homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular
that the bundle membrane segregates spatially into at least three large structural and functional domains. One membrane domain,
(protein tyrosine phosphatase Q) and polysialylated gangliosides. The taper domain forms a sharp boundary with the shaft domain,
over, a tip domain has elevated levels of cholesterol, PMCA2, and PI(4,5)P2. Protein mass spectrometry shows that bundles from chick
vestibular hair cells contain a complete set of proteins that transport, synthesize, and degrade PI(4,5)P2. The membrane domains have
functional significance; radixin, essential for hair-bundle stability, is activated at the taper–shaft boundary in a PI(4,5)P2-dependent
manner, allowing assembly of protein complexes at that site. Membrane domains within stereocilia thus define regions within hair
Hair cells, neuroepithelial cells in the inner ear that transduce
auditory and vestibular stimuli to electrical currents, provide a
remarkable example of correlation of structure with function.
a precise staircase; each stereocilium contains a paracrystal of
actin filaments, sheathed by the hair cell’s plasma membrane
dle and open transduction channels, which admit K?and Ca2?
from the apical extracellular fluid, endolymph, that bathes the
bundle. Bundles remove Ca2?using the plasma membrane
concentrated in stereocilia (Lumpkin and Hudspeth, 1998; Ya-
4,5-bisphosphate [PI(4,5)P2], a known regulator of PMCA2
(Hilgemann et al., 2001), also controls transduction and adapta-
tion by hair cells (Hirono et al., 2004).
ingly nonuniform pattern; it is present in stereocilia shafts and
concentrated at tips, but is absent from the taper region at stere-
ocilia bases and from the soma’s apical surface (Hirono et al.,
phosphatidylinositol phosphatase (Oganesian et al., 2003), pres-
ents a near-perfect reciprocal localization pattern to PI(4,5)P2
(Hirono et al., 2004); PTPRQ may therefore maintain low levels
of PI(4,5)P2in the apical surface and basal taper region. Steady-
state degradation of PI(4,5)P2at tapers by PTPRQ would be a
very inefficient way to maintain PI(4,5)P2distribution in stereo-
cilia; more likely, PI(4,5)P2is segregated into a separate mem-
brane domain (McLaughlin et al., 2002).
In many circumstances, members of the ezrin–radixin–moe-
tion that allows activating phosphorylation (Fehon et al., 2010).
Radixin is required for normal hearing in mice (Kitajiri et al.,
potentially interacts with many functionally significant proteins
present in stereocilia (J.-B. Shin and P. G. Gillespie, unpublished
observations), little is known about the mechanism of activation
We show here that the lipid composition of the hair bundle’s
membrane resembles most cellular endomembranes, except that
ceramide lipids are unusually rich in N-palmitoyl (16:0) chains.
scale membrane domain at the stereocilia basal tapers that is
physically segregated from the shaft/tip PI(4,5)P2domain; this
4600 • TheJournalofNeuroscience,March28,2012 • 32(13):4600–4609
domain is stable even when cholesterol is extracted. These mem-
brane domains are coextensive with protein domains; PTPRQ
and PMCA2 are found respectively in the ganglioside and
PI(4,5)P2domains. Moreover, radixin, essential for hair-cell
hair bundles have two large membrane domains, at least one of
which may contain additional lipid microdomains, which are
likely responsible for compartmentalization of actin dynamics,
protein targeting, and mechanotransduction.
Materials. Sigma-Aldrich was the source for protease type XXIV, chol-
era toxin B subunit (CTB; catalog #C9903), neuraminidase (catalog
#N2876), DNase I, carbenicillin, BSA, FITC-phalloidin, TRITC-pha-
lloidin, filipin (type III), methyl-?-cyclodextrin (M?C), and phenylars-
ine oxide (PAO; catalog #P3075). The mouse anti-cholera toxin B
antibody was from AbD Serotec (catalog #9540-0108). Formaldehyde
(16% stock in sealed ampules) and glutaraldehyde (8% stock in sealed
ampules) were obtained from Electron Microscopy Sciences. DME/F-12
medium was from Thermo Scientific (HyClone; catalog #SH30023.01).
