Large Membrane Domains in Hair Bundles Specify Spatially Constricted Radixin Activation

Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, OR 97239, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 03/2012; 32(13):4600-9. DOI: 10.1523/JNEUROSCI.6184-11.2012
Source: PubMed
The plasma membrane of vertebrate hair bundles interacts intimately with the bundle cytoskeleton to support mechanotransduction and homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular bundles. While the bundle glycerophospholipids and acyl chains resemble those of other endomembranes, bundle ceramide and sphingomyelin nearly exclusively contain short-chain, saturated acyl chains. Confocal imaging of isolated bullfrog vestibular hair cells shows that the bundle membrane segregates spatially into at least three large structural and functional domains. One membrane domain, including the stereocilia basal tapers and ∼1 μm of the shaft, the location of the ankle links, is enriched in the lipid phosphatase PTPRQ (protein tyrosine phosphatase Q) and polysialylated gangliosides. The taper domain forms a sharp boundary with the shaft domain, which contains the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]; moreover, a tip domain has elevated levels of cholesterol, PMCA2, and PI(4,5)P(2). 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)P(2). The membrane domains have functional significance; radixin, essential for hair-bundle stability, is activated at the taper-shaft boundary in a PI(4,5)P(2)-dependent manner, allowing assembly of protein complexes at that site. Membrane domains within stereocilia thus define regions within hair bundles that allow compartmentalization of Ca(2+) extrusion and assembly of protein complexes at discrete locations.
Large Membrane Domains in Hair Bundles Specify Spatially
Constricted Radixin Activation
Hongyu Zhao,
Diane E. Williams,
Jung-Bum Shin,
Britta Bru¨gger,
and Peter G. Gillespie
Oregon Hearing Research Center and Vollum Institute, Oregon Health & Science University, Portland, Oregon 97239 and
University of Heidelberg,
D-69120 Heidelberg, Germany
The plasma membrane of vertebrate hair bundles interacts intimately with the bundle cytoskeleton to support mechanotransduction and
homeostasis. To determine the membrane composition of bundles, we used lipid mass spectrometry with purified chick vestibular
bundles. While the bundle glycerophospholipids and acyl chains resemble those of other endomembranes, bundle ceramide and sphin-
gomyelin nearly exclusively contain short-chain, saturated acyl chains. Confocal imaging of isolated bullfrog vestibular hair cells shows
that the bundle membrane segregates spatially into at least three large structural and functional domains. One membrane domain,
including the stereocilia basal tapers and 1
m of the shaft, the location of the ankle links, is enriched in the lipid phosphatase PTPRQ
(protein tyrosine phosphatase Q) and polysialylated gangliosides. The taper domain forms a sharp boundary with the shaft domain,
which contains the plasma membrane Ca
-ATPase isoform 2 (PMCA2) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P
]; more-
over, a tip domain has elevated levels of cholesterol, PMCA2, and PI(4,5)P
. 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)P
. The membrane domains have
functional significance; radixin, essential for hair-bundle stability, is activated at the taper–shaft boundary in a PI(4,5)P
manner, allowing assembly of protein complexes at that site. Membrane domains within stereocilia thus define regions within hair
bundles that allow compartmentalization of Ca
extrusion and assembly of protein complexes at discrete locations.
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.
Transduction takes place in a dedicated subcellular organelle, the
hair bundle, which is composed of 30 –300 stereocilia arranged in
a precise staircase; each stereocilium contains a paracrystal of
actin filaments, sheathed by the hair cell’s plasma membrane
(Gillespie and Mu¨ller, 2009). Mechanical stimuli deflect the bun-
dle and open transduction channels, which admit K
and Ca
from the apical extracellular fluid, endolymph, that bathes the
bundle. Bundles remove Ca
using the plasma membrane
-ATPase isoform 2 (PMCA2), a calcium pump that is highly
concentrated in stereocilia (Lumpkin and Hudspeth, 1998; Ya-
moah et al., 1998; Dumont et al., 2001). Phosphatidylinositol (PI)
4,5-bisphosphate [PI(4,5)P
], a known regulator of PMCA2
(Hilgemann et al., 2001), also controls transduction and adapta-
tion by hair cells (Hirono et al., 2004).
