Hoege, C. | bioRxiv Science

bioRxiv2023

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes

The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

biophysics↗

bioRxiv2021

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

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. SIGNIFICANCE STATEMENTLiving cells rely on a continuous flux of energy to spatially organize biochemical processes. It remained unclear whether cells can achieve this spatial organization via thermodynamic principles. Here, we report the striking behavior of a cold-blooded organism that reacts to environmental temperature changes similar to a thermodynamic system at local equilibrium. Our key finding is that protein-rich droplets form and dissolve reversibly with temperature due to changes in the organism?s entropy. We show that the organism uses a specific molecule to extend droplet stability to the natural temperature range of the organisms habitat. Due to the relevance of such protein droplets for the organism?s fertility, our works shed light on how molecular components could facilitate biological functions via thermodynamic principles.

biophysics↗