MTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

An improved macromolecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under stressed conditions

Biological cells need to structure their interior in space and time. One way this is done are containers enclosed by a membrane as a physical barrier that can control which molecules enter and leave the container. Another, recently discovered, class are biomolecular condensates. These are liquid like droplets that form via liquid liquid phase separation. Although they have no membrane, they do have distinctly different composition from the surrounding. Weak attractive interactions between the molecules in the condensate prevent them from diffusing out of the condensate. To control the formation and dissolution of these condensates, the cell can change the attractive interaction between molecules via chemical reactions. In this thesis, we develop a theory of phase separation with chemical reactions based on thermodynamic arguments. The chemical reactions switch between two states of a protein, one state phase separates and forms droplets, while the other state is soluble in the solvent. The aim of this thesis was to analyze how such simple reactions can control the phase separation process, for example, the formation, dissolution, and size control of droplets. In the first part of the thesis, we investigate equilibrium reactions. In this case, the system relaxes to thermodynamic equilibrium. Unlike two component fluids, fluids consisting of multiple components with equilibrium reactions can form droplets, depending on the system parameters. We find that equilibrium reactions introduce a new parameter to control phase separation, the internal energy difference between the two protein states. This internal energy difference can control how much protein is in the phase separating state and thereby, if droplets form or not. We show that the droplet size is very sensitive to changes in the internal energy difference. However, the parameter range for control of droplets is narrow. In addition, the internal energy difference is an equilibrium property of the proteins, thus, it can not be changed fast or in a specific manner. In the second part of the thesis, we extend our model to non-equilibrium reactions. In this case, the reaction is coupled to fuel molecules, which introduce external energy into the system and drive the reaction away from thermodynamic equilibrium. The external driving strength is a new parameter, which describes how strong the system is driven from equilibrium. We find, that driven reactions alone can be mapped onto an effective equilibrium system with rescaled internal energy difference that depends on the driving strength. This is different if both reaction pathways, the driven and equilibrium reaction, are present. In this case, the total amount of phase separating proteins depends on the reaction kinetics, i.e. on the relative reaction rates of the two pathways. We show that this allows precise, fast and specific control over droplet formation and dissolution. The reason is, that the kinetic parameters can be tuned by enzymes that act only on specific reactions. Finally, motivated by experimental observations, we investigate what happens if enzymes that catalyze the driven reaction are enriched in the droplet phase. We find that the enzymatic enrichment can control individual droplet size and stabilize multiple droplets of the same size against their thermodynamic tendency to form one big droplet. We show that size control of droplets by reactions is based on three specific features of the reactions. (i) A protein exists in a soluble and a phase separating state and the transition between the two states can be described as a chemical reaction. (ii) There are at least two reaction pathways for the transition and at least one has to be driven out of equilibrium. (iii) The reaction rates in droplet and solvent phase need to be different, for example, due to enrichment of enzymes in the droplet. More generally, our results highlight that chemical reactions in phase separating environments can not be described by standard mass action kinetics. The reason is that phase separating systems are inherently non-ideal and mass action kinetics are only valid in ideal, dilute solutions. Instead, a thermodynamic treatment of reactions is necessary, which takes into account that droplets formed by phase separation are chemically different from the solvent phase due to enthalpic interactions.

The regulation and preservation of distinct intracellular and extracellular solute microenvironments is crucial for the maintenance of cellular homeostasis. In mammals, the kidneys control bodily salt and water homeostasis. Specifically, the urine-concentrating mechanism within the renal medulla causes fluctuations in extracellular osmolarity, which enables cells of the kidney to either conserve or eliminate water and electrolytes, depending on the balance between intake and loss. However, relatively little is known about the subcellular and molecular changes caused by such osmotic stresses. Advances have shown that many cells, including those of the kidney, rapidly (within seconds) and reversibly (within minutes) assemble membraneless, nano-to-microscale subcellular assemblies termed biomolecular condensates via the biophysical process of hyperosmotic phase separation (HOPS). Mechanistically, osmotic cell compression mediates changes in intracellular hydration, concentration and molecular crowding, rendering HOPS one of many related phase-separation phenomena. Osmotic stress causes numerous homo-multimeric proteins to condense, thereby affecting gene expression and cell survival. HOPS rapidly regulates specific cellular biochemical processes before appropriate protective or corrective action by broader stress response mechanisms can be initiated. Here, we broadly survey emerging evidence for, and the impact of, biomolecular condensates in nephrology, where initial concentration buffering by HOPS and its subsequent cellular escalation mechanisms are expected to have important implications for kidney physiology and disease.

Targeting of biomolecular condensates to the autophagy pathway.

WNK1, a molecular crowding sensor, links phase separation to cellular physiological stress.

