Session B49: Biophysics of Cellular Organization and Dynamics Across Multiple Spatial Scales - I

How cells measure length: clocks, rulers, and diffusion

The mechanisms by which living cells are able to build structures of defined size and geometry remain poorly understood, despite extensive knowledge of the molecular building blocks. We are using the flagellum of the model organism Chlamydomonas as a test-bed to explore possible mechanisms by which cells can measure the length of a simple organelle. Our approach is to formulate simple physical models and then test them them by taking advantage of the powerful genetics of this model system, in combination with quantitative microscopy and mathematical modeling. We are currently exploring length-measurement schemes based on molecular timers, ion channels, and diffusion of motors. The approaches used to formulate and test these models for flagella should be generally applicable to probing size control mechanisms of other organelles.   

Effect of Glucose-dependent Motility on Spatial Organization of Mitochondria in a Neuron

Cellular metabolism has been a focus of intense study since the early days of molecular biology. The standard quantitative methods that have been developed for modeling metabolic response generally neglect intracellular spatial organization. Carefully regulated distribution of metabolic components is especially critical for the time-sensitive energy demands of hyper-extended cells like the neuron. Recent experimental results have shown that the transport of mitochondria is regulated by glucose concentration, and that glucose itself is heterogeneously distributed in neurons. We use analytical methods and simulations to model glucose-dependent mitochondrial motility in a neuron with spatially varying permeability to extracellular glucose. Our model encapsulates biochemical pathways coupling the action of molecular motors responsible for mitochondrial transport to glucose concentration via nonlinear kinetics. The resulting distribution of mitochondria suggests that transport-based regulation can be made sensitive to a broad range of glucose levels under sufficiently rapid rates of glucose consumption by mitochondria. Our predictions for these sensitivity ranges are parameterized by and validated against in vivo data on neuronal mitochondria distribution.   

Effect of boundary conditions on the transverse thermal fluctuations of filaments and its implications for microrheological tesnsion mapping of filamentous networks

Understanding the mechanics of tissues at the scale of one to hundreds of microns is a
forefront challenge in biomechanics. At this scale, the filamentous network of the extracellular
matrix (ECM) cannot be treated as an elastic continuum; forces propagate through it along
complex paths encompassing only a small fraction of the fibers. To map tensions in such
networks under load, we propose to monitor the transverse thermal fluctuations of multiple
fibers. To that end, we discuss our calculations of the fiber fluctuations with various boundary
conditions, including complex junctions with other fibers. We also discuss fluctuations of fibers
near to their buckling transition. Finally, we address a self-consistent model of a fluctuating
fiber in a network of similar fibers. These calculations should inform a new sort of
microrheological tension mapping of the ECM via confocal fiber tracking.   

Lattice Light Sheet Microscopy of Endocytosis in Macrophages

Live cell, volumetric fluorescence imaging using a Lattice Light Sheet microscope, developed in collaboration with HHMI Janelia Farm Research Campus[1] is used to visualize endocytosis of B cells by macrophages derived from mouse bone marrow. Cells are gene edited to express fluorescence of specific organelles and/or labeled with fluorescent antibodies. Volumetric, multi-color fluorescence imaging reveals cellular level dynamical details of this process. Discussion of major findings, instrumentation, and data visualization will be presented[2].
  

Physical Control of Actin Wave Dynamics and Migration of Neutrophil-like HL60 Cells

Neutrophils, immune cells which play an important role in inflammation control, are known to respond to chemical gradients and contact guidance cues inside the body. However, neutrophil response to electric fields is not well understood. In this work, we explore actin wave dynamics and cell migration in HL60 cells exposed to a combination of DC electric fields and contact cues provided by textured surfaces. We show these coupled physical stimuli applied to neutrophil-like HL60 cells result in alignment of actin waves with field polarity and directed cell migration. The existence of coupled responses in actin polymerization waves suggest that the cytoskeletal dynamics play an important role in electrotaxis and that DC electric fields strongly affect neutrophil guidance.   

Statistical Thermodynamics of Cellular Metabolism and Growth

Cell metabolism is modeled using a maximum entropy production rate assumption from which rate parameters can be inferred for use in simulating the mass action kinetics of metabolism. Simulation predictions of metabolite levels of central metabolism of Neurospora crassa and Yarrowia lipolytica then allow for inference of enzyme regulation for both fungi. Subsequent simulations with regulation provide predictions of metabolite levels that are comparable to experimental measurements and can be used to create free energy maps of metabolic pathways. Simulations elucidate the dissipative role of the Crabtree/Warburg effect of overflow metabolism, and provide a more complete understanding of biological cells as adaptive, dissipative structures. Statistical thermodynamics is also combined with data analysis to measure the work required to create a cell (in kJ/gm cells or kJ/mol cells) and the power (in Watts) generated by cells during growth. It is estimated that a bacterial cell produces has approximately the same power/weight ratio as the most efficient fuel cells.   

A genetically encoded toolbox of orthogonal adhesins for bacterial self-assembly

In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. In particular, to our knowledge there exists no convenient method for engineering cell-cell adhesion. Towards filling this gap in synthetic biology's toolbox, here we report the first 100% genetically encoded self-assembly platform, based on modular cell-cell adhesion in Escherichia coli. Adhesive selectivity is provided by a library of outer membrane-displayed peptides with orthogonal intra-library specificities, while affinity is provided by intrinsic adhesin affinity, media conditions, and inducible expression across the entire library. We demonstrate this tool by building well-defined multicellular patterns, including multiple cell types in cluster-, mesh-, and lattice-like arrangements, even during cell growth and division. We further quantify these structures using nearest-neighbor graphs, fractal dimension, and density distribution. This adhesion system will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, in tissue engineering, and in controlled study of minimal multicellular systems.   

