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Cell Motility and the Cytoskeleton 39:159–165 (1998)
Immunolocalization of Myosin Ib in the Hair
Cell’s Hair Bundle
Anne B. Metcalf*
Laboratory of Sensory Neuroscience, Rockefeller University, New York, New York
The hair bundle, the hair cell’s sensory organelle, transduces acoustical or
vestibular stimulation into a change in membrane potential. The actin-based
stereociliary processes of the hair bundle contain a number of myosin isoforms that
may be important to the bundle’s function. One of these isoforms, myosin Ib, has
been proposed to constitute an adaptation motor controling sensitivity of the hair
bundle to mechanical displacement. To gain insight into myosin Ib’s function, its
distribution within the hair bundle was examined. A polyclonal antibody was
produced that recognizes a protein surface loop within the head domain of myosin
Ib. This antibody was used to localize myosin Ib in the hair cell by indirectimmunofluorescence microscopy and indirect-immunoelectron microscopy. Within
the hair bundle, myosin Ib immunoreactivity was located along the sides of the
stereociliary actin core, concentrated in the distal two-thirds of the stereocilia.
Within the hair cell soma, myosin Ib immunoreactivity was located throughout the
cytoplasm exclusive of the cuticular plate. Cell Motil. Cytoskeleton 39:159–165,
1998. r 1998 Wiley-Liss, Inc.
Key words: adaptation; auditory system; molecular motor; vestibular system
The sensory cells of the ear are hair cells. These
cells detect mechanical deflection of the hair bundle
induced by acoustical or vestibular stimulation. The hair
bundle is a beveled conical array of long cylindrical
extensions from the cell’s apical surface, composed of
20–300 actin-based stereocilia and one tubulin-based
kinocilium located at the bundle’s edge. (For a review of
hair cell structure, see Jacobs and Hudspeth 1990.) Rows
of stereocilia farther from the kinocilium are progressively about 500 nm shorter, which gives the top edge of
the bundle its beveled appearance. Stereocilia are connected by tip links, extracellular linkages found only in
the bundle’s plane of mechanical sensitivity and stretching from the tip of each stereocilium to a higher point on
the side of its tallest neighbor. The top of each tip link
terminates in a pucker in the plasma membrane backed by
the insertional plaque, an electron-dense structure between the plasma membrane and the stereociliary actin
core. The membrane of each stereocilium is connected to
the parallel, cross-linked actin filaments within its core by
the insertional plaque and by thin linkages located along
its height.
r 1998 Wiley-Liss, Inc.
Each stereocilium has a uniform diameter along
most of its length but narrows to 100 nm at a constriction
called the pivot within 1 µm of its insertion into the soma.
The cluster of about 50 actin filaments that pass through
the stereociliary pivot descend a few nanometers and
anchor each stereocilium in the cuticular plate, a thick,
highly cross-linked meshwork of cortical actin analogous
to a terminal web. The cuticular plate’s stiffness and
concave surface stabilize the conical shape of the hair
When the bundle is deflected toward the tallest
stereocilia, the hair cell depolarizes. This leads to an
increase in the rate of neurotransmission at the cell’s
afferent synapses. Conversely, when the bundle is deflected toward the shortest stereocilia, the cell hyperpolarContract grant sponsor: National Institutes of Health; Contract grant
number: DC00241
*Correspondence to: Anne B. Metcalf (current address): Department of
Cell Biology and Neuroscience, University of Texas, Southwestern
Medical Center, Dallas, TX 75235-9111
Received 6 August 1997; accepted 20 October 1997
izes and the rate of neurotransmission decreases. When
the hair bundle is held deflected at a constant position, the
electrical response of the cell diminishes over time to a
steady level; this process is termed adaptation.
