Bulletin of the American Physical Society
APS March Meeting 2023
Volume 68, Number 3
Las Vegas, Nevada (March 5-10)
Virtual (March 20-22); Time Zone: Pacific Time
Session B10: Quantitative Cell Physiology I - Shape and Size |
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Sponsoring Units: DBIO Chair: Suckjoon Jun, University of California, San Diego Room: Room 202 |
Monday, March 6, 2023 11:30AM - 11:42AM |
B10.00001: Robust control of replication initiation in the absence of DnaA-ATP ? DnaA-ADP regulatory elements in Escherichia coli Suckjoon Jun Replication initiation, governed by the widely conserved master initiator protein DnaA, is one of the most well-studied biological processes in bacteria. Specifically, conversion between the ATP and ADP form of DnaA during growth is critical for initiation control. In Escherichia coli, the regulatory elements for conversion have been discovered and extensively characterized over decades. However, they are not widely conserved in bacteria, raising questions on how to generalize the findings in E. coli to other organisms. In this work, we show that the intrinsic ATPase activity of DnaA itself is sufficient for robust and precise initiation control. We constructed and studied E. coli mutants lacking the extrinsic control of either DnaA-ATP → DnaA-ADP (by hda and datA) or DnaA-ADP → DnaA-ATP (by DARS1 and DARS2). These cells showed distinct and opposing characteristics in initiation timing, intrinsic noise (the degree of initiation asynchrony), and extrinsic noise (cell-to-cell variability). Strikingly, when all four regulatory elements were deleted, E. coli exhibited a near wild-type phenotype, with only mildly increased intrinsic and extrinsic initiation noise. By further characterizing the DnaA variants with increased and decreased ATPase activity, we conclude that DnaA is the only requirement for robust initiation, shedding new evolutionary light on cell-cycle control in bacteria. |
Monday, March 6, 2023 11:42AM - 11:54AM |
B10.00002: Beyond G1/S regulation: how cell size homeostasis is tightly controlled throughout the cell cycle? Xili Liu, Jiawei Yan, Marc Kirschner Proliferating cells develop mechanisms to counteract stochastic noise in order to stabilize the cell mass distribution in a population. It is widely believed that cell size is controlled by size-dependent timing of the G1/S transition. However, a model based only on such a size checkpoint cannot explain why cell lines with deficient G1/S control are able to maintain a very stable cell mass distribution and how cell mass is maintained throughout the S and G2 phases of the cell cycle. To answer such questions, we applied a recently developed form of computationally enhanced Quantitative Phase Microscopy (ceQPM), which provides much-improved accuracy of cell mass measurement for individual cells. ceQPM allows us to investigate the factors contributing to cell mass homeostasis. We find that cell mass homeostasis is robustly maintained, despite disruptions of the normal G1/S transition or cell growth rate. Cell mass control is exerted throughout the cell cycle, where the coefficient of variation in cell mass for the population declines well before the G1/S transition and throughout the cell cycle in both transformed and non-transformed cells. Furthermore, the detailed response of cell growth rate to cell mass differs in different cell types. This size-dependent growth rate modulation occurs through both mTORC1-dependent and mTORC1-independent processes, which are independently regulated. The reduction of mass accumulation rate, slightly below that of exponential growth, is used to effectively reduce cell mass variation in the population. Both size-dependent cell cycle regulation and size-dependent growth rate modulation contribute to cell mass homeostasis by strictly controlling the coefficient of variation of cell mass in the population. These findings expose new principles in cell size control for normal and pathological proliferating cells, as well as terminally differentiated cells. Further, they suggest new avenues for the discovery of the underlying molecular mechanisms. |
Monday, March 6, 2023 11:54AM - 12:06PM |
B10.00003: Cell-cycle dependent growth regulation in bacterial cells Callaghan A Cylke, Shiladitya Banerjee Proliferating bacterial cells exhibit stochastic growth and shape dynamics, but the regulation of bacterial growth and morphogenesis remains poorly understood. A quantitative understanding of the mathematical equations driving single cell growth, and how they change under different growth conditions, would provide better insights into cell-to-cell variability and intergenerational fluctuations in cell physiology. Using multigenerational growth and shape data of single Escherichia coli and Bacillus subtilis cells find that both deviate from exponential growth within the cell cycle. In particular, while the exponential growth rate of E. coli increases during the cell cycle irrespective of nutrient or temperature conditions, the behavior of B. subtilis is non-monotonic. We propose a mechanistic model that explains the emergence of these growth patterns from autocatalytic production of ribosomes, coupled to the rate of cell elongation and surface area synthesis. Using this model in the context of established proteomic modeling and statistical inference on large datasets, the behavior of B. subtilis can be explained by dynamic cellular resource allocation. Connecting the mechanistic modeling to the underlying transcription allows for inferences about gene expression directly from morphological data. |