Collective chemotaxis of cells with long-range communication

Cells use a variety of methods to navigate chemical gradients in order to move toward attractants or away from repellents. In addition, many cell types have been shown to improve the accuracy of their chemotaxis by acting as a collective and/or utilizing cell-cell communication, which can help reduce measurement noise. The ‘many wrongs’ model of collective migration is a well studied mechanism by which a collective tracks a gradient more precisely than an individual agent. However, when applied to cells, the many wrongs model typically assumes that cells interact via spatial contact. It remains unclear whether the benefits of cooperation can be extended to spatially separated cells that maintain such a coupling through long range communication. Using stochastic modeling and agent-based simulation, we study such an extension in which the cells communicate using a combination of secreted attractor and repulsor molecules. Surprisingly, we find that the accuracy of chemotaxis is optimized not when cells are tightly packed, but rather when cells are spaced at a nonzero average nearest-neighbor distance, resulting in a sparsely packed swarm. We discuss the mechanism behind the sparse packing and the implications for collective chemotaxis.   

Dynamic switching enables efficient bacterial colonization in flow

Bacteria colonize environments that contain networks of moving fluids including blood vasculature in animals and flow networks in plants. Here, bacteria form distinct biofilm structures that have an important role in pathogenesis. The physical mechanisms that determine the spatial organization of bacteria in flow are not understood. We show that the bacterium P. aeruginosa colonizes flow networks using a cyclical process involving surface attachment, motility, and transport. This process, which we have termed dynamic switching, distributes bacterial sub-populations upstream and downstream in flow through two phases: on surfaces and via the bulk. The model equations that describe dynamic switching are identical to those that describe dynamic instability, a process that enables microtubules in eukaryotic cells to search space efficiently to capture chromosomes. Our results show that dynamic switching enables bacteria to explore flow networks efficiently, which maximizes dispersal in flow networks and establishes the organizational structure of biofilms. A number of eukaryotic cells also exhibit movement in two phases in flow, which suggests that dynamic switching is a modality that enables efficient dispersal for a broad range of cell types.   

Physical determinants of 3D bacterial biofilm architectures

In many situations bacteria aggregate to form biofilms: dense, surface-associated, three-dimensional structures populated by cells embedded in matrix. Biofilm architectures are sculpted by mechanical processes including cell growth, cell-cell interactions and external forces. Using single-cell live imaging in combination with simulations we characterize the cell-cell interactions that generate Vibrio cholerae biofilm morphologies. Fluid shear is shown to affect biofilm shape through the growth rate and orientation of cells, despite spatial differences in shear stress being balanced by cell-cell adhesion. Our results demonstrate the importance of cell dynamics mediated by adhesion proteins and matrix generation in determining the global architecture of biofilm structures.   

Spatial gradient sensing and wave rectification via excitability

The social amoeba Dictyostelium discoideum perfoms chemotaxis under starvation conditions, aggregating towards emergent groups of cells that spontaneously emit cAMP. Cells sense extracellular cAMP, producing internal caches of cAMP to be released. These events lead to traveling waves of cAMP washing over the entire population of cells. While research has been performed to understand elements of the chemotaxis network in Dictyostelium, limited work has been proposed to link components of the signal relay network with the chemotaxis network to provide a holistic view of the system. We take inspiration from Dictyostelium and propose a model that links the relaying of a chemical message to the directional sensing of that signal. Utilizing excitable dynamical systems, our model provides signal amplification and perfect adaptation while also automatically matching internal time scales of adaptation to the naturally occurring periodicity of the traveling chemical waves. We show that noise plays a vital role in the system, where stochastic tunneling of transient bursts biases the system towards accurate gradient sensing. Numerical simulations were performed to study the qualitative phenomenology of the system and explore how the system responds to diverse dynamic spatiotemporal stimuli.   

Using Topographical Guidance to Investigate Cytoskeletal Excitability

The extracellular environment provides mechanical, electrical, and chemical inputs that play key roles in cellular development, intracellular signaling, and cell migration. When responding to the extracellular environment, the cell can be viewed as an excitable system that can be driven into several distinct “states.” These states, which consist of particular morphologies created by combinations of physical inputs, can be induced across a range of cell types and can be leveraged as platforms for isolating and perturbing a parameter of interest. In this talk, we use topographical features of a size comparable to collagen fibers to place MCF10A and HEK293T/17 cells into distinct states in which cytoskeletal polymerization waves are guided by and align with the underlying topography. We then use an array of techniques, including Raman spectroscopy and optical flow analysis, to investigate links between these polymerization waves and other signaling factors.   

Subdiffusion Arising from Intracellular Phase Separation

The intracellular environment of the cell, or cytoplasm, is a multicomponent mixture that is home to many active biological processes. It has been experimentally shown for a range of systems and contexts, including Saccharomyces cerevisiae, Escherichia coli, and mammalian cell lines, that proteins and nucleic acids in the cytoplasm undergo non-Gaussian anomalous diffusion even in the absence of evident viscoelastic cytoskeletal networks. One potential mechanism that could lead to these dynamics is liquid-liquid phase separation. Here, we report the properties of a system in and near the regime of phase separation, using molecular dynamics and Monte Carlo lattice simulations. We recover subdiffusion with exponentially decaying step-size distributions as have been observed in experiment and describe slowing in the vicinity of a critical point. Finally, we propose that phase separation allows the cell to actively tune and organize its interior.