notably, in the coacervate solution, the diffusion coefficient of H⁺ decreases more\nsignificantly than that of OH⁻ when compared to their respective values in water. This\nfinding aligns with the RDF results, which reveal greater recruitment of H⁺ to the\npeptide condensates and a higher contact number of H⁺ with peptides relative to OH⁻.\nTogether, these results suggest that H⁺ may have an even more preferential partitioning\naffinity in the R10/D10 coacervate system over OH⁻.”\nComment 4: The partitioning of enzymes was demonstrated to show electrostatic\nselectivity, with no significant binding and recruitment noted between the D10 peptides\nand the FITC-GOx or other negatively charged systems (dsDNA/PAA). Although the\ncorresponding MD simulations for the favorable interaction are presented\n(GOx/Urease-R10), the manuscript/supplementary information would benefit from a\nsimilar simulation with D10 indicating exclusion/poor interaction. Furthermore, this\nwould also rule out peptide-enzyme interactions as arising from excluded volume\neffects.\nResponse: We appreciate the reviewer’s valuable suggestion. In response, we have\nadded simulations and RDF data in the revised manuscript to examine the interactions\nbetween D10 and GOx/urease. (Revised in Figure 4) These results support the MST\nfindings, confirming that the negatively charged D10 peptides do not exhibit significant\nbinding or recruitment of the enzymes.\na. Snapshot of the final configuration of the D10 and GOx system after 5 μs of CG\nmolecular dynamics simulations. Water molecules are omitted for clarity. b. D10-GOx\nRDF obtained from the simulation. c. Snapshot of the final configuration of the D10\nand urease system after 5 μs of CG molecular dynamics simulations. Water molecules\nare omitted for clarity. d. D10-urease RDF obtained from the simulation.\nComment 5: The Michaelis-Menten plots in Figure 5 b-c show slower reaction rates\ninside the coacervates than in PBS. Condensates that strongly partition the substrates\nand enzymes typically improve such reaction rates but the opposite effect can be noted\nhere. Is this due to lower diffusion inside the condensates or do some of the enzymes\nlose activity upon being bound by peptides?\nResponse: We appreciate the reviewer’s insightful comment. A similar trend in the\nchanges of enzyme V within coacervates has also been observed by Jan C. M. van\nmax\nHest and colleagues (Physicochemical Characterization of Polymer-Stabilized\nCoacervate Protocells. ChemBioChem, 2019). The slower enzymatic reaction rates\nobserved inside the coacervates may result from several contributing factors. Enzymes\nmight exhibit lower catalytic activity within the coacervate phase, leading to a reduced\nVₘₐₓ. This effect could arise from the unique microenvironment of the condensate,\nwhich may increase local viscosity or alter enzyme conformation, thereby restricting\nsubstrate diffusion and access to the enzyme active sites. Together, these factors could\ncontribute to the overall decrease in catalytic efficiency compared with that in a\nhomogeneous solution.\nComment 6: The introduction and conclusions would generally benefit from a rewrite\nthat emphasizes the pH differences, the ability to regulate enzymatic reactions and\nconsequently control IVVT or cascade reactions rather than the direct connection to pH\nregulation in biological MLOs. The mechanisms of this are not entirely revealed in this\nmanuscript, as the fundamental reason of H+ enrichment in coacervates is not\ncompletely deciphered or explained but rather simply noted, observed and exploited.\nResponse: We thank the reviewer for this constructive suggestion. In response, we have\nrefined the related sections to place greater emphasis on how pH differences within\ncoacervate-based protocells may be utilized to modulate enzymatic reactions, while\nadjusting the focus from detailed mechanistic discussions of pH regulation in biological\nMLOs to the functional implications observed in our artificial system. (Revised in\nIntroduction and Discussion)\nIntroduction\n“Cellular functions rely on numerous enzyme-catalyzed reactions that occur\nspatial-temporally correct under physiological conditions. The maintenance of enzyme\nactivity is essential for metabolism, energy production, and the synthesis of various\nsubstances. A key factor that affects enzyme activity is pH, as each enzyme functions\noptimally within a