Polysome collapse and RNA condensation fluidize the cytoplasm.

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.

Development and Characterization of 50 nanometer diameter Genetically Encoded Multimeric Nanoparticles.

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.

Macromolecular interactions and geometrical confinement determine the 3D diffusion of ribosome-sized particles in live cells.

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 cells.

Macromolecular crowding: Sensing without a sensor.

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.

Mesoscale molecular assembly is favored by the active, crowded cytoplasm.

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells. Principles of biomolecular condensation have been revealed through reconstitution. 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 processes.

mRNA condensation fluidizes the cytoplasm.

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.

Epithelial tissue confinement inhibits cell growth and leads to volume-reducing divisions.

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.

How it feels in a cell.

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 environmental stress response regulates ribosome content in cell cycle-arrested .

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 to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase.

Condensation of LINE-1 is critical for retrotransposition.

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.

Increased mesoscale diffusivity in response to acute glucose starvation.

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 ( -PP7) in response to acute glucose starvation.

Controlling the crowd with a WNK.

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.

Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding.

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.

Control of nuclear size by osmotic forces in .

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.

Synthetic regulatory reconstitution reveals principles of mammalian cluster regulation.

Precise gene expression is crucial for embryonic patterning. Intra- transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling gene expression. However, quantifying their relative contributions has remained elusive.

Z-α-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state.

Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration.

Macromolecular crowding limits growth under pressure.

Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure (GIP) decreases cell growth. GIP is important in a multitude of contexts from cancer, to microbial infections, to biofouling, yet our understanding of its origin and molecular consequences remains limited.

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

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.

SWI/SNF senses carbon starvation with a pH-sensitive low-complexity sequence.

It is increasingly appreciated that intracellular pH changes are important biological signals. This motivates the elucidation of molecular mechanisms of pH sensing. We determined that a nucleocytoplasmic pH oscillation was required for the transcriptional response to carbon starvation in .

Reciprocal regulation of cellular mechanics and metabolism.

Metabolism and mechanics are intrinsically intertwined. External forces, sensed through the cytoskeleton or distortion of the cell and organelles, induce metabolic changes in the cell. The resulting changes in metabolism, in turn, feed back to regulate every level of cell biology, including the mechanical properties of cells and tissues.

Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly.

Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix.

Microtubules Enhance Mesoscale Effective Diffusivity in the Crowded Metaphase Cytoplasm.

Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle.

Spatial heterogeneity of the cytosol revealed by machine learning-based 3D particle tracking.

The spatial structure and physical properties of the cytosol are not well understood. Measurements of the material state of the cytosol are challenging due to its spatial and temporal heterogeneity. Recent development of genetically encoded multimeric nanoparticles (GEMs) has opened up study of the cytosol at the length scales of multiprotein complexes (20-60 nm).