Buffers, salts, and other solution components were of the highest quality
available. The PTPRQ antibody was a gift from Guy Richardson, (Uni-
versity of Sussex, Brighton, UK); PMCA2a was detected using antibody
antibody from Abnova (catalog #H00005962-M06), and phospho-ERM
was detected using #3149 from Cell Signaling Technology.
Lipid mass spectrometry. Lipid and protein mass spectrometry used
embryonic day 20 (E20)–E21 chick utricles of either sex. Hair bundles
were purified from utricles by the twist-off technique (Gillespie and
Hudspeth, 1991; Shin et al., 2007). To obtain utricular sensory epithelia,
the epithelium was then peeled off the basement membrane using an
Lipids were extracted from hair bundles and epithelial fractions using
an acidic organic phase (Bligh and Dyer, 1959) in all cases except for
tive analyses of lipids by nanoelectrospray ionization tandem mass spec-
trometry (MS/MS) were performed as described previously (Bru ¨gger et
al., 2006). Lipid analysis was done in positive ion mode on a QII triple
quadrupole mass spectrometer (Waters) equipped with a nano Z-spray.
elin (SM) detection was performed by precursor ion scanning for frag-
ment ion 184 Da at a collision energy of 32 eV. Neutral loss scanning of
sitol, where a collision energy of 30 eV was applied. Precursor ion
scanning of m/z 364, 390 and 392 Da was used for detection of plasmal-
ogen species, using a collision energy of 20 eV. Hexosylceramide and
ceramide were detected by precursor ion scanning for fragment ion 264
Da at collision energy 35 and 30 eV, respectively. Cholesterol was ana-
lyzed as an acetate derivate as described (Liebisch et al., 2006).
spectrometry as described previously (Shin et al., 2010). Label-free pro-
tein quantitation used MS2 intensities (Spinelli et al., 2012) divided by
molecular mass, normalized to the sum of all intensity/molecular mass;
these normalized molar intensities (im) are proportional to the mole
fraction of each protein (J.-B. Shin and P. G. Gillespie, unpublished
observations). Data analyzed here were from a SEQUEST–X! Tandem
analysis (J.-B. Shin and P. G. Gillespie, unpublished observations).
Hair cell isolation and immunocytochemistry. Hair cells were isolated
previously (Hirono et al., 2004) in low-Ca2?saline containing 112 mM
HEPES, pH 7.4. Briefly, sacculi were treated with 1 mM EGTA for 15
treatment with 100 ?g/ml DNase I, the cells were isolated from the epi-
thelium using an eyelash.
dehyde in low-Ca2?saline; washed; blocked in PBS with 1% normal
donkey serum, 1% BSA, and 0.2% saponin; and then incubated over-
?M FITC-phalloidin. All samples were observed with an Olympus
FV1000 confocal microscope equipped with a 60?, 1.42 NA oil plan-
The rabbit anti-PTPRQ antibody (affinity purified, against the C ter-
minus) was used at 1:250; PMCA2a was detected using 10 ?g/ml F2a,
Hair-bundle structural domains and lipid composition. A, Lipid composition of chick utricle hair bundles. The inner light blue pie graph indicates distribution of phospholipids,
Zhaoetal.•Hair-BundleMembraneDomainsJ.Neurosci.,March28,2012 • 32(13):4600–4609 • 4601
generated in rabbit. Radixin was detected with
5 ?g/ml H00005962-M06 mouse monoclonal
antibody (Abnova). Phospho-ERM was de-
tected using 0.65 ?g/ml Cell Signaling Tech-
nology #3149, generated in rabbit.
Cholera toxin B subunit labeling and neur-
aminidase treatment. Isolated cells were fixed
with 4% formaldehyde in low-Ca2?saline,
washed thoroughly with low-Ca2?saline, and
then treated with neuraminidase for 30 min.