Cells are constantly exposed to the risk of volume perturbation under physiological conditions. The increase or decrease in cell volume accompanies intracellular changes in cell membrane tension, ionic strength/concentration and macromolecular crowding. To avoid deleterious consequences caused by cell volume perturbation, cells have volume recovery systems that regulate osmotic water flow by transporting ions and organic osmolytes across the cell membrane. Thus far, a number of biomolecules have been reported to regulate cell volume. However, the question of how cells sense volume change and modulate volume regulatory systems is not fully understood. Recently, the existence and significance of phaseseparated biomolecular condensates have been revealed in numerous physiological events, including cell volume perturbation. In this review, we summarize the current understanding of cell volume-sensing mechanisms, introduce recent studies on biomolecular condensates induced by cell volume change and discuss how biomolecular condensates contribute to cell volume sensing and cell volume maintenance. In addition to previous studies of biochemistry, molecular biology and cell biology, a phase separation perspective will allow us to understand the complicated volume regulatory systems of cells.

Controllable Protein Phase Separation and Modular Recruitment to Investigate Biochemical Compartmentalization in Membraneless Organelles

的脂质和亲水的蛋白孔道使它们具有半通透、半开放 的特性.无膜区室包括P颗粒(P granules)、压力颗粒 (stress granules)、加工小体(processing bodies)等无膜 细胞器 [1,2] , 还包括在一些生化路径和细胞通路中发现. 根据近些年的研究, 这些无膜区室都通 过多价相互作用驱使的"液-液相分离"(liquid-liquid phase separation, 常简称为相分离或称为相变)进行组 装, 并被统一命名为"生物分子凝集体"(biomolecular condensates) [7,8] .关于生物分子凝集体的组装与溶解、 组成、物理性质、生化和细胞功能等方面的研究已成 为生物学及与相关交叉领域的热点.本文将综述相关 重要进展, 并进行讨论.

Zinc ions trigger the prion protein liquid-liquid phase separation.

Cells are segregated into two distinct compartment groups to optimize cellular function. The first is characterized by lipid membranes that encapsulate specific regions and regulate macromolecular flux. The second, known collectively as membraneless organelles (MLOs), lacks defining lipid membranes and exhibits self-organizing properties. MLOs are enriched with specific RNAs and proteins that catalyze essential cellular processes. A prominent sub-class of MLOs are known as nuclear bodies, which includes nucleoli, paraspeckles, and other droplets. These microenvironments contain specific RNAs, exhibit archetypal liquid-liquid phase separation characteristics, and harbor intrinsically disordered, multivalent hub proteins. We present an analysis of nuclear body protein disorder that suggests MLO proteomes are significantly more disordered than structured cellular features. We also outline common MLO ultrastructural features, exemplified by the three sub-compartments present inside the nucleolus. A core-shell configuration, or phase within a phase, is displayed by several nuclear bodies and may be functionally important. Finally, we summarize evidence indicating extensive RNA and protein sharing between distinct nuclear bodies, suggesting functional cooperation and similar nucleation principles. Considering the substantial accumulation of specific coding and noncoding RNA classes inside MLOs, evidence that RNA buffers specific phase transition events, and the absence of a clear correlation between total intrinsic protein disorder and MLO accumulation, we conclude that RNA biogenesis may play a key role in MLO formation, internal organization, and function. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Phase Separation of Disordered Protein in the Formation of Membrane-Less Organelles

Simple SummaryThe cytoplasm of a living cell is a crowded place, containing hundreds of types of protein and other macromolecules. Cells reliably and continually perform thousands of biochemical reactions to maintain their health. Biomolecular condensates are fluid protein compartments that provide distinct local environments, within which they carry out cellular functions. How they prevent their contents mixing with the external environment without being encapsulated inside a lipid membrane is not fully understood. Many researchers approach this question by studying simpler systems in a test tube that contain only a few protein types although it is hard to relate their results to the complex cellular milieu. Computer simulations are used to explore the predictions of simple models of cellular behavior, but are also limited by the ability of human experimenters to recreate important aspects of the cytoplasm, in particular, its crowded nature. We have used a novel computer framework to perform dozens of simultaneous simulations that map out the influence of macromolecular crowding on the formation and structure of a biomolecular condensate. We find that the spatial structure of the model condensate is surprisingly insensitive to the composition and concentration of external macromolecules, even when its formation is assisted by steric repulsion from its environment.The crowded interior of a living cell makes performing experiments on simpler in vitro systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically disordered proteins into biomolecular condensates, which is proposed to underlie the membrane-less compartmentalization of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally-varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behavior of model biomolecular condensates, but the number of relevant parameters increases as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in their high-dimensional parameter space. Here, we have studied the phase behavior of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein/crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the model’s high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist the formation of biomolecular condensates, while relying on the dense phase to reliably provide a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

Liquid-liquid phase separation of intrinsically disordered proteins: Effect of osmolytes and crowders.