Actinosomes: Condensate-Templated Containers for Engineering Synthetic Cells

Abstract

Engineering synthetic cells has a broad appeal, from understanding living cells to designing novel biomaterials for therapeutics, biosensing, and hybrid interfaces. A key prerequisite to creating synthetic cells is a three-dimensional container capable of orchestrating biochemical reactions. In this study, we present an easy and effective technique to make cell-sized porous containers, coined actinosomes, using the interactions between biomolecular condensates and the actin cytoskeleton. This approach uses polypeptide/nucleoside triphosphate condensates and localizes actin monomers on their surface. By triggering actin polymerization and using osmotic gradients, the condensates are transformed into containers, with the boundary made up of actin filaments and polylysine polymers. We show that the guanosine triphosphate (GTP)-to-adenosine triphosphate (ATP) ratio is a crucial parameter for forming actinosomes: insufficient ATP prevents condensate dissolution, while excess ATP leads to undesired crumpling. Permeability studies reveal the porous surface of actinosomes, allowing small molecules to pass through while restricting bigger macromolecules within the interior. We show the functionality of actinosomes as bioreactors by carrying out in vitro protein translation within them. Actinosomes are a handy addition to the synthetic cell platform, with appealing properties like ease of production, inherent encapsulation capacity, and a potentially active surface to trigger signaling cascades and form multicellular assemblies, conceivably useful for biotechnological applications.

pH-Responsive Elastin-Like Polypeptide Designer Condensates

“…The final concentration of each ELP in the mixture was 25 μM with 10% fluorescently labeled ELP (22.5 μM unlabeled and 2.5 μM labeled). To trigger condensation, 1 μL freshly dissolved GDL 300 mg/mL was added (15 mg/mL final concentration) to 19 μL of PRE mixture in a custom polydimethylsiloxane (PDMS) well prepared as previously described . Time-lapse images were acquired using a Prime BSI Express sCMOS camera connected to a Nikon-Ti2-Eclipse inverted fluorescence microscope, equipped with a pE-300 ultra illumination light source.…”

“…The droplets generated were collected and stored in a glass vial at 4 °C. During experimentation, 5 μL of double emulsion dispersion and 5 μL of PBS 100 at pH 7.4 was added to a custom PDMS well . To trigger PRE condensation, 10 μL of PBS 100 at pH 2 was added to the solution and images were taken at an interval of 15 s.…”

Coacervate Droplets for Synthetic Cells

“…For instance, a 50‐fold enhancement in actin filament assembly rate was reported in complex coacervates. [ 137 ] The interface of complex coacervate has also been reported to play a critical role in promoting the formation of actin filaments [ 138 ] or α‐synuclein (αSyn) fibrils (Figure 5d). [ 139 ] Yet, other examples have shown that the coacervate interface could play a protective role against protein aggregation during folding via the sequestration of partly folded aggregation‐prone intermediates.…”

Methods to mechanically perturb and characterize GUV-based minimal cell models

“…Thus, one of the initial tasks in bottom-up reconstitution was to identify a confining substrate that closely mimics the function and chemical composition of the cell membrane. Among other protocell compartments such as droplets, coacervates enriched with charged molecules [10] , [11] , capsules made of polymeric amphiphiles (including block copolymers and peptides) [12] , [13] , [14] , [15] and proteinosomes [16] , [17] , GUVs made of lipid bilayers have been largely used to create a cell-sized (1–100 µm) confinement to encapsulate numerous cellular components. Like natural cell membranes, GUVs can be made from various compositions of lipids, mainly phospholipids and cholesterol, whose amphiphilic nature allow them to self-assemble into spherical compartments in an aqueous solution.…”