The gating-spring model of mechanoelectrical transduction and adaptation, formulated to explain current
experimentation but not verified in detail, delineates how
the hair bundle converts mechanical displacement to a
change in ion-channel open probability [Howard and
Hudspeth, 1987, 1988; Assad and Corey, 1992; Hudspeth
and Gillespie, 1994]. Acoustical and vestibular stimulation causes deflection of the hair bundle and alters
transduction channel open probability, resulting in a fast
change in receptor current. It is proposed that the tip link,
the extracellular linkage connecting the tip of each
stereocilium to the insertional plaque on its tallest neighbor, is connected directly to transduction channels and
functions as a gating spring. When the bundle is displaced, stereociliary tips slide with respect to one another,
and the resulting change in tip-link tension is hypothesized to control gating. Additionally, when bundle
deflection is prolonged, the receptor current adapts.
This adaptation is thought to be achieved by a motor
element, the adaptation motor, which is proposed to
comprise a group of myosin molecules positioned at the
insertional plaque. The adaptation motor slipping down
and climbing up the stereociliary core sets the resting
position of the insertional plaque and therefore the
tension under which the tip link is held in the absence of
hair bundle deflection. During adaptation, a disequilibrium in motor slipping and climbing alters the insertionalplaque position and the tip-link tension. Consistent with a
myosin-based adaptation motor, adaptation is blocked by
dialysis of hair cells with the nucleotide analog ADPbS as
well as the phosphate analogues vanadate, sulfate, and
beryllium fluoride [Gillespie and Hudspeth, 1993; Yamoah and Gillespie, 1996].
Biochemical and immunohistochemical analysis
indicates that hair bundles contain several members of the
myosin family, a large group of proteins that convert the
binding and hydrolysis of ATP to force production or
directional movement along actin filaments [Gillespie et
al., 1993; Hasson et al., 1997]. Photocross-linking of frog
hair-bundle proteins to vanadium-ion-trapped nucleotide
under a variety of conditions identified three potential
myosin isotypes [Gillespie et al., 1993]. One of these, a
protein of molecular mass 120 kD, is recognized by the
monoclonal antibody mT2, which was raised to bovine
myosin Ib [Gillespie et al., 1993; Wagner et al., 1992;
Reizes et al., 1994]. Subsequent molecular cloning of the
frog myosin Ib (amphibian myosin Ib or AMIb) and
epitope mapping of the mT2 binding site have confirmed
the identification of the 120-kD hair-bundle myosin as
myosin Ib [Metcalf et al., 1994; Solc et al., 1994;
Metcalf, 1995]. Immunofluorescence experiments indicated that myosin Ib is located throughout the stereocilia
of the hair bundle and concentrated near the bundle’s
beveled top edge, in the vicinity of the tip links [Gillespie
et al., 1993; Hasson et al., 1997].
To confirm and expand the study of myosin Ib’s
location and function within the hair bundle, affinitypurified polyclonal antibody was produced as a reagent
for both immunolocalization of myosin Ib in ultrastructural experiments and inhibition of myosin Ib’s mechanochemical cycle in functional experiments. Antiserum was
raised to a peptide with a sequence identical to that of
residues 485–505 of AMIb, a segment of the protein
corresponding to a flexible, surface-exposed loop in the
crystal structures of the head domain of myosin II
[Rayment et al., 1993b; Fisher et al., 1995]. Modeling of
the interaction of myosin II with F-actin suggested a
potential interaction between this loop and the actin
monomer one actin-helical turn away from the primary
site of actomyosin interaction on the actin filament
[Rayment et al., 1993a; Schroder et al., 1993]. This paper
presents immunolocalization studies describing the subcellular distribution of myosin Ib in the hair cell of the
Antibody Production
which corresponds to amino acids 485–505 of AMIb plus
a terminal cysteine, was synthesized. The peptide was
reduced by passage through an immobilized reductant
column (Reduce-Imm; Pierce, Rockford, IL) following
the manufacturer’s instructions and coupled to maleimideactivated keyhold-limpet hemocyanin (KLH) in 0.085 M
sodium phosphate, 0.9 M sodium chloride, 0.05 M EDTA,
and 0.02% sodium azide at pH 7.2. Maleimide-activated
KLH is KLH coupled to the heterobifunctional cross-linker
sulfo succinimidyl 4-[N-maleimidomethyl]cyclohexane1-carboxylate (Pierce). Every 2 weeks for 18 weeks,
rabbits were bled and immediately injected with 100 µg
peptide-coupled KLH in Freund’s adjuvant, 0.041 M
sodium phosphate, and 0.45 M sodium chloride at pH 7.2.
