Solving a long-standing mystery, scientists identify principal protein sensor for touch

A team led by biologists at The Scripps Research Institute (TSRI) has solved a long-standing mystery in neuroscience by identifying the "mechanoreceptor" protein that mediates the sense of touch in mammals.

Mice that lack the Piezo2 ion-channel protein in their skin cells and nerve endings lose nearly all their sensitivity to ordinary light touch, but retain a mostly normal sensitivity to painful mechanical stimuli.

"We can say with certainty that Piezo2 is the principal touch sensor in mammals," said Ardem Patapoutian, professor at TSRI and investigator with Howard Hughes Medical Institute.

Patapoutian and his colleagues report their discovery in the December 4, 2014 issue of Nature.

Unraveling the Clues

By the 1980s scientists have known the identity and sequence of the main protein photoreceptor that underlies the mammalian sense of sight. Since the early 1990s they have been identifying smell and taste receptors. But the mechanoreceptor protein that mediates the sense of touch has been more elusive. "It works in very few specialized cells and isn't abundant in those cells," Patapoutian said, "and at the start we didn't have many clues about what it would look like."

Four years ago, with the help of advanced genomics techniques, Patapoutian and members of his laboratory were able to identify two mechanically activated ion-channel proteins, Piezo1 and Piezo2, in mouse cells. Physical force, enough to distort a cell membrane in which one of these ion channels was embedded, could effectively switch the ion-channel from closed to open, allowing sodium or other positively charged electrolytes to flow inward. In a sensory nerve, that could trigger an electrical nerve impulse -- thus tranducing physical force into a neural signal.

Of the two newly identified ion-channel proteins, only Piezo2 was expressed significantly in the touch-sensing neurons that are based in the dorsal root ganglia of the spine and extend their nerve processes into the skin. That led Patapoutian to focus on it as the likely transducer for the mammalian sense of touch.

Last year, Patapoutian and colleagues reported that Piezo2 works as the touch sensor on Merkel cells, specialized cells that lie at touch-sensitive nerve terminals in the skin and augment the sense of touch in mice.

One Ion Channel for One Type of Touch

In the new study, the researchers extended these findings to the touch-sensitive nerve terminals themselves. These are often shaped to detect different types and directions of force, and for extra sensitivity may be attached to other force-responsive structures such as Merkel cells or hair follicles.

To start, the scientists made use of special mice, bred in the earlier Merkel cell study led by postdoctoral fellow Seung-Hyun Woo, which produce Piezo2 linked to a fluorescent protein. That allowed them to verify, via the resulting fluorescence, that Piezo2 is expressed in a broad range of "low-threshold mechanoreceptor" nerve terminals, which are embedded in both hairy and hairless areas of mouse skin.

The next step was to delete the Piezo2 gene from mice and observe whether the animals still responded normally to touch stimuli. But mice bred without Piezo2 all died at birth. Thus, postdoctoral fellow Sanjeev S. Ranade, lead author of the paper, had to accomplish the tricky task of developing a "conditional knockout" mouse line, in which the Piezo2 gene could be almost completely deleted from already-mature mice, and just from their dorsal root ganglia sensory neurons and Merkel cells.

Electrical tests of these neurons cultured from the mice, performed by staff scientist Adrienne Dubin, showed that they lost virtually all the responsiveness to mechanical stimuli that would be required for sensing ordinary light touch. Collaborator Gary Lewin and members of his laboratory at Berlin's Max-Delbrück Center for Molecular Medicine found the same profound loss of mechanosensitivity in special tests of intact skin nerves from the mice.

The mice lacking Piezo2 in their nerve endings and Merkel cells also showed a clear behavioral difference from normal mice. "Across a range of tests, we observed a dramatic reduction in their responsiveness to ordinary light touch stimuli," said Ranade.

Remarkably, these touch-insensitive mice remained responsive to skin-applied stimuli that are normally painful, such as heat, cold and pinching. Painful mechanical sensations such as pinching are thought to be mediated by high-threshold mechanoreceptor nerve terminals, which require more force to activate. "The functions of these high-threshold mechanoreceptor nerves seemed unaffected in the Piezo2 conditional knockout mice," Patapoutian said.

