The nervous system is often considered the body’s wiring, sending electrical signals to communicate needs and hazards between different parts of the body. However, researchers at the University of Massachusetts at Amherst have now also measured bioelectronic signals propagating from cultured epithelial cells, as they respond to a critical injury.

“Cells are pretty amazing in terms of how they are making collective decisions, because it seems like there is no centre, like a brain,” says researcher Sunmin Yu, who likens epithelial cells to ants in the way that they gather information and solve problems. Alongside lab leader Steve Granick, Yu reports this latest finding in Proceedings of the National Academy of Sciences, suggesting a means for the communication between cells that enables them to coordinate with each other.

While neurons function by bioelectric signals, and punctuated rhythmic bioelectrical signals allow heart muscle cells to keep the heart pumping blood throughout our body, when it comes to intercell signals for any other type of cell, the most common hypothesis is the exchange of chemical cues. Yu, however, had noted from previous work by other groups that the process of “extruding” wounded epithelial cells to get rid of them involved increased expression of the relevant proteins at some distance from the wound itself.

“Our thought process was to inquire about the mechanism by which information could be transmitted over the necessary long distance,” says Yu. She realised that common molecular signalling mechanisms, such as extracellular signal-regulated kinase 1/2 (ERK), which has a speed of around 1 mm/s, would be rather slow as a potential conduit.

Epithelial signals measure up

Yu and Granick grew a layer of epithelial cells on a microelectrode array (MEA). While other approaches to measuring electrical activity in cultured cells exist, an MEA has the advantage of combining electrical sensitivity with a long range, enabling the researchers to collect both temporal and spatial information on electrical activity. They then “wounded” the cells by exposing them to an intense focused laser beam.

Following the wound, the researchers observed electrical potential changes with comparable amplitudes and similar shapes to those observed in neurons, but over much longer periods of time. “The signal propagation speed we measured is about 1000 times slower than neurons and 10 times faster than ERK,” says Yu, expressing great interest in whether the “high-pitch speaking” neurons and heart tissue cells communicate with these “low-pitch speaking” epithelial cells, and if so, how.

The researchers noted an apparent threshold in the amplitude of the generated signal required for it to propagate. But for those that met this threshold, propagation of the electric signals spanned regions up to 600 µm for as long as measurements could be recorded, which was 5 h. Given the mechanical forces generated during “cell extrusion”, the researchers hypothesized the likely role of mechanosensitive proteins in generating the signals. Sure enough, inhibiting the mechanosensitive ion channels shut down the generation of electrical signals.

Yu and Granick highlight previous suggestions that electrical potentials in epithelial cells may be important for regulating the coordinated changes that take place during embryogenesis and regeneration, as well as being implicated in cancer. However, this is the first observation of such electrical potentials being generated and propagating across epithelial tissue.

“Yu and Granick have discovered a remarkable new form of electrical signalling emitted by wounded epithelial cells – cells traditionally viewed as electrically passive,” says Seth Fraden, whose lab at Brandeis University in Massachusetts in the US investigates a range of soft matter topics but was not involved in this research.

Fraden adds that it raises an “intriguing” question: “What is the signal’s target? In light of recent findings by Nathan Belliveau and colleagues, identifying the protein Galvanin as a specific electric-field sensor in immune cells, a compelling hypothesis emerges: epithelial cells send these electric signals as distress calls and immune cells – nature’s healers – receive them to rapidly locate and respond to tissue injuries. Such insights may have profound implications for developing novel regenerative therapies and bioelectric devices aimed at accelerating wound healing.”

Adam Ezra Cohen, whose team at Harvard University in the US focuses on innovative technology for probing molecules and cells, and who was not directly involved in this research, also finds the research “intriguing” but raises numerous questions: “What are the underlying membrane voltage dynamics?  What are the molecular mechanisms that drive these spikes? Do similar things happen in intact tissues or live animals?” he asks, adding that techniques such as patch clamp electrophysiology and voltage imaging could address these questions.

