ABSTRACT
The mechanisms that regulate the physical properties of the cell interior remain poorly understood, especially at the mesoscale (10nm-100nm). Changes in these properties have been suggested to be crucial for both normal physiology and disease. Many crucial macromolecules and molecular assemblies such as ribosomes, RNA polymerase, and biomolecular condensates span the mesoscale size range. Therefore, we need better tools to study the cellular environment at this scale. A recent approach has been to use genetically encoded multimeric nanoparticles (GEMs), which consist of self-assembling scaffold proteins fused to fluorescent tags. After translation of the fusion protein, the monomers self-assemble into bright and stable nanoparticles of defined geometry that can be visualized by fluorescence microscopy. Physical properties of the cell can then be inferred through analysis of the motion of these particles, an approach called nanorheology. Previously, 40nm-GEMs elucidated TORC1 kinase as a regulator of cytoplasmic crowding. However, extremely sensitive microscopes were required. Here, we describe the development and characterization of a 50 nm diameter GEM that is brighter and probes a larger length scale. 50nm-GEMs will make high-throughput nanorheology accessible to a broader range of researchers and reveal new insights into the biophysical properties of cells.
ABSTRACT
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
Subject(s)
Cytoplasm , Polyribosomes , Ribonucleoproteins , Polyribosomes/metabolism , Cytoplasm/metabolism , Humans , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Molecular Dynamics Simulation , RNA, Messenger/metabolism , RNA, Messenger/genetics , Protein Biosynthesis , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Biomolecular Condensates/metabolism , Stress Granules/metabolism , Stress Granules/geneticsABSTRACT
The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.
ABSTRACT
All living cells are crowded with macromolecules. Crowding can directly modulate biochemical reactions to various degrees depending on the sizes, shapes, and binding affinities of the reactants. Here, we explore the possibility that cells can sense and adapt to changes in crowding through the widespread modulation of biochemical reactions without the need for a dedicated sensor. Additionally, we explore phase separation as a general physicochemical response to changes in crowding, and a mechanism to both transduce information and physically restore crowding homeostasis.
Subject(s)
Cell Physiological Phenomena , Macromolecular Substances/metabolismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Cell proliferation is a central process in tissue development, homeostasis, and disease, yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. Using MDCK epithelial monolayers, we show that a limiting rate of tissue expansion creates confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell volume. Division becomes arrested at a minimal cell volume, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches the minimum volume capable of packaging the genome. Loss of cyclin D1-dependent cell-volume regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell-volume regulation.
Subject(s)
Epithelial Cells , Epithelial Cells/metabolism , Cell Cycle/physiology , Cell Division , Epithelium , Cell ProliferationABSTRACT
Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved.
ABSTRACT
LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p, both essential for retrotransposition. ORF2p has reverse transcriptase and endonuclease activities, while ORF1p is a homotrimeric RNA-binding protein with poorly understood function. Here, we show that condensation of ORF1p is critical for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that electrostatic interactions and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 ribonucleoprotein (RNP) complex formation in cells. Furthermore, we relate the dynamics of ORF1p assembly and RNP condensate material properties to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1p condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that dynamic ORF1p oligomerization on L1 RNA drives the formation of an L1 RNP condensate that is essential for retrotransposition.
Subject(s)
Long Interspersed Nucleotide Elements , Retroelements , Humans , Retroelements/genetics , Long Interspersed Nucleotide Elements/genetics , Mutation , RNA-Binding Proteins/metabolism , RNA/metabolismABSTRACT
Macromolecular crowding is an important property of cells that impacts multiple biological processes. Passive microrheology using single particle tracking is a powerful means of studying macromolecular crowding. Here we monitored the diffusivity of self-assembling fluorescent nanoparticles (µNS) and mRNPs ( GFA1 -PP7) in response to acute glucose starvation. mRNP diffusivity was reduced upon glucose starvation as previously reported. By contrast, we observed increased diffusivity of µNS particles. Our results suggest that, upon glucose starvation, mRNP granule diffusivity may be reduced due to increased physical interactions, whereas macromolecular crowding in the cytoplasm may be globally reduced.
ABSTRACT
In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in1-3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease.4-7 During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, â¼25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.
Subject(s)
Biomolecular Condensates , Disease , Phase Transition , HumansABSTRACT
Macromolecular crowding is an important parameter that impacts multiple biological processes. Passive microrheology using single particle tracking is a powerful means of studying macromolecular crowding. Here we monitored the diffusivity of self-assembling fluorescent nanoparticles (µNS) in response to acute glucose starvation. mRNP diffusivity was reduced upon glucose starvation as previously reported. In contrast, we observed increased diffusivity of µNS particles. Our results suggest that, upon glucose starvation, mRNP granule diffusivity may be reduced due to changes in physical interactions, while global crowding in the cytoplasm may be reduced.
ABSTRACT
Volume control is a fundamental challenge for all cells, the mechanisms of which have been long debated. In this issue of Cell, Boyd-Shiwarski et al. find that increased molecular crowding drives condensation of WNK kinase, allowing cells to sense and respond to cell volume loss.
Subject(s)
Cell SizeABSTRACT
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.
Subject(s)
Signal Transduction , tau Proteins , Cyclin-Dependent Kinase 2/metabolism , Cytoplasm/metabolism , Humans , Macromolecular Substances/metabolism , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Active Transport, Cell Nucleus/physiology , Cell Nucleus/metabolism , Nuclear Envelope/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.
Subject(s)
Body Patterning , Gene Expression Regulation, Developmental , Genes, Homeobox , Homeodomain Proteins , Animals , Body Patterning/genetics , Enhancer Elements, Genetic , Genome , Homeodomain Proteins/genetics , Mice , Rats , Transcription, GeneticABSTRACT
Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.
ABSTRACT
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 in vivo 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 in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.