The finding suggests that the detection of light, innocuous touch -- which we commonly think of as the "sense of touch" -- is mediated principally by one set of nerve ends using piezo2 ion channels. By contrast, stronger, pain-causing touch sensations appear to be mediated by a less force-sensitive set of nerve ends with their own ion channel proteins, which have yet to be discovered.

Potential Applications

Patapoutian now plans to investigate how much "crosstalk" exists between these two mechanosensitive nerve systems. It is well known that chronic pain conditions can make even light touch stimuli feel painful. "This discovery now allows us to test the relationship between touch and pain," he said.

He and his colleagues also are investigating the role of Piezo2 in other parts of the body where it is expressed, including the lungs.

Funding for the research came partly from the National Institutes of Health (R01 DE022358).

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Identifying the cellular origin of fibrosis

A Gli1 progenitor cell (red) located in a healthy kidney. During fibrosis these cells differentiate into myofibroblasts, causing scarring and organ failure.

Researchers from Brigham and Women's Hospital (BWH) have identified what they believe to be the cells responsible for fibrosis, the buildup of scar tissue. Fibrotic diseases, such as chronic kidney disease and failure, lung disease, heart failure and cirrhosis of the liver, are estimated to be responsible for up to 45 percent of deaths in the developed world.

The findings are published online in the journal Cell Stem Cell.

"Previous research indicated that myofibroblasts are the cells responsible for fibrosis," said Benjamin Humphreys, MD, PhD, senior author of the research paper and a physician scientist in the Renal Division at BWH. "But there was controversy around the origin of this cell. Identifying the origin could lead to targeted therapies for these very common diseases."

With the knowledge that fibrosis appears to radiate from blood vessels, Humphreys and colleagues examined the hedgehog signaling pathway, which normally regulates organ development but whose roles in the adult are less clear. They noticed that in adult mice, a hedgehog pathway gene called Gli1 was specifically expressed in a rare group of cells located around blood vessels in all solid organs. This pattern suggested that the cells might play a role in fibrosis. To test this hypothesis the researchers tagged this protein in tissue with varying forms of fibrosis, and found that these cells proliferated by almost 20-fold under chronic injury and turned into myofibroblasts.

"We believe that this cell population is responsible for about 60 percent of all organ myofibroblasts," said Humphreys.

After identifying these cells, first author Rafael Kramann, MD, Humphreys and colleagues set out to determine whether removing these cells would lead to improvement in organ function.

Using a genetic strategy in mice, the researchers were able to ablate these Gli1 cells, while leaving other cells unharmed. In mice with kidney and cardiac fibrosis, the ablation of these cells resulted in reduction in fibrosis and rescued heart function.

"We've found that these Gli1 progenitor cells differentiate into myofibroblasts, and in fibrotic disease, when they are ablated, we can rescue organs and organ function," said Humphreys.

Researchers note that the genetic strategy employed in the preclinical model is not feasible in humans. For this reason, future research involves the exploration of drugs that could specifically target and shut off these fibrosis-causing stem cells with the hope that either an existing drug or a new drug could translate to a potential therapy for humans.

Humphreys also notes that this cell population plays a role in the aging process. "Most organs develop fibrosis as we age," he said. "Specifically, in the kidney we lose one percent of kidney function as a result of fibrosis for each year that we age. We look forward to future research, using human tissue, to confirm our findings in humans and work to develop a potential therapy."

This research was supported by the Harvard Stem Cell Institute, the National Institutes of Health, the National Institute of Diabetes and Digestive and Kidney Diseases, the American Heart Association, and the Deutsche Forschungsgemeinschaft.

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Revealed: How bacteria drill into our cells and kill them

A team of scientists has revealed how certain harmful bacteria drill into our cells to kill them. Their study shows how bacterial 'nanodrills' assemble themselves on the outer surfaces of our cells, and includes the first movie of how they then punch holes in the cells' outer membranes. The research, published in the journal eLife, supports the development of new drugs that target this mechanism, which is implicated in serious diseases. The team brings together researchers from UCL, Birkbeck, University of London, the University of Leicester, and Monash University (Melbourne).

