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The composition and role of cross links inmechanoelectrical transduction in vertebrate sensoryhair cells
Carole M. Hackney1 and David N. Furness2,*1Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, South Yorkshire, S10 2TN, UK2School of Life Sciences and Institute for Science and Technology in Medicine, Keele University, Staffordshire, ST5 5BG, UK
*Author for correspondence ([email protected])
Journal of Cell Science 126, 1–11� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.106120
SummaryThe key components of acousticolateralis systems (lateral line, hearing and balance) are sensory hair cells. At their apex, these cells have
a bundle of specialized cellular protrusions, which are modified actin-containing microvilli, connected together by extracellularfilaments called cross links. Stereociliary deflections open nonselective cation channels allowing ions from the extracellularenvironment into the cell, a process called mechanoelectrical transduction. This produces a receptor potential that causes the release ofthe excitatory neurotransmitter glutamate onto the terminals of the sensory nerve fibres, which connect to the cell base, causing nerve
signals to be sent to the brain. Identification of the cellular mechanisms underlying mechanoelectrical transduction and of some of theproteins involved has been assisted by research into the genetics of deafness, molecular biology and mechanical measurements offunction. It is thought that one type of cross link, the tip link, is composed of cadherin 23 and protocadherin 15, and gates the
transduction channel when the bundle is deflected. Another type of link, called lateral (or horizontal) links, maintains optimal bundlecohesion and stiffness for transduction. This Commentary summarizes the information currently available about the structure, functionand composition of the links and how they might be relevant to human hearing impairment.
Key words: Acousticolateralis systems, Cadherin 23, Lateral links, Mechanoelectrical transduction, Protocadherin 15, Stereocilia, Tip links
IntroductionHair cells are the sensory receptors of the vertebrate
acousticolateralis system. These mechanosensors have an apical
bundle of cellular protrusions (the ‘hairs’), which are actually
modified microvilli and are called stereocilia (Fig. 1A). These
are stiff structures filled with a longitudinal array of actin
filaments. In most sensory hair bundles, the exception being
those of the adult mammalian cochlea, there is also a single
cilium, called the kinocilium, located to one side of the bundle. In
all vertebrate hair-cell epithelia, the stereocilia on individual hair
cells have a precise organization within the bundle, forming rows
that increase in height from short to tall across it. Displacement
of the bundle modulates the opening of mechanoelectrical
transduction (MET) channels; deflection in the direction of the
tallest row depolarizes the hair cell by means of K+ and Ca2+ ions
entering through the MET channels. (Other cations such as Na+
and Mg2+ can pass through the MET channels but are not the
main cations found around the bundle in vivo.) Deflections in the
direction towards the shortest row close the channels and reduce
the flow of ions, resulting in hyperpolarization of the cell. Thus,
the MET channel is a mechanically gated nonselective cationic
channel (see review by Furness and Hackney, 2006).
The operating mechanism is ascribed to a mechanical element
in the bundle that is attached to the MET channel, the gating
spring (Corey and Hudspeth, 1983), which, in effect, ‘pulls open’
the channel during positive deflections. The current produced
then declines in two phases when the deflection is maintained, a
process that is Ca2+ sensitive; the first phase is a rapid adaptation
that might reflect an intrinsic property of the channel itself (Ricci
et al., 1998; Beurg et al., 2006) or, potentially, in some hair cells,
requires myosin XVa, an unconventional myosin (Stepanyan and
Frolenkov, 2009). (Myosins are molecules that interact with actin,
for instance in muscles, and are generally involved in motile
processes in cells; unconventional myosins are those found in
nonmuscle cells.) The second phase is a slower adaptation
(Stauffer and Holt, 2007) for which both myosin XVa, in
cochlear inner hair cells (Stepanyan and Frolenkov, 2009), and
myosin 1c (see review by Gillespie and Cyr, 2004) are candidates.
Although defects in myosin VIIa also affect adaptation they do not
abolish it (Kros et al., 2002), so this myosin is not considered to be
a candidate for the adaptation motor.
A fundamental feature of all hair bundles is the presence of
extracellular filaments that hold the stereocilia together
(Fig. 1B–G) and also link the tallest stereocilia to the
kinocilium. The precise distribution of these filaments varies
between hair cells of different types, but there are two main
categories, tip links and lateral links. The tip link comprises a
filament that connects each of the shorter stereocilium tips
with the neighbouring longer stereocilium located behind it
along the excitatory axis of the bundle (Pickles et al., 1984;
Furness and Hackney, 1985). The lateral links are horizontally
directed links connecting the rows of stereocilia, both along a
row and between rows (Pickles et al., 1984; Furness and
Hackney, 1985) and also connecting the bundle to the
Commentary 1
JCS Advance Online Article. Posted on 2 May 2013
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kinocilium (Takumida et al., 1988). It has been suggested that
the gating spring is represented by the tip links because they
are situated in such a way that depolarizing deflections would
stretch them, thereby putting tension on the MET channel gate,
and opposing deflections would relax them, releasing tension
(Box 1). The function of the lateral links is more likely to be in
holding the stereocilia together. However, there are still
uncertainties with this basic view of the role of tip links and
lateral links, such as the nature of the connection between the
channel and the tip link, and there is also the idea that its
structure and composition might indicate it is too stiff to be the
spring (Kachar et al., 2000; Sotomayor et al., 2005). This
article will discuss the structure and function of stereociliary
linkages and what is currently known about their molecular
composition, concentrating primarily on tip links. It will also
indicate some human and animal mutations that have provided
further information about the molecular basis of the linkages
and their role in hearing.