is localized in hair cell plasma membranes in a strik-
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.,
2004). Protein tyrosine phosphatase receptor type Q (PTPRQ), a
phosphatidylinositol phosphatase (Oganesian et al., 2003), pres-
ents a near-perfect reciprocal localization pattern to PI(4,5)P
(Hirono et al., 2004); PTPRQ may therefore maintain low levels
of PI(4,5)P
in the apical surface and basal taper region. Steady-
state degradation of PI(4,5)P
at tapers by PTPRQ would be a
very inefficient way to maintain PI(4,5)P
distribution in stereo-
cilia; more likely, PI(4,5)P
is segregated into a separate mem-
brane domain (McLaughlin et al., 2002).
In many circumstances, members of the ezrin–radixin–moe-
sin (ERM) family depend on PI(4,5)P
for triggering a conforma-
tion that allows activating phosphorylation (Fehon et al., 2010).
Radixin is required for normal hearing in mice (Kitajiri et al.,
2004) and humans (Khan et al., 2007). Although radixin has been
localized to the taper region in stereocilia (Pataky et al., 2004) and
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
in stereocilia.
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.
Strikingly, polysialylated gangliosides are found in a micrometer-
scale membrane domain at the stereocilia basal tapers that is
physically segregated from the shaft/tip PI(4,5)P
domain; this
Received Dec. 5, 2011; revised Feb. 3, 2012; accepted Feb. 16, 2012.
Author contributions: P.G.G. designed research; H.Z., D.E.W., J.-B.S., and B.B. performed research; H.Z., D.E.W.,
B.B., and P.G.G. analyzed data; H.Z. and P.G.G. wrote the paper.
This work was supported by NIH grants R01 DC002368, R01 DC007602, and P30 DC005983 (P.G.G.). We thank
Ulrich Mu¨ller and Peter Mayinger for comments on earlier versions of this manuscript.
D. E. Williams’s present address: Wilfrid Laurier University, Waterloo, Ontario, Canada, N2L 3C5.
J.-B. Shin’s present address: Department of Neuroscience, University of Virginia, Charlottesville, VA 22908.
Correspondence should be addressed to Peter G. Gillespie, Oregon Hearing Research Center, L335A/3181 South-
west Sam Jackson Park Road, Portland, OR 97239. E-mail:
Copyright © 2012 the authors 0270-6474/12/324600-10$15.00/0
4600 The Journal of Neuroscience, March 28, 2012 32(13):4600 4609
Page 1
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
domains. Moreover, radixin, essential for hair-cell
function, is poised at the taper–shaft boundary and is activated at
the border of the PI(4,5)P
domain. These experiments show that
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 and Methods
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
F2a (Dumont et al., 2001). Radixin was detected with mouse monoclonal
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,
otoconia and otolithic membranes were removed from dissected utricles;
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
plasmalogens, which were extracted under neutral conditions. Quantita-
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.
Cone voltage was set to 30 V. Phosphatidylcholine (PC) and sphingomy-
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
m/z 141, 185, 189, or 277 Da, respectively, was applied for the analyses of
phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphati-
dylinositol, using a collision energy of 20 eV, except for phosphatidylino-
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).
Protein mass spectrometry. Purified hair bundles were analyzed by mass
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 (i
) 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
from saccular epithelia of bullfrogs of either sex using methods described
previously (Hirono et al., 2004) in low-Ca
saline containing 112 mM
NaCl, 2 mM KCl, 2 mM MgCl
, 100
M CaCl
,3mMD-glucose, and 10 mM
HEPES, pH 7.4. Briefly, sacculi were treated with 1 mM EGTA for 15
min and then 75
g/ml protease XXIV (Sigma) for 30 min. Aftera5min
treatment with 100
g/ml DNase I, the cells were isolated from the epi-
thelium using an eyelash.
