Session T37: Quantitative Cell Physiology II

Multimodal Characterization of Systems-Level Robustness in the Cellular Organelle Network

A distinguishing characteristic of eukaryotic cells is their compartmentalization into membrane bound organelles. While each type of organelle has their own individual functions, there are vital cellular processes that come from the interactions between them, such as organelle biogenesis and metabolic regulation. A central question in cellular biophysics is to what degree is cellular function robust or fragile to breaks in the organelle network. Here I aim to address the systems-level role organelle interactions have in regulating organelle composition and metabolic flows. These interactions can be explored by genetically deleting interorganelle protein bridges called organelle contact sites. Using hyperspectral confocal microscopy on a strain of Saccharomyces cerevisiae engineered with fluorescent labels for six types of organelles, we can simultaneously measure organelle size, morphology, and spatial components at a single cell resolution. To investigate metabolic regulation, we take transcriptomic and metabolomic data and infer their fluxes with optimization modeling. The parallel information from microscopy and omics is compared across genetic mutants to highlight altered physiology that stems from perturbations in the network. Our imaging and functional genomics results suggest that mitochondria represent particularly general fragile node in the organelle network, while attendant organelle deficiencies appear in reduced subsets of breaks to the organelle contact network.   

Optimality in organelle number and size control under a limiting pool of resources

Among the hallmarks of the eukaryotic cell is its organization into spatially defined subcompartments known as organelles. Organelles provide specialized environments for otherwise incompatible biochemical reactions within the cell. In order to tailor organelle biogenesis to the needs of the cell, the cell can regulate the size and number of many of its organelles. Organelle biogenesis, however, is fundamentally constrained by the limited available pool of resources available to the cell to synthesize its organelles. This begs the question: what principles dictate how much of the cell’s limited resources are devoted to increasing the number versus the size of a given organelle? We find that the solutions to the constrained optimization fall into two regimes separated by a critical point, suggesting that cells face an unexpectedly sharp tradeoff between organelle number and size in resource-limited contexts. In particular we find that the mechanisms governing the biogenesis of a given organelle coincides with whether cells grow the organelle in number versus size. In organelles that are created via de-novo synthesis, the cell appears to grow the total volume of the organelle by increasing the number of organelles with minimal concomitant increase in average organelle size.  The situation is reversed in organelles that undergo cycles of fission and fusion. Our theoretical and experimental results are consistent with the idea that cells attempt to allocate organelle number and size to maximize usage of cellular volume available to organelles.   

The coordination of global macromolecular synthesis and organelle biogenesis during cellular growth

Over the course of the growth and maintenance of the eukaryotic cell, the cell is tasked with managing the flow of material to its component parts in a controlled fashion. Among both the chief consumers and sources of these materials in the cell, including proteins and lipids, are its organelles. Despite remarkable progress in our molecular understanding of how the cell can change the rate of global protein and lipid synthesis, it is still unclear how organelles react, both alone and in concert with the other organelles in the cell, upon perturbations to these global material fluxes. Here, we tuned the rates of global protein, lipid, and nucleic acid synthesis in the model organism Saccharomyces cerevisiae and used hyperspectral confocal microscopy to monitor the effects of these perturbations on the systems-level organelle architecture of individual cells. Far from having uniform effects on systems-level organelle biogenesis, reduction in global protein synthesis appears to reveal prioritization of certain organelles over others. At the broadest level, protein synthesis inhibition reduces the fraction of the cell allocated to mitochondria, lipid droplets and Golgi, while increasing vacuoles and peroxisomes, a pattern of reallocation reminiscent of our previous findings documenting systems-level organelle changes in response to altered cell size. Our results thus point to a framework in which we may infer how the cell interprets global material fluxes in coordinating organelle biogenesis with cellular growth.    