Structure and function of tip links and laterallinksIn this section, the main anatomical features of the tip links and
lateral links, as described primarily from ultrastructural studies,
will be discussed along with suggestions for their respective
functions. The basic features of the hair bundle and links are
illustrated in Fig. 1A–F and summarized in Fig. 1G.
Tip links
The idea that an extracellular link between stereocilia could
represent the MET channel gating spring stems from suggestions
by Thurm (Thurm, 1981) and Hudspeth (Hudspeth, 1982). This
led to anatomical studies attempting to identify specific structures
in the hair bundle that could act as a gating spring, and a study
using high-resolution scanning electron microscopy identified the
tip link in adult hair bundles, a single filament (the upwards-
pointing link) connecting shorter to taller stereocilia in the row
behind (Pickles et al., 1984). During early stages of hair bundle
development, however, the tips of stereocilia have many
filaments connecting them to the adjacent taller stereocilia, and
it is thus not always possible to identify an individual tip link. As
development progresses, the number of links at the tips
diminishes to one main tip link (Furness et al., 1989). In
vestibular organs, multiple links can still be observed at the tips
in some adult hair cells (Furness and Hackney, 2006), making
individual tip links difficult to identify. Thus, it is possible that
the tip link is not absolutely distinct from other links.
Nevertheless in many hair bundles, a single tip link is present
in an ideal location to represent a gating spring, provided the
MET channels are located near one or other end of the link.
Electrophysiological studies (Beurg et al., 2006) and high-speed
Ca2+ imaging (Beurg et al., 2009) indicate that there are 1 or 2
MET channels located at the tips of the shorter stereocilia, thus at
the lower end of the tip link (Box 1) but this technique is of
Box 1. Simplified tip-link model of mechanotransduction
The most widely accepted model for the mechanism underlying the opening of the mechanotransduction channels is that the MET channels are
located at the ends of the tip links with recent evidence favouring the lower end, as shown in the figure (Beurg et al., 2009). The schematic
illustration below shows the links that connect the tall (T) to the intermediate (I), and the intermediate to the short (S) row of stereocilia. Deflection
of the bundle in the excitatory direction (indicated by the arrow) puts tension on the tip link (represented as a gating spring), which mechanically
pulls open the channel (depicted in red in the inset) to allow K+ and Ca2+ ions to flow in (arrow, inset) and depolarise the cell. Opposing
deflections relax the spring and allow the channels to close. Figure adapted from Furness et al., 2010.
Deflection Tiplink
K+
Ca2+
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insufficient resolution to indicate the precise location. To address
this question using higher resolution electron microscopy, an
approach based on detecting the presence of amiloride-binding
sites on the channels was attempted. Although of relatively low
sensitivity compared with other amiloride-sensitive channels, the
MET channel is blocked by amiloride, an effect which was
exploited by using antibodies to amiloride-binding proteins and
immunoelectron microscopy to localize the MET channel. The
data suggest that the MET channel is located below the tip, ,50–
100 nm away from the tip link (Hackney et al., 1992; Furness
et al., 1996). Kinematic modeling suggests that channels in this
location would still be efficiently activated by bundle deflections
(Furness et al., 1997) but not necessarily by the tip link, although
if the latter stretched the membrane at the stereociliary tip, it
might pull on MET channels that are in this location. As the
identity of the MET channels remains uncertain, there are no
specific reagents such as antibodies that are available to
determine their exact localization and distribution. There have
been a number of candidate proteins suggested as possible MET
channels, most recently two that are known to form channels,
transmembrane channel-like protein 1 (TMC1) and TMC2
(Kawashima et al., 2011).
Despite the difficulties of localizing the MET channel, there is
developmental and physiological evidence that it is operated by
the tip link. Identifiable tip links appear during development
concurrently with the ability of hair cells to transduce (Geleoc
and Holt, 2003). Treatment with the Ca2+-chelating agent 2-bis-
(o-aminophenoxy)ethane-N,N,N9,N9-tetra-acetic acid (BAPTA)
Fig. 1. Organisation of stereocilia and cross links in hair bundles. (A) Scanning electron micrograph (SEM) of the stereociliary bundle of an inner hair cell
from a rat cochlea. The stereocilia form well-defined rows that increase in height along the excitatory axis of the bundle. (B) SEM showing that shorter stereocilia
are connected to taller stereocilia in the adjacent row by fine filaments called tip links (arrows). (C) Close up of a tip link. These links frequently appear to
bifurcate towards the upper attachment site. (D) Transmission electron microscopy (TEM) of a tip link from a guinea-pig cochlear hair cell, showing the fine
filament (arrow) connecting the short and tall stereocilia, within each of which is a dense patch at either end of the link. Note also the region of close approach
(contact region) between stereocilia (arrowhead). (E) TEM of a horizontal section of a group of rat hair-cell stereocilia showing filamentous lateral links (arrows)
connecting those in adjacent rows and within a row. (F) TEM of the three rows of guinea-pig stereocilia, in which the different cross links are visible (left) and a
schematic illustration of interrelationship between the different cross links (right). The contact region is shown, an area that may coincide with a region of ‘top
connectors’ but in mammals also appears to label with antibodies to amiloride sensitivity sodium channels (Hackney et al., 1992). Another region of top
connectors is the lateral links at the tip of the tallest stereocilia, some of which extend into the overlaying tectorial membrane (Verpy et al., 2011). Scale bars:
A53 mm; B5600 nm; C550 nm; D5100 nm; E and F5250 nm.
