Nucleation landscape of biomolecular condensates

Abstract

Forming at the right place and time is important for all structures within living cells. This includes condensates such as the nucleolus, Cajal bodies, and stress granules, which form via liquid-liquid phase separation (LLPS) of biomolecules, particularly proteins enriched in intrinsically-disordered regions (IDRs) 1, 2 (Fig. 1a). In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modeled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, while the location at which droplets appear is controlled by interfacial heterogeneities 3,4 . In living cells, however, it is unknown whether this framework applies, due to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear [5][6][7][8] . Here we show that despite this complexity, nucleation within living cells occurs through a physical process not unlike that within inanimate materials, but where the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates within living cells, we found that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be significantly enhanced by compatible biomolecular (IDR) seeds, while the kinetics of cellular processes can impact condensate nucleation rates and location specificity. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time.

A rich get richer effect governs intracellular condensate size distributions

“…However, in more typical rapidly dividing mammalian cells, nuclear bodies, such as nucleoli and speckles 8,17 , tend to appear following mitosis and coalesce 18 , but then maintain relatively stable sizes until the next cell division, likely in part due to strong mechanical constraints from chromatin [19][20][21][22] . This picture is consistent with other recent work that has taken advantage of synthetic nuclear condensates, which form quickly 23 but grow and coalesce slowly due to their constrained, subdiffusive motion 20 .…”

“…Here, we have shown that both native nuclear speckles and synthetic condensates form via a quench-then-coalesce mechanism, which may present a common mechanism for size control of condensates. Indeed, several native organelles within the nucleus, including nuclear speckles 8 but also nucleoli 23 , Cajal bodies 23 , histone locus bodies, paraspeckles, and PML bodies, all disassemble or scatter into the cytoplasm during mitosis 18 , likely due to a combination of dilution and post–translational modifications 19,41 , and then nucleate and regrow in the resulting daughter nuclei. Our results show that a relatively narrow, exponentially decaying size distribution will be attained for simple regrowth driven by coalescence.…”

Spatial organization of chromosomes leads to heterogeneous chromatin motion and drives the liquid- or gel-like dynamical behavior of chromatin

“…Recent progresses in multiple loci (Zhou et al 2017) and in super-resolution (Alexander et al 2019;Chen et al 2018;Brandão et al 2020;Barth et al 2020) live imaging would make it possible to test quantitatively the relations between mobility, coherent motion and compaction predicted by our polymer models and the role of basic mechanisms such as phase separation or loop extrusion in regulating chromosome dynamics. For example, live-imaging the coupled structural and dynamical responses of chromatin after rapid in vivo perturbations of the amount of key chromatin-binding complexes using the auxin-inducible-degron system (Schwarzer et al 2017;Dobrinić et al 2021) or after modifications of their self-interacting capacities using optogenetics (Shin et al 2019;Shimobayashi et al 2021) would allow to directly investigate how chromatin dynamics respond to changes in chromatin compaction.…”

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

“…Above a critical radius a * , the free energy of cluster formation overcomes the interfacial penalty, and the new dense phase grows in a thermodynamically downhill fashion. Recently, these ideas from classical nucleation theory were applied to analyze and interpret the dynamics of phase separation in supersaturated solutions in cells (12, 13, 15).…”