Biology subjects

Keating, C. D.

Publications and source records attributed to Keating, C. D..

12 recordsLinked to original sources

bioRxiv2026

Coacervates protect RNA against hydrolysis under wet-dry cycling conditions

RNA is crucial to all extant biology and thought to have played an essential role in lifes origins. However, hydrolysis of RNA under common environmental conditions such as heating, drying, and exposure to divalent cations presents challenges for its persistence and effectiveness in prebiotic contexts. RNAs with less stable secondary and tertiary structures are especially susceptible to degradation. In living cells, sequestration in biopolymer-rich membraneless organelles helps shield RNA from damage and enzymatic degradation. Here, we report that compartmentalization into polyelectrolyte-based complex coacervate droplets protects RNA from hydrolysis during wet-dry cycling at elevated temperatures in the presence of Mg2+. This protection can be understood in terms of the coacervate microenvironment under varied salt concentrations and how it changes during sample drying. After gaining insight into this prebiotically-plausible mechanism using an unstructured model RNA, we demonstrate coacervate-based protection of a ligase ribozyme, supporting the functional relevance of the protection.

biophysics↗

bioRxiv2026

Increasing the compositional heterogeneity of single-chain amphiphile membranes supported by coacervate cores alters stability and properties of the hybrid protocells

Coacervate droplets and lipid vesicles are two classes of self-assembled compartments that have been proposed as protocell models. Hybrid protocells, in which a coacervate core is surrounded by a lipid membrane, can integrate the advantages of both protocell systems while overcoming their limitations. Although hybrid protocell membranes have been produced with a variety of diacyl phospholipids related to modern biology and some single-chain amphiphiles inspired by prebiotic scenarios, little is known about how mixtures of single-chain amphiphiles impact hybrid protocell membrane formation and properties. Given the plausible diversity of amphiphiles in the prebiotic milieu, the resulting membranes would have inherently incorporated multiple lipids of different types, potentially altering the properties and viability of hybrid protocells in their environment. Here, we systematically increased the compositional heterogeneity of hybrid protocell membranes by using different prebiotically relevant single-chain amphiphiles of varying head groups and alkyl chain lengths. These membranes were assembled around model coacervate droplets generated from polyallylamine hydrochloride and adenosine diphosphate, and the effect of heterogeneity on membrane properties and stability was evaluated. Compared to protocells with homogeneous membranes, those with heterogeneous amphiphile membranes exhibited higher yields, smaller sizes, and greater sub-compartment formation. Also, they showed increased membrane order, retained similar lateral lipid diffusion, and showed population-level variability in permeability to small anionic molecules. Notably, heterogeneous membranes showed enhanced structural stability under acidic conditions, retaining key properties like size and sub-compartment heterogeneity, thereby broadening the pH range over which hybrid protocells remain intact. These findings suggest that amphiphile diversity not only would have influenced the structural properties of hybrid protocells but also created diversity within the protocell population and enhanced their robustness, thereby playing a crucial role in protocell evolution on early Earth.

biochemistry↗

bioRxiv2025

Dynamic and Catalytic Multiphase Coacervates

Subcellular organization is critical for myriad cellular functions, including spatiotemporal regulation of biocatalysts. Liquid-liquid phase separation contributes to intracellular organization by providing microenvironments that sequester biomolecules and control their activities. Although catalytic reactions in multiphase coacervate systems are important for living cells, provide functionality to artificial systems, and may aid understanding of how metabolism first arose, they are not well understood. Here we report an artificial multiphase system in which catalytically active droplets containing three distinct coexisting coacervate phases undergo reorganization in response to pH, enabling control over chemical reactions. Catalysis is performed by an inner, polyhistidine-dense coacervate phase with inherent nonenzymatic activity. We show how phase composition, in addition to pH, affects catalytic activity, and how the addition of enzymes that alter local pH provides a means to regulate composition and catalysis. By exploiting competitive interplay between intramolecular self-association and intermolecular complexation among polymers within multiphase coacervate systems, it was possible to (re)organize and tune the reactivity of dynamic synthetic biological systems beyond what is possible for single-phase coacervates. Such approaches could prove valuable in designing artificial cells and related microreactor systems capable of catalyzing and controlling complex chemical and biochemical processes.

biochemistry↗

bioRxiv2025

Residue-level mapping of crowding effects on protein phase separation

Proteins can undergo liquid-liquid phase separation to form condensates within the crowded cellular environment. In vitro, polymer-based crowding agents are widely used to mimic this effect, but how their chemical environments influence phase separation compared to native cytosol remains unclear. Here, we use NMR to probe the chemical influences on the intrinsically disordered region of RNA polymerase II under different crowding conditions, including polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol. We find a general trend of enhanced protein self-interactions across all conditions, but also distinct chemical environments that depend on crowder identity, reflecting changes in preferential interactions. Given the widespread use of polymer crowders in phase separation studies, our results establish a strategy to dissect the microscopic role of crowders and lay the foundation for designing more physiologically relevant in vitro crowding models of protein phase separation. More broadly, this framework enables systematic probing of residue-level environmental influences in complex settings including within the cellular milieu.