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While the biology of how an entire organism develops from a single cell has long been a source of fascination, recent research has increasingly highlighted the role of mechanical forces. “If we want to have rigorous predictive models of morphogenesis, of tissues and cells forming organs of an animal,” says Konstantin Doubrovinski at the University of Texas Southwestern Medical Center, “it is absolutely critical that we have a clear understanding of material properties of these tissues.”

Now Doubrovinski and his colleagues report a rheological study explaining why the developing fruit fly (Drosophila melanogaster) epithelial tissue stretches as it does over time to allow the embryo to change shape.

Previous studies had shown that under a constant force, tissue extension was proportional to the time the force had been applied to the power of one half. This had puzzled the researchers, since it did not fit a simple model in which epithelial tissues behave like linear springs. In such a model, the extension obeys Hooke’s law and is proportional to the force applied alone, such that the exponent of time in the relation would be zero.

They and other groups had tried to explain this observation of an exponent equal to 0.5 as due to the viscosity of the medium surrounding the cells, which would lead to deformation near the point of pulling that then gradually spreads. However, their subsequent experiments ruled out viscosity as a cause of the non-zero exponent.

For their measurements, the researchers had exploited a convenient feature of Drosophila epithelial cells – a small hole, through which they could manipulate a droplet of ferrofluid to enter using a permanent magnet. Once inside the cell, a magnet acting on this droplet could exert forces on the cell to stretch the surrounding tissue.

For the current study, the researchers first tested the observed scaling law over longer periods of time. A power law gives a straight line on a log–log plot but as Doubrovinski points out, curves also look like straight lines over short sections. However, even when they increased the time scales probed in their experiments to cover three orders of magnitude – from fractions of a second to several minutes – the observed power law still held.

Understanding the results

One of the post docs on the team – Mohamad Ibrahim Cheikh – stumbled upon the actual relation giving the power law with an exponent of 0.5 while working on a largely unrelated problem. He had been modelling ellipsoids in a hexagonal meshwork on a surface, in what Doubrovinski describes as a “large” and “relatively complex” simulation. He decided to examine what would happen if he allowed the mesh to relax in its stretched position, which would model the process of actin turnover in cells.

Cheikh’s simulation gave the power law observed in the epithelial cells. “We totally didn’t expect it,” says Doubrovinski. “We pursued it and thought, why are we getting it? What’s going on here?”

Although this simulation yielded the power law with an exponent of 0.5, because the simulation was so complex, it was hard to get a handle on why. “There are all these different physical effects that we took into account that we thought were relevant,” he tells Physics World.

To get a more intuitive understanding of the system, the researchers attempted to simplify the model into a lattice of springs in one dimension, keeping only some of the physical effects from the simulations, until they identified the effects required to give the exponent value of 0.5. They could then scale this simplified one-dimensional model back up to three dimensions and test how it behaved.

According to their model, if they changed the magnitude of various parameters, they should be able to rescale the curves so that they essentially collapse onto a single curve. “This makes our prediction falsifiable,” says Doubrovinski, and in fact the experimental curves could be rescaled in this way.

When the researchers used measured values for the relaxation constant based on the actin turnover rate, along with other known parameters such as the size of the force and the size of the extension, they were able to calculate the force constant of the epithelial cell. This value also agreed with their previous estimates.

Doubrovinski explains how the ferrofluid droplet engages with individual “springs” of the lattice as it moves through the mesh. “The further it moves, the more springs it catches on,” he says. “So the rapid increase of one turns into a slow increase with an exponent of 0.5.” Against this model, all the pieces fit into place.

“I find it inspiring that the authors, first motivated by in vivo mechanical measurements, could develop a simple theory capturing a new phenomenological law of tissue rheology,” says Pierre Françoise Lenne, group leader at the Institut de Biologie du Development de Marseille at L’Universite d’Aix-Marseille. Lenne specializes in the morphogenesis of multicellular systems but was not involved in the current research.

Next, Doubrovinski and his team are keen to see where else their results might apply, such as other developmental stages and other types of organisms, such as mammals, for example.

The research is reported in Physical Review Letters and bioRxiv.

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