Monday, March 6, 2023 12:06PM - 12:18PM |
B10.00004: A Preservation of Macromolecular Densities Defines Cell Width in Escherichia coli Griffin Chure, Roshali T De Silva, Jonas Cremer All microbes demonstrate exquisite control over their shape and size. While a fundamental aspect of microbial life, our understanding of the biological and physical principles that underlie this control remains enigmatic despite its storied history of research in bacterial physiology. Seminal studies on cell size in rod-shaped, gram-negative organisms such as E. coli have culminated in a series of descriptive phenomenological models which capture important scaling relations, but remain separate from molecular details. Here, we present a novel model where the cell width is precisely adjusted such that macromolecular density within the periplasmic space is maintained across growth conditions. We test this hypothesis experimentally through a series of genetic and environmental perturbations which reveal this principle to be maintained. We quantitatively explore this hypothesis with a simple mathematical model that recapitulates classical scaling relationships, cementing the importance of cell width control in determining cell size. Finally, we consider explicit molecular players which may direct this control and its relationship and hypothesize how cell width- and length-control are interconnected. |
Monday, March 6, 2023 12:18PM - 12:30PM |
B10.00005: Adder minimizes cell size noise in bacteria Motasem ElGamel, Andrew Mugler Cells employ control strategies to maintain a stable size. Dividing at a target size (the 'sizer' strategy) is thought to produce the tightest size distribution. However, this result follows from phenomenological models that ignore the molecular mechanisms required to implement the strategy. Here we investigate a simple mechanistic model for exponentially growing cells whose division is triggered at a molecular abundance threshold. We find that size noise inherits the molecular noise and is consequently minimized not by the sizer but by the 'adder' strategy, where a cell divides after adding a target amount to its birth size. We derive a lower bound on size noise that agrees with publicly available data from six microfluidic studies on Escherichia coli bacteria. |
Monday, March 6, 2023 12:30PM - 12:42PM |
B10.00006: Dynamic proteome trade-offs regulate cell size and growth in fluctuating nutrient conditions Josiah Kratz, Shiladitya Banerjee Bacteria dynamically regulate cell size and growth rate to thrive in changing environments. While much work has been done to characterize bacterial growth physiology and cell size control during steady-state exponential growth, a quantitative understanding of how bacteria dynamically regulate cell size and growth in time-varying nutrient environments is lacking. Here we develop a dynamic coarse-grained proteome sector model which connects growth rate and division control to proteome allocation in time-varying environments in both exponential and stationary phase. In such environments, growth rate and size control is governed by trade-offs between prioritization of biomass accumulation or division, and results in the uncoupling of single-cell growth rate from population growth rate out of steady-state. Specifically, our model predicts that cells transiently prioritize ribosome production, and thus biomass accumulation, over production of division machinery during nutrient upshift, explaining experimentally observed size control behaviors. Strikingly, our model predicts the opposite behavior during downshift, namely that bacteria temporarily prioritize division over growth, despite needing to upregulate costly division machinery and increasing population size when nutrients are scarce. Importantly, when bacteria are subjected to pulsatile nutrient concentration, we find that cells exhibit a transient memory of the previous metabolic state due to the slow dynamics of proteome reallocation. This phenotypic memory allows for faster adaptation back to previously-seen environments when nutrient fluctuations are short-lived. |
Monday, March 6, 2023 12:42PM - 12:54PM |
B10.00007: Using simulations to investigate the mechanical properties of peptidoglycan Marco Mauri, Abimbola F. Adedeji Olulana, Jamie K. Hobbs, Sheila Hoshyaripour, Rosalind J Allen In bacteria, the peptidoglycan (PG) cell wall counteracts the internal turgor pressure and maintains cell shape. PG consists of a mesh of glycan strands crosslinked by short peptides. Maintaining the integrity of this PG mesh is necessary to prevent cell lysis; indeed, many antibiotics target PG synthesis. The mechanical properties of the PG mesh are important for understanding the biophysics of cell growth, cell shape and antibiotic action: yet these properties are hard to measure experimentally. |
Monday, March 6, 2023 12:54PM - 1:06PM |