Neuraminidase was diluted to 0.8 U/ml with
0.1 M potassium acetate, pH 4.5, and then
hair cells, so the final concentration was ?0.4
U/ml, and the final pH was ?4.6. After cells
were washed, they were treated with 10 ?g/ml
CTB in PBS for 15 min. The cells were washed
tibody in PBS for 15 min. The cells were
washed, postfixed with 4% formaldehyde in
PBS for 15 min, washed again, and then
blocked, permeabilized, and treated with sec-
ondary reagents as above.
frog hair cells was performed as described
previously (Hirono et al., 2004). For PAO
saline. After letting cells settle for 15–20 min at
room temperature (RT), cells were washed
with 75% DME/F-12 medium with 18.75
?g/ml carbenicillin. Hair cells were treated
with 30 ?M PAO in the same medium for 1.5 h
at RT (21?22°C), then washed three times
stock solution in DMSO. Control cells were
treated with 0.15% DMSO.
Filipin was made as a 5 mg/ml stock in
DMSO. Hair cells were isolated as usual and
then fixed with 0.75% glutaraldehyde, 2.25%
formaldehyde in PBS. After washing three
times with PBS, cells were stained for 2 h with
was excited using a 405 nm laser. M?C was
diluted from a 2 M stock in water to a final
concentration of 10 mM.
To determine the lipid composition
of hair bundles, purified bundles from
E20–E21 chick utricles (Gillespie and
Hudspeth, 1991; Shin et al., 2007) were
ing nanoelectrospray ionization tandem
mass spectrometry (Fig. 1). Despite the
high sensitivity of mass spectrometry
analysis, only by pooling hair bundles
from many dissections were we able to readily detect bundle lip-
ids. Each analysis (n ? 6) used bundles from 100 chicken ears
(?1 ?g protein for each preparation); given the average size of a
chick utricle stereocilium (0.25 ? 5 ?m), number of stereocilia
per cell (?60), and bundles recovered per ear (?8000, or 40%),
pmol per ear. PC, cholesterol, and PE accounted for 86% of the
dylglycerol, ceramide (Cer), and hexosylceramide were also de-
accounted for 96% of the total lipid species (Fig. 1).
overall lipid class composition of hair bundles did not differ sig-
in the epithelium (Fig. 1B). However, within individual lipid
4602 • J.Neurosci.,March28,2012 • 32(13):4600–4609Zhaoetal.•Hair-BundleMembraneDomains
dles, SM and Cer species nearly exclusively contained short,
saturated N-palmitoyl (16:0) acyl chains. For example, the 16:0
epithelium, but only 2% in porcine brain (B. Bru ¨gger, unpub-
broad distribution of sphingolipid species. For PC and PE, hair
bundles were enriched in arachidonoyl-containing species (36:4,
38:4, and 40:4), while docosahexaenoic-containing species (38:6
We localized lipid domains of hair bun-
dles using isolated bullfrog hair cells; the
eter (?0.4 ?m), permitting unusually
clear visualization of individual stereo-
cilia, basal stereocilia tapers, and other
structures. We confirmed that PI(4,5)P2
segregates within hair bundles, as we re-
ported previously (Hirono et al., 2004).
Because bullfrog hair cells are recalcitrant
to transfection, we used immunostain-
ing to show that PI(4,5)P2was absent
from the basal taper region but found
throughout the remainder of the hair
bundle (Fig. 2C).
Gangliosides, which are sialic acid-
modified, ceramide-based glycosphingo-
lipids, have often been associated with
cell signaling and membrane domains
(Sonnino et al., 2007) (see Fig. 4D). To
probe for gangliosides in stereocilia, we
used CTB, which binds to many ganglio-
sides but particularly tightly to GM1
(Kuziemko et al., 1996). Under standard
from hair bundles (Fig. 2A); however,
pretreatment of isolated hair cells with
neuraminidase, which converts polysialy-
lated gangliosides to GM1 (Rauvala,
1979), markedly increased the ability of
CTB to label bundles (Fig. 2B). Neura-
minidase-dependent labeling extended
through the stereocilia taper region, ter-
minating a micrometer or so above the
tapers in the region of the ankle links; the
rocal of the PI(4,5)P2domain. This pat-
tern was observed in at least 95% of
isolated hair cells, in ?15 separate exper-
from the apical membrane by the rem-
nants of the tight junctions, had much
lower levels of neuraminidase-dependent
Both boiling the CTB and preincuba-
tion with excess GM1 ganglioside elimi-
nated labeling in bundles. While not
labeling as strongly as CTB, an antibody
specific for GM1 ganglioside gave a simi-
lar pattern after neuraminidase treatment
(Fig. 3A). We were unable to identify the
specific ganglioside species responsible for the taper labeling. Al-
though antibodies against GD1a and GT1a, two polysialylated
GM1 relatives, gave no hair-bundle signal, cells potentially have
(Fig. 4D). M?C, which extracts cholesterol and often disrupts
ganglioside-containing lipid domains (Simons and Sampaio,
2011), did not disrupt the basal ganglioside domain (Fig. 3E).