For standard immunocytochemistry, cells were fixed with 4% formal-
dehyde in low-Ca
saline; washed; blocked in PBS with 1% normal
donkey serum, 1% BSA, and 0.2% saponin; and then incubated over-
night at 4°C with primary antibodies in the blocking solution. Cells were
washed and then treated with secondary antibodies (7.5
g/ml) and 0.25
M FITC-phalloidin. All samples were observed with an Olympus
FV1000 confocal microscope equipped with a 60, 1.42 NA oil plan-
apochromat objective.
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,
Figure 1. 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,
sphingomyelin, and cholesterol (mol% indicated); “Other” includes ceramide, hexosylceramide, and phosphatidylglycerol. Dark blue pie graphs indicate acyl chain compositions for the indicated
lipid species (for phospholipids, the sum of the two acyl chains(denoted bynumber oftotal Catoms inboth fattyacids/number oftotal double bonds in both fatty acids). In addition to the indicated
acyl chain, sphingolipids also contain a C18 sphingosine backbone. B, Species distributions for hair bundles and utricular epithelium. The “Mol%” in the lipid class panel is calculated as moles of
indicatedlipid divided bymoles ofalllipids analyzed; percentagesign inindividuallipid panels refersto molesofindicated acyl chainspecies dividedbytotal moles ofthat lipidclass.Values are given
as mean SEM (n 6 for each).
Zhao et al. Hair-Bundle Membrane Domains J. Neurosci., March 28, 2012 32(13):4600 4609 4601
Page 2
generated in rabbit. Radixin was detected with
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-Ca
washed thoroughly with low-Ca
saline, and
then treated with neuraminidase for 30 min.
Neuraminidase was diluted to 0.8 U/ml with
M potassium acetate, pH 4.5, and then
mixed 1:1 with the low-Ca
saline bathing the
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
CTB in PBS for 15 min. The cells were washed
and then incubated with a mouse anti-CTB an-
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.
Other methods. Labeling of PI(4,5)P
in bull
frog hair cells was performed as described
previously (Hirono et al., 2004). For PAO
treatment, hair cells were isolated in low-Ca
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 (2122°C), then washed three times
with PBS and fixed. PAO was stored as a 20 m
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
g/ml filipin and TRITC-phalloidin. Filipin
was excited using a 405 nm laser. M
C was
diluted from a 2
M stock in water to a final
concentration of 10 m
Lipid composition of hair-bundle
To determine the lipid composition
of hair bundles, purified bundles from
E20 –E21 chick utricles (Gillespie and
Hudspeth, 1991; Shin et al., 2007) were
subjected to quantitative lipid analysis us-
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
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%),
we calculated a theoretical amount of 10 pmol per ear, which is in
good agreement with the experimentally determined value of 8
pmol per ear. PC, cholesterol, and PE accounted for 86% of the
lipid detected; PS, SM, and PI together made up 12%. Phosphati-
dylglycerol, ceramide (Cer), and hexosylceramide were also de-
tected as minor species. We detected 182 lipid species, 76 of which
accounted for 96% of the total lipid species (Fig. 1).
Comparison with lipids of utricular epithelia revealed that the
overall lipid class composition of hair bundles did not differ sig-
nificantly from the whole organs, except for PI, which was higher
in the epithelium (Fig. 1B). However, within individual lipid
classes, significant differences were observed in the species distri-
Figure 2. Membrane domains in bullfrog hair bundles. A, Little CTB labeling without neuraminidase treatment. B, Pretreat-
ment with neuraminidase greatly enhances CTB labeling, particularly inbasal taper region (delineated by arrows); hair-cell apical
surface labeling was also enhanced. CTB antibody used for detection in A and B. C, PI(4,5)P
antibody labeling. In this and other
figures, PI(4,5)P
is abbreviated PIP
. D, Filipin labeling. A–D show isolated bullfrog hair cells; panel widths are all 12.5
m. E,
Approximate positionsof regions used for profile averaging. F,Profile averages for CTB (n 7), actin (n 7), andPI(4,5)P
6). Colored lines indicate means, and the gray shading shows SEM. Profiles of individual cells were aligned to the dip in actin
staining at thetaper; thispoint was defined tobe zero onthe abscissa.G, Profile averagesfor filipin-stainedcells (n 11). Profiles
were aligned to the peak of actin staining, which is near stereocilia tips; this point was defined to be zero on the abscissa.
