Translating the physical code of life

Abstract

The cytoplasm is a highly crowded and complex environment, and the regulation of its physical properties has only recently begun to be revealed. In this issue of Cell, Delarue et al. demonstrate that the control of ribosome concentration through mTORC1 sets limits on the diffusion of large particles and controls phase separation in eukaryotic cells.

Summary

In the 1940s, Francis Crick measured the movement of phagocytosed magnetic particles in chick embryos and concluded that the cytoplasm was not strictly elastic, as had been thought, but rather behaved as a gel (Crick and Hughes, 1950). Today, we know that the cytoplasm is a highly crowded environment, in which at least 20% of the volume is composed of proteins; the viscosity of the cytoplasm has been estimated to be at least several times that of water (Luby-Phelps et al., 1986; van den Berg et al., 2017). The intracellular density of cells is often assumed to be tightly regulated to ensure proper biosynthetic coordination and molecular mobility, but the extent to which and how this regulation occurs have remained unclear. Proteins that have a stronger propensity to self-associate than to interact with the solvent can undergo a phase transition, where a large number of interacting proteins coalesce into a condensed liquid phase that is separate from the surrounding bulk liquid solvent, and have recently emerged as a central player in several biological processes (Boeynaems et al., 2018). Whether such phase transitions are regulated by cells remains speculative, but it is clear that macromolecular crowding plays a key role in promoting the demixing of molecules in solution (Boeynaems et al., 2018). In the current issue of Cell, Delarue et al. (Delarue et al., 2018) introduce a powerful tool for probing cytoplasmic properties at length scales previously challenging to study across a broad range of organisms, revealing that ribosome concentration regulates the diffusion of large particles and controls phase separation.

Common methods for measuring diffusive behavior in living cells generally involve tracking the movement of fluorescently labeled particles. These techniques are limited by the fixed or undefined sizes of tracer particles and the number of emission cycles that the fluorophore can undergo before photobleaching, and changes in the motion of native structures may be due to direct regulation rather than the biophysical properties of the cell. To address these issues, Delarue et al. developed Genetically Encoded Multimeric nanoparticles (GEMs) as a biologically orthogonal tool for microrheology. These GEMs are homomultimer protein scaffolds derived from bacteria and archaea that self-assemble into spheres with diameters of 40 nm (120-mer) and 15 nm (60-mer), respectively. Each monomer is fused to a fluorescent protein, yielding stable diffraction-limited spots of high intensity, which allows for a high imaging frame-rate (100 Hz) and longer tracking before the signal extinguishes due to bleaching (Figure 1A). Tracking GEMs enables the measurement of cytoplasmic properties like molecular diffusion constants and viscosity. Delarue et al. found that the rapamycin complex 1 (mTORC1) pathway modulates the diffusion of particles >20 nm in size nearly two-fold by controlling ribosome concentration (Figure 1B), which also affects phase separation.

Figure 1: Ribosome concentration controls phase separation through crowding, as revealed by GEMs.

(A) Genetically encoded multimers (GEMs) are protein scaffolds used to study the diffusive behavior of mesoscopic particles (15-40 nm) in the cytoplasm. Ribosome production and autophagy counterbalance for crowding homeostasis, but inhibiting mechanistic target of rapamycin 1 (mTORC1), responsible for ribosome synthesis, increases GEM diffusion rates.

(B) This crowding homeostasis is also necessary for liquid phase separation.

In contrast to known viscosity increases during energy depletion (Parry et al., 2014), Delarue et al. determined that the diffusion constant of GEMs in yeast increased after entry into stationary phase, and that amino-acid depletion was responsible for the reduction in cytoplasmic crowding. Since the mechanistic target of rapamycin complex 1 (mTORC1) is responsible for amino-acid sensing in eukaryotes, when the authors treated cells with the mTORC1 inhibitor rapamycin, the diffusion constant of GEMs dropped significantly in both budding yeast and mammalian HEK293 cells (Figure 1A). Surprisingly, cryo-electron tomography of yeast cells revealed that the ribosome concentration was nearly halved under mTORC1 inhibition (from 23 μM to 13 μM). Intriguingly, when the motion of tracer particles (including GEMs) over a range of sizes was measured, only those ≥20 nm were affected by rapamycin treatment (Figure 1B), indicating that the mobility of individual proteins is unaffected by changes in ribosome concentrations.