specific pH range. Deviations from this range can impair the\nstructure and function of enzymes, which even leads to diseases or disorders. It has\nbeen well established that various membrane exchangers and transporters play\nsignificant roles in regulating intracellular pH. In contrast, membrane-less organelles\n(MLOs), which host diverse biochemical reactions and play essential roles in cellular\nregulation, lack such membrane-based control mechanisms. Recent studies have\nrevealed that MLOs can exhibit local pH variations, which may influence the kinetics\nand selectivity of enzymatic reactions. However, given the complexity of living cells,\nit is challenging to directly investigate the mechanisms of pH regulation in vivo by\ncontrolling conditions across the MLOs interface.\nIn this study, we found that a natural pH difference exists between the nucleolus\nand nucleoplasm in several tested cell lines, which can be modulated by drug treatments\nto alter their structure and properties (Figure 1a). Since coacervates are liquid-liquid\nphase separation (LLPS) systems driven by noncovalent interactions, and they share\nsimilar properties to MLOs such as molecular organization and compartmentalization,\nwe then present peptide-based coacervate droplets as protocells to mimic the pH\ndifference found in the nucleus. We use microfluidics to prepare these coacervate\ndroplets to ensure their uniformity in size, thus allowing for the generation of a similar\npH difference across the LLPS interface under precisely controlled conditions. Through\nmolecular dynamics simulations and quantitative experiments, we conclude that the\ndense phase of coacervates, rich in charged polymers, tends to attract a higher\nconcentration of H+/OH- ions than the dilute phase. This results in an uneven apparent\npH distribution between the interior and exterior of the coacervates. More importantly,\nsuch pH difference weakens or even disappears following destruction of LLPS, a\nphenomenon observed in both nucleolus and coacervate droplets. Based on these\nfindings, we demonstrate that the pH-regionalized environment of the coacervate\ndroplets can be customized by selectively accommodating enzymatic reactions (as\nshown in Figure 1b), and such feature can be further employed to control cascade\nreactions and perform polymerase chain reaction (PCR) and in vitro transcription and\ntranslation reaction (IVTT), thereby recapitulating various cellular activities (as shown\nin Figure 1c). Our findings suggest that coacervate-based protocells offer a valuable\nplatform to explore how physicochemical compartmentalization influences\nbiochemical reactions.”\nDiscussion\n“This study employs coacervate droplet-based protocells as a minimal model to\nexplore how phase separation can give rise to and utilize local pH differences for\nbiochemical reaction regulation. We first identified a pH difference across the nucleolus\nin several tested cell lines and found that treatment with CX-5461 or Act D altered\nnucleolar structure and function, accompanied by changes in the pH difference between\nthe nucleolus and nucleoplasm. We then developed an in vitro R10/D10 coacervate\nsystem to mirror such pH difference and examine its response to environmental changes.\nNotably, uniform coacervate droplets are generated through the microfluidic technique,\nwhich allows precise control of the droplet composition as well as environmental\nparameters. We observe that a pH difference can be established between the condensate\nphase and the dilute phase of coacervates with the presence of acid or base added to the\nsystem. Through molecular dynamics simulations, we conclude that these pH\ndifferences likely arise from the enrichment of H+/OH- in the condensate phase rich in\ncharged polymers. Intriguingly, we find that the pH difference no longer exists after the\ndisassembly of the coacervate by adding salt, which mirrors the same phenomenon\nobserved after the disassembly of the nucleolus. Based on the pH-regionalized feature\nof the coacervate droplets, we further demonstrate a way of finely tuning the local pH\nby incorporating GOx- and urease-catalyzed reactions by leveraging the proteins’\npartitioning affinity behavior. The acidic and alkaline byproducts of the enzymatic\nreactions can alter the local pH in dependence of the substrate concentration, which\nfurther impacts the activity of another, downstream enzymatic reaction. With this, the\ncoacervate droplets can serve as protocells accommodating various biological-relevant\nprocesses such as PCR and IVTT.