4602 J. Neurosci., March 28, 2012 32(13):4600 4609 Zhao et al. Hair-Bundle Membrane Domains
Page 3
butions for sphingolipids and glycerophospholipids. In hair bun-
dles, SM and Cer species nearly exclusively contained short,
saturated N-palmitoyl (16:0) acyl chains. For example, the 16:0
species of Cer accounted for 73% of all Cer in bundles and 33% in
epithelium, but only 2% in porcine brain (B. Bru¨gger, unpub-
lished observations). Moreover, epithelial lipids show an unusual
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
and 40:6) of PS were elevated in bundles compared to epithelium.
Membrane domains in hair bundles
We localized lipid domains of hair bun-
dles using isolated bullfrog hair cells; the
stereocilia of these cells have a large diam-
eter (0.4
m), permitting unusually
clear visualization of individual stereo-
cilia, basal stereocilia tapers, and other
structures. We confirmed that PI(4,5)P
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)P
was 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. 4 D). 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
conditions, CTB binding sites were absent
from hair bundles (Fig. 2 A); 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. 2 B). Neura-
minidase-dependent labeling extended
from the apical surface of the hair cell and
through the stereocilia taper region, ter-
minating a micrometer or so above the
tapers in the region of the ankle links; the
region labeled with CTB was exactly recip-
rocal of the PI(4,5)P
domain. This pat
tern was observed in at least 95% of
isolated hair cells, in 15 separate exper-
iments. The lateral membrane, segregated
from the apical membrane by the rem-
nants of the tight junctions, had much
lower levels of neuraminidase-dependent
CTB labeling.
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
many polysialylated gangliosides that can be converted into GM1
(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
Figure 3. Ganglioside domaincontrols. A, Taper domain isvisible with antibodyagainst GM1ganglioside (after neuraminidase
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-
phosphatidylethanolamine (Rh-PE) to label membranes and Alexa 488-CTB (488-CTB) to label gangliosides. C, Additional treat-
mentofcellinB with anti-cholera toxin antibodydoesnot change the 488-CTB pattern appreciably.D,Control hair cell labeled with
CTB after neuraminidase treatment. CTB antibody used for detection. E, Hair cell treated with 10 m
C for 40 min to extract
cholesterol.Thetaper ganglioside domain is notdisrupted. As seen occasionally,the lower halves ofthe kinociliary bulbs of thecells
in D and E were also labeled with CTB.
Zhao et al. Hair-Bundle Membrane Domains J. Neurosci., March 28, 2012 32(13):4600 4609 4603
Page 4
useful for its sensitivity, the method can generate artificially large
ganglioside domains due to antibody cross-linking (Harder et al.,
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. 2 D,G).
Filipin-detected cholesterol was present in stereocilia shafts, but
appeared more abundant in the soma’s apical surface.
pathway in hair-bundle membranes
As PI(4,5)P
is 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-
thesis in hair bundles (Fig. 4A). As with lipid mass spectrometry,
we used bundles purified from E20–E21 chick utricles. Using
intensity-weighted spectral counting (Shin et al., 2007; Spinelli et
al., 2012), we estimated that the 4
plasma membrane of
each stereocilium contained 7,000 transmembrane proteins,
10,000 peripheral membrane proteins, and 1,000 lipid trans-
fer molecules.
Proteins associated with PI(4,5)P
metabolism were readily
detected in hair bundles (Fig. 4B,C,E). Lipid transfer proteins
included 120 molecules per stereocilium of phosphatidylinosi-
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)P
; 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
3000 molecules of PTPRQ, the principal PI(4,5)P
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)P
is present in
hair bundles (Fig. 4C).
