The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells1. Principles of biomolecular condensation have been revealed through in vitro reconstitution2. However, intracellular environments are much more complex than test-tube environments: They are viscoelastic, highly crowded at the mesoscale, and are far from thermodynamic equilibrium due to the constant action of energy-consuming processes3. We developed synDrops, a synthetic phase separation system, to study how the cellular environment affects condensate formation. Three key features enable physical analysis: synDrops are inducible, bioorthogonal, and have well-defined geometry. This design allows kinetic analysis of synDrop assembly and facilitates computational simulation of the process. We compared experiments and simulations to determine that macromolecular crowding promotes condensate nucleation but inhibits droplet growth through coalescence. ATP-dependent cellular activities help overcome the frustration of growth. In particular, actomyosin dynamics potentiate droplet growth by reducing confinement and elasticity in the mammalian cytoplasm, thereby enabling synDrop coarsening. Our results demonstrate that mesoscale molecular assembly is favored by the combined effects of crowding and active matter in the cytoplasm. These results move toward a better predictive understanding of condensate formation in vivo.

The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.

Life emerges from thousands of biochemical processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochemical reactions. However, the reaction medium in test tubes is typically simple and diluted. The cell interior is far more complex: macromolecules occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromolecules, with an emphasis on mesoscale particles (10-1000 nm diameter). We describe methods to probe and analyze the biophysical properties of cells and highlight how changes in these properties can impact physiology and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration.

Phase separation can concentrate biomolecules and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects associated with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concentration within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromolecular crowding. Finally, the phosphorylation of the Alzheimer's-disease-associated protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophysical properties of the cytoplasm.

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleus because previous methods were labor-intensive and perturbative.

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

Synaptic vesicle fusion is mediated by SNARE proteins—VAMP2 on the vesicle and Syntaxin‐1/SNAP25 on the presynaptic membrane. Chaperones Munc18‐1 and Munc13‐1 cooperatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin‐1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13‐1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single‐molecule optical tweezer studies. Detailed structure–function analyses show that the binding is mediated by the Munc13‐1 MUN domain and is specific for the SNAP25 ‘linker’ region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~ 1 : 1 stoichiometry by the self‐assembled Munc13‐1 nanoclusters.

Recent studies have shown that liquid-liquid phase separation (LLPS) organizes cellular biochemical reactions through the formation of membrane-less organelles. Cells are extremely crowded, with up to 40% of the cell volume taken up by macromolecules. This molecular crowding can strongly affect LLPS. Moreover, the intracellular environment is far from equilibrium due to many energy-dependent active processes. We sought to understand the influence of molecular crowding and active processes on LLPS by combining both experimental and computational studies of an engineered synthetic LLPS system. We discovered that macromolecular crowding increases the kon rate of biochemical bond formation, shifts the critical point for LLPS, affects the kinetics of initial droplet formation, and stabilizes the condensed state. in addition, active processes outside the droplets in the cytoplasm promote the coarsening of individual liquid droplets. Our work highlights the importance of molecular crowding and active processes for LLPS.

Significance Neurons in the brain communicate with each other by release of neurotransmitters. Neurotransmitter release is mediated by 3 membrane-anchored SNARE proteins and various regulatory proteins, including Munc13-1 and Munc18-1. SNAREs couple their folding and assembly to membrane fusion in a regulatory protein-dependent manner. The physiological pathway of the regulated SNARE assembly, however, is unclear. We found that the MUN domain of Munc13-1, Munc18-1, and 2 SNAREs—syntaxin 1 and VAMP2—associate into a weak tetrameric complex. The third SNARE protein, SNAP-25B, rapidly binds the 2 SNAREs in the complex to form a ternary SNARE complex likely with a displacement of the regulatory proteins. Therefore, Munc13-1 and Munc18-1 cooperatively chaperone SNARE assembly, a process required for neurotransmitter release.