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destroys tip links and abolishes transduction (Assad et al., 1991)and causes hair-bundle stiffness to decline substantially, which is
thought to be due to loss of the gating spring (Marquis andHudspeth, 1997). Loss of tip links and their regeneration in vitro
is accompanied by loss and recovery of transduction (Zhao et al.,1996). However, one study suggests that destroying the tip link
leaves the MET channel open rather than closed, prompting theidea that the gating spring is actually a distinct structure thatconnects the MET channel to the internal cytoskeleton (Meyer
et al., 1998). In fact, this idea may well be consistent with theproposed molecular model of the tip link whereby the link mightnot itself be the gating spring (see below), an idea supported by
mathematical models (Powers et al., 2012). Determining thestructure and molecular composition of the tip link and how it iscoupled to the MET is therefore crucial to determining themechanism of transduction.
Structural studies in guinea pig cochlea suggest that the tip linkhas a mean length of about 185 nm under conditions of relativelylow Ca2+ concentrations (50 mM) (Furness et al., 2008), similar
to those found in endolymph, the fluid that bathes the apex of thehair cell. In conditions of higher Ca2+, the tip-link length isshorter, ,150 nm. The same study showed that tip-link lengths in
turtle cochlear hair cells are similar to those of mammals whenthey are fixed under similar conditions. However, based on dataobtained from bullfrog saccular hair cells, it has been suggested
that tip links fall into two populations that have different lengths(Auer et al., 2008). These differences might reflect speciesdifferences, differences in location of the hair cells (in thecochlea, the bundles vary systematically in shape and height
along the length of the hair-cell epithelium, the organ of Corti) ordifficulties in finding a sufficient amount of intact tip links forquantitative analysis. However, when we measured a greater
number of tip links than did Auer et al. (Auer et al., 2008), weonly found one population of tip links with regard to their length(Furness et al., 2008).
A number of morphological studies suggest that the tip link isnot a single strand but that it is composed of helically woundstrands (Kachar et al., 2000; Tsuprun and Santi, 2000). The linkalso frequently bifurcates partway along its length so that two
filaments connect to the membrane of the taller stereocilia(Fig. 1C; Hackney and Furness, 1995; Kachar et al., 2000). Thebifurcation has been observed in tip links from all vertebrate
classes except teleost fish (roach) hair cells where onlyunbifurcated tip links have so far been observed (Furness andHackney, 2006). This may be resolved by examining more
samples or different fish species. In addition, not all links in anysample are bifurcated. The molecular architecture of thebifurcation also remains uncertain but its presence or absence
may depend on physiological conditions. For example, itspresence may be affected by Ca2+ sensitivity or changes intension on the tip link. The number of strands that form the link isalso uncertain as some studies suggest that there are two (Kachar
et al., 2000), whereas one study suggests possibly three (Tsuprunand Santi, 2000). The more likely model is that the tip linkconsists of two spirally wound strands, which perhaps unwind
into two separate strands near the top of the link; see below forevaluation of the evidence supporting this model. Alternatively, ithas been suggested that one of the branches is a thinner auxiliary
link (Auer et al., 2008; Rzadzinska and Steel, 2009).
At both the lower (Pickles et al., 1984) and upper terminationsof the tip link, electron-dense patches can be observed beneath
the membrane in transmission electron microscopy (Fig. 1D,F;
Furness and Hackney, 1985; Little and Neugebauer, 1985).Immunocytochemical data suggest these patches contain avariety of proteins. The patch at the lower end of the tip link
lies over the end of the core of actin filaments and containswhirlin (Delprat et al., 2005), a protein encoded by the deafnessgene DFNB31 (Mburu et al., 2003; Mburu et al., 2006), themouse ortholog of which produces a whirling behaviour (Holme
et al., 2002). Myosin XVa (Belyantseva et al., 2003), myosinVIIa, twinfilin, an actin binding protein that inhibits actinpolymerization (Rzadzinska et al., 2009), and EPS8, an actin
regulating protein (Manor et al., 2011; Zampini et al., 2011), arealso present. The patch at the tip-link’s upper end is thought tocontain myosin 1c (Steyger et al., 1998), myosin VIIa, scaffold
protein containing ankyrin repeats and sterile alpha motif domain(SANS) (Grati and Kachar, 2011) and harmonin isoform b(harmonin-b) (Grillet et al., 2009), (Fig. 2), although there is also
evidence for SANS being present at the lower end of the tip link(Caberlotto et al., 2011), where harmonin-b is also found duringdevelopment (Lefevre et al., 2008).
The upper electron-dense plaque appears to have a regular
arrangement of subunits, which might reflect some of theseproteinaceous components (Furness et al., 2008). Both patchregions are enriched in calmodulin (Furness et al., 2002), which
could be associated with the myosins therein and modulate theiractivity by Ca2+ binding and unbinding. For instance, myosinlocated in the upper attachment could change tension on the tiplinks by means of moving along the actin core, hence producing
adaptation (Howard and Hudspeth, 1987).