Although we did not detect any glycosphingolipid synthetic
enzymes in stereocilia using mass spectrometry, these glycolipids
are usually synthesized in the ER and Golgi and then transported
to plasma membrane. While glycosphingolipids are usually de-
Figure 4. Hair-bundle membrane proteins and PI(4,5)P
pathway. In all panels, proteins indicated by blue-colored short names were detected in purified hair bundles by mass spectrometry. A,
Chick hair-bundle membrane proteins.Proteins detectedby liquidchromatography (LC)-MS/MSwere quantifiedusing intensity-weightedspectral counting.The sizeof each slice is proportional to
molar abundance. Red, Proteins enriched in bundles over epithelium more than fivefold; green, less than fivefold. Only proteins enriched 0.1-fold or greater are plotted. B, Peptide coverage of
-transport and metabolizing proteins in LC-MS/MS of chick hair bundles. Peptides detected by X! Tandem analysis of the complete dataset of chick bundles were plotted against residue
number. Height of plotted bars corresponds to the number of identical peptides detected; width corresponds to peptide length, plotted at the position in sequence. Gray shading indicates peptide
log(e) (log of expectation) score, indicating statistical confidence in protein identification (key is shown in PITPNA panel). C, PI pathway in hair bundles. D, Ceramide, sphingolipid, and ganglioside
pathways. Green indicates lipids analyzed by mass spectrometry (Fig. 1); glucosylceramide and galactosylceramide are analyzed together as hexosylceramide. Italicized enzyme short names
correspondto proteins notdetected inbundles. Allsialyltransferase (SAT)reactions canbe reversedby endogenousor exogenousneuraminidase. Polysialylatedgangliosides thatcould beconverted
to GM1 ganglioside with neuraminidase treatment are indicated with gray shading. E, Bundle abundance (green) and bundle-to-epithelium enrichment (magenta) of PI(4,5)P
and ganglioside
metabolic proteins.
4604 J. Neurosci., March 28, 2012 32(13):4600 4609 Zhao et al. Hair-Bundle Membrane Domains
Page 5
graded in lysosomes, we detected several enzymes of the pathway
for metabolizing polysialylated gangliosides, including GLB1 (
galactosidase), HEXA (
), and NAGA (N-
acetylgalactosaminidase) (Fig. 4D, E).
Membrane proteins respect the glycosphingolipid domain
The stereocilia transmembrane proteins PMCA2 and PTPRQ lo-
calized, respectively, to the PI(4,5)P
and glycosphingolipid do
mains. In isolated bullfrog hair cells, PMCA2 labeling extended
through the upper part of stereocilia shafts, but was reduced sub-
stantially in the glycosphingolipid zone, the bottom 2
stereocilia (Fig. 5 A,C). This PMCA2 localization was not an ar-
tifact of cell isolation, as cells in whole-mount bullfrog sacculus
tissue, folded to allow high-resolution imaging, displayed similar
localization (Fig. 5B). When cells were colabeled with the PMCA2
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-
reocilia tips and then increased near the taper region. This pattern
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-
beling pattern appeared continuous
between adjacent stereocilia, as if localiza-
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.
at the taper–shaft boundary
activates radixin
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. 6 A). 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.,
2004), starting at the base of a hair bundle,
radixin rose in concentration to a point
m from the apical surface and then
fell exponentially toward stereociliary tips
(Fig. 6 D,E).
Radixin interacts with membranes and
membrane proteins only after activation,
which requires sequential PI(4,5)P
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).
Remarkably, phosphorylated radixin was only detected above the
basal tapers (Fig. 6B–D). The boundary was sharp and corre-
sponded to the taper–shaft membrane-domain boundary. Above
the boundary, phosphorylated radixin was elevated in a band
about 0.5
m wide and then declined exponentially toward
stereocilia tips; taller stereocilia had more intense, more extensive
labeling (Fig. 6B–D). Notably, this band was located near the
ankle links and a concentration of myosin-VIIA (MYO7A) (Has-
son et al., 1997), although the phosphoradixin band only partially
overlapped with the MYO7A band (Fig. 6D).