Antipeptide antibody was purified from serum by
affinity chromatography on a protein-A affinity column,
followed by affinity chromatography on a peptide-affinity
column. Antibody was applied to a prewashed protein-A
column by three gravity-flow passages of serum. The
column was washed with 10 mM Tris at pH 8.2, and the
bound proteins were eluted with 100 mM sodium citrate
at pH 3.0. After neutralization to pH 8.0 with NaOH, the
pooled protein-containing eluant fractions were applied
to washed peptide columns, which were produced by
coupling immunizing peptide to a sulfo-link matrix
Immunolocalization of Myosin Ib in Hair Cell’s Hair Bundle
(Pierce) following the manufacturer’s instructions. Peptide-specific antibody was eluted from the peptideaffinity column and neutralized as described above for the
protein-A affinity column.
The specificity of the affinity-purified polyclonal
antibody, called I4, was analyzed by assessing its reactivity with SDS solubilized protein from saccular maculae in
immunoblot analysis and comparing this reactivity to that
of the monoclonal antibody mT2. The saccular macula is
the sensory epithelium of the sacculus, one of the
vestibular organs. Saccular-macula protein was resolved
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF in 10
mM CAPS at pH 11. The membrane-bound protein was
detected by first blocking with 5% nonfat dried milk in
phosphate-buffered saline (PBS) and then incubating
sequentially with primary antibody followed by goatantirabbit IgG antibody conjugated to alkaline phosphatase or, in the case of mT2, goat-antimouse IgG antibody
conjugated to alkaline phosphatase. After each antibody
incubation, the membranes were washed with PBS.
Chemiluminescent detection was performed using CDPstar as phosphatase substrate (Tropix, Bedford, MA).
Immunofluorescence Experiments
Immunofluorescence experiments were performed
on both epithelial sheets and isolated hair cells from the
frog’s sacculus. To produce isolated epithelia, saccular
maculae were dissected in standard saline solution (108
mM NaCl, 2 mM KCl, 4 mM CaCl2, 3 mM D-glucose,
and 5 mM HEPES at pH 7.25) and digested for 1 hr in 500
µg/ml adipocyte collagenase (Sigma type II) in standard
saline solution (Sigma, St. Louis, MO). The epithelium
was peeled from the basement membrane with an eyelash
and attached to coverslips coated with Cell-tak (Collaborative Biomedical Products, Bedford, MA). The otolithic
membrane was removed from the hair bundles with
forceps after the epithelial sheet was attached to the
Hair cells were isolated from saccular maculae by a
previously described procedure, with some modifications
[Lumpkin and Hudspeth, 1995]. Maculae were dissected
into saline solution lacking added Ca21, then incubated
for 12 min in 1 mM EGTA, 1 mM MgCl2, and 25 µg/ml
bacterial protease (Sigma type XXIV) in the above saline
solution. After transfer to fresh saline solution lacking
added Ca21, the procedure was finished by removing the
otolithic membrane with forceps and brushing hair cells
from the epithelium onto coverslips coated with concanavalin A, using an eyelash.
Both preparations were processed similarly. The
tissue or cells were fixed with 1.3 M formaldehyde in
PBS for 15–120 min. The specimen was rinsed 5–6 times
with PBS, then permeabilized and blocked for 30–120
min with PBS containing 12.5% normal goat serum, 1.5
M NaCl, and 0.16% Triton X-100. Samples were exposed
sequentially to primary and secondary antibodies diluted
in blocking solution containing 0.024% Tween-20 and
lacking Triton X-100. Each antibody incubation lasted
60–120 min and was followed by PBS rinses. RhodamineTRITC conjugated goat-antirabbit IgG antibody was used
as a secondary antibody.