\nIn conclusion, our work emphasizes how pH heterogeneity in coacervate-based\nprotocells can be harnessed to regulate biochemical reactivity. Our findings also\nhighlight the potential of coacervate droplets as pH-regionalized protocells to simulate\ncellular environments, study phase separation-related cellular activities, and perform\ncomplex bio(chemical) reactions.”\nResponse to reviewers\nDear Editor:\nWe hereby submit the revised manuscript (NCOMMS-25-41534-T), entitled\n“Coacervate Droplets as pH-Regionalized Protocells” by Chong Wang, Zhonglin Fang,\nLinyi Zhang, Yuanjin Zhao, and Luoran Shang, for your consideration as a Research\nArticle in Nature Communications. We sincerely appreciate your thorough evaluation\nof our manuscript and are grateful for the insightful and constructive suggestions\nprovided by the reviewers. In response to the reviewers’ comments, we have made the\nfollowing revisions:\nReviewer #1\nComment: I appreciate the significant effort the authors have made in performing\nseveral additional experiments and effectively addressing the vast majority of my points.\nThe value of the work has significantly strengthened. However, a few points remain\nbefore final recommendation for acceptance.\nResponse: We sincerely thank the reviewer for the careful evaluation and constructive\ncomments, and for acknowledging the additional experiments and revisions. We are\npleased that the work is considered strengthened and valuable, and we have addressed\nthe remaining points to further improve the manuscript.\nComment 1: Response to Comment 3: Minor point. The authors have not fully\nanswered the question. They stated, “we consider that transcriptional inhibition and\nstructural disruption are not mutually exclusive processes but rather interrelated\nphenomena.” This indeed raises the question of whether the observed pH change is a\nresult of the inhibition of the reaction, the structural disruption, or a combination of\nboth. If it is not possible to determine this, it should be clearly articulated in the\nmanuscript.\nResponse: We thank the reviewer for this helpful comment. In the revised manuscript,\nwe clarify that the drug treatments may influence both transcriptional activity and\nnucleolar organization. Although clear pH changes were observed, the current data do\nnot allow us to separate the contributions of these two processes. This potential\nlimitation is now explicitly acknowledged in the revised manuscript (Revised in\nIdentification of the pH difference between nucleolus and nucleoplasm, paragraph\n4).\n“Taken together, these observations clarify that a pH difference exists across the\nnucleolus interface and suggest that this difference is closely linked to the LLPS status\nand biological function of the nucleolus. These experiments imply that the\ntranscriptional activity and structural integrity of the nucleolus might both contribute to\nmaintaining this pH difference. However, as transcriptional inhibition and structural\ndisruption may take place simultaneously under drug treatment, the available data are\nnot sufficient to distinguish which of these processes predominantly drives the observed\npH changes.”\nComment 2: Response to Comment 7: Regarding Figure 3: Major point. The difference\nbetween the two conditions is smaller than the error bars, and there is considerable\noverlap in the data distributions. In particular, the dilute phase exhibits much greater\nvariability than the dense phase (no explanation provided). Moreover, the statistical\nsignificance at 0.8, 1.2 and 2.0 umol added ion is small (*). Overall, the statistical\nanalysis requires more careful consideration, particularly regarding the determination\nof the required sample size.\nResponse: We thank the reviewer for the insightful comments on the statistical analysis\nin Figure 3d. In response, we performed additional measurements to strengthen the\nrobustness of our dataset. Specifically, we repeated the experiments and collected 30\nindependent measurements for each condition, using identical sample preparation and\nmeasurement procedures to minimize technical variability. To more accurately assess\nthe significance of the observed differences, we analyzed the paired measurements\nusing a paired t-test, as the dense-phase and dilute-phase pH values in each replicate\nwere obtained from the same coacervate solution. The updated analysis confirms that\nthe experimental groups with 0.8, 1.2, and 2.0 μmol of added ion show statistically\nsignificant pH differences. These results have been incorporated into the revised\nmanuscript (Revised in Figure 3d). The raw data for the repeated measurements have\nbeen uploaded named as “Revised Figure 3d data.xlsx” as a “Supplementary Dataset”\nfile.