Mechanochemical feedback underlies coexistence of qualitatively distinct cell polarity patterns within diverse cell populations.

Cell polarization and directional cell migration can display random, persistent, and oscillatory dynamic patterns. However, it is not clear whether these polarity patterns can be explained by the same underlying regulatory mechanism. Here, we show that random, persistent, and oscillatory migration accompanied by polarization can simultaneously occur in populations of melanoma cells derived from tumors with different degrees of aggressiveness. We demonstrate that all of these patterns and the probabilities of their occurrence are quantitatively accounted for by a simple mechanism involving a spatially distributed, mechanochemical feedback coupling the dynamically changing extracellular matrix (ECM)-cell contacts to the activation of signaling downstream of the Rho-family small GTPases. This mechanism is supported by a predictive mathematical model and extensive experimental validation, and can explain previously reported results for diverse cell types. In melanoma, this mechanism also accounts for the effects of genetic and environmental perturbations, including mutations linked to invasive cell spread. The resulting mechanistic understanding of cell polarity quantitatively captures the relationship between population variability and phenotypic plasticity, with the potential to account for a wide variety of cell migration states in diverse pathological and physiological conditions.

Directed migration of cancer cells guided by the graded texture of the underlying matrix.

Living cells and the extracellular matrix (ECM) can exhibit complex interactions that define key developmental, physiological and pathological processes. Here, we report a new type of directed migration-which we term 'topotaxis'-guided by the gradient of the nanoscale topographic features in the cells' ECM environment. We show that the direction of topotaxis is reflective of the effective cell stiffness, and that it depends on the balance of the ECM-triggered signalling pathways PI(3)K-Akt and ROCK-MLCK. In melanoma cancer cells, this balance can be altered by different ECM inputs, pharmacological perturbations or genetic alterations, particularly a loss of PTEN in aggressive melanoma cells. We conclude that topotaxis is a product of the material properties of cells and the surrounding ECM, and propose that the invasive capacity of many cancers may depend broadly on topotactic responses, providing a potentially attractive mechanism for controlling invasive and metastatic behaviour.

High content cell screening in a microfluidic device.

A comprehensive, systems level understanding of cell signaling networks requires methods to efficiently assay multiple signaling species, at the level of single cells, responding to a variety of stimulation protocols. Here we describe a microfluidic device that enables quantitative interrogation of signaling networks in thousands of individual cells using immunofluorescence-based readouts. The device is especially useful for measuring the signaling activity of kinases, transcription factors, and/or target genes in a high throughput, high content manner. We demonstrate how the device may be used to measure detailed time courses of signaling responses to one or more soluble stimuli and/or chemical inhibitors as well as responses to a complex temporal pattern of multiple stimuli. Furthermore we show how the throughput and resolution of the device may be exploited in investigating the differences, if any, of signaling at the level of a single cell versus at the level of the population. In particular, we show that NF-kappaB activity dynamics in individual cells are not asynchronous and instead resemble the dynamics of the population average in contrast to studies of cells overexpressing p65-EGFP.

Information transduction capacity of noisy biochemical signaling networks.

Molecular noise restricts the ability of an individual cell to resolve input signals of different strengths and gather information about the external environment. Transmitting information through complex signaling networks with redundancies can overcome this limitation. We developed an integrative theoretical and experimental framework, based on the formalism of information theory, to quantitatively predict and measure the amount of information transduced by molecular and cellular networks. Analyzing tumor necrosis factor (TNF) signaling revealed that individual TNF signaling pathways transduce information sufficient for accurate binary decisions, and an upstream bottleneck limits the information gained via multiple integrated pathways. Negative feedback to this bottleneck could both alleviate and enhance its limiting effect, despite decreasing noise. Bottlenecks likewise constrain information attained by networks signaling through multiple genes or cells.

Using a microfluidic device for high-content analysis of cell signaling.

Quantitative analysis and understanding of signaling networks require measurements of the location and activities of key proteins over time, at the level of single cells, in response to various perturbations. Microfluidic devices enable such analyses to be conducted in a high-throughput and in a highly controlled manner. We describe in detail how to design and use a microfluidic device to perform such information-rich experiments.