The tip links appear to insert through the membrane and mightbe anchored into the electron-dense plaques as the link remains in
contact with both attachment sites after detergent treatment,which removes the membrane (Furness et al., 2008). Although, atthe upper end, the attachment might be direct, at the lower end anumber of fine strands have been detected (Osborne et al., 1988;
Kachar et al., 2000; Furness et al., 2008), which potentiallyconnect the tip link from the membrane to the dense material thatcaps the actin core of the stereocilium.
Lateral links
There are various types of horizontal cross links or lateral linksbetween the shafts of the stereocilia (Fig. 1E–G), which project
in all directions, attaching the nearest neighbours to each other(Pickles et al., 1984; Furness and Hackney, 1985). The preciselocation and appearance of these links differs in different species
and types of hair cells (see review by Furness and Hackney,2006). Nevertheless, there are particular regions of horizontalconnections (top connectors) between the tops of adjacent tallstereocilia (another category of top connectors occurs just below
the tips of shorter stereocilia) and close to the lower tip linkinsertion (Verpy et al., 2011). Occasionally a region of closeapproach (the contact region) between the short and taller
stereocilia is seen in this location (Fig. 1; Hackney et al., 1992).Lower down, shaft links exist with a medial stripe where theyoverlap (Fig. 1E), and these link stereocilia both between rows
and within rows (Furness and Hackney, 1985). The medial stripemight represent the joining of links that emanate separately fromeach stereociliary shaft. The extent of these shaft links varies
even within one organ. A particular set of links occurs at theankles of the stereocilia that are present only during developmentin mammalian cochlear hair bundles (Goodyear et al., 2005) but
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are retained in adult non-mammalian vertebrates, such as chicken(Goodyear and Richardson, 1999), as well as in adult mammalianvestibular hair cells (Jeffries et al., 1986).
How the different types of lateral link are segregated in thebundle is also another interesting question. According to thedisposition of membranes with different structural and functional
properties, there are three different bundle domains (Zhao et al.,2012), which could be associated with where and how differentkinds of lateral links are distributed.
Lateral links probably tie the stereocilia together to enable thebundle to act as a unit, increasing its stiffness and preventing it
from splaying. Measurements of bundle motion imply that thestereocilia are tightly constrained so that there is very little lagbetween the motion of stereocilia on either side of the bundle andthey all move coherently resulting in maximally efficient gating
of the MET channels (Kozlov et al., 2007).
Approximately 48% of the bundle stiffness in chick vestibular
hair cells has been attributed to shaft and upper shaft links, and,43% to ankle and tip links (Bashtanov et al., 2004). Adultmammalian cochlear hair cells appear to lack the ankle links and,
therefore, an even greater proportion of their stiffness mightdepend on the shaft and upper shaft links. However, in mammalsa substantial amount of stiffness has been attributed to the
rootlets that anchor the stereocilia in to the apex of the hair cellbased on studies of a mouse engineered to lack the actin-bundlingprotein TRIOBP (trio and actin binding protein), in which therootlets fail to form. Defects in TRIOBP underlie human deafness
type DFNB28 (Kitajiri et al., 2010).
Nevertheless, mathematical modeling suggests that variation in
the quantity of lateral links within the bundle is likely to impacton stiffness (Silber et al., 2004) and that upper shaft connectorsmight contribute most to bundle stiffness (Cotton and Grant,
2004). This might explain observations that loss of topconnectors substantially affects cochlear non-linearity (Verpyet al., 2011). It has also been reported that the top connectors may
function to reduce viscous drag on the bundle (Kozlov et al.,2011). The extent to which links contribute to stiffness andbundle motion therefore deserves further investigation in avariety of bundle types; for example, a candidate protein
composing the shaft connectors PTPRQ, is absent in maturebasal (high frequency) hair cells of the cochlea, but present in theapical (low frequency) ones (Goodyear et al., 2003). Whether the
lateral links contribute to transduction more directly remains tobe discovered, but there is some evidence that the MET channelsmight be associated with horizontal connections or top
connectors below the tips of shorter stereocilia (Hackney et al.,1992). In addition, bundles lacking the staircase arrangement,such as in myosin-XVa-deficient mice, which do not possessidentifiable tip links, still transduce mechanical stimuli
(Stepanyan and Frolenkov, 2009).
Molecular composition of the linksTip links
Evidence from immunocytochemistry, mutations and knockoutsconfirms that members of the cadherin superfamily are
components of tip links. Cadherin 23 (CDH23) (Sollner et al.,2004) and protocadherin 15 (PCDH15) (Ahmed et al., 2006) havebeen proposed to interact to form tip links (Kazmierczak et al.,
2007) and both proteins are mutated in Usher type I syndrome, anautosomal recessive condition that affects human hearing andvision (Di Palma et al., 2001; Alagramam et al., 2001b).