To demonstrate the dependence of the phosphoradixin zone
on PI(4,5)P
, we depleted PI(4,5)P
using the PI(4)P kinase in
hibitor PAO. As reported previously (Hirono et al., 2004), PAO
Figure 5. PMCA2 and PTPRQ segregate to distinct domains delineated by CTB labeling. A, PMCA2 localization in isolated cell
labeledwith F2a antibody.Notenear absence ofPMCA2in basal taperregion(arrows), which appearsredin the colormergepanel.
No neuraminidase,CTB, or CTB antibody wereused. B, PMCA2 absence frombasal taper region is alsoclear in whole-mount tissue
(arrows). C, Reciprocal PMCA2 and CTB labeling. D, PTPRQ and CTB labeling overlap. CTB antibody was used for detection in C and
D. Panel widths: A, C, D, 12.5
m; B,65
Zhao et al. Hair-Bundle Membrane Domains J. Neurosci., March 28, 2012 32(13):4600 4609 4605
Page 6
effectively reduced PI(4,5)P
levels in hair
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)P
. In addi
tion, PAO treatment destabilized radixin,
as total radixin in bundles declined by
50%. This result suggests that a substan-
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).
Hair-bundle lipid composition
The lipid composition of stereocilia mem-
branes is similar to that of other cellular
membranes; PC and cholesterol make up
the bulk of the lipids, with PE, PS, SM, and
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
chains predominant in PE, PS, and PI. Al-
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-
sponsible for translocating lipids from the
extracellular to intracellular leaflet, is es-
sential for hearing (Stapelbroek et al.,
2009) and is readily detected by mass
spectrometry in chick utricle hair bundles
(J.-B. Shin and P. G. Gillespie, unpublished
Several key lipids were not detected in our analysis. PI(4,5)P
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)P
, and PTPRQ may exhaust remaining PI(4,5)P
before bundles can be isolated and degradation stopped. Thus the
concentration of PI measured likely reflects the total PI plus
PI(4)P plus PI(4,5)P
in intact bundles. Glycosphingolipids are
not readily detected by mass spectrometry because of their scar-
city and diversity; together, they account for only a few percent of
all lipids, and 100 distinct glycosphingolipid species have been
identified (Hakomori, 2004). Although lipidomics with high-
resolution mass spectrometers allows direct detection of ganglio-
sides (Sampaio et al., 2011), the total amount of lipid in bundles
and levels of gangliosides are too low for detection at present.
Two membrane domains in stereocilia
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)P
and PMCA2 are enriched
in the shaft domain; however, PI(4,5)P
and 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
detection techniques, as the two-step detection could cluster CTB
Figure 6. Radixin is activated at the PI(4,5)P
–PTPRQ boundary. A, Radixin and PTPRQ are located in similar, but not entirely
coextensive, patterns at the baseof thehair bundle.B, Antibody for activated ERM proteins(including pRDX)only labels above the
stereocilia tapers. Note pRDX punctae throughout bundle. Arrows indicate the pRDX-free taper region. C, pRDX labeling is shifted
toward stereocilia tips from CTB labeling. Arrows indicate the pRDX-free taper region. D, The MYO7A band is more basal than the
pRDX band. Arrows indicate the basal MYO7A band. E–G, Intensity profiles for RDX/pRDX (n 5; E), RDX/PTPRQ (n 5; F ), and
CTB/pRDX (n 5; G) averaged from individual cells aligned at the apical-surface actin dip. Panel widths: A–C, 12.5
4606 J. Neurosci., March 28, 2012 32(13):4600 4609 Zhao et al. Hair-Bundle Membrane Domains
Page 7
pentamers by antibody cross-linking. However, the glycosphin-
golipid domain was readily visible using CTB alone, with fixed or
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).
Physical basis of membrane domains
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. 1 B). 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
and sphingomyelin.