Tight Control Over Cytoplasmic and Membrane Protein Densities Defines Regulation of Cell Geometry in Escherichia coli

The interplay between gene expression, macromolecular composition, and cell size control has been a central topic in the study of microbial physiology for the better part of a century. However, we lack a mechanistic understanding of how cells so tightly coordinate biosynthesis and cell size control across diverse environments. In this work, we present a simple theory of cellular resource allocation that quantitatively predicts how rod-shaped bacterial cells control their surface-area-to-volume across a broad range of growth conditions. Central to this theory is a biochemical constraint that the protein density within the cell membranes and the macromolecular density within the cell cytoplasm are strictly controlled and kept at a constant ratiometric value. As a result, this theory predicts a sublinear scaling relationship between the cellular surface-to-volume ratio and ribosome content, linking empirical “growth laws” in a predictive manner.  We compare these predictions to a broad array of literature data and our own measurements in E. coli. Furthermore, we test this theory through genetic perturbations of cellular ribosome content and demonstrate that cell size and bulk growth rate can be effectively decoupled, challenging a long-held hypothesis that the former is set by the latter.   

Cell Division and Motility Enable Hexatic Order in Biological Tissues

Biological tissues transform between solid-like and liquid-like states in many fundamental physiological events. Recent experimental observations further suggest that in two-dimensional epithelial tissues these solidliquid transformations can happen via intermediate states akin to the intermediate hexatic phases observed in equilibrium two-dimensional melting. The hexatic phase is characterized by quasi-long-range (power-law) orientational order but no translational order, thus endowing some structure to an otherwise structureless fluid. While it has been shown that hexatic order in tissue models can be induced by motility and thermal fluctuations, the role of cell division and apoptosis (birth and death) has remained poorly understood, despite its fundamental biological role. Here we study the effect of cell division and apoptosis on global hexatic order within the framework of the self-propelled Voronoi model of tissue. Although cell division naively destroys order and active motility facilitates deformations, we show that their combined action drives a liquid-hexatic-liquid transformation as the motility increases. The hexatic phase is accessed by the delicate balance of dislocation defect generation from cell division and the active binding of disclination-antidisclination pairs from motility. We formulate a meanfield model to elucidate this competition between cell division and motility and the consequent development of hexatic order.   

Amphiphilic Nanoparticles Induce Membrane Capacitance and Tension Reductions in planar lipid membranes

While amphiphilic gold nanoparticles (NPs) that have been modified with a striped arrangement of hydrophilic sulfonate and hydrophobic octanethiol (OT) ligands demonstrate potential for improved drug delivery due to their passive cellular uptake capabilities, precise methods by which they traverse biological membranes remain elusive. Herein, we utilize the planar lipid bilayer, which allows for subsequent electrophysiological characterization. Electrical measurements and image processing reveal that the utilization of NPs containing 15 mol% OT ligands leads to a reduction in the area-normalized capacitance and contact angle of the bilayer. The extent of this reduction is contingent upon the concentration of NPs employed in the experiment. When integrated with additional electrical data, this observed pattern suggests that these NPs exhibit a spontaneous tendency to insert themselves into the hydrophobic core of the bilayer, resulting in an overall increase in its average thickness and a decrease in its capacitance per unit area. In contrast, the introduction of hydrophilic NPs without hydrophobic ligands did not result in any noticeable alterations in the specific capacitance of the bilayer. This suggests that hydrophobic ligands play a crucial role in facilitating the incorporation of NPs into the bilayer. It was also observed that NPs with 30 mol% hydrophobic ligands exhibited more significant reductions in specific capacitance at a lower concentration of NPs, compared to ones with 15 mol% OT.   

MreB mechanically couples the cytoplasmic membrane to the cell wall in Escherichia coli

MreB is a prokaryotic homolog of actin that is present in many rod-shaped bacteria and plays a key role in cell shape maintenance. MreB filaments orient the insertion of cell-wall precursors via circumferential movement along with the cell-wall synthesis machinery. How mechanical forces are involved in the localization and function is largely unknown. In this study, we probed the mechanical contribution of MreB to the cell wall with a microfluidic hyperosmotic shock assay combined with treatment of A22, an MreB-targeting antibiotic. Interestingly, disruption of MreB filaments did not alter cell wall deformation under moderate osmotic shocks, suggesting that MreB does not directly strengthen the cell wall. In the absence of A22, repeated large osmotic shocks led to gradual reduction in cytoplasmic contraction under hyperosmolar conditions, whereas depolymerizing MreB allowed cells to freely plasmolyze, which together indicate that MreB mechanically couples the cytoplasmic membrane and the cell wall. GFP labeling of MreB confirmed that relocation of MreB to the cell poles was key to such phenotypical differences. Based on these data, we propose a physical model in which MreB, together with the other components of the cell-wall synthesis machinery, serves as the major linkage between the cytoplasmic membrane and the cell wall.   

