Local thermodynamics governs the formation and dissolution of protein condensates in living cells

Abstract

Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm C. elegans which play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams which are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intra-cellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the non-equilibrium nature of the cytoplasm is manifested on larger scales.

Biomolecular condensates at sites of DNA damage: More than just a phase

“…As living cells are not at equilibrium, the definition of a phase broadens, yet without losing its general meaning or applicability. Accordingly, a polymer core is not per se incompatible with multi-layered condensate organization through percolation (an example for a cellular compartment with multiple co-existing phases being the nucleolus [ 20 ]), and local thermodynamic equilibria exist at mesoscopic scales despite living cells being out of equilibrium [ 120 – 122 ]. Phase separation and the regulation of intracellular condensates by active processes are therefore not mutually exclusive mechanisms, and a number of energy-consuming processes, including different post-translational modifications (PTMs), have been shown to participate in the spatio-temporal regulation of phase separation [ 9 , 117 ].…”

Metabolic resilience is encoded in genome plasticity

“…Formation of self-organized assemblies synchronizes with metabolic fluctuations to better use thermodynamic states favouring phase transitions (8,9). Coupling the degree of self-organization with favourable states such as cold and fasting has implications on their tuning, and more broadly on cellular and evolutionary adaptations (8,9). Cold and fasting, through reduced cytosolic volume, promote cell crowding and less molecular diffusion, which increases molecular interactions and assembly of condensates (9)(10)(11)(12)(13).…”

“…Coupling the degree of self-organization with favourable states such as cold and fasting has implications on their tuning, and more broadly on cellular and evolutionary adaptations (8,9). Cold and fasting, through reduced cytosolic volume, promote cell crowding and less molecular diffusion, which increases molecular interactions and assembly of condensates (9)(10)(11)(12)(13). Since their formation is driven by entropic-forces and less by ATP (9,14,15), elucidating their range of parametrization is crucial to understand cellular adaptations (8).…”

“…Formation of self-organized assemblies synchronizes with metabolic fluctuations to better use thermodynamic states favouring phase transitions (8,9). Coupling the degree of self-organization with favourable states such as cold and fasting has implications on their tuning, and more broadly on cellular and evolutionary adaptations (8,9).…”

“…Cold and fasting, through reduced cytosolic volume, promote cell crowding and less molecular diffusion, which increases molecular interactions and assembly of condensates (9)(10)(11)(12)(13). Since their formation is driven by entropic-forces and less by ATP (9,14,15), elucidating their range of parametrization is crucial to understand cellular adaptations (8). Indeed, cellular states condition the material properties of condensates, which impact the collective behavior of interactions and their viscoelasticity (16).…”