We usually detected gangliosides with a two-step procedure,
first labeling with CTB and then amplifying the signal with an
anti-CTB antibody (Fra et al., 1994; Harder et al., 1998). While
treatment). As seen with some cells labeled with CTB (e.g., D, E), the lower half of the kinociliary bulb was labeled. B, After
neuraminidase treatment, the taper ganglioside domain is visible in live hair cells treated with rhodamine-
Zhaoetal.•Hair-BundleMembraneDomainsJ.Neurosci.,March28,2012 • 32(13):4600–4609 • 4603
1998). The taper-region ganglioside domain was readily de-
tected, however, when live (or fixed) (data not shown) hair
cells were labeled with CTB modified with Alexa 488, without
anti-CTB antibody (Fig. 3B). Cross-linking CTB molecules
with anti-CTB had little effect on the taper ganglioside do-
main, although some increase in punctate labeling was seen in
stereocilia shafts (Fig. 3C).
Stereocilia tips also show lipid segregation. We used the anti-
biotic filipin, a fluorescent cholesterol-binding molecule, to lo-
calize cholesterol within hair cells (Bornig and Geyer, 1974). As
seen in a previous electron microscopy study (Jacobs and Hud-
speth, 1990), filipin strongly stained stereocilia tips (Fig. 2D,G).
Filipin-detected cholesterol was present in stereocilia shafts, but
appeared more abundant in the soma’s apical surface.
As PI(4,5)P2is usually synthesized locally within cells, we used
protein mass spectrometry to identify membrane proteins, as
well as proteins involved in membrane trafficking and lipid syn-
we used bundles purified from E20–E21 chick utricles. Using
al., 2012), we estimated that the 4 ?m?2plasma membrane of
each stereocilium contained ?7,000 transmembrane proteins,
Proteins associated with PI(4,5)P2metabolism were readily
detected in hair bundles (Fig. 4B,C,E). Lipid transfer proteins
tol transfer protein ? (PITPNA), which is thought to shuttle PI
within cells. We also detected ?10 molecules per stereocilium
each of type III? phosphatidylinositol 4-kinase (PIK4CA) and
type II? phosphatidylinositol-5-phosphate 4-kinase (PIP5K2B),
kinases that sequentially transform PI to PI(4,5)P2; type I?
phosphatidylinositol-5-phosphate 4-kinase (PIP5K1A) was also
detected. In yeast, the PIK4CA ortholog Stt4 is anchored to the
membrane by the scaffolding protein Ypp1 and the integral
membrane protein Efr3a (Baird et al., 2008); we detected the
orthologs TTC7A (by SEQUEST only) and EFR3A (by both
search algorithms) in bundles. Finally, stereocilia contained
in hair bundles. The relative abundances of these proteins was
also reflected by the spectral count tally for each in the com-
plete dataset (Fig. 4B). Although antibodies against PIK4CA
and PIP5K2B were insufficiently sensitive to detect these pro-
teins by immunocytochemistry, we readily detected PIPTNA,
present in a punctate pattern throughout stereocilia (data not
shown), and PTPRQ (Fig. 5). Thus, a complete pathway for
transport, synthesis, and hydrolysis of PI(4,5)P2is present in
hair bundles (Fig. 4C).
