RESUMEN

The construction of a protocell with dynamic hierarchical structures via spontaneous phase separation sheds light on the mechanisms of life processes. Taking advantage of the transition from the liquid to solid phase, we built composite droplets with PLL/oligo/oligocomp solid particles randomly distributed inside a PLL/oligo liquid coacervate. The circulation and vacuolization under an electric field drive the particles into a fibrous structure and even clusters. A PLL/oligo/oligocomp solid phase can also form on the interface of the PLL/oligo coacervate, turning the droplet into a vesicular structure.

RESUMEN

Construction of protocells with hierarchical structures and living functions is still a great challenge. Growing evidence demonstrates that the membraneless organelles, which facilitate many essential cellular processes, are formed by RNA, protein, and other biopolymers via liquid-liquid phase separation (LLPS). The formation of the protocell in the early days of Earth could follow the same principle. In this work, we develop a novel coacervate-based protocell containing membraneless subcompartments via spontaneous liquid-liquid phase separation by simply mixing single-stranded oligonucleotides (ss-oligo), quaternized dextran (Q-dextran), and poly(l-lysine) (PLL) together. The resulting biphasic droplet, with PLL/ss-oligo phase being the internal subcompartments and Q-dextran/ss-oligo phase as the surrounding medium, is capable of sequestering and partitioning biomolecules into distinct regions. When the droplet is exposed to salt or Dextranase, the surrounding Q-dextran/ss-oligo phase will be gradually dissociated, resulting in the chaotic movement and fusion of internal subcompartments. Besides, the external electric field at a lower strength can drive the biphasic droplet to undergo a deviated circulation concomitant with the fusion of localized subcompartments, while a high-strength electric field can polarize the whole droplet, resulting in the release of daughter droplets in a controllable manner. Our study highlights that liquid-liquid phase separation of biopolymers is a powerful strategy to construct hierarchically structured protocells resembling the morphology and functions of living cells and provides a step toward a better understanding of the transition mechanism from nonliving to living matter under prebiotic conditions.

RESUMEN

Construction of protocell models from prebiotically plausible components to mimic the basic features or functions of living cells is still a challenge. In this work, we prepare a hybrid protocell model by coating sodium oleate on the coacervate droplet constituted by poly(l-lysine) and oligonucleotide and investigate the transport of different molecules under electric field. Results show that sodium oleate forms a layered viscoelastic membrane on the droplet surface, which is selectively permeable to small, polar molecules, such as oligolysine. As the droplet is stimulated at 10 V cm-1, the oleate membrane slips along the direction of electric field while maintaining its integrity. Most of the molecules are still excluded under such conditions. As repetitive cycles of vacuolization occur at 20 V cm-1, all molecules are internalized and sequestrated in the droplet through their specific pathways except enzyme, which anchors in the oleate membrane and is immune to electric field. Cascade enzymatic reactions are then carried out, and the product generated from the membrane exhibits a time-dependent concentration gradient across the droplet. Our work makes a step toward the nonequilibrium functionalization of synthetic protocells capable of biomimetic operations.

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RESUMEN

A hybrid protocell is constructed to investigate the membrane transport of neutral, cationic, and anionic molecules under non-equilibrium conditions. Each model molecule follows a specific pathway to be internalized and generates different distributions in the droplets. This work provides a step towards functionalization of synthetic protocells capable of biomimetic operations.

RESUMEN

Artificial protocells operating under non-equilibrium conditions offer a new approach to achieve dynamic features with life-like properties. Using coacervate micro-droplets comprising polylysine (PLL) and a short single-stranded oligonucleotide (ss-oligo) as a membrane-free protocell model, we demonstrate that circulation and vacuolization can occur simultaneously inside the droplet in the presence of an electric field. The circulation is driven by electrohydrodynamics and applies specifically to the major components of the protocell (PLL and ss-oligo). Significantly, under low electric fields (E = 10 V cm-1) the circulation regulates the movement of the vacuoles, while high levels of vacuolization produced at higher electric fields can deform or reshape the circulation. By taking advantage of the interplay between vacuolization and circulation, we achieve dynamic localization of an enzyme cascade reaction at specific droplet locations. In addition, the spatial distribution of the enzyme reaction is globalized throughout the droplet by tuning the coupling of the circulation and vacuolization processes. Overall, our work provides a new strategy to create non-equilibrium dynamic behaviors in molecularly crowded membrane-free synthetic protocells.

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RESUMEN

Protein-mediated endocytosis of membrane is a key event in biological system. The mechanism, however, is still not clear. Using a de novo designed bola-type peptide KKKLLLLLLLLKKK (K3L8K3) as a protein mimic, we studied how it induced giant unilamellar vesicle (GUV) to form inward buds or endocytosis at varying conditions. Results show that the inward budding is initiated as the charged lipids are neutralized by K3L8K3, which results in a negative spontaneous curvature. If the charged lipids have unsaturated tails, the buddings are slim fibrils, which can further wrap into a spherical structure. In the case of saturated charged lipids, the buddings are rigid tubules, stable in the studied time period. The unsaturated lipid to saturated lipid ratio in the mother membrane is another key parameter governing the shape and dynamics of the buds. A complete endocytosis is observed when K3L8K3 is attached with a hydrophobic moiety, suggesting that hydrophobic interaction helps the buds to detach from the mother membrane. The molecules in the surrounding medium, such as negatively charged oligonucleotides, are engulfed into the GUV via endocytosis pathway induced by K3L8K3. Our study provides a novel strategy for illustrating the endocytosis mechanism by using peptides of simple sequence.

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