Unlike drills from a DIY kit, which twist and grind their way through a surface, bacterial nanodrills do not contain rotating parts. Rather, they are ring-like structures (similar to an eyelet) built out of self-assembling toxin molecules. Once assembled, the toxins deploy a blade around the ring's inside edge that slices down into the cell membrane, forming a hole.
To determine how these rings are built, team member Natalya Dudkina made several thousand images of artificial cell membranes coated with toxins, using an electron microscope. Dudkina is a member of Helen Saibil's group at Birkbeck, University of London, which specialises in mapping biological structures using electron microscopy.

"Each ring was formed of around 37 copies of the toxin molecule. But aside from complete rings, we also observed arc-shaped, incomplete rings," Dudkina said. "One problem we had, though, was that our method can only record snapshots of the membrane perforation process frozen at different intermediate stages."

The solution to this was to produce a 'movie' of what happens when the toxins are placed on a cell membrane. This was carried out with atomic force microscopy (AFM) at Bart Hoogenboom's lab at the London Centre for Nanotechnology at UCL. AFM uses an ultrafine needle to feel, rather than see, a surface. This needle repeatedly scans the surface to produce a moving image that refreshes fast enough to show how the toxins move over the membrane and then cut holes in the membrane as they sink in.

"It was quite spectacular to look at," said Carl Leung, a member of Hoogenboom's lab at UCL. "After the initial assembly of the toxins into arcs and rings, they kept skating over the membrane surface. Then they stopped, sank into the membrane, and started spitting out the material they had drilled through, like sawdust when you drill holes in wood."

A big surprise for the team was that complete rings aren't needed to pierce the cell membrane: even relatively short fragments are still able to cut holes, albeit smaller ones, and hold them open, allowing bacteria to feed on the cell's contents.

Together, these findings give a detailed view of how these bacterial toxins drill holes in cell membranes. The snapshots from the electron microscopy show how the rings are structured at the start and the end of the drilling process, and the moving images from the AFM show the process as it unfolds.

The discovery supports the development of new drugs that can target bacterial nanodrills and help treat the diseases in which they are implicated. These include pneumonia, meningitis and septicaemia. Extensive research into such drugs that is ongoing at the University of Leicester, which also provided a genetically modified form of the toxin to help identify the different steps in the hole-drilling process.

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Ciliopathies lie behind many human diseases

In recent years, cilia, microscopic, tentacle-like extensions from biological cells, have risen from relative obscurity and are now considered important to the understanding of many human afflictions. In a December BioScience article, George B. Witman, of the University of Massachusetts Medical School, and Jason M. Brown, of Salem State University, describe recent discoveries involving cilia-related diseases (called "ciliopathies") and highlight "model" species that could be useful for systematic study of ciliopathies.

Cilia perform a broad range of functions, including a starring role in cell signalling. Motile ones wiggle and so move fluids within the body, including cerebrospinal fluid in the brain. In humans, cilia are found on almost every cell in the body. Because of this, ciliopathies often make themselves known as syndromes with widely varying effects on a number of tissue types. For instance, the ciliopathy Jeune asphyxiating thoracic dystrophy involves the development of abnormally short ribs, accompanied by short limbs and, occasionally, the development of extra digits.

In primary ciliary dyskinesia, motile cilia are dysfunctional and fail to beat. This can lead to bronchitis resulting from the failure to clear mucus from the sufferer's airways. Male patients with primary ciliary dyskinesia are infertile because of impaired motility of the sperm's flagellum (flagella and cilia are structurally similar).

The article's authors point to a number of other human diseases in which cilia may play a role; for example, some cancers and neurological diseases may be related to ciliopathies. Because of the limitations placed on research involving humans, the authors propose the use of model species ranging from the green alga Chlamydomonas to the house mouse to further study the role of cilia. They write, "We can anticipate that new and improved techniques will open new avenues for gaining further insight into these immensely important and ever more fascinating cell organelles."

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