Although we did not detect any glycosphingolipid synthetic
to plasma membrane. While glycosphingolipids are usually de-
pathways. Green indicates lipids analyzed by mass spectrometry (Fig. 1); glucosylceramide and galactosylceramide are analyzed together as hexosylceramide. Italicized enzyme short names
4604 • J.Neurosci.,March28,2012 • 32(13):4600–4609 Zhaoetal.•Hair-BundleMembraneDomains
galactosidase), HEXA (?-hexosaminidase ?), and NAGA (N-
acetylgalactosaminidase) (Fig. 4D,E).
calized, respectively, to the PI(4,5)P2and glycosphingolipid do-
mains. In isolated bullfrog hair cells, PMCA2 labeling extended
stantially in the glycosphingolipid zone, the bottom 2 ?m of the
stereocilia (Fig. 5A,C). This PMCA2 localization was not an ar-
tifact of cell isolation, as cells in whole-mount bullfrog sacculus
antibody and CTB, PMCA2 and gangliosides did not overlap
significantly (Fig. 5C).
While PMCA2 was always excluded from the glycosphingo-
lipid domain, the pattern of labeling seen in the upper domain
varied remarkably. Stereocilia tip labeling was usually stronger
than that of shafts; labeling often diminished ?1 ?m below ste-
was seen with monoclonal and polyclonal
antibodies against PMCA2, and was not
seen with antibody against NHE9, an-
other stereocilia membrane protein (Hill
et al., 2006). Remarkably, the PMCA2 la-
tion was coordinated across the gap.
As reported previously (Hirono et al.,
2004), PTPRQ was located at the base of
the stereocilia; PTPRQ and CTB labeling
overlapped extensively (Fig. 5D), al-
though CTB punctae seen in upper parts
of stereocilia shafts apparently contained
little or no PTPRQ.
The membrane-to-actin cross-linker ra-
dixin, a member of the ERM family, is
concentrated at basal tapers (Pataky et al.,
2004), although not as exclusively as is
PTPRQ (Fig. 6A). Mass spectrometry in-
dicates that the ?6000 molecules of ra-
dixin per stereocilium account for ?97%
of total bundle ERM proteins (J.-B. Shin
and P. G. Gillespie, unpublished observa-
tions). As shown previously (Pataky et al.,
radixin rose in concentration to a point
?1 ?m from the apical surface and then
membrane proteins only after activation,
which requires sequential PI(4,5)P2bind-
ing and phosphorylation on T564 (Fehon
et al., 2010). Once activated, radixin not
only links membrane and cytoskeleton,
but coordinates cellular activities by scaf-
folding signaling components (Neisch
and Fehon, 2011). In hair bundles, radixin may interact with a
large network of candidate partners identified by network analy-
sis, including overlapping interaction with SLC9A3R2 (NH-
ERF2) and Ras homology family member A (RHOA) networks
(J.-B. Shin and P. G. Gillespie, unpublished observations).
To examine phosphoradixin distribution in stereocilia, we
used a phosphospecific antibody selective for ERM proteins
phosphorylated on the activating threonine (T564 for radixin).
basal tapers (Fig. 6B–D). The boundary was sharp and corre-
the boundary, phosphorylated radixin was elevated in a band
about ?0.5 ?m wide and then declined exponentially toward
labeling (Fig. 6B–D). Notably, this band was located near the
overlapped with the MYO7A band (Fig. 6D).
To demonstrate the dependence of the phosphoradixin zone
on PI(4,5)P2, we depleted PI(4,5)P2using the PI(4)P kinase in-
hibitor PAO. As reported previously (Hirono et al., 2004), PAO
Zhaoetal.•Hair-BundleMembraneDomains J.Neurosci.,March28,2012 • 32(13):4600–4609 • 4605
bundles (Fig. 7G–I). Likewise, PAO re-
duced the level of phosphoradixin by al-
most 60% (Fig. 7A,B,I). Although PAO
also affects enzymes other than PI(4)P ki-
nase, our result is consistent with the hy-
pothesis that localized phosphoradixin
formation depends on PI(4,5)P2. In addi-
tion, PAO treatment destabilized radixin,
as total radixin in bundles declined by
tial fraction of radixin in bundles is phos-
phorylated, and when dephosphorylated,
it exits bundles. PAO had no effect on the
distribution or abundance of PMCA2 or
PTPRQ (Fig. 7C–F).