B10.00008: Teichoic acids as organizing centers for growth and shape in Bacillus subtilis Felix Barber, Zhe Yuan, Enrique Rojas The Gram-positive cell wall is a rigid, sugar-peptide meshwork that constrains the cell's immense turgor pressure and confers cell shape. Specifically, rod shape is maintained through the circumferential orientation of the cell wall's peptidoglycan backbone by the Rod complex. Wall teichoic acids (WTAs) contribute up to 50% of the Gram-positive cell wall by mass and are known to assist with divalent cation homeostasis. However, a longstanding mystery is why preventing the first dedicated step of wall teichoic acid synthesis causes the complete loss of rod-shape. We used dynamical, quantitative microscopy to demonstrate that inhibiting wall teichoic acid synthesis causes a fundamental re-organization of peptidoglycan synthesis. Inhibiting WTA synthesis prompts a rapid decline in Rod complex activity coincident with a growth rate decrease. We further show that subsequent growth and the loss of rod-shape are then sustained by a separate pathway that inserts peptidoglycan isotropically. We posit that WTAs provide a fulcrum that quantitatively tunes the balance between oriented and isotropic cell wall synthesis, thereby maintaining cell shape and growth rate. |
Monday, March 6, 2023 1:06PM - 1:18PM |
B10.00009: Direct measurement of turgor pressure in E. coli and B. subtilis Octavio Albarran, Christoph F Schmidt, renata garces, Jeff D Eldredge, Harold P Erickson The ability to maintain turgor pressure, i.e. osmotic imbalance, across the cell envelope, is a requirement for |
Monday, March 6, 2023 1:18PM - 1:30PM |
B10.00010: Coarse-grained simulations of bacterial cell-wall mechanics and failure under extreme conditions Xiaoxuan Jian, Christoph F Schmidt, renata garces, Jeff D Eldredge, Octavio Albarran, Giacomo Po Bacterial cell walls have to contain high internal turgor pressures of ~1atm in gram-negative bacteria and >10 atm in gram-positive bacteria. At the same time the wall has to be continuously expanded while a bacterium grows. For most bacterial cell walls a covalently crosslinked polymer network, the peptidoglycan (PG) layer, provides mechanical toughness. The PG layer is a thin porous polymer network - made of rigid glycan strands crosslinked by flexible oligopeptides. Bacteria achieve mechanical toughness while the wall is growing by careful control of defect generation, material insertion and network repair mechanisms. Many antibiotics act by interfering with these mechanisms. In order to understand the mechanisms of mechanical wall failure under extreme challenges, we model the PG layer as an anisotropic elastic network composed of two types of nonlinear springs (glycans and oligopeptides) using parameters from E-coli. The model assigns different structural, linear and non-linear elastic properties to the network constituents: Glycan strands are rigid and long, while peptides are flexible and short. We characterize stress-strain relationships, anisotropy, pore size distributions, and failure susceptibility and geometry as a function of the crosslink density, length distribution of glycan strands and angular alignment. |
Monday, March 6, 2023 1:30PM - 1:42PM |
B10.00011: Mapping nanostructural changes in E.coli Peptidoglycan Abimbola F Olulana, Jacob Biboy, Oliver Meacock, Laia Pasquina-Lemonche, William M Durham, Simon J Foster, Waldemar Vollmer, Jamie K Hobbs E.coli is a rod-shaped Gram-negative bacterium whose shape is maintained by a biopolymer known as Peptidoglycan (PG)1. The chemical composition of PG is well understood2 but the following questions remain unanswered; 1) what is the detailed molecular organization of the PG; 2) is its organization location-dependent or not; 3) In the case of antibiotic-induced shape change and death, what happens to the PG organization? To this end, we utilized high-resolution atomic force microscopy (AFM) to map the changes in the PG organization from the pole to the cylindrical section of the rod. We extend this location-dependent imaging to interrogate different areas of the PG under different antibiotic treatment times. |
Monday, March 6, 2023 1:42PM - 1:54PM |
B10.00012: Systems-Level Robustness and Fragility in the Cellular Organelle Network Aline Arra A defining feature of the eukaryotic cell is its compartmentalization into organelles. While individual organelles have important functions on their own, they also make up a dynamic network whose interactions are responsible for vital cellular processes. A fundamental question in cellular biophysics is the degree to which the function of the cell is fragile or robust to perturbations to the organelle network. Here we aim to address the systems-level role organelle interactions play in regulating organelle composition and metabolic flows in the model system Saccharomyces cerevisiae. To dissect the organelle network, we have genetically broken interorganelle links formed by protein bridges called organelle contact sites. To assess the effects on organelle properties, we visualize a strain of budding yeast that expresses fluorescent labels for six organelles with hyperspectral confocal microscopy. This allows for simultaneous measurement of organelle size, number, and morphology at single cell resolution. Our data suggests that while metabolic hubs such as the ER, vacuoles, and peroxisomes are robust to network perturbations, mitochondria are a fragile node, with cells exhibiting divergent mitochondrial morphologies upon disruption of even non-mitochondrial network links. To examine the role robust organelles play in mitigating mitochondrial dysfunction, we present our initial characterization of the cell’s metabolic flexibility to disruptions in the organelle contact network. |