Immunoelectron Microscopy Experiments
Samples for electron microscopy were labeled with
antibody prior to fixation with glutaraldehyde and processing for electron microscopy. Two separate formaldehyde
fixation and permeabilization protocols were used prior to
antibody labeling. In most experiments, isolated maculae
were fixed for 30 min with 1.3 M formaldehyde and 1.3
µM phalloidin in PBS. After rinsing, the tissue was
permeabilized and blocked for 90 min with PBS containing 12.5% normal goat serum, 1.5 M NaCl, 0.16% Triton
X-100, and 1.3 µM phalloidin. Saccular maculae were
then incubated overnight at 4°C in primary antibody in
the same solution lacking phalloidin. After rinsing with
PBS, samples were incubated for 4 hr with goat-antirabbit
IgG antibody coupled to colloidal gold. This procedure
removed most or all of the plasma membrane and gave
good antibody access to the cytoplasm.
Some samples were fixed and permeabilized in an
hypo-osmotic solution, followed by antibody incubations
in a milder detergent. This procedure led to a better
preservation of permeabilized membranes. Samples were
fixed and permeabilized for 30 min with 1.3 M formaldehyde, 0.1% Triton X-100, 1.3 mM phalloidin, and 50 mM
phosphate buffer at pH 6.3, then blocked and permeabilized for 30 min with 12.5% goat serum, 1.5 mM MgCl2,
0.1% Triton X-100, and 20 mM Pipes at pH 6.8. Samples
were then incubated with antibody as described above,
except that 0.024% Tween-20 was used for the antibody
incubations instead of 0.16% Triton X-100.
Samples from both labeling protocols were transfered to 100 mM cacodylate buffer and fixed overnight at
room temperature in 100 mM glutaraldehyde, 500 mM
formaldehyde, 3 mM tannic acid, and 100 mM cacodylate
buffer at pH 7.4. Saccular maculae were subsequently
fixed for 1 hr at 4°C in 50 mM OsO4 and 100 mM
cadodylate buffer at pH 7.4. Samples were then dehydrated and stained at room temperature. Dehydration was
accomplished by immersion in progressively increasing
concentrations of ethanol to 70%. At this point, the
samples were stained by incubation in 0.5% uranyl
acetate, 70% ethanol for 60 min. Then dehydration was
completed by immersion in progressively increasing
concentrations of ethanol to 100%. The 100% ethanol
incubation was extended to 60 min and was followed by
two incubations in 100% propylene oxide for 60 min
Fig. 1. Immunoblot analysis of saccular macula protein labeled with
antibody I4 (A,B). Each lane contains SDS-solubilized protein from
0.75 macula separated on a 12% acrylamide SDS-PAGE gel. mT2 (A)
and I4 (B) recognize the 120-kD myosin Ib in saccular-maculae
protein. The molecular weight markers between the two blots show
144, 87, 44.1, 32.7, and 17.7 kD. C–N: Indirect-immunofluorescence
localization of myosin Ib in hair cells. Pairs of light microscopic
images obtained with a Zeiss Axiovert-135, 363 1.4 NA lens are
shown. I4 labeling of hair cells visualized with a rhodamine-TRITC
conjugated secondary is shown in the left panel of each pair. The right
panel contains the corresponding differential interference contrast
(DIC) image. C,D: Apical surface of a saccular macula and were
acquired with a charge-coupled-device camera. E–L: Views of hair
bundles from isolated hair cells. M,N: Whole cell images of an isolated
hair cell. E–N: Acquired using a confocal microscope with an 363, 1.4
numerical aperture (NA) lens. Scale bars 5 5 µm.
each. Saccular maculae were embedded by rotating for 24
hr in 50% propylene oxide, 50% plastic mixture comprising 45.5% Embed-812 resin (Electron Microscopy Sciences), 27.7% dodecenyl succinic anhydride, 25.6%
nadic methyl anhydride, and 1.2% 2,4,6-tri(dimethylaminomethyl)phenol. After incubation in 100% plastic mixture for 24 hr, samples were transfered to fresh plastic
mixture and cured at 50°C for 2 days.
tissue known to contain at least nine other myosin
isoforms [Solc et al., 1994]. Preimmune sera did not bind
any proteins in the saccular macula (data not shown). In
initial immunofluorescence experiments, I4 labeled hair
bundles more strongly and consistently than did mT2.