Extensive on the apical surface, it is cu-
rious that gangliosides do not extend fully
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
to be localized on convex surfaces, as their
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
surface of the hair cell, this highly concave
region is quite small and likely cannot be
resolved by light microscopy. Localization
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
within the basal taper region could plausibly
depend on PTPRQ, particularly if the struc-
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
domain formed, it was relatively stable.
Spatial constriction of radixin activation
A primary role for the hair-bundle membrane domains may be to
allow precise spatial activation of radixin at the position where
the ganglioside and PI(4,5)P
domains meet. While total radixin
was abundant in the stereocilia taper region, we only saw phos-
Figure 7. Radixin activation depends on polyphosphatidylinositols. A, B, RDX and pRDX (detected with anti-pERM antibody)
immunolabeling without (A) and with (B)30
M PAO for 1 h. pRDX and RDX both decline after PAO treatment. C–F, PMCA2 and
PTPRQ distribution and intensity are not affected by PAO treatment. G, H, PI(4,5)P
immunolabeling with (G) and without (H )30
M PAO (1 h). PAO completely abolishes PI(4,5)P
immunoreactivity. I, Mean SEM for whole-bundle pixel intensity; regions of
interest used formeasurement includedthe entire bundle.Significance: **p 0.01; ***p 0.001.Panel widths: A–H,12.5
Figure8. Membranedomainsin hair bundles. A, Relationshipbetween radixin, phosphoradixin, and PI(4,5)P
from differentsets of cells, aligned atthe apical surface (small peak in each profile).The black curve is obtainedby multiplying the
square of the relativeradixin concentrationby thesquare ofthe relative PI(4,5)P
concentration and then normalizingto apeak of
1.0. The approximate stereocilia profile is indicated by diagram. B, Scanning electron micrograph of bullfrog hair bundle pseudo-
colored to indicate structural and functional domains. Purple, Taper domain; green, shaft/tip domain; yellow,kinocilium domain.
Inset, Transmission electron micrograph of taper region.
Zhao et al. Hair-Bundle Membrane Domains J. Neurosci., March 28, 2012 32(13):4600 4609 4607
Page 8
phoradixin beginning at the ganglioside-PI(4,5)P
only there should PI(4,5)P
be 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]
(Fig. 8
A), suggesting that phosphoradixin forma-
tion depends steeply on the concentrations of radixin and
. 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).
Based on recruitment of the PDZ-domain protein SLC9A3R1
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
upregulate the paralog ezrin, partially compensating for radixin’s
loss (Kitajiri et al., 2004).
Corralling PI(4,5)P
with glycosphingolipids
The bundle contains far more PTPRQ, which degrades PI(4,5)P
than it does the PI(4,5)P
synthetic enzymes PIK4CA, PIP5K2B,
and PIP5K1A. While turnover numbers for these enzymes are not
known, if PI(4,5)P
freely interacted with PTPRQ, present at a
concentration 100-fold greater than the synthetic enzymes, it
would be readily hydrolyzed. The glycosphingolipid domain may
therefore act as a physical barrier to prevent PI(4,5)P
between the hair bundle and apical surface; PI(4,5)P
might enter
the domain infrequently because of structural mismatch with
glycosphingolipid domain, but PTPRQ would be present to mop
up those PI(4,5)P
molecules that did manage to penetrate the
basal taper compartment.
Are there reasons for compartmentalization of PI(4,5)P
stereocilia beyond phosphoradixin activation? PI(4,5)P
levels in
the apical surface may fluctuate as exocytosis occurs, as fusion of
vesicles with the plasma membrane is associated with PI(4,5)P
synthesis. In contrast, PI(4,5)P
controls 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)P
domain using the glycosphingolipid physical barrier thus allows
precise activity control through PI(4,5)P
Gangliosides play an essential role in the inner ear; mice with
a null mutation in GM3 synthase, which is essential for formation
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
basal taper domain, consisting of glycosphingolipids and PTPRQ
(Fig. 8 B), is essential for hair-cell function, presumably by segre-
gating PI(4,5)P
, PMCA2, and other stereocilia components
away from the soma’s apical surface and allowing radixin activa-
tion in a spatially precise manner.
