Membrane potential mediates an ancient mechano-transduction mechanism for multi-cellular homeostasis.

Membrane potential is a property of all living cells1. However, its physiological role in non-excitable cells is poorly understood. Resting membrane potential is typically considered fixed for a given cell type and under tight homeostatic control2, akin to body temperature in mammals. Contrary to this widely accepted paradigm, we found that membrane potential is a dynamic property that directly reflects tissue density and mechanical forces acting on the cell. Serving as a quasi-instantaneous, global readout of density and mechanical pressure, membrane potential is integrated with signal transduction networks by affecting the conformation and clustering of proteins in the membrane3,4, as well as the transmembrane flux of key signaling ions5,6. Indeed, we show that important mechano-sensing pathways, YAP, Jnk and p387-121314, are directly controlled by membrane potential. We further show that mechano-transduction via membrane potential plays a critical role in the homeostasis of epithelial tissues, setting tissue density by controlling proliferation and cell extrusion of cells. Moreover, a wave of depolarization triggered by mechanical stretch enhances the speed of wound healing. Mechano-transduction via membrane potential likely constitutes an ancient homeostatic mechanism in multi-cellular organisms, potentially serving as a steppingstone for the evolution of excitable tissues and neuronal mechano-sensing. The breakdown of membrane potential mediated homeostatic regulation may contribute to tumor growth.

A universal mechanism of biomass density homeostasis via ribosomal counterions.

In all growing cells, the cell envelope must expand in concert with cytoplasmic biomass to prevent lysis or molecular crowding. The complex cell wall of microbes and plants makes this challenge especially daunting and it unclear how cells achieve this coordination. Here, we uncover a striking linear increase of cytoplasmic pressure with growth rate in E. coli. Remarkably, despite this increase in turgor pressure with growth rate, cellular biomass density was constant across a wide range of growth rates. In contrast, perturbing pressure away from this scaling directly affected biomass density. A mathematical model, in which endopeptidase-mediated cell wall fluidization enables turgor pressure to set the pace of cellular volume expansion, not only explains these confounding observations, but makes several surprising quantitative predictions that we validated experimentally. The picture that emerges is that changes in turgor pressure across growth rates are mediated by counterions of ribosomal RNA. Profoundly, the coupling between rRNA and cytoplasmic pressure simultaneously coordinates cell wall expansion across growth rates and exerts homeostatic feedback control on biomass density. Because ribosome content universally scales with growth rate in fast growing cells, this universal mechanism may control cell wall biosynthesis in microbes and plants and drive the expansion of ribosome-addicted tumors that can exert substantial mechanical forces on their environment.

Multi-parametric functional imaging of cell cultures and tissues with a CMOS microelectrode array.

Electrode-based impedance and electrochemical measurements can provide cell-biology information that is difficult to obtain using optical-microscopy techniques. Such electrical methods are non-invasive, label-free, and continuous, eliminating the need for fluorescence reporters and overcoming optical imaging's throughput/temporal resolution limitations. Nonetheless, electrode-based techniques have not been heavily employed because devices typically contain few electrodes per well, resulting in noisy aggregate readouts. Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs) have sometimes been used for electrophysiological measurements with thousands of electrodes per well at sub-cellular pitches, but only basic impedance mappings of cell attachment have been performed outside of electrophysiology. Here, we report on new field-based impedance mapping and electrochemical mapping/patterning techniques to expand CMOS-MEA cell-biology applications. The methods enable accurate measurement of cell attachment, growth/wound healing, cell-cell adhesion, metabolic state, and redox properties with single-cell spatial resolution (20 µm electrode pitch). These measurements allow the quantification of adhesion and metabolic differences of cells expressing oncogenes versus wild-type controls. The multi-parametric, cell-population statistics captured by the chip-scale integrated device opens up new avenues for fully electronic high-throughput live-cell assays for phenotypic screening and drug discovery applications.

ArcA overexpression induces fermentation and results in enhanced growth rates of E. coli.

Overflow metabolism in the presence of oxygen occurs at fast growth rates in a wide range of organisms including bacteria, yeast and cancer cells and plays an important role in biotechnology during production of proteins or metabolic compounds. As recently suggested, overflow metabolism can be understood in terms of proteome allocation, since fermentation has lower proteome cost for energy production than respiration. Here, we demonstrate that ArcA overexpression in aerobic conditions, results in downregulation of respiratory pathways and enhanced growth rates on glycolytic substrates of E. coli, coinciding with acetate excretion and increased carbon uptake rates. These results suggest that fermentation enables faster growth and demonstrate that fermentation on many glycolytic carbon sources is not limited by carbon uptake. Hence, these findings are difficult to reconcile with many alternative hypotheses that have been proposed for the origin of overflow metabolism and the growth rate dependence of fermentation and respiration, which are based on limited capacity of respiration or limitations in uptake rates and catabolic pathways. Instead, as suggested by increased lag phases of ArcA overexpression strains, respiratory energy metabolism may be related to a general preparatory response, observed for decreasing growth rates, but with limited advantages for maximizing steady-state growth rate.