biophysics↗

bioRxiv2025

Primitive Molecular Buffering by Low-Multivalency Coacervates

Coacervate droplets formed by liquid-liquid phase separation serve as models for intracellular biomolecular condensates and as potential protocellular compartments during the emergence of life. Changes in availability of molecular components can be anticipated for intracellular and prebiotic milieu, and protocells may have also faced fluctuations in salinity and pH. Compartments able to maintain their molecular composition, i.e. homeostasis, under such conditions would be better able to preserve internal functions. Phase separation could in principle provide resistance to local changes in molecular composition. To evaluate this possibility, we investigated the impact of non-stoichiometric charge ratios of coacervate molecules on coacervate formation and RNA compartmentalization in oligoarginine (R10)/ATP coacervates across salinity and pH conditions relatable to plausible prebiotic environments. These R10/ATP coacervate systems resisted changes in oligoarginine concentration in both phases under freshwater and ocean-relevant salt conditions, providing a primitive molecular buffering function. Moreover, RNA accumulation was observed in the coacervates over a range of pH, salinity, and R10/ATP stoichiometry. We also observed salt-dependent differences in molecular buffering and compartmentalization that can be understood in terms of how salinity impacts the relative strengths of intermolecular binding modes that drive coacervation and RNA uptake. By varying relative phase volumes and altering which intermolecular binding modes dominate, LLPS provides general mechanisms for resisting changes in molecular availability and environmental conditions, even without the active homeostasis of living cells. Such primitive molecular buffering could have aided the emergence of life and may find utility in biotechnological or commercial applications based on molecular compartmentalization.

biophysics↗

bioRxiv2024

Hybrid Protocells based on Coacervate-Templated Fatty Acid Vesicles combine Improved Membrane Stability with Functional Interior Protocytoplasm

Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets formed by liquid-liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in todays living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, we describe protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes. Coacervate-supported membranes formed over a range of coacervate and lipid compositions, with membrane properties impacted by charge-charge interactions between coacervates and membranes. Vesicles formed by coacervate-templated membrane assembly exhibited profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without coacervate interiors, particularly in the presence of Mg2+ ions. While fatty acid and blended membrane vesicles were disrupted by addition of 25 mM MgCl2, the corresponding coacervate-supported membranes remained intact and impermeable to externally-added solutes even in the presence of MgCl2. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, could be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate-mediated abiotic hydrolysis to fluorescein.

biophysics↗

bioRxiv2023

Cell-free expressed membraneless organelles sequester RNA in synthetic cells

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

synthetic biology↗

bioRxiv2023

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life. One Sentence SummaryWe demonstrate that RNA folds into native secondary and tertiary structures in protocell models and that this is favored by covalent modifications, which is critical for the origins of life.

biochemistry↗

bioRxiv2021

Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles

Liquid-liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving different putative biochemical roles; the mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here, we show that adjacent phases of decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Additionally, their coexistence introduces new equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA binding sites nor full-length proteins. As such, they are general and point to more primitive versions of mechanisms operating in extant biology that could aid understanding and enable design of functional artificial membraneless organelles.

biochemistry↗

bioRxiv2021

Phospholipid membrane formation templated by coacervatedroplets

We report formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully-coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated with what appear to be continuous lipid bilayers. Within the same population, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and still others are coated with multilayered membranes. These membranes surrounding protein-based coacervate droplets provided protection from a protease added to the external solution. The simplicity of producing artificial cells having a coacervate model cytoplasm surrounded by a model membrane is at the same time interesting as a potential mechanism for prebiotic protocell formation and appealing for biotechnology. We anticipate that such structures could serve as a new type of model system for understanding interactions between intracellular phases and cell- or organelle membranes, which are implicated in a growing number of processes ranging from neurotransmission to signaling.

biophysics↗

bioRxiv2020

Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments

Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluated the impact of low, prebiotically-relevant polyion multivalency in coacervate performance as functional compartments. As model polyions, we used positively and negatively charged homopeptides with one to 100 residues, and adenosine mono-, di-, and triphosphate nucleotides. Polycation/polyanion pairs were tested for coacervation, and resulting membraneless compartments were analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the relatively shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes. Hence, reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, but they can offer advantages over higher molecular weight analogues.

biophysics↗

bioRxiv2018

Bacterial division FtsZ forms liquid condensates with nucleoid-associated Z-ring inhibitor SlmA

Macromolecular condensation resulting from biologically regulated liquid-liquid phase transitions is emerging as a mechanism to organize the intracellular space in eukaryotic systems, with broad implications in cell physiology and pathology. Here we show that FtsZ, central element of the division ring in most bacteria, forms condensates when in complex with SlmA, the protein preventing septal ring assembly nearby the chromosome in E. coli. The formation of condensates is promoted by crowding and enhanced by sequence-specific binding of SlmA to DNA. These structures are dynamic and FtsZ within them remains active for GTP-triggered fiber formation. Their location is sensitive to compartmentalization and to the presence of a membrane boundary in microfluidics-based cell mimetic systems, likely affecting their reactivity. We propose that reversible condensation may play a role in the modulation of FtsZ assembly and/or location by SlmA and, hence, in the regulation of ring stability, constituting a singular example of a prokaryotic nucleoprotein complex exhibiting this kind of phase transition.

biochemistry↗