Faculty Bibliography

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.

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 and an increase in crowding 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.

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.

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.

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.

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 Saccharomyces cerevisiae. The SWI/SNF chromatin remodeling complex is a key mediator of this transcriptional response. A glutamine-rich low complexity domain (QLC) in the SNF5 subunit of this complex, and histidines within this sequence, were required for efficient transcriptional reprogramming. Furthermore, the SNF5 QLC mediated pH-dependent recruitment of SWI/SNF to an acidic transcription factor in a reconstituted nucleosome remodeling assay

Cells that grow in confined spaces eventually build up mechanical compressive stress. This growth-induced pressure decreases cell growth. Growth-induced pressure is important in a multitude of contexts, including cancer^1"“3, microbial infections⁴ and biofouling⁵; yet, our understanding of its origin and molecular consequences remains limited. Here we combine microfluidic confinement of the yeast Saccharomyces cerevisiae⁶ with rheological measurements using genetically encoded multimeric nanoparticles⁷ to reveal that growth-induced pressure is accompanied with an increase in a key cellular physical property: macromolecular crowding. We develop a fully calibrated model that predicts how increased macromolecular crowding hinders protein expression and thus diminishes cell growth. This model is sufficient to explain the coupling of growth rate to pressure without the need for specific molecular sensors or signalling cascades. As molecular crowding is similar across all domains of life, this could be a deeply conserved mechanism of biomechanical feedback that allows environmental sensing originating from the fundamental physical properties of cells.

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. Here we examine the links between metabolism and mechanics, highlighting signalling pathways involved in the regulation and response to cellular mechanosensing. We consider how forces and metabolism regulate one another through nanoscale molecular sensors, micrometre-scale cytoskeletal networks, organelles and dynamic biomolecular condensates. Understanding this cross-talk will create diagnostic and therapeutic opportunities for metabolic disorders such as cancer, cardiovascular pathologies and obesity.