The twisted and forked appearance of the tip link gives some clues
to its possible structure and composition. Immunocytochemicallabeling suggests that tip links are composed of a homodimer ofCDH23, which forms the upper part of the tip link, and a homodimer
of PCDH15, which forms the lower part (Box 2; Kazmierczak et al.,2007). CDH23 has a short intracellular domain, a transmembranedomain and an ectodomain composed of 27 cadherin repeats(Kazmierczak et al., 2007). PCDH15 has cytoplasmic and
transmembrane domains with an ectodomain that consists of 11cadherin repeats (Kazmierczak et al., 2007). This hypothesis issupported by the fact that regeneration of the links following
chemical disruption in immature hair cells, but not in adults, can beinhibited by PCDH15 and CDH23 fragments (Lelli et al., 2010).There is a Ca2+-binding motif in each cadherin repeat (Sotomayor
et al., 2005; Sotomayor et al., 2010) and the terminal regions ofCDH23 and PCDH15 probably also interact with each other bymeans of a Ca2+-sensitive binding site (Kazmierczak et al., 2007;
Sotomayor et al., 2010; Elledge et al., 2010).
The model of CDH23 being coupled to PCDH15 at the N-termini predicts a length for the tip link of about 180 nm, basedon measurements of the molecules in trans, which is in close
agreement with measurements made by electron microscopy ofguinea pig and turtle hair-cell tip links (Furness et al., 2008) andsimilar to those measured for chick tip links (150–200 nm)
(Tsuprun et al., 2004). The model is also supported byobservations that CDH23 forms dimers in trans, which canpartially unwind into two separate branches at one end if mediumcontaining lower Ca2+ levels are used (Kazmierczak et al., 2007).
On the basis of the biochemical data (Kazmierczak et al.,2007) and our own anatomical data, which show that increasingCa2+ levels decrease the length of the tip link, we have proposed
the hypothesis that the bifurcation represents a varying degree ofunwinding of the two interwoven strands of the CDH23 dimer(Furness et al., 2008). This hypothesis could be tested byanalyzing the dimensions of the forked part of the tip link under
different Ca2+ conditions, which, however, is technically difficultto achieve.
As noted earlier, myosin VIIa and harmonin-b are concentrated
in the stereociliary tips of developing hair bundles of mice. Resultsfrom immunofluorescence and confocal microscopy suggest that,soon after birth, harmonin-b relocates to the position of the upperend of the tip link and that this relocalisation does not occur in
CDH23- or PCDH15-deficient mice (Lefevre et al., 2008). Myosin1c receptors in the stereocilia are also absent in the absence ofCDH23 and after Ca2+ chelation (Phillips et al., 2006), which is
known to destroy the tip links and eliminate transduction (Assadet al., 1991). Another protein that binds to CDH23 is themembrane-associated guanylate kinase MAGI1, which shows a
punctate staining pattern corresponding to that shown by CDH23on the stereocilia both during development and in adults. Thisprotein might be involved in anchoring the tip link to the
cytoskeleton (Xu et al., 2008). During development, the relatedmembrane-associated guanylate kinase (MAGUK) p55 is localizedto the stereociliary tips region, suggesting that these proteins arepart of the regulatory process that forms the actin core and the
transduction apparatus (Mburu et al., 2006).
At the C-terminal ends of both cadherins, there aretransmembrane regions (Kazmierczak et al., 2007), which allow
the two sets of homodimers to pass through the membrane andanchor the tip link to the cytoskeleton (Furness et al., 2008). Atthe upper end of the tip link, CDH23 is likely to interact with
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myosins, harmonin-b and SANS, with harmonin-b linking
CDH23 to the actin filaments in the stereocilium core (Grati
and Kachar, 2011). At the lower end, it is assumed that PCDH15
interacts somehow with the MET channel, enabling it to be gated
by tension in the link. The linkers between the lower end of the
tip link and the dense material at the top of the actin cytoskeleton
that have been reported in a number of studies (Osborne et al.,
1988; Kachar et al., 2000; Furness et al., 2008) might allow the
tip link to interact with the MET channels or might simply anchor
the channels and/or the tip link to the lower electron-dense patch.
Recent studies of a stereociliary tetraspan protein (tetraspan
membrane protein of hair cell stereocilia, or THMS), mutations
in which cause deafness in humans (Shabbir et al., 2006) and
defects in tip-link development and transduction in mice, suggest
that this protein couples PCDH15 to the channel (Box 2; Xiong
et al., 2012). THMS appears to bind preferentially with the
transmembrane domain and a conserved membrane proximal
domain on the cytoplasmic side of PCDH15 so that it could
occupy the region between the latter and the channel.
The evidence suggesting that the tip link is composed of two
homodimers of PCDH15 and CDH23 is fairly strong, at least in
mammals; in avians (chick), a similar distribution of the two
homodimers has been indicated by immunogold labeling
(Goodyear et al., 2010). Whether such a composition is also
present in other vertebrates remains to be determined, but there
are indications that the tip link structure is highly conserved. For
example, turtle tip links appear morphologically identical to
those in mammals (Furness et al., 2008), and CDH23 has been
detected in fish tip links (Sollner et al., 2004). Fish also have
PCDH15a, which is similar to mammalian PCDH15 and is
necessary for hearing (Seiler et al., 2005).
