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Knowing When to Stop: NIDCD Researchers Discover Unique Protein That Appears to Control Stereocilia Length

If there were a Seven Wonders of the human body, stereocilia would definitely be one of them. Perched like a bristly, V-shaped battlement atop the sensory cells that line the inner ear, their formation is a marvel of micro-engineering. Grouped in precisely ordered rows rising in staircase fashion from the shortest in the front to the tallest in the back, these bundles of super-sensitive fibers are the structures (or organelles) responsible for converting the vibrations that enter the ear into electrical signals that travel to the brain and say "sound."

Stereocilia sensory cells that line the inner ear
Mouse stereocilia without Eps8 (bottom) are
much shorter than normal (top).
Credit: Uri Manor and Leonardo Andrade

Scientists know quite a bit about stereocilia. They know that each stereocilium is composed of bundled filaments of a protein called actin. Actin filament bundles also appear in many other types of cellular structures, such as the microvilli that line the small intestine and help us absorb nutrients from our food. They know that stereocilia are linked by a system of horizontal filaments that connect the shorter ones to their taller neighbors so that the whole bundle moves as one unit when it's stimulated by sound. What isn't known, however, is how stereocilia know when to stop growing. Their precise height and staircase formation are essential to hearing, which means there must be strong regulatory functions within the cell to police their length.

Now a discovery by NIDCD scientist Bechara Kachar, M.D., his Laboratory of Cell Structure and Dynamics, and a cadre of international researchers has identified a new protein that appears to work together with two already known proteins to regulate stereocilia length. This important discovery builds on decades of work in this and other intramural laboratories at the NIDCD to understand the complex structure and assembly of stereocilia. The findings have been published in the January 25, 2011 issue of Current Biology.

"Stereocilia lengths are regulated with incredible precision,"says Uri Manor, Ph.D., who was a graduate student in Dr. Kachar's lab who has since successfully defended his thesis, "with no more than a few nanometers of difference among those in the same row."(A nanometer is a measure of length so tiny it would take a billion of them to stretch almost the length of a yardstick.)

The scientists' search for a mechanism that could make and maintain stereocilia at such an extraordinary level of precision was built on previous studies conducted in mice hair cells, in their lab and others, of proteins that are unique to stereocilia, including the motor protein myosin XVa and another protein called whirlin. Gene mutations associated with the two proteins are known to cause several different forms of hearing loss and vestibular dysfunction.

Previous experiments have established that stereocilia lacking either myosin XVa or whirlin are abnormally short, indicating that the two proteins must be somehow involved in stereocilia growth. Also, researchers have shown that the two proteins cluster in abundant numbers at the tips of normal stereocilia. However, neither protein appears to have the ability to regulate actin bundle lengths, so they were unlikely candidates to be the ones responsible for determining stereocilia length.

Dr. Kachar and his colleagues turned to another protein—Eps8—that was known from earlier studies to regulate actin bundle growth in other kinds of cells. "No one had thought about looking at this protein in hair cells,"says Dr. Kachar, "but a group in Italy that had been involved in characterizing its function contacted us about Eps8, so we thought it was worth a try to see if we could find Eps8 in stereocilia.”

In collaboration with researchers at The FIRC Institute of Molecular Oncology Foundation and the Universita degli Studi di Milano in Milan, Italy, they were able to show that, sure enough, Eps8 localized at the tips of stereocilia. Even more interesting, it was present in amounts that were proportional to the length of each stereocilium—there was more of it in long ones and less of it in short ones. Further experimentation established that hair cells from mice that lacked Eps8 have very short stereocilia. This made it clear that Eps8 was regulating the lengths of stereocilia.

A series of experiments using immunofluroescence markers to make the proteins visible showed striking similarities in the localization of Eps8, myosin XVa, and whirlin in stereocilia tips, as well as the shortening of stereocilia when the proteins were absent. Notable was that without myosin XVa, Eps8 could not be found in the stereocilia tips. However, if whirlin was absent, Eps8 could be found, but only in smaller amounts. This gave Dr. Kachar and his colleagues enough information to hypothesize how the three proteins might interact to regulate actin bundle elongation and therefore the length of stereocilia.

According to Dr. Kachar, myosin XVa, Eps8, and whirlin are part of a system of regulated protein assembly. "Myosin XVa is the motor element that transports Eps8 and whirlin to the tips,"he says. "Eps8 is the actin-regulating element that determines stereocilia length, and whirlin has scaffolding properties that help accumulate this complex at the tips."

In a mouse model, the researchers have already shown that mice without Eps8 are deaf. It is likely that the absence of Eps8 in humans could also cause loss of hearing. Next they'll be looking at the molecular details involved in exactly how Eps8 regulates stereocilia length. Dr. Manor says that Eps8 has several regulatory domains—areas of the protein that contain actin-controlling activities. "One domain caps filaments,"he explains, "which would normally be expected to shorten actin filaments. Another domain crosslinks filaments, which could be one way of stabilizing and elongating actin bundles. It will be interesting to see how the cell uses a specific molecule with seemingly contradictory activities to so dramatically regulate the length of an actin protrusion.”