Therefore, I4 was used exclusively for the rest of the
Myosin Ib’s location within the hair cell was first
investigated with light-microscopy. Observation of the
apical surface of the sensory epithelium using a compound microscope allowed views of the three major actin
networks of the hair cell: the hair bundle, the cuticular
plate, and the actin ring at the adhering belt. Experiments
with isolated sensory epithelia indicated that myosin Ib
immunoreactivity was present in hair bundles and absent
from, or present at a very low level in, cuticular plates
(Fig. 1C–D). Although myosin Ib immunoreactivity was
present in the region of the adhering belt, it was not
enriched there with respect to the surrounding cytoplasm
(Fig. 1C–D).
Confocal images of isolated hair cells afford an
excellent view of hair bundles. Myosin Ib immunoreactivity was present in the stereocilia but not in the kinocilium
of hair bundles (Fig. 1E–L). Within stereocilia, myosin Ib
To determine myosin Ib’s distribution within the
hair bundle, a polyclonal antibody was produced, affinitypurified, and used in immunolocalization studies. Antibodies were raised in rabbit to a peptide corresponding to
amino acids 485–505 of AMIb. The peptide sequence is
quite specific for myosin Ib; the antibody, therefore, is
not expected to recognize other proteins, including other
myosins. The specificity of the polyclonal antibody I4
was assessed by analyzing its reactivity with frog saccularmaculae proteins by immunoblot analysis and comparing
this reactivity with that of the monoclonal antibody mT2
[Wagner et al., 1992]. Both mT2 (Fig. 1A) and I4 (Fig.
1B) recognized only myosin Ib in the saccular macula, a
Immunolocalization of Myosin Ib in Hair Cell’s Hair Bundle
immunoreactivity was concentrated in the top two-thirds
of the bundle. In some cells, myosin Ib immunoreactivity
was concentrated in just the top third of the bundle (Fig.
1E–H). The image plane in Figure 1E–K is close to the
surface of the cell and passes mostly or entirely above the
cuticular plate. The immunoreactivity seen in the vicinity
of the cuticular plate is within the cytoplasm surrounding
the cuticular plate. Full-cell images of isolated hair cells
showed myosin Ib immunoreactivity throughout the
cytoplasm but absent from the nucleus (Fig. 1M–N).
These images confirmed a dearth of myosin Ib immunoreactivity in the cuticular plate and in the region immediately above the stereociliary pivots. Hair cells were not
labeled by an irrelevant primary, preimmune serum, or
secondary antibody alone (data not shown). Immunoreactivity could be eliminated by preincubation of antibody
with an excess of the immunizing peptide (data not
Myosin Ib’s localization within the stereocilia was
investigated at higher resolution by immunoelectron
microscopy. Within stereocilia, myosin Ib immunoreactivity was strictly limited to the edge of the actin core (Fig.
2A–E). Immunoreactivity was scattered along the length
of the stereocilia, with more labeling found in the top
two-thirds than in the bottom third of the bundle. The low
level of labeling seen in the bottom third of stereocilia
was significant as hair cells were not labeled in this or
other regions by secondary antibody alone (Fig. 2F). No
myosin Ib immunoreactivity was seen at the pivots, in the
kinocilium, or in the cuticular plate. This pattern of
labeling is entirely consistent with the results described
above from light microscopy. Very little labeling was
observed at the tips of stereocilia, where the actin
filaments terminate. Some immunoreactivity was seen in
a position where insertional plaques might be located,
about 100–500 nm above the next shorter stereocilium
along the beveled edge (Fig. 2A–C). However, the
clusters of gold seen at these positions were not larger,
nor were they observed more frequently than those seen
elsewhere along the sides of stereocilia. The two fixation
protocols yielded qualitatively equivalent results but a
uniformly lower degree of labeling was found when more
membrane was preserved.