branes is similar to that of other cellular
membranes; PC and cholesterol make up
PI each contributing 3% or more to the
total. Acyl chains are mixed between the
relatively short and saturated 34:1 PC and
16:0 SM chains, and longer unsaturated
though mass spectrometry cannot deter-
mine the leaflet distribution of each
component, stereocilia devote substantial
effort to properly maintaining phospho-
lipid asymmetry (Shi et al., 2007; Good-
year et al., 2008). Indeed, the ATP8B1
P-type transporter, proposed to be re-
extracellular to intracellular leaflet, is es-
sential for hearing (Stapelbroek et al.,
2009) and is readily detected by mass
Several key lipids were not detected in our analysis. PI(4,5)P2
is typically present at much lower levels than PI; moreover, iso-
lated hair bundles likely deplete ATP rapidly, preventing synthe-
sis of PI(4,5)P2, and PTPRQ may exhaust remaining PI(4,5)P2
concentration of PI measured likely reflects the total PI plus
PI(4)P plus PI(4,5)P2in intact bundles. Glycosphingolipids are
not readily detected by mass spectrometry because of their scar-
all lipids, and ?100 distinct glycosphingolipid species have been
identified (Hakomori, 2004). Although lipidomics with high-
sides (Sampaio et al., 2011), the total amount of lipid in bundles
and levels of gangliosides are too low for detection at present.
We show here that stereocilia membranes are divided into at
least three large domains, each containing specific lipids and
proteins. Glycosphingolipids and PTPRQ are enriched in the
taper domain, which extends from a micrometer or so above the
stereocilia tapers to the apical surface of the hair cell. While
glycosphingolipids are prominent in so-called membrane rafts
(Edidin, 2003; Simons and Sampaio, 2011), insensitivity of the
taper domain to cyclodextrins suggests that cholesterol is not
necessary for its stability. Although cholesterol is typically a
component of rafts, gangliosides can form separate domains
with sphingomyelin but without cholesterol (Ferraretto et al.,
1997). Above basal tapers, PI(4,5)P2and PMCA2 are enriched
in the shaft domain; however, PI(4,5)P2and PMCA2 both
appear clustered within stereocilia shafts and are further con-
centrated at stereocilia tips along with cholesterol, suggesting
that additional segregation of membrane components occurs.
Distribution of PMCA2 and PTPRQ into shaft and taper do-
mains did not depend on CTB, CTB antibody, neuraminidase,
or cell dissociation.
The membrane domains reported here are unusually large.
Although lipid domains have long been detected in artificial ves-
icles and in native cells (Klausner et al., 1980), stable lipid clus-
tering on a micrometer scale is not typically seen in native cells
(Simons and Sampaio, 2011). The extent and appearance of the
stereocilia membrane domains could have been affected by our
4606 • J.Neurosci.,March28,2012 • 32(13):4600–4609Zhaoetal.•Hair-BundleMembraneDomains
pentamers by antibody cross-linking. However, the glycosphin-
live cells, which demonstrates that glycosphingolipids were clus-
tered before CTB treatment. If preexisting ganglioside domains
were not present, CTB could not induce formation of a continu-
ous, large-scale phase in stereocilia (Lingwood et al., 2008).
Lateral lipid segregation can occur due to
structural dissimilarity of domains’ lipid
acyl chains. Hair-bundle gangliosides con-
sist of an unusually high fraction of N-pal-
mitoyl (16:0) species; both ceramide, the
precursor for all gangliosides, and its me-
tabolite sphingomyelin predominantly
have a 16:0 N-acyl chain (Fig. 1). In con-
trast, brain ceramide lipids are predomi-
nantly composed of 18:0 or longer N-acyl
chains (Ben-David and Futerman, 2010).