Monday, March 6, 2023 1:54PM - 2:06PM |
B10.00013: Spatial allocation rules governing systems-level organelle biogenesis Shankar Mukherji, Deepthi Kailash Uncovering the functional rules by which the eukaryotic cell controls the size of its organelles is a fundamental problem in cellular biophysics. Here we aim to elucidate one such rule, namely how the budding yeast Saccharomyces cerevisiaecoordinates the size of its organelles with the size of the cytoplasm within which they reside. By combining quantitative fluorescence microscopy with tools from synthetic cell biology, namely an artificial biomolecular condensate derived from the bacterium Escherichia coli, we directly engineer cells to exhibit a wide range of cytoplasmic spatial availability and monitor the response of organelle size to varying spatial constraints. In contrast to the mitochondria, lipid droplets, and endoplasmic reticulum, we observe that vacuolar sizes exhibit a strong response to decreasing cytoplasmic availability. We observe that with a significant amount of condensate in cells, the vacuolar volume starts to decrease as cell size increases, the opposite relationship to what is observed in the cells with an insignificant amount of condensate and previously reported observations. Our data suggest the hypothesis that the cell actively controls the volume of available cytoplasm even as the size of the cell as a whole changes and that the vacuole potentially plays a key role in buffering variation in cytoplasmic volume. |
Monday, March 6, 2023 2:06PM - 2:18PM |
B10.00014: How Type 1 Fimbriae Help E. coli Adhere to Interfaces? Udayanidhi Ramesh Kumar, Jacinta C Conrad, Patrick C Cirino, Nam T Nguyen Adhesion of bacteria to interfaces is the initial step in the formation of biofilms, in pathogenic infection, and in the bioremediation of oil spills. Bacteria use a variety of surface appendages to attach to surfaces, and the level of expression of these appendages varies in response to environmental conditions and external stimuli. Type 1 fimbriae, one such appendage, help bacteria attach to cells and to evade antibiotics during initial infection and hence are widely studied as a critical factor in pathogenic virulence. Here, we present a tunable approach to quantify how changes in the expression level of type 1 fimbriae alter the ability of bacteria to adhere to interfaces. A plasmid that enables inducible expression of E. coli MG1655 type 1 fimbriae was transformed into fimbriae-deficient mutant MG1655ΔfimA. The level of fimH gene expression in the engineered strain was tuned by changing the concentration of inducer isopropyl β-D-1- thiogalactopyranoside (IPTG). We find that increasing the degree of fimbriation results in a significant decrease in cell motility, but enhances the ability of bacteria to adhere to solid surfaces and to oil-water interfaces. The tunable extent of fimbriation accessible with these engineered strains may prove useful for physical measurements probing the effects of adhesin expression on biofilm formation over surfaces and on biodegradation of hydrocarbons. |
Monday, March 6, 2023 2:18PM - 2:30PM |
B10.00015: The role of surface-surface interactions and growth media in antibacterial action of microscale ZnO Yuri M Strzhemechny, Dustin A Johnson, John M Reeks, Alexander Caron, Iman Ali, Shauna M McGillivray, Abagael Speights Robust antimicrobial action of ZnO is well known, thoroughly documented and actively studied, especially for the nano- and microscale geometries. Several driving mechanisms have been proposed behind this phenomenon; however, the most fundamental physical and chemical processes are still not well identified. Particularly, the nature of interactions between ZnO surfaces, cellular membranes and bacterial growth media remain unambiguous. To address this matter, we employ ZnO microparticles synthesized hydrothermally and subjected to Staphylococcus aureus biological assays with different microbial growth media. The ZnO microcrystals are produced using growth parameters for reliable control of morphology, and specifically surface polarity. The biological assays are used to examine the antibacterial action and also to run pre- and post-assay comparative studies of the ZnO crystals themselves. For the latter we employ a variety of characterization techniques, such as electron microscopy, energy-dispersive X-ray spectroscopy, time and wavelength dependent surface photovoltage, temperature-dependent photoluminescence spectroscopy, etc. Our experiments suggest that structural and optoelectronic changes of the ZnO surfaces strongly depend on the growth media type. There are indications of the influence of crystalline surface polarity, in particular in regard to the relevant concentration of surface defects and surface charge dynamics. |
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