Abstracts for parallel session on Thursday May 19th

Compartments and confinements (I&II)

3.1: Polymeric biomimetic multicompartment vesicles

Tsvetomir Ivanov¹, Shoupeng Cao¹, Shuai Jiang², Katharina Landfester¹, Lucas Caire da Silva¹

1: Max Planck Institute for Polymer Research, Germany;  2: Ocean University of China, China

Biomimetic multicompartment vesicles (BMVs) are artificial systems that mimic the architecture of eukaryotic cells. BMVs consist of synthetic sub-compartments encapsulated within a larger compartment that serves as a host. In this way, a multicompartment structure is created that, like in cells, enables the organization of chemical reactions and other processes with high spatiotemporal control. Vesicles made of amphiphilic copolymers have demonstrated superior mechanical properties and unmatched chemical versatility compared to lipids, making them excellent materials for engineering robust BMVs with tunable properties. However, few methods exist for producing well-defined polymeric BMVs. In this presentation, we will describe the engineering of polymeric BMVs using a microfluidic approach that allows the precise encapsulation of structural and active material into giant polymeric vesicles. Micron-sized polymer vesicles were produced with controllable membrane permeability, narrow size distribution, and high throughput. These well-defined microcompartments were used to construct BMV systems with different functions determined by the encapsulation of active components. In the first example, we will describe a hybrid microreactor constructed via the precise encapsulation of enzyme-containing silica nanoreactors that served as internal organelles. We will also discuss the construction of photo-responsive adaptive microreactors with sub-compartments made of coacervates droplets that can be dynamically formed and dissolved in a stimuli-responsive manner. The combination of precise microfluidic methods with robust polymeric compartmentalization offers a powerful platform for the construction of multi-functional BMVs.

3.2: Bacterial microcompartments follow substrate gradients by the mechanisms of stabilitaxis

Jan Steinkühler¹, Charlotte H. Abrahamson¹, Jaime Agudo-Canalejo², Ramin Golestanian², Danielle Tullman-Ercek¹, Neha P. Kamat¹

1: Northwestern University, United States of America;  2: Max Planck Institute for Dynamics and Self-Organization, Germany

Chemotaxis, or the directed movement of substance or particle in a concentration gradient, is a prevalent phenomenon across a wide range of systems and scales that include organisms, eukaryotic cells, bacteria, colloids, enzymes. The mechanism for such behavior can vary depending on the identity of the moving particle as well as its environment. A recently proposed mechanism, known as stabilitaxis, results in particle drift towards regions in which particle-particle interactions are favored, and the presence of chemical gradients can modulates such interactions. Here, we study bacterial microcompartments (MCP), enzyme loaded protein capsules of 40 to 200 nm diameter. Considered a kind of bacterial organelle, these structures consist of shell proteins and can be genetically designed to encapsulate enzymes and fluorescent proteins. Because they are amenable to genetic engineering, they are an ideal platform to study the physics of active compartments. We first test our design strategy using a computational model that is based on MCP-MCP interactions in a substrate gradient. After identifying important design parameters, we show that bacterial microcompartments are indeed chemotactic by a stabilitactic mechanism. The modular character of MCPs then allowed us to study the effect of activity in the experimental system by designing MCPs that catalyze a substrate or are inactive, respectively. Through comparison to our computational model, we show how enzymatic activity protects MCPs against large-scale aggregation. In this way we introduce a modular chemotactic platform that allows us to control and direct chemical activity that should provide new capabilities in the design of synthetic cells.

3.3: Assembly of synthetic organelles in shrinking lipid vesicles modulated by osmotic stress

Clémence Schvartzman, Hang Zhao, Emmanuel Ibarboure, Vusala
Ibrahimova, Elisabeth Garanger, Sébastien Lecommandoux

Laboratoire de Chimie des Polymères Organiques (LCPO), France

Most membraneless organelles form through the liquid-liquid phase separation (LLPS) of proteins and RNA, related to cellular organization. In nature, cells are regularly confronted to osmotic stress. As a response to these variations in osmolarity, they have developed complex mechanisms to release their excess content of water [1], avoiding them to burst and die. As water is expelled, cells shrink, concentrating and inducing the liquid-liquid phase separation of the inner biomolecules into membraneless organelles.

We aim to mimic this intrinsic property of cells by incorporating functionalized thermoresponsive Elastin-Like Polypeptides (ELPs) into vesicles as simple models of artificial cells. By inducing a hypertonic shock onto our vesicles, the increase of ELP-PEG [2] bioconjugates’ concentration inside of the vesicles induces a shift of its cloud point (Tcp) to lower temperatures. Consequently, the bioconjugates assemble into coacervates, mimicking cellular organelles (Figure 1). The use of such osmotic variation rather than temperature to trigger this phenomenon is an elegant way to prevent any damage on biological materials such as enzymes. Finally, we moved from using non-functional ELP-PEG bioconjugates to functional ones involving HRP as a model enzyme (ELP-HRP). As synthetic organelles are formed through coacervation, HRP concentration will locally increase and enhance the enzymatic reaction kinetics.

References
[1] S. Majumder and A. Jain, Mol. Cell, 2020, 79, 876–877.
[2] H. Zhao, E. Ibarboure, V. Ibrahimova, Y. Xiao, E. Garanger and S. Lecommandoux, Adv. Sci., 2021, 2102508, 2102508.