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    • "Among these proteins, radixin is important for auditory development and maintenance (Khan et al. 2007). Radixin is mainly expressed along the length of hair cell stereocilia from both the organ of Corti and the vestibular system (Zhao et al. 2012). In the mouse, knockout of the radixin gene (Rdx) is associated with early postnatal progressive degeneration of cochlear stereocilia and subsequent deafness (Kitajiri et al. 2004). "
    [Show abstract] [Hide abstract] ABSTRACT: Our previous work has suggested that traumatic noise activates Rho-GTPase pathways in cochlear outer hair cells (OHCs), resulting in cell death and noise-induced hearing loss (NIHL). In this study, we investigated Rho effectors, Rho-associated kinases (ROCKs), and the targets of ROCKs, the ezrin-radixin-moesin (ERM) proteins, in the regulation of the cochlear actin cytoskeleton using adult CBA/J mice under conditions of noise-induced temporary threshold shift (TTS) and permanent threshold shift (PTS) hearing loss, which result in changes to the F/G-actin ratio. The levels of cochlear ROCK2 and p-ERM decreased 1 h after either TTS- or PTS-noise exposure. In contrast, ROCK2 and p-ERM in OHCs decreased only after PTS-, not after TTS-noise exposure. Treatment with lysophosphatidic acid, an activator of the Rho pathway, resulted in significant reversal of the F/G-actin ratio changes caused by noise exposure and attenuated OHC death and NIHL. Conversely, the down-regulation of ROCK2 by pretreatment with ROCK2 siRNA reduced the expression of ROCK2 and p-ERM in OHCs, exacerbated TTS to PTS, and worsened OHC loss. Additionally, pretreatment with siRNA against radixin, an ERM protein, aggravated TTS to PTS. Our results indicate that a ROCK2-mediated ERM-phosphorylation signaling cascade modulates noise-induced hair cell loss and NIHL by targeting the cytoskeleton. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Full-text · Article · Feb 2015 · Journal of Neurochemistry
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    • "Our observation that PTPRQ and RDX exhibit a more diffuse pattern but still with a base-to-tip gradient in hair bundles of jbg mice suggests that the localization of these proteins during bundle maturation is regulated not only by CLIC5 but also by other proteins such as MYO6. Conceivably, differences in localization of individual proteins could reflect differences in protein-protein interactions, or their dynamics at particular membrane microdomains having specific protein and lipid compositions [Zhao et al., 2012]. The compartmentalization of GFP-CLIC5 at the base of the hair bundle is compatible with a mechanism driven by actin filament minus end-directed MYO6 motor activity [Sakaguchi et al., 2008] , comparable to tip protein compartmentalization by plus end-directed motors [Salles et al., 2009]. "
    [Show abstract] [Hide abstract] ABSTRACT: Chloride intracellular channel 5 protein (CLIC5) was originally isolated from microvilli in complex with actin binding proteins including ezrin, a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-cytoskeletal linkers. CLIC5 concentrates at the base of hair cell stereocilia and is required for normal hearing and balance in mice, but its functional significance is poorly understood. This study investigated the role of CLIC5 in postnatal development and maintenance of hair bundles. Confocal and scanning electron microscopy of CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Radixin (RDX), protein tyrosine phosphatase receptor Q (PTPRQ), and taperin (TPRN), deafness-associated proteins that also concentrate at the base of stereocilia, were mislocalized in fused stereocilia of jbg mice. PTPRQ and RDX were dispersed even prior to stereocilia fusion. Biochemical assays showed interaction of CLIC5 with ERM proteins, TPRN, and possibly myosin VI (MYO6). In addition, CLIC5 and RDX failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we propose a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at the base of stereocilia. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Jan 2014 · Cytoskeleton
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    [Show abstract] [Hide abstract] ABSTRACT: Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.
    Full-text · Article · Jan 2013 · Nature Neuroscience
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