Lateral links
The main categories of lateral links – top connectors, horizontal
links along the shaft and ankle links (and kinociliary links when a
kinocilium is present) – appear to contain different molecules (Box
3). There are a number of candidate molecules, although more might
yet be discovered. Among the lateral links, a subset of top
connectors has been found in mammalian hair cells of the organ of
Corti, the outer hair cells (OHC), that contain the protein stereocilin,
which was first described in a search for candidate deafness genes in
mice that are linked to the human deafness locus DFNB16 (Verpy
et al., 2001; Verpy et al., 2011). Stereocilin is a member of the
mesothelin protein superfamily, which contains proteins that
have been suggested to function as superhelical lectins that
bind to carbohydrate moieties of extracellular glycoproteins
(Sathyanarayana et al., 2009). The region of the top connectors
stains with antibodies against stereocilin and, in a stereocilin-
knockout mouse, the OHC hair bundle shape is normal until
postnatal day 9 (P9), but because the top connectors fail to form
properly, bundle cohesion progressively deteriorates after P9 and tip
links disappear after P15, so that by P60, the stereocilia are no longer
Box 2. A putative model of the molecular composition of the tip link
Studies by immunoelectron microscopy and in trans observations of PCDH15 and CDH23 suggest that the upper part of the tip link is composed
of a dimer of CDH23 (coloured green), wrapped into a helix, and the lower part of a dimer of PCDH15 (coloured blue), also wound helically. The
Ca2+-dependent splitting of the CDH23 dimer (Kazmierczak et al., 2007) is a possible explanation of the bifurcation seen in EM studies of the tip
link. At the upper end, the electron-dense plaque (blue) consists of a number of subunits (dark blue area) that might represent several of the
known protein constituents, which are listed at the upper attachment point of the link. At the lower end, the link is shown attached to two channels
linked to PCDH15 via THMS (inset) as proposed by Xiong et al. (Xiong et al., 2012). The attachment of the TMHS is here depicted as
intracellular, but it is not known where the channel gate is located. Proteins known to be present in the lower tip link dense plaques are also listed
(some of these such as harmonin-b and MAGUK are in involved in development of the stereocilia).
Lower tip link density(EPS8, harmonim b, myosin VIIa,whirlin, twinfilin,calmodulin)
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linked together. There is also evidence that these stereocilin links
connect the tall OHC stereocilia to the overlying gelatinous flap, the
tectorial membrane. This flap in the organ of Corti contacts the
stereocilia to stimulate these hair cells. Stereocilin thus appears to be
required to form the top connectors and, in their absence, the bundle
cannot retain a normal shape. This in turn affects the ability of the
hair cell to maintain intact tip links.
The shaft links appear to contain protein tyrosine phosphatase
receptor type Q, in the absence of which the lateral links become
elongated (Nayak et al., 2011). As noted above, the extent of
PTPRQ, and therefore shaft links, differs both between different
organs and in different frequency regions of the mammalian
cochlea.
A component of the ankle links is the very large G-protein-
coupled receptor (VLGR1; McGee et al., 2006), which has been
found in the ankle regions together with the transmembrane
proteins usherin and vezatin, and the submembrane protein
whirlin (Kussel-Andermann et al., 2000; Michalski et al., 2007).
VLGR1 is defective in type IIc Usher syndrome, whereas usherin
is defective in type IIa Usher syndrome. Together with vezatin
and whirlin, these proteins might be targeted to ankle links by
myosin VIIa during the development of hair cells (Michalski
et al., 2007). Usherin is also present in adult vestibular hair cells,
which retain ankle links (Adato et al., 2005).
Another protein that might be involved in the development of
the lateral links is transient receptor potential mucolipin 3
(TRPML3), a member of the TRP ion channel protein family. Its
loss in the varitint-waddler mouse causes stereociliary splaying
(Atiba-Davies and Noben-Trauth, 2007).
Box 3. Composition of different categories of laterallink in mature bundles
The diagram illustrates the three categories of inter-stereociliary
lateral link presently recognized as structurally and compositionally
distinct from studies on a variety of different bundle types. Not all
categories are present in all bundles; for example, ankle links are
present in all bundles examined except mature mammalian
cochlear hair bundles. Note that the top connectors on tall
stereocilia in mammalian cochlea connect to the overlying
tectorial membrane. The tip links are shown to illustrate their
relationship with the other links. Other than the aforementioned
mammalian cochlear ankle links, transiently present components
of lateral links (protocadherin 15 and cadherin 23) are not
illustrated. The molecular details of these links are significantly
less well known than that of the tip link.
Box 4. Hearing loss caused by gene defects
Genetic hearing loss contributes to ,50% of the cases of hearing
loss in humans, with the remainder being attributed to
environmental factors (Bitner-Glindzicz, 2002). Studies of genetic
hearing loss have led to the identification of a number of candidate
proteins that have been found to be crucial for mechanotransduction
and forming or maintaining the links between stereocilia (El Amraoui
and Petit, 2005). Usher syndrome, a disease with three clinical
subtypes, Usher types 1–3, each based on separate defects in
several genes, combines deafness and blindness (see review by
Williams, 2008) and thus is a major disability. The most common
form, Usher syndrome type IIa, results in people developing
moderate to severe hearing impairment and arises from mutations
in the USH2A gene that encodes usherin, which has previously
been considered to be a basement membrane protein
(Bhattacharya et al., 2002). However, in rodents, several larger
USH2A transcripts encode transmembrane isoforms of different
lengths, one of which has a cytoplasmic form that is predominantly
expressed in the inner ear but not in the retina, where it is also
typically found. Usherin has been found to localize to the base of
stereocilia, where it binds to harmonin and whirlin, two additional
proteins whose defects lead to the genetic deafness types,
nonsyndromic deafness and Usher type I, respectively (Adato
et al., 2005).