Immunolabeling with the affinity-purified antibody
I4 indicates that myosin Ib is located along the sides of
the stereociliary actin core and at positions that might
correspond to those of the insertional plaque. Myosin Ib
may therefore contribute to the lateral linkages seen
between the actin core and the stereociliary plasma
membrane [Hirokawa and Tilney, 1982]. The function of
Fig. 2. Immunoelectron-microscopic localization of myosin Ib in
stereocilia. Transmission electron micrographs of hair bundles subjected to pre-embedding, indirect immunolabeling of myosin Ib with
the polyclonal serum I4. Images are of sections in the plane of
mechanical sensitivity. The kinocilia are located to the right in the
images shown. The beveled edge of the bundle is in or above the upper
left corner of the panels. Arrowheads indicate the positions of gold
particles. Myosin Ib immunoreactivity is located along the sides of the
actin core of stereocilia (D,E) and in positions that might correspond to
those of the insertional plaque (A–C). No labeling was seen when
primary antibody was omitted (F). The experiments shown in B,C, and
F were fixed and permeabilized by the second procedure, which
resulted in the preservation of more membrane (see Methods). The
overall distribution of the label was similar, using either permeabilization procedure, although the panels presented only show labeling at
positions that might correspond to those of the insertional plaque. Scale
bar 5 200 nm.
these lateral linkages is unclear, although they may
contribute to the mechanical integrity of the stereocilium.
The gating-spring model of mechanoelectrical transduction and adaptation proposes a specific function for an
unconventional myosin located at the insertional plaque
[Howard and Hudspeth, 1987, 1988; Assad and Corey,
1992; Hudspeth and Gillespie, 1994]. Results from
mechanical and electrophysiological studies of transduction and adaptation have been used to formulate a
quantitative model that predicts that the adaptation motor
is formed by an accumulation of myosin molecules at the
insertional plaque; this model predicts that 50 or more
myosin molecules constitute the adaptation motor [Howard
and Hudspeth, 1987, 1988; Assad and Corey, 1992;
Hudspeth and Gillespie, 1994]. In the current study, the
permeabilization conditions required to allow antibody
access to the cytoplasm removed the tip links between
adjacent stereocilia, precluding direct identification of the
insertional plaques. Myosin Ib immunoreactivity was
found at positions that might correspond to those of the
insertional plaque, but there was no compelling increase
in myosin Ib immunoreactivity at these positions when
compared to other points along the sides of the stereocilium.
However, two technical effects related to epitope
masking could potentially reduce the sensitivity of myosin Ib immunolabeling at the position of the insertional
plaque compared to elsewhere. First, the accessibility of
the I4 epitope may depend on the myosin Ib molecule’s
interaction with actin filaments. Structural models of
myosin II interaction with actin suggest that the I4
binding site could be located in close apposition to actin
when myosin is tightly bound to actin [Rayment et al.,
1993a; Schroder et al., 1993]. In the absence of ATP and
in the presence of actin, myosin enters the rigor state
where it is tightly bound to actin. The insertional plaque
may hold the myosin molecules of the adaptation motor
in register with the actin filaments, making these myosin
molecules more likely to bind tightly to actin in our
permeabilized, fixed preparation compared to the myosin
molecules scattered elsewhere along the sides of stereocilia. The sensitivity of myosin immunolabeling at the
insertional plaque could also be reduced as a result of
dense packing in the cluster of myosin Ib molecules,
which is predicted by the adaptation-motor model. If this
prediction is correct, the close packing of myosin Ib
molecules could potentially restrict access to the I4
epitope on the side of the myosin head in some molecules
in the cluster. However, although these epitope masking
effects could reduce the sensitivity of AMIb immunolabeling at the insertional plaque, there is no proof that such
effects are operative in the experiments reported here.
