Faculty Bibliography

Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.

Quasisymmetric icosahedral viral capsids achieve larger sizes than possible with strictly symmetric icosahedra by tessellating pentagons and hexagons using a single subunit that adopts different conformations in symmetrically non-equivalent locations^1,2. Recapitulating such quasisymmetric architectures through computational design is a considerable challenge in nanomaterials engineering. Here we introduce a computational design strategy based on geometric frustration to generate two-component, quasisymmetric protein cages with customizable properties. We designed complementary trimeric and dimeric protein components that co-assemble into positively curved local hexagonal assemblies. Hexagonal lattices cannot tile spherical surfaces; instead, the components form closed sphere-like cage assemblies through incorporation of curvature-inducing pentagonal defects, as evidenced by electron microscopy. By designing dimers that encode different local curvatures, we programmed cage dimensions ranging from 40 to over 200 nm in diameter and with molecular weights from 2 MDa to over 50 MDa, comparable with natural virus capsids. We further functionalized these large cages with additional protein domains to enable ribonucleoprotein cargo loading and cellular uptake. Fluorescently labelled cage assemblies expressed in mammalian cells function as rheological probes and cargo recruiters, enabling a systematic study of size-dependent cytoplasmic diffusion and protein localization. Thus, the quasi-symmetry that has long fascinated structural biologists can now be achieved by computational protein design, with immediate applications to biologics delivery and molecular cell biology.

Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties and the processes they impact remain poorly understood, especially in the nucleus. We hypothesized that some viruses might change the biophysical properties of the nucleus to favor virus survival and replication and found that herpes simplex virus 1 (HSV-1) increases the mesoscale fluidity of the nucleus. The HSV-1 protein ICP4 (infected cell protein 4) caused fluidization and enabled the growth of synthetic nuclear condensates. Conversely, conditions that decreased nuclear fluidity inhibited the growth of viral replication compartment condensates and reduced infectious virus production. Together, our data suggest that ICP4 increases nuclear fluidity to promote the formation of condensates that drive the progression of the HSV-1 life cycle. We speculate that a key function of ICP4 is to overcome the crowding and elastic confinement within cell nuclei that are a fundamental barrier to virus replication.

Understanding how cells control their biophysical properties during development remains a fundamental challenge. While macromolecular crowding affects multiple cellular processes in single cells, its regulation in living animals remains poorly understood. Using genetically encoded multimeric nanoparticles for in vivo rheology, we found that Caenorhabditis elegans tissues maintain mesoscale properties that differ from those observed across diverse systems, including bacteria, yeast species, and cultured mammalian cells. We identified two conserved mechanisms controlling particle mobility: Ribosome concentration, a known regulator of cytoplasmic crowding, works in concert with a previously unknown function for the giant KASH (Klarsicht/ANC-1/SYNE homology) protein ANC-1 in providing structural constraints through associating with the endoplasmic reticulum. These findings reveal mechanisms by which tissues establish and maintain distinct mesoscale properties, with implications for understanding cellular organization across species.

The crowded bacterial cytoplasm is composed 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 (-3,240 to +2,700 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 subdiffusive 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 subdiffusion arises from geometrical confinement within the nucleoid and by the cell boundary.

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 in vitro 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. 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, stirring of the cytoplasm by actomyosin dynamics is the dominant mechanism that potentiates droplet growth in the mammalian cytoplasm by reducing confinement and elasticity. 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.

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.

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.

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.

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.