This latter finding suggested that pathways for both synthesizing and degrading ribosomes are upstream regulators of macromolecular diffusion. Exploiting the relative ease of incorporating GEMs into distinct genetic backgrounds, Delarue et al. screened a set of candidate deletions in budding yeast. Cells lacking SFP1, a transcription factor involved in RNA biogenesis, exhibited an increase in GEM diffusion greater than that obtained with rapamycin treatment, consistent with the argument that ribosomal concentration determines the viscosity encountered by mesoscopic particles. Further, deletions of autophagy and ribophagy genes decreased diffusion by as much as 20% (Figure 1A). The authors observed similar phenomena in HEK293 cells by stimulating or inhibiting autophagy through drug targeting. Moreover, changes to cell volume, protein synthesis, and the cytoskeleton were unable to account for the large increase in diffusion under mTORC1 inhibition. Collectively, these observations provide strong evidence that ribosomes are indeed the major crowding agent in the eukaryotic cytoplasm.

Beyond altering cytoplasmic diffusion, what other physical effects result from changing ribosome concentration? To investigate whether ribosomal crowding impacts phase separation in cells, Delarue et al. examined the behavior of the well-characterized SUMO/SIM model system, which forms phase-separated droplets at appropriate concentrations (Banani et al., 2016). Both in vitro and in vivo, ribosomal crowding induced droplet formation, while droplets dissolved under mTORC1 inhibition (Figure 1A). Thus, ribosomes can act as a crowding agent to induce phase separation. It will be fascinating to explore the links between ribosomes and the native systems that exhibit phase transitions in organisms that are beginning to be identified throughout the kingdoms of life.

The future design of GEMs of a wide range of sizes and their application to diverse species should help to answer fundamental questions about whether certain diffusion behaviors are general or species-specific. Delarue et al. observed subdiffusive behavior with a species-dependent exponent that may point to differences in how the mesh-like properties of the cytoskeleton limit diffusion at large length scales. Another outstanding question is how the diffusion of a particle depends on its size. Under the assumption of simple diffusion in a Newtonian fluid, the Stokes-Einstein equation predicts that the diffusion constant should scale as R⁻¹, where R is the particle radius. The authors reported a dependence of R⁻², intermediate between the predicted scaling for diffusion within a polymer mesh and that in a simple fluid, suggesting a further impact of the cytoskeleton. Cells may therefore exhibit spatial variations in diffusive behavior dependent on cytoskeletal density. In bacterial cells, the diffusion constant of fluorescent proteins has been reported to scale as R⁻⁶ (Kumar et al., 2010); it remains to be seen whether this scaling comparison reflects intrinsic differences between the bacterial and eukaryotic cytoplasms. The challenge to understand the molecular and physical underpinnings of these phenomena may motivate new theoretical models and the identification of other contributing cytoplasmic factors; unexamined in this study was the contribution to diffusion of active processes, which can use energy to drive random transport throughout the cell (Brangwynne et al., 2009).

During the typical lifespan of any cell, the osmolarity of the environment can change drastically, producing large and rapid fluctuations in cell volume and, in turn, cytoplasmic viscosity. Delarue et al. demonstrated that normal diffusion can be recovered under rapamycin treatment by exposing cells to a hypertonic solution to reduce cell volume. Do cells dynamically alter their volume to regulate cytoplasmic rheology? Theoretical studies have suggested the existence of an optimal cellular concentration, which cells may actively regulate to balance the need for efficient biosynthesis and overcrowding (van den Berg et al., 2017). In support of this hypothesis, in Escherichia coli, ribosome concentration during steady-state growth is maintained across a wide range of temperatures (Farewell and Neidhardt, 1998).

GEMs constitute a powerful tool not only due to their ease of application, but also how quickly data can be generated (< 10 s), making them highly useful for screens. Delarue et al. also discuss the possibility of future work on measuring GEM diffusion in tissues to uncover possible links between cytoplasmic physical properties and large-scale tissue mechanics and pathology. The use of GEMs in this study highlights the need for a greater understanding of the role of ribosomes beyond setting protein-synthesis rates in the cell. How quickly are cells able to tune ribosome concentration and how robustly? If ribosome concentration increases based on a need for rapid protein production, does crowding act against the necessary movement of molecules? In this case, phase separation may be one solution for selectively condensing the cytoplasm; paraphrasing Yogi Berra, “no molecules go there anymore, it’s too crowded.” Regardless, it seems the mysteries of the cytoplasm are soon to be uncovered.