Previous studies with antibodies that bind to other
regions of the myosin Ib molecule gave immunohisto-
chemical results with significant differences compared to
those observed with I4. However, in addition to epitope
masking, antigenic cross-reactivity, and nonspecific reactivity are also potential hazards in immunolocalization
studies, and all these factors can result in different
staining patterns between antibodies. Therefore, it is
essential to compare the staining patterns of a number of
antibodies to the same molecule. The monoclonal antibody mT2 binds amino acids 813–904 within the tail
domain of AMIb [Wagner et al., 1993; Metcalf, 1995].
By immunofluorescence localization, mT2 immunoreactivity was found scattered throughout the bundle as well
as concentrated near the bevel [Gillespie et al., 1993]. In
contrast, I4 immunoreactivity was also scattered within
the bundle but was not concentrated near the bevel. The
position of mT2 immunoreactivity with respect to the
insertional plaque has not been reported at higher resolution.
The affinity-purified polyclonal antibody rafMIb is
directed against a fusion protein containing amino acids
899–1028 of AMIb, which comprises the molecule’s tail
domain between the mT2 binding site and the C-terminus
[Hasson et al., 1997]. The distribution of rafMIb immunoreactivity was investigated using both light and electron
microscopy. Within stereocilia, it was found concentrated
in the distal third, a pattern similar to that observed with
I4. However, in contrast with I4 staining, rafMIb immunoreactivity was seen in two other locations within
stereocilia. First, rafMIb immunoreactivity, but never I4
immunoreactivity, was seen at the pivots. Second, immunofluorescence experiments showed intense rafMIb immunoreactivity at the tips of the stereocilia, and EM
studies indicated that this concentration of immunoreactivity was located at the very top of the stereocilium,
above the ends of the actin filaments. The function of a
population of myosin Ib molecules clustered in this
position is unclear, although it might represent motor
molecules no longer associated with actin or motor
molecules which have re-associated with internal filaments at the very top of the stereocilium after dissociating
from their physiological tracks at the outer edge. If this
population is not associated with actin, conformational
masking of the I4 epitope is unlikely, making it unclear
why I4 would not detect an AMIb population in this
position. On the other hand, partial degradation of the
myosin Ib in this location could account for the differences in the pattern of immunoreactivity if the head is
degraded more quickly than the tail. On the sides of the
stereocilium, some clustering of rafMIb was seen at
positions that might correspond to those of the insertional
plaque, although this staining was not as prominent as
that at the very tips of stereocilia and was not responsible
for the increased immunoreactivity seen at the tips by
light microscopy.
Immunolocalization of Myosin Ib in Hair Cell’s Hair Bundle
An additional difference between the staining pattern of the two antibodies is observed in the cell body.
The immunoreactivity of both I4 and rafMIb immunoreactivity was seen throughout the cytoplasm exclusive of
the nucleus and the cuticular plate. However, rafMIb
immunoreactivity was concentrated between the cuticular
plate and the actin ring of the adhering belt, and electron
microscopic investigation indicated this immunoreactivity was clustered on vesicles. Although I4 immunoreactivity was also seen on vesicles in this region, there was no
net increase in I4 immunoreactivity between the cuticular
plate and the adhering belt when compared to the rest of
the cytoplasm.
Although significant questions remain as to the
exact pattern and density of the subcellular distribution of
myosin Ib, the results presented here and in the other
works described above are consistent with a myosin
Ib-based adaptation motor. Further investigation of myosin Ib’s role in adaptation would be aided by an inhibitory
antibody. I4 binding to myosin Ib might be expected to
inhibit myosin function. If this is the case, I4 could be a
useful reagent for functional inhibition studies of myosin
Ib both in vitro and in vivo.
I thank Dr. A.J. Hudspeth for support and encouragement throughout this project. Thanks to Dr. E.A. Lumpkin for help in acquiring confocal images and to Drs. S.
Burlacu, A.J. Hudspeth, J.F. Hunt, H. Krämer, and E.A.
Lumpkin for critically reading the manuscript. This work
was supported by grant DC00241 from the National
Institutes of Health (to A.J.H.).
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