Artificial Compartments Research Articles

Advancing Artificial Cells with Functional Compartmentalized Polymeric Systems - In Honor of Wolfgang Meier.

The fundamental building block of living organisms is the cell, which is the universal biological base of all living entities. This micrometric mass of cytoplasm and the membrane border have fascinated scientists due to the highly complex and multicompartmentalized structure. This specific organization enables numerous metabolic reactions to occur simultaneously and in segregated spaces, without disturbing each other, but with a promotion of inter- and intracellular communication of biomolecules. At present, artificial nano- and microcompartments, whether as single components or self-organized in multicompartment architectures, hold significant value in the study of life development and advanced functional materials and in the fabrication of molecular devices for medical applications. These artificial compartments also possess the properties to encapsulate, protect, and control the release of bio(macro)molecules through selective transport processes, and they are capable of embedding or being connected with other types of compartments. The self-assembly mechanism of specific synthetic compartments and thus the fabrication of a simulated organelle membrane are some of the major aspects to gain insight. Considerable efforts have now been devoted to design various nano- and microcompartments and understand their functionality for precise control over properties. Of particular interest is the use of polymeric vesicles for communication in synthetic cells and colloidal systems to reinitiate chemical and biological communication and thus close the gap toward biological functions. Multicompartment systems can now be effectively created with a high level of hierarchical control. In this way, these structures can not only be explored to deepen our understanding of the functional organization of living cells, but also pave the way for many more exciting developments in the biomedical field.

An Artificial Liposome Compartment with Size Exclusion Molecular Transport.

The cellular compartment plays an essential role in organizing the complex and diverse biochemical reactions within the cell. By mimicking the function of such cellular compartments, the challenge of constructing artificial compartments has been taken up to develop new biochemical tools for efficient material production and diagnostics. The important features required for the artificial compartment are that it isolates the interior from the external environment and is further functionalized to control the transport of target chemicals to regulate the interior concentration of both substrate and reaction products. In this study, an artificial compartment with size-selective molecular transport function was constructed by using a DNA origami-guided liposome prepared by modifying the method reported by Perrault et al. This completely isolates the liposome interior, including the DNA origami skeleton, from the external environment and allows the assembly of a defined number of molecules of interest inside and/or outside the compartment. By incorporating a bacterial membrane protein, OmpF, into the liposome, the resulting artificial compartment was shown to transport only the molecule of interest with a molecular weight below 600 Da from the external environment into the interior of the compartment.

Light energy transduction in liposome-based artificial cells.

In this work we review the latest strategies for the bottom-up assembly of energetically autonomous artificial cells capable of transducing light energy into chemical energy and support internalized metabolic pathways. Such entities are built by taking inspiration from the photosynthetic machineries found in nature which are purified and reconstituted directly in the membrane of artificial compartments or encapsulated in form of organelle-like structures. Specifically, we report and discuss recent examples based on liposome-technology and multi-compartment (nested) architectures pointing out the importance of this matter for the artificial cell synthesis research field and some limitations and perspectives of the bottom-up approach.

Controlled Assembly of Fluorophores inside a Nanoliposome.

Cellular compartmentalization plays an essential role in organizing the complex and multiple biochemical reactions in the cell. An artificial compartment would provide powerful strategies to develop new biochemical tools for material production and diagnosis, but it is still a great challenge to synthesize the compartments that encapsulate materials of interest while controlling their accurate locations, numbers, and stoichiometry. In this study, we evaluated chemical characteristics of a liposome-encapsulated compartment, which has great potential to locate various materials of interest with precise control of their locations and numbers in the compartment. A nanoliposome was constructed inside a ring-shaped DNA origami skeleton according to the method of Yang et al., and further equipped with a double-stranded DNA platform to assemble molecules of interest in the nanoliposome. Upon formation of the nanoliposome, a pH-sensitive fluorophore on the bridged platform showed little or no response to the pH change of the outer buffer, ensuring that the molecules assembled on the platform are effectively shielded from the outer environment. The ring-shaped DNA skeleton equipped with a double-stranded DNA platform allows spatial assembly of several functional molecules inside the nanoliposome to isolate them from the outer environment.

