T. -Y. Dora Tang

Phytochelatin is a heavy metal-binding biogenic peptide found across different organisms that helps cells mitigate cytotoxicity by chelating metal ions. Yeasts produce phytochelatin to chelate intracellular cadmium and sequester it as cadmium metal sulfide nanoparticles. These nanoparticles were proposed to be stored within cytoplasmic microcompartments, although it remains unclear whether they are stored within membrane-bound or membraneless compartments. Here, we focused on the structure of phytochelatin and hypothesized that it can form membraneless compartments with positively charged biomolecules, which are capable of internal cadmium sulfide sequestration. Indeed, mixing phytochelatin with natural polysaccharide chitosan led to the instant formation of membraneless condensates. Moreover, Cd and S were preferentially accumulated within the condensates relative to other elements and CdS nanoparticles formed inside them. These nanoparticles exhibited crystalline features, with sizes of 2–3 nm and structures comparable to those of the CdS particles observed in cells, which predominantly correspond to the hawleyite structure. This study demonstrated that heavy metal-binding peptides can participate in the formation of membraneless compartments and internal sequestration of nanoparticles. Our findings further indicate a potential role of phytochelatin-based membraneless compartments in spatially controlled intracellular biomineralization and heavy metal sequestration for cellular detoxification, which remains to be explored in vivo.

Phytochelatin is a heavy metal-binding biogenic peptide found across different organisms that helps cells mitigate cytotoxicity by chelating metal ions. Yeasts produce phytochelatin to chelate intracellular cadmium and sequester it as cadmium metal sulfide nanoparticles. These nanoparticles were proposed to be stored within cytoplasmic microcompartments, although it remains unclear whether they are stored within membrane-bound or membraneless compartments. Here, we focused on the structure of phytochelatin and hypothesized that it can form membraneless compartments with positively charged biomolecules, which are capable of internal cadmium sulfide sequestration. Indeed, mixing phytochelatin with natural polysaccharide chitosan led to the instant formation of membraneless condensates. Moreover, Cd and S were preferentially accumulated within the condensates relative to other elements and CdS nanoparticles formed inside them. These nanoparticles exhibited crystalline features, with sizes of 2–3 nm and structures comparable to those of the CdS particles observed in cells, which predominantly correspond to the hawleyite structure. This study demonstrated that heavy metal-binding peptides can participate in the formation of membraneless compartments and internal sequestration of nanoparticles. Our findings further indicate a potential role of phytochelatin-based membraneless compartments in spatially controlled intracellular biomineralization and heavy metal sequestration for cellular detoxification, which remains to be explored in vivo.

Living systems must maintain robust biochemical function despite fluctuations that span a wide range of timescales. Biomolecular condensates formed by liquid–liquid phase separation (LLPS) have been shown to buffer concentration fluctuations, but the principles governing their dynamic regulation remain unclear. We address this by probing the response of LLPS to oscillatory perturbations that mimic fluctuations across different timescales, establishing the first systematic frequency-domain analysis of concentration buffering by condensates. We find that condensates act as frequency-selective filters: The perturbed dilute phase behaves as a high-pass filter, while the dense phase attenuates both low- and high-frequency perturbations. We establish quantitative links between LLPS parameters, including interaction strength, droplet size, and interphase exchange rates, and the timescale range over which condensates effectively buffer concentration fluctuations. These findings establish fundamental dynamical limits of concentration buffering by LLPS, with implications for how cells may use LLPS to adapt to fluctuating environments and for the design of synthetic condensates with programmable control properties.

Compartmentalization via phase separation is increasingly recognized as a dynamic regulator of cellular biochemistry, yet the consequences of coupling enzymatic reactions to compartmentalization remain poorly understood. We combine a minimal, cell-free expression system with kinetic modeling to show how coupling enzymatic activity to biomolecular condensates drives emergent self-regulation via droplet formation and dissolution. In-vitro mRNA transcription induces phase separation with an intrinsically disordered protein (mutant G3BP1), forming condensates that modulate transcription and degradation kinetics. Our kinetic model shows that phase separation reduces rate constants, with slower degradation within condensates than in the mRNA-protein-poor phase. This leads to more mRNA with a prolonged lifetime of mRNA relative to the case without condensates. Extending the model to sustained and oscillatory resource supply reveals that condensates elevate mean mRNA levels and buffer deviations from the mean compared to the non-condensate scenario. These findings provide a general mechanism of cross-regulation and feedback between phase separation and enzymatic networks, highlighting condensates as active regulators of biochemical flux rather than as passive organizers.

Macromolecular assembly between biomolecules dictates the material state of chemically complex aqueous dispersions such as the cytoplasm. The formation of protein precipitates, fibers, or liquid droplets have been associated with metabolic regulation and disease. However, the effect of metabolic flux on the material properties of aqueous dispersions remains underexplored. Here, we use the protometabolic reduction of NAD⁺ to NADH by pyruvate to study the effect of NADH production on the phase separation properties of polyarginine. We show that reduction of NAD⁺ in the presence of polyarginine can tune the material properties of the dispersion between precipitates, homogeneous solution, and liquid droplets depending on the buffer concentration. In situ droplet formation results in 2-3 times higher reaction rate and NADH yield, compared to homogeneous solution. Our study provides a setting for coupling protometabolism to active protocell environments in the absence of enzymes and sheds light on the self-regulation of metabolic flux on mediating biomolecular phase separation.

Towards the goal of building synthetic cells from the bottom‐up, the establishment of micrometer‐sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein‐polymer conjugates offer a promising solution to membrane‐bound compartmentalization with an open, semi‐permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water‐in‐water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein‐polymer (BSA‐PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real‐world applications.

The front cover artwork is provided by David T. Gonzales of the Max Planck Institute of Molecular Cell Biology and Genetics. The image shows a collection of aqueous water‐in‐oil droplets with lipid bilayer interfaces. Inside each droplet is a cell‐free expression system that is capable of gene expression or intercellular communication. Read the full text of the Research Article at 10.1002/syst.202300029 .