Specific Spatial Localization of Actin and DNA in a Water/Water Microdroplet: Self‐Emergence of a Cell‐Like Structure

Abstract

The effect of binary hydrophilic polymers on a pair of representative bio‐macromolecules in a living cell has been examined. The results showed that these bio‐macromolecules exhibited specific localization in cell‐sized droplets that were spontaneously formed through water/water microphase segregation under crowding conditions with coexisting polymers. In these experiments, a simple binary polymer system with poly(ethylene glycol) (PEG) and dextran (DEX) was used. Under the conditions of microphase segregation, DNA was entrapped within cell‐sized droplets rich in DEX. Similarly, F‐actin, linearly polymerized actin, was entrapped specifically within microdroplets rich in DEX, whereas G‐actin, a monomeric actin, was distributed evenly inside and outside these droplets. This study has been extended to a system with both F‐actin and DNA, and it was found that DNA molecules were localized separately from aligned F‐actin proteins to create microdomains inside microdroplets, reflecting the self‐emergence of a cellular morphology similar to a stage of cell division.

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[55] Low-molecular-weight solutes do not usually exhibit high levels of partitioning in neutral polymer ATPSs. In PEG/dextranA TPSs, single-stranded RNA [57] and double-stranded (ds) DNA [58] preferentially accumulate in the dextran phase, with increased partition coefficients reported for longerp olynucleotides. [19,21,52] Small biomolecules have also been used as scaffold solutes to assemble membrane-free droplets.…”

“…[7] In contrast, complexc oacervate droplets have been found to sequester small solutes,i ncluding ions, [56] through complementary electrostatic interactions, together with hydrophobic effects associated with the lower dielectric constant of the droplets' interiors relative to the dilute phase. [57,58] The polynucleotide exchange rate between the dropletsa nd their environment appears to be highly system-dependent. [21,52,56] In one notable example, Koga et al demonstrated the formation of coacervate microdroplets from nucleoside triphosphates (ATP), diphosphates (ADP,F AD, NAD) or monophosphates (AMP) mixed with oppositely charged oligolysine chains,and reported a20-fold increase in ATPc oncentration relative to the dilute continuous phase.…”

“…It revealed complex and unevenp artitionp atterns depending on the polymers used. [58] Cytoskeletonr econstitution has also been investigated in dropletsf ormed by associative phase separation. [151] Spontaneous sequestration of actin filaments in PEGsuspended dextran-rich microdroplets was also reported, and the accumulation of bundled filaments at the droplets urface was shown to induce dropletdeformations.…”

“…[21] Oligo-and polynucleotides also partition unevenly in LLPSbased droplets. In PEG/dextranA TPSs, single-stranded RNA [57] and double-stranded (ds) DNA [58] preferentially accumulate in the dextran phase, with increased partition coefficients reported for longerp olynucleotides. [57,58] The polynucleotide exchange rate between the dropletsa nd their environment appears to be highly system-dependent.…”

“…In PEG/dextranA TPSs, single-stranded RNA [57] and double-stranded (ds) DNA [58] preferentially accumulate in the dextran phase, with increased partition coefficients reported for longerp olynucleotides. [57,58] The polynucleotide exchange rate between the dropletsa nd their environment appears to be highly system-dependent. Rapid exchange of RNA oligomers, for instance, was reported in aP EG/dextranA TPS and in polylysine/ATP coacervate droplets, [59] yet very little exchange of single-stranded (ss) DNA oligomers was observed for up to 48 hi nabinary poly(diallyldimethylammonium bromide) (PDDA)/ATP coacervate droplet population produced by microfluidics.…”