Overflow metabolism in Escherichia coli results from efficient proteome allocation.

Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as the Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast-growing cells, including bacteria, fungi and mammalian cells, but its origin has remained unclear despite decades of research. Here we study metabolic overflow in Escherichia coli, and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviours, and accurately predict responses to new perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry.

Intercellular stress reconstitution from traction force data.

Cells migrate collectively during development, wound healing, and cancer metastasis. Recently, a method has been developed to recover intercellular stress in monolayers from measured traction forces upon the substrate. To calculate stress maps in two dimensions, the cell sheet was assumed to behave like an elastic material, and it remains unclear to what extent this assumption is valid. In this study, we simulate our recently developed model for collective cell migration, and compute intercellular stress maps using the method employed in the experiments. We also compute these maps using a method that does not depend on the traction forces or material properties. The two independently obtained stress patterns agree well for the parameters we have probed and provide a verification of the validity of the experimental method.

Alignment of cellular motility forces with tissue flow as a mechanism for efficient wound healing.

Recent experiments have shown that spreading epithelial sheets exhibit a long-range coordination of motility forces that leads to a buildup of tension in the tissue, which may enhance cell division and the speed of wound healing. Furthermore, the edges of these epithelial sheets commonly show finger-like protrusions whereas the bulk often displays spontaneous swirls of motile cells. To explain these experimental observations, we propose a simple flocking-type mechanism, in which cells tend to align their motility forces with their velocity. Implementing this idea in a mechanical tissue simulation, the proposed model gives rise to efficient spreading and can explain the experimentally observed long-range alignment of motility forces in highly disordered patterns, as well as the buildup of tensile stress throughout the tissue. Our model also qualitatively reproduces the dependence of swirl size and swirl velocity on cell density reported in experiments and exhibits an undulation instability at the edge of the spreading tissue commonly observed in vivo. Finally, we study the dependence of colony spreading speed on important physical and biological parameters and derive simple scaling relations that show that coordination of motility forces leads to an improvement of the wound healing process for realistic tissue parameters.

Stress clamp experiments on multicellular tumor spheroids.

The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10 kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression.

Undulation instability of epithelial tissues.

Treating the epithelium as an incompressible fluid adjacent to a viscoelastic stroma, we find a novel hydrodynamic instability that leads to the formation of protrusions of the epithelium into the stroma. This instability is a candidate for epithelial fingering observed in vivo. It occurs for sufficiently large viscosity, cell-division rate and thickness of the dividing region in the epithelium. Our work provides physical insight into a potential mechanism by which interfaces between epithelia and stromas undulate and potentially by which tissue dysplasia leads to cancerous invasion.

A reaction-diffusion model of the cadherin-catenin system: a possible mechanism for contact inhibition and implications for tumorigenesis.

Contact inhibition is the process by which cells switch from a motile growing state to a passive and stabilized state upon touching their neighbors. When two cells touch, an adhesion link is created between them by means of transmembrane E-cadherin proteins. Simultaneously, their actin filaments stop polymerizing in the direction perpendicular to the membrane and reorganize to create an apical belt that colocalizes with the adhesion links. Here, we propose a detailed quantitative model of the role of cytoplasmic beta-catenin and alpha-catenin proteins in this process, treated as a reaction-diffusion system. Upon cell-cell contact the concentration in alpha-catenin dimers increases, inhibiting actin branching and thereby reducing cellular motility and expansion pressure. This model provides a mechanism for contact inhibition that could explain previously unrelated experimental findings on the role played by E-cadherin, beta-catenin, and alpha-catenin in the cellular phenotype and in tumorigenesis. In particular, we address the effect of a knockout of the adenomatous polyposis coli tumor suppressor gene. Potential direct tests of our model are discussed.

Homeostatic competition drives tumor growth and metastasis nucleation.

We propose a mechanism for tumor growth emphasizing the role of homeostatic regulation and tissue stability. We show that competition between surface and bulk effects leads to the existence of a critical size that must be overcome by metastases to reach macroscopic sizes. This property can qualitatively explain the observed size distributions of metastases, while size-independent growth rates cannot account for clinical and experimental data. In addition, it potentially explains the observed preferential growth of metastases on tissue surfaces and membranes such as the pleural and peritoneal layers, suggests a mechanism underlying the seed and soil hypothesis introduced by Stephen Paget in 1889, and yields realistic values for metastatic inefficiency. We propose a number of key experiments to test these concepts. The homeostatic pressure as introduced in this work could constitute a quantitative, experimentally accessible measure for the metastatic potential of early malignant growths.