Another protein with relevance for deafness is stereocillin, which
is found at the other end of the stereocilia compared with where
usherin is located and has been localised immunocytochemically
to the top connector region where it links to the tectorial
membrane. It is encoded by the STRC gene, which is defective
in the human deafness type DFNB16 (Verpy et al., 2001). DFNB16
is an autosomal recessive progressive condition with hearing
impairment that appears in childhood and which, in mouse models,
results in a lack of cohesiveness in the hair bundle after P9 (Verpy
et al., 2011).
Usher syndrome type 1D, in which deafness is also
accompanied by blindness, is caused by null alleles of CDH23
that presumably affect both the tip links and also the lateral links
(which contain CDH23 during development). In addition, various
missense mutations in CDH23 result in nonsyndromic deafness
(so-called DFNB12) and, according to studies of salsa mice, which
model this mutation, might cause a weakening or greater
susceptibility of the tip link to damage and affect tip-link
maintenance, thus explaining the progressive hearing loss seen
with this syndrome (Schwander et al., 2009). Other mutations in
CDH23 and PCDH15 produce non-syndromic deafness that have
been identified in Turkish (Duman et al., 2011) and Japanese
families (Nakanishi et al., 2010), confirming that these genes play
a vital role in maintaining normal auditory function. It should be
noted that nonsyndromic deafness, in which hearing loss is not
accompanied by other symptoms, is thought to result from
missense mutations that leave some residual function in the
affected proteins, whereas mutations underlying other types of
deafness cause the loss of the respective protein, resulting in both
hearing loss and other symptoms (Schultz et al., 2011).
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As well as being part of the tip link, both PCDH15 and CDH23are also present in lateral links at some stages of development. As
with other bundle proteins, interaction between PCDH15 andmyosin VIIa is necessary for hair bundles to develop properly(Senften et al., 2006). It has also been reported that a set of lateral
links that contain CDH23 is transiently present duringdevelopment (Michel et al., 2005). These lateral links mightbe recruited into tip links, for example, to repair themechanotransducing apparatus if it is damaged or has been
regenerated (Zhao et al., 1996).
The kinociliary links also contain CDH23 and PCDH15 in an
asymmetrical arrangement, as in the tip link but in the reversedirection (Goodyear et al., 2010). Such links are not present inadult mammalian cochlear hair cells, which lack the kinocilium,but they do occur in mammalian vestibular hair cells and those of
non-mammalian vertebrates. It is possible that these kinociliarylinks might represent a parallel transducer mechanism, separatefrom the usual tip link, or a mechanism for transmitting forces to
the hair bundle from the kinocilium, given that the latter isusually the point of coupling to an overlying accessory structurethat delivers a stimulus to the bundle (see discussion in Goodyear
et al., 2010).
Mutations of Cdh23 and Pcdh15 in mouse and zebra fishand their effects on hair-cell function
A variety of mutations and transgenic animals have allowed thecomposition of the links and their function to be further
investigated. Gene mutations have been found in humans thathave been investigated by using murine and zebrafish geneticexperiments. Usher syndrome, as noted above, provided some
initial clues. CDH23 was found to be one of the proteins affectedin Usher syndrome (Bork et al., 2001) and there is a mouseorthologue of this protein (Di Palma et al., 2001). Moreover, a
mutation in CDH23 was found in zebrafish, and zebrafish withthis mutation have defects in balance and hearing that involveimpaired mechanotransduction, and no tip links in their hairbundles (Sollner et al., 2004).
Studies of Usher syndrome have also implicated mutations ofPCDH15 in the disease (Alagramam et al., 2001b; Ahmed et al.,
2008), for which there is the mouse model Ames waltzer(Alagramam et al., 2001a). Genomic studies of Pcdh15 led to thediscovery of three isoforms in mice with variations in thecytoplasmic domains, CD1, CD2 and CD3, and CD1 and CD3
antibody staining of the stereocilia suggests that these might beassociated with the tip link (Ahmed et al., 2006). Knockout micethat lack CD1 and CD3 still retain tip links and hearing,
suggesting that when one isoform is absent, the others cancompensate. However, in the absence of the CD2 isoform, tiplinks remain but hearing is lost (Webb et al., 2011). Lack of CD2
also results in defects in polarity of the hair bundles, which mightexplain deafness. In these mice, vestibular function is stillretained (Webb et al., 2011).
Cdh23 and Pcdh15 mutations have been investigated in otherrodent models for their effects on hair-cell structure and function.The waltzer mouse has several mutations in CDH23 (Di Palma
et al., 2001; Holme and Steel, 2002, Holme and Steel, 2004), Anexample is the v2J waltzer variant, in which the hair bundlesshow developmental defects and become disturbed [although in
early development, stereociliary ranking (i.e. the increase in rowheight from the modular side to the strial side of the bundle incochlear hair cells) can still be present in portions of the bundle].
The transducer currents measured in organ cultures from theseP2–P3 mice are also abnormal in that the cells respond with
depolarisation to displacements in the normally negative(inhibitory) direction (Alagramam et al., 2011).