Strikingly, the utricular epithelium as a
whole is far more enriched than bundles
in long-chain species of ceramide and
sphingomyelin (Fig. 1B). Sphingolipids
(e.g., sphingomyelin, ceramide, and gan-
gliosides) readily form segregated mem-
brane domains due to ceramide-moiety
hydrogen bonding, polar head-group
interaction, and acyl chain mismatch
with glycerophospholipids (Masserini
and Ravasi, 2001); the preponderance of
N-palmitoyl species in bundles would
enhance this latter effect (Holopainen et
al., 2001). Together these physical fea-
tures may promote lateral membrane
segregation of gangliosides in hair bun-
dles, presumably along with ceramide
throughout the stereocilia, as shown with
CTB labeling. Some mechanism must
control the balance of sphingolipids and
glycerophospholipids in the apical mem-
brane. Gangliosides are typically thought
bulky head group and compact acyl chains
gives them a wedge-like shape. Although
the stereocilia external leaflet is highly
concave where the taper enters the apical
region is quite small and likely cannot be
of PTPRQ to stereocilia bases has been
proposed to depend on active transport by
myosin-VI (MYO6) (Sakaguchi et al.,
2008), so presence of glycosphingolipids
ture of PTPRQ’s transmembrane domain
favored binding of short, saturated acyl
chains. The glycosphingolipid domain re-
mained even when PTPRQ was internal-
ized, however, suggesting that once the
allow precise spatial activation of radixin at the position where
the ganglioside and PI(4,5)P2domains meet. While total radixin
was abundant in the stereocilia taper region, we only saw phos-
Zhaoetal.•Hair-BundleMembraneDomains J.Neurosci.,March28,2012 • 32(13):4600–4609 • 4607
phoradixin beginning at the ganglioside-PI(4,5)P2boundary;
only there should PI(4,5)P2be present at high enough levels to
preactivate radixin, allowing activating phosphorylation by an
unknown kinase. Indeed, the phosphoradixin profile seen in
bundles can be modeled accurately as the [pRDX] ? [RDX]2?
[PI(4,5)P2]2(Fig. 8A), suggesting that phosphoradixin forma-
tion depends steeply on the concentrations of radixin and
PI(4,5)P2. Moreover, the presence of ceramide in the taper do-
main could also promote radixin dephosphorylation, as is the
case for ezrin (Canals et al., 2010). This phosphoradixin activa-
tion zone recalls the concentration of phospho-ERM proteins
toward microvillar tips, despite the presence of total ERM pro-
teins throughout a microvillus (Hanono et al., 2006).
by ERM proteins in microvilli (Reczek et al., 1997), once stereo-
cilia radixin is activated, we speculate that it recruits the paralog
SLC9A3R2, which is present at a concentration close to that of
radixin (J.-B. Shin and P. G. Gillespie, unpublished observa-
tions). SLC9A3R2, in turn, may bind to many important stereo-
cilia proteins (J.-B. Shin and P. G. Gillespie, unpublished
observations). In addition, as in other systems (Fehon et al.,
2010), activated radixin may bind directly to other membrane
proteins, serving as a actin-membrane connector. The role of
ERM proteins is so important that in radixin’s absence, hair cells
loss (Kitajiri et al., 2004).
than it does the PI(4,5)P2synthetic enzymes PIK4CA, PIP5K2B,
known, if PI(4,5)P2freely interacted with PTPRQ, present at a
concentration ?100-fold greater than the synthetic enzymes, it
therefore act as a physical barrier to prevent PI(4,5)P2exchange
the domain infrequently because of structural mismatch with
up those PI(4,5)P2molecules that did manage to penetrate the
basal taper compartment.
Are there reasons for compartmentalization of PI(4,5)P2in
the apical surface may fluctuate as exocytosis occurs, as fusion of
vesicles with the plasma membrane is associated with PI(4,5)P2
synthesis. In contrast, PI(4,5)P2controls transduction and adap-
tation (Hirono et al., 2004), as well as other critical molecules
such as PMCA2. Formation of a discrete stereocilia PI(4,5)P2
domain using the glycosphingolipid physical barrier thus allows
precise activity control through PI(4,5)P2levels.
Gangliosides play an essential role in the inner ear; mice with
of most ganglioside species, transiently show some responses in
an auditory brainstem response assay; however, all knockout
mice are deaf by postnatal day 17 (Yoshikawa et al., 2009). The
ganglioside defect could be in stereocilia; likewise, PTPRQ null
mice show progressive hearing loss that is complete by several
weeks of age (Goodyear et al., 2003). We thus suggest that the
gating PI(4,5)P2, PMCA2, and other stereocilia components
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