3.4: Programmable zwitterionic droplets as model of membraneless organelles

Umberto Capasso Palmiero, Paolo Arosio

ETH Zurich, Switzerland

It is emerging that cells can regulate biochemical functions in time and space by generating membraneless compartments with well-defined composition. One important mechanism underlying this control is simple coacervation driven by disordered protein regions that encode multivalent interactions. Inspired by these observations, here we develop programmable droplets based on simple coacervation of synthetic responsive polymers that mimic the architecture of these biological disordered protein regions. We have adopted a bottom-up approach starting from zwitterionic polymers, demonstrating that they can form liquid droplets that largely exclude most molecules. Starting from this reference material we have progressively added different functional groups in the polymer architecture to induce an increasing number of different intermolecular interactions. We show that with this strategy we can independently control multiple mesoscopic properties of the droplets, such as stimulus responsiveness, polarity, selective uptake of client molecules, fusion time, and miscibility. By exploiting this high programmability we reproduce a model of cellular compartmentalization and generate droplets capable to localize different molecules in space without physical barriers. Moreover, we demonstrate that these biomolecular sorters can localize, separate and enable the detection of target molecules even within complex mixtures, opening attractive applications in bioseparation and in diagnostics.

Reference:
U. Capasso Palmiero et al. Advanced Materials 34(4) (2022): 2104837

3.5: Light-responsive coacervates with non-enzymatic ligase activity

Nicolas Martin¹, Tommaso Fraccia²

1: University Bordeaux, CNRS, France;  2: ESPCI Paris, PSL Univ., CNRS, France

Compartmentalization is essential for the coordination of biochemical reactions in living cells. Membraneless organelles are increasingly recognized as a mean to organize intracellular contents. These condensates are dynamically formed and dissolved in response to environmental stimuli, which allows cells to orchestrate reactions in space and time. Coacervate droplets produced by associative liquid-liquid phase separation between oppositely charged polyions represent promising models of membraneless organelles.[1] It is yet still challenging to precisely control the formation/dissolution and function of coacervate droplets in space and time. Recently, we have designed oligonucleotide-based photoswitchable coacervates that are reversibly formed and dissolved in response to light.[2] Using chemically reactive oligonucleotides, we have shown that coacervates droplets significantly enhance non-enzymatic oligonucleotide polymerization.[3] Dynamical modulation of coacervate assembly and dissolution with light is used to demonstrate on/off cycles of oligonucleotide ligation. Excitingly, changes in the length distribution of oligonucleotides during polymerization induce the formation of multiphase droplets via the segregation of longer polynucleotides into sub-domains. Overall, our light-responsive coacervates with ligase activity provide a novel general route to the enzyme-free synthesis of polynucleotides that could be used as verstaile modules to build advanced functional synthetic cells.

References:
[1]. N. Martin, ChemBioChem 2019, 20, 2553-2568.   [2]. N. Martin, et al., Angew. Chem. Int. Ed. 2019, 58, 14594-14598.   [3]. T.P. Fraccia, N. Martin, ChemRxiv, 2021 (https://doi.org/10.26434/chemrxiv-2021-zg20r).

3.6: Minimal surfactant-free microfluidic setup for the generation of polymeric cell-like bioreactors

Tsvetomir Ivanov, Lucas Caire da Silva^*, Nitin Bohra, Shoupeng Cao, Katharina Landfester^*

Max Planck Institute for Polymer Research, Germany

Compartmentalization is an essential feature of cells. The ability to control the formation, distribution, and function of compartments is essential for life. In recent years, polymersomes have emerged as viable synthetic compartments for basic and applied studies of synthetic cells. Their chemical versatility and robustness make them stable compartments. However, due to the usually high molecular weight of the blocks used to make them, polymersomes are characterized by thick, non-permeable membranes that limit their application as biomimetic compartments. In this study, we developed a novel minimal microfluidics method to prepare monodisperse semipermeable polymersomes. The polymeric compartments were prepared without the addition of surfactants by using low molecular weight diblock copolymers as membrane components. We have shown that low molecular weight polymers can play the dual role of surfactant and membrane component when a suitable organic phase is used in the formation of double emulsion droplets by microfluidics. The developed method allowed the preparation of monodispersed polymersomes within only one minute at high throughput. The versatility of the low molecular weight polymersomes was demonstrated by their application as biomimetic compartments in the construction of cell-like bioreactors. The bioreactors were based on the encapsulation of biomolecules and pH-sensitive coacervate droplets. The method described in this contribution provides a versatile platform for the development of polymer-based cell-like systems, such as delivery systems, bioreactors, and synthetic cells.

3.7: Actinosomes: condensate-templated proteinaceous confinements for engineering synthetic cells

Ketan Ganar, Siddharth Deshpande

Wageningen University and Research, the Netherlands

Engineering synthetic cells has a broad appeal, from understanding the existing biological cells in a bottom-up manner to designing novel biomaterials for therapeutics, biosensing, and hybrid interfacing systems. Our vision of synthetic cell is a functional three-dimensional micro-confinement capable of organizing biochemical reactions as well as communicating with the environment. While membranous vesicles like liposomes and polymersomes have been widely used as cell-mimicking containers, they suffer from various limitations regarding encapsulation, permeability, and stability. In this study, we exhibit an easy and robust technique to make proteinaceous porous containers crafted using actin cytoskeleton, hence the name actinosome. Our approach takes the advantage of biomolecular condensates comprised of polylysine and nucleotides (ATP + GTP) to sequester and spatially organize monomeric actin. By triggering actin polymerization at the expense of ATP hydrolysis, we obtain hollow confinements whose boundary is made up of actin filaments and poly-L-Lysine polymers. Upon systematic investigation, we find that ATP:GTP ratio is crucial to forming actinosomes. The inherent property of condensate to sequester biomolecules facilitates easy and efficient encapsulation of complex biological mixtures. We demonstrate this by carrying out protein expression inside the actinosomes. We believe actinosome is a handy addition to the synthetic cell platform, with appealing properties like the ease of production, high sequestration capacity, and an active surface that holds the potential to trigger signaling cascades, forming multicellular assemblies, and biomedical applications.

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