Unveiling the orientation and dynamics of enzymes in unstructured artificial compartments of metal-organic frameworks (MOFs).

Confining enzymes in well-shaped MOF compartments is a promising approach to mimic the cellular environment of enzymes and determine enzyme structure-function relationship therein. Under the cellular crowding, however, enzymes can also be confined in unstructured spaces that are close to the shapes/outlines of the enzyme. Therefore, for a better understanding of enzymes in their physiological environments, it is necessary to study enzymes in these unstructured spaces. However, practically it is challenging to create compartments that are close to the outline of an enzyme and probe enzyme structural information therein. Here, for proof-of-principle, we confined a model enzyme, lysozyme, in the crystal defects of a MOF via co-crystallization, where lysozyme served as the nuclei for MOF crystal scaffolds to grow on so that unstructured spaces close to the outline of lysozyme are created, and determined enzyme relative orientation and dynamics. This effort is important for understanding enzymes in near-native environments and guiding the rational design of biocatalysts that mimic how nature confines enzymes.

Lipidoid Artificial Compartments for Bidirectional Regulation of Enzyme Activity through Nanomechanical Action.

Photoresponsive inhibitor and noninhibitor systems have been developed to achieve on-demand enzyme activity control. However, inhibitors are only effective for a specific and narrow range of enzymes. Noninhibitor systems usually require mutation and modification of the enzymes, leading to irreversible loss of enzymatic activities. Inspired by biological membranes, we herein report a lipidoid-based artificial compartment composed of azobenzene (Azo) lipidoids and helper lipids, which can bidirectionally regulate the activity of the encapsulated enzymes by light. In this system, the reversible photoisomerization of Azo lipidoids triggered by UV/vis light creates a continuous rotation-inversion movement, thereby enhancing the permeability of the compartment membrane and allowing substrates to pass through. Moreover, the membrane can revert to its impermeable state when light is removed. Thus, enzyme activity can be switched on and off when encapsulating enzymes in the compartments. Importantly, since neither mutation nor modification is required, negligible loss of activity is observed for the encapsulated enzymes after repeated activation and inhibition. Furthermore, this approach provides a generic strategy for controlling multiple enzymes by forgoing the use of inhibitors and may broaden the applications of enzymes in biological mechanism research and precision medicine.

Formation of subcellular compartments by condensation-prone protein OsJAZ2 in Oryza sativa and Nicotiana benthamiana leaf cells.

OsJAZ2 protein has a propensity to form condensates, possibly by multivalent interactions, and can be used to construct artificial compartments in plant cells. Eukaryotic cells contain various membraneless organelles, which are compartments consisting of proteinaceous condensates formed by phase separation. Such compartments are attractive for bioengineering and synthetic biology, because they can modify cellular function by the enrichment of molecules of interest and providing an orthogonal reaction system. This study reports that Oryza sativa JAZ2 protein (OsJAZ2) is an atypical jasmonate signalling regulator that can form large condensates in both the nucleus and cytosol of O. sativa cells. TIFY and Jas domains and low-complexity regions contribute to JAZ2 condensation, possibly by multivalent interaction. Fluorescence recovery after photobleaching (FRAP) analysis suggests that JAZ2 condensates form mostly gel-like or solid compartments, but can also be in a liquid-like state. Deletion of the N-terminal region or the TIFY domain of JAZ2 causes an increase in the mobile fraction of JAZ2 condensates, moderately. Moreover, JAZ2 can also form liquid-like condensates when expressed in Nicotiana benthamiana cells. The recombinant JAZ2 fused to the green fluorescent protein (GFP) forms condensate in vitro, suggesting that the intermolecular interaction of JAZ2 molecules is a driving force for condensation. These results suggest the potential use of JAZ2 condensates to construct artificial membraneless organelles in plant cells.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Complex coacervates as artificial membraneless organelles and protocells.

