Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds

Abstract

Summary Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce an oligomerizing biomimetic system, “Corelets”, and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs, and the mode of phase separation. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

“…[5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains. [12,20,21] Owing to their liquid-like nature and the weak molecular interactions,s ynthetic coacervate droplets can dynamically respond to external stimuli.…”

“…[7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains.…”

“…[15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains. [39][40][41] Here,w ee xploit the attractive electrostatic interactions between double stranded DNA(dsDNA) and alight-responsive azobenzene cation (trans-azobenzenetrimethylammonium bromide; trans-azoTAB) to produce liquid-like coacervate microdroplets that can be disassembled and reassembled within afew seconds with UV and blue light, respectively ( Figure 1a).…”

Expression and phase separation potential of heterochromatin proteins during early mouse development

“…Molecular interactions between positively charged amino acids with nucleic acids also certainly play a role in the establishment of membrane-less organelles enriched in RNA and RNA-binding proteins [55,62]. In agreement with the importance of electrostatic interactions between macromolecules with different charges, phosphorylation and acetylation have been shown to perturb phase separation and dissolve membrane-less organelles [62][63][64][65]. Hydrophobic interactions have also been suggested to play an important role in phase separation [35,66].…”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…[113] Al ight-responsive aqueous two-phases ystem based on PEG and spiropyran-functionalised dextran macromolecules was also reported. In one notable example, Brangwynne and co-workersd esigned protein constructs capable of self-association and subsequentp hotostimulated LLPS in cellulo,t hus resulting in spatiotemporal control of biomolecular condensation in living cells.…”