Liquid-liquid phase separation of α-Synuclein: A new mechanistic insight for α-Synuclein aggregation associated with Parkinson's disease pathogenesis.

The origin of life on Earth remains one of the biggest open questions in science. Despite recent advances in molecular mechanism of cell biology, a large blind spot still exists between non-living matter and the emergence of life which cannot be filled by biology alone. The quest to comprehend the cell origin inspires the construction of synthetic analogs (protocells) to mimic their life-like functionality and structural complexity. Among all kinds, coacervates formed by liquid-liquid phase separation featured by their dynamic structure, molecularly crowded interior and molecular sequestration capability, have been regarded as a protocell model for exploring the origin of life. Their biological counterparts in natural cells are also found to facilitate subcellular organization and spatiotemporal regulation of biological molecules. In this tutorial review, we summarize the recent progress on engineering coacervate protocells to potentially reproduce the function and structure of primitive life by multiphase organization, membranization and structural hybridization. Routes to high-ordered protocellular system comprising spatially arranged coacervates, as well as to the construction of tissue-like structures are also described. Finally, we deduce some perspectives of coacervate engineering in the direction of the emergence of life, from molecular scale to the emergence of integrated multicellular system. 

Engineering coacervate droplets towards the building of multiplex biomimetic protocells

A rapidly growing body of work in recent years has resulted in exciting advances in our understanding of the importance of biomolecular condensates (or, more generally, various forms of mesoscale to macroscale biological matter) and their transitions in biology and disease (1–3). At first glance, the physics of how liquids percolate through porous/granular materials or related concepts in network connectivity may not seem relevant to furthering our mechanistic understanding of biomolecular condensates. Interestingly, however, percolation theory has been extensively used in the related areas of polymer physics (4, 5) and phase transitions (as well as in numerous other fields). Now, in an exciting advance, Kar et al. (6) describe a combination of experimental, conceptual, and computational work that explores the connection between percolation physics and an important class of biomolecular condensation with links to neurodegenerative diseases. Conceptual understanding in the biomolecular condensate field has been extensively guided by simple forms of nucleation and Flory–Huggins-type theories. A prediction of this type of theory is that, below a saturation concentration (csat) of a single macromolecule (e.g., protein) in a solvent, the macromolecule will exist mainly as monomers and very small clusters, because there is a size-dependent energy penalty for cluster formation. It is only above the saturation concentration that phase separation (a density transition) will occur, resulting in the formation of a dense phase (aka micrometer-sized droplets). Now, Kar et al. (6) describe a broad set of data that can provide a test of this prediction. The proteins studied in this work are FET (FUS, EWSR1, TAF15) family proteins, with links to neurodegenerative disease, which have been extensively investigated in the field. Using a combination of imaging, dynamic light scattering (DLS), and single-particle (tracking, multiparameter fluorescence, and microfluidics-based) experiments, Kar et al. (6) show that, while phase separation is not observed in solutions below an effective csat, subsaturated solutions of these proteins contain a range of nanoscale clusters. The data indicate that clusters follow a heavytailed distribution, with low abundance of larger mesoscale clusters and distributions changing with total protein concentration. It is only above csat that larger micrometer-sized bodies that display coarsening appear. Fluorescence resonance energy transfer/DLS data show that cluster formation is reversible, and that protein exchanges between clusters. Together, these data draw a sharp contrast with the predictions based on nucleation theory discussed above. The authors then go on to invoke percolation theory–based ideas to offer an explanation for these observations, building on their and other previous work (4, 5, 7, 8). In percolation theory, there exists a critical connectivity probability (pc) at which the system undergoes a connectivity (geometric) transition, forming a system-spanning network (Fig. 1A). In the present work, the proteins are represented in a stickers and spacers model of associative polymers, with stickers contributing specific protein–protein interactions and spacers contributing generalized excluded volume/ solvation effects. In the framework of the percolation model, this system can undergo a percolation-type connectivity transition (via specific sticker–sticker interactions) at a critical protein concentration cperc (Fig. 1B). Thus, specific sticker– sticker interactions give rise to an additional energy scale class that contributes to system behavior. As previously described by the Pappu laboratory (8), this scenario can give rise to an interesting coupling between density and percolation transitions if the system/conditions result in cperc being greater than csat but less than the dense phase concentration cden. Under these conditions, a density transition results in a coupled percolation transition in the dense phase, since cden is greater than cperc. How does this relate to the authors’ experimental findings of molecular clusters below csat (6)? The key result from the percolation approach is that, even below the percolation threshold, smaller network clusters are still formed (Fig. 1), and the size distribution of these clusters shifts to larger sizes as the connectivity (concentration) is increased. As noted above, this is just what was observed in the authors’ measurements. The authors then looked at ways to test the coupling between the two types of transitions. Indeed, using small-molecule solutes or mutations as perturbations, they find either coupled or differential effects on formation of clusters and macroscopic phase separation. These results are consistent with the existence of separate types of interactions governing generalized solubility and specific connectivity effects, and the idea that these can be perturbed selectively but can also be coupled. The above work is also complemented with simulations whose results are in keeping with the above model. Overall, the work serves to inspire a number of lines of thinking and inquiry.

Percolation physics and density transition frameworks converge in biomolecular condensation

Conformational Freedom and Topological Confinement of Proteins in Biomolecular Condensates.

Conformational Freedom and Topological Confinement of Proteins in Biomolecular Condensates

Phase transitions and coacervates play key roles in natural and synthetic soft matter. In particular, the past few years have seen a rapid expansion in studies of these phenomena in the context of dynamic cellular compartmentalization. In this brief review, we mainly focus on a few concepts and selected in vitro and cellular examples of recent developments in the areas of dynamics and multicomponent systems. Topics covered include the flexibility and conformational dynamics of polymeric species involved in phase separation, valence and non-monotonic effects, noise modulation and feedback loops, and multicomponent systems and substructure. The fundamental concepts discussed in this review are widely applicable, including in the context of cellular function and the development of materials with novel properties.

Complex dynamics of multicomponent biological coacervates

RNA–RNA interactions have increasingly been recognized for their potential to shape the mesoscale properties of biomolecular condensates, influencing morphology, organization, and material state through networking interactions. While most studies have focused on networking via Watson–Crick base pairing interactions, previous work has suggested a potential for noncanonical RNA–RNA interactions to also give rise to condensation and alter overall material state. Here, we test the phase separation of short polyA RNA (polyrA) homopolymers. We discover and characterize the potential for short polyrA sequences to form RNA condensates at lower Mg2+ concentrations than previously observed, which appear as internally arrested droplets with slow polyrA diffusion despite continued fusion. Our work also reveals a negative cooperativity effect between the effects of Mg2+ and Na+ on polyrA condensation. Finally, we observe that polyrA sequences can act as promoters of phase separation in mixed sequences. These results demonstrate the potential for noncanonical interactions to act as networking stickers, leading to specific condensation properties inherent to polyrA composition and structure, with implications for the fundamental physical chemistry of the system and function of polyA RNA in biology.

Jenna K.A. Tom

Papers

Complex dynamics of multicomponent biological coacervates

Short PolyA RNA Homopolymers Undergo Mg2+-Mediated Kinetically Arrested Condensation

Non-canonical interactions tune condensation and kinetically arrested material states of short RNA homopolymers.