Complex coacervates are water droplets dispersed in water, which are formed by spontaneous liquid-liquid phase separation of an aqueous solution of two oppositely charged polyelectrolytes. Similar to the membraneless organelles that exist in biological cells, complex coacervate droplets are membraneless and have a myriad of features including easy formation, high viscosity, selective encapsulation of biomolecules, and dynamic behaviors in response to environmental stimuli, which make coacervates an excellent option for constructing artificial membraneless organelles. In this article, I first summarize recent advances in artificial compartments that are built from coacervates and their response to changes in the surrounding environment and then show the advantages of microfluidic techniques in the preparation of monodisperse coacervates and encapsulation of coacervates in droplets and liposomes to construct complex cell-like compartments, and finally discuss the future challenges of such membraneless aqueous compartments in cell mimics and origin of life.

The interplay of spatial organization and biochemistry in building blocks of cellular signalling pathways.

Biochemical pathways and networks are central to cellular information processing. While a broad range of studies have dissected multiple aspects of information processing in biochemical pathways, the effect of spatial organization remains much less understood. It is clear that space is central to intracellular organization, plays important roles in cellular information processing and has been exploited in evolution; additionally, it is being increasingly exploited in synthetic biology through the development of artificial compartments, in a variety of ways. In this paper, we dissect different aspects of the interplay between spatial organization and biochemical pathways, by focusing on basic building blocks of these pathways: covalent modification cycles and two-component systems, with enzymes which may be monofunctional or bifunctional. Our analysis of spatial organization is performed by examining a range of 'spatial designs': patterns of localization or non-localization of enzymes/substrates, theoretically and computationally. Using these well-characterized in silico systems, we analyse the following. (i) The effect of different types of spatial organization on the overall kinetics of modification, and the role of distinct modification mechanisms therein. (ii) How different information processing characteristics seen experimentally and studied from the viewpoint of kinetics are perturbed, or generated. (iii) How the activity of enzymes (bifunctional enzymes in particular) may be spatially manipulated, and the relationship between localization and activity. (iv) How transitions in spatial organization (encountered either through evolution or through the lifetime of cells, as seen in multiple model organisms) impacts the kinetic module (and pathway) behaviour, and how transitions in chemistry may be impacted by prior spatial organization. The basic insights which emerge are central to understanding the role of spatial organization in biochemical pathways in both bacteria and eukaryotes, and are of direct relevance to engineering spatial organization of pathways in bottom-up synthetic biology in cellular and cell-free systems.

Adjustable Bioorthogonal Conjugation Platform for Protein Studies in Live Cells Based on Artificial Compartments.

The investigation of complex biological processes in vivo often requires defined multiple bioconjugation and positioning of functional entities on 3D structures. Prominent examples include spatially defined protein complexes in nature, facilitating efficient biocatalysis of multistep reactions. Mimicking natural strategies, synthetic scaffolds should comprise bioorthogonal conjugation reactions and allow for absolute stoichiometric quantification as well as facile scalability through scaffold reproduction. Existing in vivo scaffolding strategies often lack covalent conjugations on geometrically confined scaffolds or precise quantitative characterization. Addressing these shortcomings, we present a bioorthogonal dual conjugation platform based on genetically encoded artificial compartments in vivo, comprising two distinct genetically encoded covalent conjugation reactions and their precise stoichiometric quantification. The SpyTag/SpyCatcher (ST/SC) bioconjugation and the controllable strain-promoted azide-alkyne cycloaddition (SPAAC) were implemented on self-assembled protein membrane-based compartments (PMBCs). The SPAAC reaction yield was quantified to be 23% ± 3% and a ST/SC surface conjugation yield of 82% ± 9% was observed, while verifying the compatibility of both chemical reactions as well as enhanced proteolytic stability. Using tandem mass spectrometry, absolute concentrations of the proteinaceous reactants were calculated to be 0.11 ± 0.05 attomol/cell for PMBC surface-tethered mCherry-ST-His and 0.22 ± 0.09 attomol/cell for PMBC-constituting pAzF-SC-E20F20-His. The established in vivo conjugation platform enables quantifiable protein-protein interaction studies on geometrically defined scaffolds and paves the road to investigate effects of scaffold-tethering on enzyme activity.