Directly measuring turgor pressure in bacterial cells: effects of osmolality of growth and nutrient conditions

Bacteria generally maintain high turgor pressures (~1-20 atm). Gram positive bacteria typically have higher turgor pressures than gram negative bacteria. Maintaining turgor requires specialized machineries that balance the cytoplasmic osmolyte concentration against the embedding medium. Details of this machinery as well as the roles of the components of the multilayer cell wall are not well understood. Directly measuring turgor has been an experimental challenge, and a rapid, precise method has been lacking. We here present an experimental method based on force spectroscopy using an atomic force microscope to quantify turgor and its variations at the single cell level in vivo. We measured turgor of different gram-negative and gram-positive bacteria as well as the effect of different growth conditions and nutrient concentrations on turgor pressure.   

Modeling the peptidoglycan layer of gram-negative bacteria as an anisotropic, elastic network composed of two types of nonlinear springs

Bacteria mechanically protect themselves by covalently linked peptidoglycan (PG) cell walls that preserve cellular morphology and contain high osmotic pressures. As a bacterium grows, the cell wall undergoes continuous expansion. Despite a good understanding of the molecular components and the assembly machineries of the cell wall, it remains largely unknown how the mesoscopic mechanical properties of the cell wall emerge from the properties and arrangement of molecular components. Here, we introduce a quantitative physical model of the bacterial cell wall, based on molecular details, that predicts the mesoscopic mechanical response of the cell wall for the Gram-negative bacterium Escherichia coli. We modeled the PG layer as an anisotropic elastic network composed of two types of nonlinear springs (glycans and oligopeptides). We vary structural properties such as glycan length distribution, angular distribution, and cross-link density (pore size distribution) to accurately reproduce observed mechanical properties such as stress-strain relationships (elastic moduli), strain and stress ratios between axial and hoop directions.   

Structure, Dynamics, and Transport in Mitochondrial Networks

Mitochondria form network architectures ranging from partially fragmented to highly fused, depending on metabolic state and expression of fusion/fission proteins. Using spatially-resolved, agent-based simulations and mean-field models, we establish how experimentally tractable parameters such as local fusion/fission kinetics and mitochondrial mechanics and mobility shape network structure. We demonstrate that increased mitochondrial motion and junction flexibility inhibit the onset of a percolation transition in network connectivity. Distinct mammalian and yeast network structures are shown to arise from similar microscopic rate constants as a result of different geometric constraints. The intermediate fusion regime observed in mammalian cells is also shown to optimize rapid network rearrangement. We further employ our model to explore biomolecular spread through a mitochondrial population. In budding yeast, we highlight a potential mechanism for cellular aging in which selective mitochondrial transport and protein import lead to asymmetric content distribution between mother and daughter cells. In patterned mammalian cells, our model illustrates how the sparse transport events needed to maintain a broad mitochondrial mass distribution propel asymmetric spread of mitochondrial material.   

Finding the Last Bits of Positional Information

Small discrepancies between theory and experiment often are hints of new physics; here we provide an example in the physics of a living system, the fruit fly embryo. For the first hours of development, information about the position of cells is carried by the concentrations of a handful of molecules, all identified. These signals encode position with a precision of ~1% of the length of the embryo, smaller than the spacing between neighboring cells. But errors come from distributions, and distributions have tails. The result is that this precision, while higher than expected, is not quite enough to specify the identity of each cell uniquely. We make this “information gap” precise, in bits. We then show that the gap can be closed if positional noise is correlated over distances ~20% of the embryo length. Finally, we analyze the positions of stripes in pair rule gene expression in 100+ embryos. Correlations in positional noise are a function of distance, even though different stripes reflect the action of different transcription factors, and this dependence agrees with our prediction for what is needed to close the information gap. Direct estimates show that the total positional information, including correlations, is within ~2% of that needed for unique cellular identities.   

Osmotic Regulation of Nuclear Size in Yeast

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.