The nature of the putative tip links in the waltzer mice is
controversial. One study in early postnatal mice with the v alleleof CDH23 reported that the tip link is still present in some formand, on the basis of this observation, the authors implied that
CDH23 is not required for tip link formation (Rzadzinska andSteel, 2009). The tip links observed were not bifurcated, as theywere in normal wild-type adult mouse controls (Rzadzinska and
Steel, 2009). In the study of the v2J allele, very short non-bifurcated links were also found, and these could not be classedunequivocally as tip links, which run from the tips of the shorterstereocilia to sides of the taller row stereocilia in the next row,
whereas in age-matched controls, bifurcated tip links wereevident (Alagramam et al., 2011). It is possible to reconcile thesedata if the tip link in waltzer mice is incomplete, i.e. lacks the
upper forked part that consists of CDH23.
Another mouse mutant, salsa, suffers from hearing loss due toa missense mutation of CDH23. In contrast to waltzer mice, in
which hair cell development is abnormal, salsa mice haverelatively normal bundles, but progressively lose tip links(Schwander et al., 2009). At P7–P8, the hair cells only have a
relatively mild impairment of transducer function. Hearing inthese mice is also progressively lost; it is impaired at 3 weeks andworsens by the time the mice are 2 months old. Therefore, the
hearing loss has been primarily attributed to a loss of tip links.However, the tip links do not appear to be affected in vestibularhair cells in salsa mice. It is possible that the mutation weakensthe tip links, resulting in their damage and loss in the cochlea
(where the bundles are stimulated at greater frequencies and havedifferent morphologies than those in the vestibular system).
There are several variants of another mouse, the Ames waltzer(av), which have different PCDH15 mutations. In av6J, there isan in-frame deletion that affects the ninth cadherin repeat ofPCDH15 and in av3J, a point mutation that is expected to abolish
expression of PCDH15. Studies of these two mice mutationsshow that av6J mice have an almost normal phenotype, with tiplinks present, albeit in a reduced number compared with the av3J
mice, which have severely affected hair bundles with breaks inthe rows of stereocilia and substantial loss of tip links(Alagramam et al., 2011). The reduced number of tip links in
av6J suggests that the mutation does not prevent tip linkformation but might, as suggested for salsa mice, cause the linksto be more susceptible to damage. The hair cells of the av6J
mouse still show evidence of transduction, although this isreduced in amplitude, consistent with the presence of fewer tiplinks than in controls. The av3J mouse shows a more significantloss of mechanotransduction, consistent with the even greater
reduction in tip links than in av6J mice. Thus, tip links are lesslikely to form in the absence of PCDH15 expression, and the lossof transduction indicates that PCDH15 is likely to be essential for
channel gating.
Future perspectivesThe recent findings from mouse models and human deafness withregard to the composition and structure of tip links and lateral
links discussed above have led to a greater understanding ofmechanoelectrical transduction and to the molecular factorsinvolved. The current working model of the tip link is that it
Journal of Cell Science 126 (0)8
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consists of two conjoined homodimers of CDH23 and PCDH15,
with a possible bifurcation as the result of Ca2+-dependent
untwisting of the CDH23 dimer, which is likely to be the basis of
further investigation. Although the function of the tip link as the
gating spring of the MET channel is not indisputable, as there are
still unexplained aspects of gating [such as the structural data of
the tip link, which suggests that it may not have sufficient
elasticity to reflect that measured for the gating spring (Kachar
et al., 2000)], the biochemical and genetic evidence underlying
this model go a long way towards clarifying the role of the tip
link. Taken together they show that: (1) tip link regeneration can
be inhibited by CDH23 and PCDH15 fragments, which results in
concomitant failure of transduction; and (2) various mutations in
these proteins produce hair bundle and cross-link anomalies with
associated deafness (El-Amraoui and Petit, 2005). The location of
the MET channel, which is likely to be at the lower end of the tip
link, needs to be confirmed by directly identifying the channel at
high resolution, for instance using electron microscopy combined
with immunocytochemical methods. Further evidence about how
the channel is coupled to the tip link, perhaps via THMS, remains
to be obtained and the precise role of myosins found at the lower
and upper tip-link insertion points require further investigations.
Answering these questions is necessary to resolve important
aspects of mechanotransduction, such as the molecular
mechanisms of gating and the way in which adaptation is
achieved.
The lateral links and their contribution to transduction remain
even less well understood. For example, if their main role is in
providing stiffness to the bundle, their distribution and
composition might show systematic changes that are associated
with the different frequencies detected by hair cells in different
inner ear organs and in different cochlear locations because
cochlear hair cells detect a wide range of frequencies from a few
hundred to 100,000 Hz or more, across different mammalian
species. This possible correlation is a further avenue of research
that is worthwhile exploring.
From the human perspective, the importance of tip and lateral
links lies in their potential contribution to hearing and balance
disorders if they are lost or do develop not properly. Some of
the genes associated with deafness clearly have a role in the
establishment and maintenance of the cross links, but
the prevalence of such disorders is still not fully appreciated;
the combination of genetic studies of human deafness and
analysis of mouse models is likely to continue to bring new
insights into the structure and function of hair bundle linkages.
FundingThe authors’ research has been supported by Deafness Research UK,the Henry Smith Charity, the Wellcome Trust, the MRC, RNID (nowAction on Hearing Loss) and the Grand Charity.
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