\nThe pH changes in the condensate phase and dilute phase upon the addition of different\namounts of H+ or OH-. n=30. Data are presented as mean ± SD. Statistical significance\nis determined using a paired Student’s t-test.\nComment 3: Response to Comment 11: While the authors measured the partition\ncoefficient, they did not report the percentage of the total enzyme recruited within the\ncoacervates, which was the key request of this comment (it may require the estimation\nof the total volume fraction of the coacervate).\nResponse: We appreciate the reviewer’s helpful suggestion. In the revised manuscript,\nwe have now included the percentage of the total enzyme recruited within the\ncondensate phase. Using the condensate volume fraction determined in Figure S5b\ntogether with the partition coefficient calculated in Figure S21, we estimated the\nproportion of the enzyme population residing in the condensate phase, yielding P =\nGOx\n80.45% and P = 73.14%. This information has been added to the revised manuscript\nurease\n(Revised in Construction of pH-regionalized protocell protocells, paragraph 2).\n“In addition, with the condensate volume fraction already determined (Figure S5b),\nthese parameters enabled us to estimate the percentage of the total enzyme recruited\ninto the condensate phase, yielding P = 80.45% and P = 73.14%.”\nGOx urease\nReviewer #2\nComment: The authors did a great job revising the manuscript with rigor to address all\nthe points raised by this reviewer and other reviewers.\nResponse: We sincerely appreciate the reviewer’s positive assessment and the\nthoughtful recognition of our revision efforts. The constructive feedback provided\nthroughout the review process has been greatly helpful in improving the clarity, rigor,\nand overall quality of the manuscript. We are truly grateful for the reviewer’s time,\ncareful evaluation, and valuable guidance.\nReviewer #3\nComment: In the revised manuscript, Wang et al. have addressed the concerns that\nwere raised previously by performing replicate simulations, providing control data from\nnew reference simulations with H+/OH-, carrying out additional in silico work to\nvalidate and further substantiate their findings. The new results have substantially\nimproved the report by placing them in context. Also, the rewritten introduction and\ndiscussion section better reflects the insights from the work, improving the clarity and\nrelevance of the findings to the field. A minor clarification that would improve the\nMaterials and Methods section for the Coarse-Grained MD simulations would be to\nprovide the bead types, mass etc. used in MARTINI 3.0 for the H+ and OH- ions as\nthey are not currently found in the forcefield files available online. Once this\nclarification is added, I recommend the manuscript for publication.\nResponse: We thank the reviewer for the constructive comments and for recognizing\nthe improvements made in the revised manuscript. In response to the reviewer’s\ncomment, we have clarified how H₃O⁺ and OH⁻ ions were represented in our MD\nsimulations. Both ions were treated as single charged beads, following the approach of\na previous study (Analysis of Structural and Water Diffusional Properties of Ionomer\nThin Films by Coarse-Grained Molecular Dynamics Simulation, J. Mol. Liq., 2023).\nSpecifically, H₃O⁺ was modeled as a TQ5 bead with a net charge of +1 e, and OH⁻ as\na TQ5 bead with a net charge of −1 e. No bonded interactions were applied, as each ion\nis represented by a single bead. All other parameters were set according to the default\nMARTINI 3.0 force field. This information has been added to the revised manuscript\n(Revised in Molecular dynamics simulation of R10/D10 coacervate formation and ion\ndistribution).\n“Specifically, H₃O⁺ was modeled as a TQ5 bead with a net charge of +1 e, and OH⁻ as\na TQ5 bead with a net charge of −1 e. All other parameters were set according to the\ndefault MARTINI 3.0 force field.”"}]

Decision letter