Frontispiece: Gene Expression Inside Liposomes: From Early Studies to Current Protocols

Protein synthesis inside artificial compartments is under strong expansion in the context of synthetic biology (in bottom-up approaches), and, in particular, it strongly contributes to the construction of artificial cell-like systems. These systems, often called “synthetic cells”, can be used to model cellular processes, including membrane-centered ones. They are very innovative models that complement traditional studies and promise future applications. This article is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. For more details see Review by P. Stano on page 7798 ff.

Gene Expression Inside Liposomes: From Early Studies to Current Protocols.

Synthesizing proteins inside liposomes and other microcompartments is a well-established practice. However, the origin of this research is not from the distant past, dating back to 1999-2004, when the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in bottom-up approaches), and, in particular, it strongly contributes to the construction of artificial cell-like systems. These systems, often called "synthetic cells", can be used to model cellular processes, including membrane-centered ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular, it does not include all kinds of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is to note the importance of mastering liposome technology together with cell-free systems for the successful realization of this specific type of synthetic cell. With this aim, four currently employed protocols are compared and discussed, with a major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency.

학생의 다양성과 역사교육 : 학생의 역사 정체성 및 역사의식, ‘다원적 역사’에 대한 해외 연구 검토

This paper examines empirical studies of students from various ethnic backgrounds in Europe and North America, analyzing their historical identity and thinking about how to consider student diversity in history education. Many empirical studies conducted in the 1990s and early 2000s have examined and analyzed the relationship between the student’s historical identity and the student’s racial and ethnic backgrounds and found that the “given situation” of the student’s race, ethnicity, religion, gender influence the students’ selection of historical interpretation and narratives and their historical identities. However, recent studies have emphasized that not only students’ “given situation” but also their social awareness of social problems are important factors influencing their selections of historical significance and construction of their historical identities. Therefore, in historical education, the diversity of students needs to be redefined not only by the given situation of the student’s race, ethnicity, religion, gender but also crossing artificial compartments of such conditions.

Light-Driven Biocatalysis in Liposomes and Polymersomes: Where Are We Now?

The utilization of light energy to power organic-chemical transformations is a fundamental strategy of the terrestrial energy cycle. Inspired by the elegance of natural photosynthesis, much interdisciplinary research effort has been devoted to the construction of simplified cell mimics based on artificial vesicles to provide a novel tool for biocatalytic cascade reactions with energy-demanding steps. By inserting natural or even artificial photosynthetic systems into liposomes or polymersomes, the light-driven proton translocation and the resulting formation of electrochemical gradients have become possible. This is the basis for the conversion of photonic into chemical energy in form of energy-rich molecules such as adenosine triphosphate (ATP), which can be further utilized by energy-dependent biocatalytic reactions, e.g., carbon fixation. This review compares liposomes and polymersomes as artificial compartments and summarizes the types of light-driven proton pumps that have been employed in artificial photosynthesis so far. We give an overview over the methods affecting the orientation of the photosystems within the membranes to ensure a unidirectional transport of molecules and highlight recent examples of light-driven biocatalysis in artificial vesicles. Finally, we summarize the current achievements and discuss the next steps needed for the transition of this technology from the proof-of-concept status to preparative applications.

Characterization and application of chemical-resistant polyurethane-based enzyme and whole cell compartments

This study presents the preparation and physical-chemical characterization of chemical resistant polyurethane-based compartments for biocatalytic application. The artificial compartments were prepared from an emulsion of polymer precursor and an aqueous phase that includes a biocatalytic reaction system. After curing, highly dispersed aqueous domains were obtained, which still contain the entire biocatalytic reaction system and remain fixed in the solid polymer preparation. The tensile and compression behavior of the prepared polymeric material is not significantly affected by the incorporation and facilitates excellent stability against various organic solvents and acid solutions. Thereby, the compartments can be used not only for enantioselective alcohol-dehydrogenase catalyzed reduction but also for a whole cell catalyzed hydrolysis of esters. Moreover, the compartmented whole-cell system was considerably stable to allow multiple reuses without a noticeable loss of catalytic activity of the incorporated whole cell catalytic reaction system.

Biomimetic artificial organelles with in vitro and in vivo activity triggered by reduction in microenvironment

Despite tremendous efforts to develop stimuli-responsive enzyme delivery systems, their efficacy has been mostly limited to in vitro applications. Here we introduce, by using an approach of combining biomolecules with artificial compartments, a biomimetic strategy to create artificial organelles (AOs) as cellular implants, with endogenous stimuli-triggered enzymatic activity. AOs are produced by inserting protein gates in the membrane of polymersomes containing horseradish peroxidase enzymes selected as a model for natures own enzymes involved in the redox homoeostasis. The inserted protein gates are engineered by attaching molecular caps to genetically modified channel porins in order to induce redox-responsive control of the molecular flow through the membrane. AOs preserve their structure and are activated by intracellular glutathione levels in vitro. Importantly, our biomimetic AOs are functional in vivo in zebrafish embryos, which demonstrates the feasibility of using AOs as cellular implants in living organisms. This opens new perspectives for patient-oriented protein therapy.

Experiments on and Numerical Modeling of the Capture and Concentration of Transcription-Translation Machinery inside Vesicles.

Synthetic or semi-synthetic minimal cells are those cell-like artificial compartments that are based on the encapsulation of molecules inside lipid vesicles (liposomes). Synthetic cells are currently used as primitive cell models and are very promising tools for future biotechnology. Despite the recent experimental advancements and sophistication reached in this field, the complete elucidation of many fundamental physical aspects still poses experimental and theoretical challenges. The interplay between solute capture and vesicle formation is one of the most intriguing ones. In a series of studies, we have reported that when vesicles spontaneously form in a dilute solution of proteins, ribosomes, or ribo-peptidic complexes, then, contrary to statistical predictions, it is possible to find a small fraction of liposomes (<1%) that contain a very large number of solutes, so that their local (intravesicular) concentrations largely exceed the expected value. More recently, we have demonstrated that this effect (spontaneous crowding) operates also on multimolecular mixtures, and can drive the synthesis of proteins inside vesicles, whereas the same reaction does not proceed at a measurable rate in the external bulk phase. Here we firstly introduce and discuss these already published observations. Then, we present a computational investigation of the encapsulation of transcription-translation (TX-TL) machinery inside vesicles, based on a minimal protein synthesis model and on different solute partition functions. Results show that experimental data are compatible with an entrapment model that follows a power law rather than a Gaussian distribution. The results are discussed from the viewpoint of origin of life, highlighting open questions and possible future research directions.

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: Engineering attempts using a modified SNARE protein

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging.Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking.The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

Stochastic Turing patterns: analysis of compartment-based approaches.

Turing patterns can be observed in reaction-diffusion systems where chemical species have different diffusion constants. In recent years, several studies investigated the effects of noise on Turing patterns and showed that the parameter regimes, for which stochastic Turing patterns are observed, can be larger than the parameter regimes predicted by deterministic models, which are written in terms of partial differential equations (PDEs) for species concentrations. A common stochastic reaction-diffusion approach is written in terms of compartment-based (lattice-based) models, where the domain of interest is divided into artificial compartments and the number of molecules in each compartment is simulated. In this paper, the dependence of stochastic Turing patterns on the compartment size is investigated. It has previously been shown (for relatively simpler systems) that a modeler should not choose compartment sizes which are too small or too large, and that the optimal compartment size depends on the diffusion constant. Taking these results into account, we propose and study a compartment-based model of Turing patterns where each chemical species is described using a different set of compartments. It is shown that the parameter regions where spatial patterns form are different from the regions obtained by classical deterministic PDE-based models, but they are also different from the results obtained for the stochastic reaction-diffusion models which use a single set of compartments for all chemical species. In particular, it is argued that some previously reported results on the effect of noise on Turing patterns in biological systems need to be reinterpreted.