Invasion and Defense Interactions between Enzyme‐Active Liquid Coacervate Protocells and Living Cells

The design and construction of mutual interaction models between artificial microsystems and living cells have the potential to open a wide range of novel applications in biomedical and biomimetic technologies. In this study, an artificial form of invasion‐defense mutual interactions is established in a community of glucose oxidase (GOx)‐containing liquid coacervate microdroplets and living cells, which interact via enzyme‐mediated reactive oxygen species (ROS) damage. The enzyme‐containing coacervate microdroplets, formed via liquid–liquid phase separation, act as invader protocells to electrostatically bind with the host HepG2 cell, resulting in assimilation. Subsequently, the glucose oxidation in the liquid coacervates initiates the generation of H₂O₂, which serves as an ROS resource to block cell proliferation. As a defense strategy, introduction of catalase (CAT) into the host cells is exploited to resist the ROS damage. CAT‐mediated decomposition of H₂O₂ leads to the ROS scavenging and results in the recovery of cell viability. The results obtained in the current study highlight the remarkable opportunities for the development of mutual interacting communities on the interface of artificial protocells/living cells. They also provide a new approach for engineering cellular behaviors through exploiting artificial nonliving microsystems.     

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes,and Nucleosides Bind Better than Individual Bases Do

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or – with the addition of phosphate – a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.     

Spatial Organization in Proteinaceous Membrane‐Stabilized Coacervate Protocells

As a model protocell, the membrane‐free coacervate microdroplet is widely utilized in functional studies to provide insights into the physicochemical properties of the cell and to engineer cytomimetic soft technologies; however, the lack of a discrete membrane contributes to its instability and limits further application. Herein, a strategy is developed to fabricate a hybrid protocell based on the self‐assembly of a proteinaceous membrane at the surface of coacervate microdroplets driven by a combination of electrostatic adhesion and steric/hydrophilic surface buoyancy. The semipermeable proteinaceous membrane can enhance coacervate stability obviously without compromising sequestration behavior. Significantly, such hybrid protocells demonstrate spatial organization whereby various functional enzymes can be located in discrete regions, which facilitates an on/off modulation for a cascade enzymatic reaction along with enhanced chemical communication between subpopulations.     

Toward Artificial Cells: Novel Advances in Energy Conversion and Cellular Motility

The demand to discover every single cellular component has been continuously increasing along with the development of biological techniques. The bottom‐up approach to construct a cell‐mimicking system from well‐defined and tunable compositions is accelerating, with the ultimate goal of comprehending a biological cell. From among the available techniques, the artificial cell has been increasingly recognized as one of the most powerful tools for building a cell‐like system from scratch. This review summarizes the development of artificial cells, from a pure giant unilamellar vesicle (GUV) to a controllable, self‐fueled proteoliposome, both of which are highly compartmentalized. The basic components of an artificial cell, as well as the optimal conditions required for successful, reproducible GUV formation and protein reconstitution, are discussed. Most importantly, progress in studying the metabolic reactions in and the motility of a reconstituted artificial cell are the main focus of the review. The ability to perform a complicated reaction cascade in a controllable manner is highlighted as a promising perspective to obtaining an autonomous and movable GUV.     

Minimalist Protocell Design: A Molecular System Based Solely on Proteins that Form Dynamic Vesicular Membranes Embedding Enzymatic Functions

Life in its molecular context is characterized by the challenge of orchestrating structure, energy and information processes through compartmentalization and chemical transformations amenable to mimicry of protocell models. Here we present an alternative protocell model incorporating dynamic membranes based on amphiphilic elastin-like proteins (ELPs) rather than phospholipids. For the first time we demonstrate the feasibility of combining vesicular membrane formation and biocatalytic activity with molecular entities of a single class: proteins. The presented self-assembled protein-membrane-based compartments (PMBCs) accommodate either an anabolic reaction, based on free DNA ligase as an example of information transformation processes, or a catabolic process. We present a catabolic process based on a single molecular entity combining an amphiphilic protein with tobacco etch virus (TEV) protease as part of the enclosure of a reaction space and facilitating selective catalytic transformations. Combining compartmentalization and biocatalytic activity by utilizing an amphiphilic molecular building block with and without enzyme functionalization enables new strategies in bottom-up synthetic biology, regenerative medicine, pharmaceutical science and biotechnology.     

Spontaneous Growth and Division in Self‐Reproducing Inorganic Colloidosomes

Self‐reproduction in compartmentalized chemical ensembles is a central issue for the development of new materials and processes capable of autonomous behavior, self‐amplification and artificial evolution. Current approaches to synthetic cellularity focus primarily on self‐assembled soft matter systems such as membrane‐bounded lipid vesicles, which have sufficient structural plasticity to undergo growth and division. Steps towards inorganic protocells are being advanced, but self‐reproduction in these more structurally robust micro‐compartments has not been demonstrated. Here, a primitive form of growth and division involving inorganic colloidosomes (Pickering emulsions), comprising aqueous micro‐droplets enclosed by an ultrathin membrane of silica nanoparticles, is shown. Growth of the colloidosomes is induced by organosilane‐mediated methanol formation, and results in a localized rupture of the inorganic membrane followed by outgrowth and separation of a second‐generation protocell, which is stabilized by de novo nanoparticle assembly. These observations provide a first step towards synthetic cell‐like inorganic materials capable of chemically induced self‐reproduction.     

Chemical Signaling and Functional Activation in Colloidosome‐Based Protocells

An aqueous‐based microcompartmentalized model involving the integration of partially hydrophobic Fe(III)‐rich montmorillonite (FeM) clay particles as structural and catalytic building blocks for colloidosome membrane assembly, self‐directed membrane remodeling, and signal‐induced protocell communication is described. The clay colloidosomes exhibit size‐ and charge‐selective permeability, and show dual catalytic functions involving spatially confined enzyme‐mediated dephosphorylation and peroxidase‐like membrane activity. The latter is used for the colloidosome‐mediated synthesis and assembly of a temperature‐responsive poly(N‐isopropylacrylamide)(PNIPAM)/clay‐integrated hybrid membrane. In situ PNIPAM elaboration of the membrane is coupled to a glucose oxidase (GOx)‐mediated signaling pathway to establish a primitive model of chemical communication and functional activation within a synthetic “protocell community” comprising a mixed population of GOx‐containing silica colloidosomes and alkaline phosphatase (ALP)‐containing FeM‐clay colloidosomes. Triggering the enzyme reaction in the silica colloidosomes gives a hydrogen peroxide signal that induces polymer wall formation in a coexistent population of the FeM‐clay colloidosomes, which in turn generates self‐regulated membrane‐gated ALP‐activity within the clay microcompartments. The emergence of new functionalities in inorganic colloidosomes via chemical communication between different protocell populations provides a first step toward the realization of interacting communities of synthetic functional microcompartments.     

Cyclophospholipids Increase Protocellular Stability to Metal Ions

Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and extant life is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here, prebiotically plausible monoacyl cyclophospholipids are shown to form robust vesicles that survive a broad range of pH and high concentrations of Mg²⁺, Ca²⁺, and Na⁺. Importantly, stability to Mg²⁺ and Ca²⁺ is improved by the presence of environmental concentrations of Na⁺. These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution.