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The Journal of Neuroscience, June 15, 1998, 18(12):4603-4615
Myosin I
Is Located at Tip Link Anchors in Vestibular Hair
Bundles
Peter S.
Steyger1,
Peter G.
Gillespie2, and
Richard A.
Baird1
1 R. S. Dow Neurological Sciences Institute,
Legacy Good Samaritan Hospital, Portland, Oregon 97209, and
2 Department of Physiology, Johns Hopkins University,
Baltimore, Maryland 21205
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ABSTRACT |
Recent studies have suggested that myosin I
mediates the
adaptation of mechanoelectrical transduction in vestibular hair cells.
An important prediction of this hypothesis is that myosin I should
be found in the side insertional plaque, an osmiophilic hair bundle
structure that anchors tip links and is thought to house the adaptation
motor. To determine whether myosin I was situated properly to
perform adaptation, we used immunofluorescence and immunoelectron
microscopy with the monoclonal antibody mT2 to examine the distribution
of myosin I in hair bundles of the bullfrog utricle. Although
utricular hair cells differ in their rates and extent of adaptation
[Baird RA (1994) Comparative transduction mechanisms of hair cells in
the bullfrog utriculus. II. Sensitivity and response dynamics to hair
bundle displacement. J Neurophysiol 71:685-705.], myosin I
was present in all hair bundles, regardless of adaptation kinetics.
Confirming that, nevertheless, it was positioned properly to mediate
adaptation, myosin I was found at significantly higher levels in the
side insertional plaque. Myosin I was also present at elevated
levels at the second tip link anchor of a hair bundle, the tip
insertional plaque, found at the tip of a stereocilium. These data
support myosin I as the adaptation motor and are consistent with the
suggestion that the motor serves to restore tension applied to
transduction channels to an optimal level, albeit with different
kinetics in different cell types.
Key words:
vestibular; hair cell; myosin; adaptation; mechanosensory
transduction; stereocilia
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INTRODUCTION |
Hair cells of the bullfrog's
vestibular otolith organs respond to gravity, vibration, and linear
acceleration. Saccular hair cells detect with particular sensitivity
substrate-borne vibration (Lewis et al., 1982
); to ensure optimal
sensitivity to their favored stimulus, most cells adapt rapidly to
static stimuli (Eatock et al., 1987). By contrast, the utricle responds
to both static gravitational and dynamic linear acceleration;
accordingly, this organ has several hair cell types, each with a
characteristic rate and extent of adaptation (Lewis and Li, 1975;
Baird, 1994a,b).
An elastic gating spring controls mechanically sensitive transduction
channels of hair cells (Corey and Hudspeth, 1983). The best candidate
for this spring is the tip link, an extracellular filament that
connects adjacent stereocilia along the axis of sensitivity of a hair
cell (Pickles et al., 1984; Furness and Hackney, 1985; Assad et al.,
1991). A tip link terminates in osmiophilic structures at the
kinociliary side of a stereociliary tip and along the side of the
adjacent stereocilium (Furness and Hackney, 1985). The structure at the
upper end of the tip link, the side insertional plaque, probably houses
the adaptation motor, a force-producing mechanism hypothesized to
encompass a cluster of myosin molecules. In this view, myosin molecules
respond to changes in gating spring tension and intracellular
Ca2+ by moving the insertional plaque along the
stereociliary cytoskeleton, restoring tension to near the original
level (Howard and Hudspeth, 1987a,b; Assad and Corey, 1992; Jaramillo
and Hudspeth, 1993; Hudspeth and Gillespie, 1994; Gillespie and Corey,
1997).
Although it may operate on different time scales in different hair cell
types, adaptation optimizes the tension applied to transduction
channels (Howard et al., 1988). For maximal sensitivity to
low-frequency stimuli, transduction channels should be poised at the
point of maximum slope on the tension-open probability curve, a two-
or three-state Boltzmann relation (Corey and Hudspeth, 1983; Hudspeth,
1992). Alternatively, cells responding to high-frequency stimuli should
circumvent low-pass membrane filtering by positioning channels where
the slope of the curve changes most rapidly (Hudspeth, 1983). Because
several piconewtons must be applied through a gating spring before the
open probability of a transduction channel reaches either of these
positions (Hudspeth, 1992), a mechanism that applies force at rest to
gating springs must be a universal feature of sensitive hair cells.
Although the adaptation motor applies resting tension and effects
adaptation to sustained stimuli, other mechanisms of adaptation also
may function simultaneously (Ricci and Fettiplace, 1997).
Thirteen myosin transcripts are expressed in the bullfrog saccule (Solc
et al., 1994). Although myosin I, VI, and VIIa reside in hair
bundles (Gillespie et al., 1993; Hasson et al., 1997), only myosin I
is situated appropriately to mediate adaptation. To test more
definitively whether myosin I could be the adaptation motor, we
sought to determine whether it was located within or near the side
insertional plaque. Because the adaptation motor should be a universal
feature in sensitive hair cells, an adaptation motor myosin should be
found in all hair cells, regardless of adaptation kinetics. Hair cells
of widely ranging adaptation kinetics contained myosin I, and in all
cell types this isozyme was associated with tip link anchors, the
expected position of the adaptation motor.
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MATERIALS AND METHODS |
Materials. Nonimmune antisera, proteolytic enzymes,
and reagents used in fixatives and immunochemical buffers were obtained from Sigma (St. Louis, MO), unless otherwise stated.
Rhodamine-conjugated phalloidin was purchased from Sigma or Molecular
Probes (Eugene, OR). Biotinylated horse anti-mouse secondary antibodies
and rhodamine-conjugated streptavidin were obtained, respectively, from
Vector Laboratories (Burlingame, CA) and Amersham (Arlington Heights,
IL). Donkey 
-globulin was purchased from Jackson ImmunoResearch
(West Grove, PA), Tween 20 from Bio-Rad (Hercules, CA), Immobilon P
from Millipore (Bedford, MA), and
sulfosuccinimidyl-3-(4-hydroxyphenyl)-propionate (sulfo-SHPP) from
Pierce (Rockford, IL). We obtained alkaline phosphatase-coupled goat
anti-mouse secondary antibodies from Southern Biotechnology
(Birmingham, AL) and
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl
phosphate (CSPD) from Tropix (Bedford, MA). Cell-Tak was purchased from
Collaborative Biomedical Products, Becton Dickinson (Bedford, MA).
Formaldehyde, glycol methacrylate (JB4), Fluoromount, and Aquamount
were obtained from Polysciences (Warrington, PA). Electron microscopy
supplies, including gold-conjugated antibodies and glutaraldehyde,
osmium tetroxide, propylene oxide, Eponate 12, and uranyl acetate, were
purchased from Ted Pella (Redding, CA).
Dissection of the utricular macula. Healthy adult bullfrogs
(Rana catesbeiana) were anesthetized with 0.2%
3-aminobenzoic acid ethyl ester (MS-222), immersed in crushed ice, and
decapitated. The lower jaw was removed, and the utricular maculae were
dissected from the membranous labyrinth in chilled, oxygenated
HEPES-buffered saline (HBS) containing (in mM): 110 Na+, 2 K+, 4 Ca2+, 120 Cl
, 3 D-glucose, and 5 HEPES, pH 7.25. Utricular maculae were
immersed for 30-45 min in oxygenated HBS containing 50 µg/ml
subtilisin Carlsberg (Sigma protease type XXIV) to loosen the otolith
membrane; then the digested remains of the otolith membrane were
removed with gentle mechanical agitation.
Actin and myosin I labeling. To visualize filamentous
actin, we fixed utricular maculae for 2 hr with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.25, rinsed them in phosphate
buffer, and incubated them for 30 min with 0.5% Triton X-100 and 5 µM rhodamine-conjugated phalloidin in PBS (0.09% NaCl in
0.02 M phosphate buffer, pH 7.8). Phalloidin-labeled
maculae were mounted in Fluoromount in depression slides and were
observed with fluorescence microscopy.
The visualization of myosin I required an abbreviated fixation
protocol to preserve myosin I antigenicity. In these experiments the
utricular maculae were fixed for 15 min with 3% formaldehyde in 0.1 M phosphate buffer, rinsed in PBS, and permeabilized for 10 min in 0.5% Triton X-100 in PBS. Nonspecific binding sites were
blocked by immersion of the tissue for 30 min in 5% normal horse serum
and 1% bovine serum albumin in PBS (PBS + 1% BSA). Utricular maculae
then were incubated for 1 hr in blocking solution containing 10 µg/ml
mT2 (also known as M2; Wagner et al., 1992), a mouse monoclonal
antibody raised against bovine adrenal myosin I, and were rinsed in
PBS + 1% BSA. For fluorescence microscopy, bound primary antibodies
were detected by indirect immunolabeling. Utricular maculae were
incubated for 30 min with biotinylated horse anti-mouse antibodies
(1:100 in PBS + 1% BSA), were washed several times with PBS + 1% BSA,
and were incubated with streptavidin conjugated with Texas Red (1:100
in PBS + 1% BSA). In some experiments the utricular maculae were
embedded in glycol methacrylate (JB4), serially sectioned at 7 µm;
individual cross sections were observed with fluorescence
microscopy.
For immunocytochemical controls, primary antibody was replaced with (1)
nonimmune mouse serum or (2) primary antibody preadsorbed for 30 min
with a synthetic peptide containing the epitope for mT2, located within
the tail region of frog myosin I (PVVKYDRKGYKPRRRQLLLT; Metcalf,
1996).
Fluorescence microscopy. Whole-mount maculae and macular
cross sections were examined through rhodamine or Texas Red filter sets
(Chroma Technology, Brattleboro, VT), using 40× (numerical aperture,
0.9) or 63× (numerical aperture 1.25) oil-immersion Zeiss Plan Apo
objectives in a Zeiss Standard 16 microscope (Oberkochen, Germany).
Low-power photomicrographs of phalloidin-labeled maculae or myosin I
immunoreactivity were taken with T-Max film (Kodak, Rochester, NY)
exposed at 400 or 1600 ASA. For images at higher magnifications, we
obtained video images of labeled material via a cooled CCD video camera
(model 72, Dage-MTI, Michigan City, IN). Gain and black level controls
were set identically for both control and experimental samples.
Individual video images were integrated for 50-150 msec to increase
sensitivity and averaged 64-128 times with an image acquisition board
(Imagraph, Precision Monoscan, Natick, MA) to reduce noise. Resultant
video images were corrected with a defocused background image and were
adjusted for optimal brightness and contrast.
Hair bundle isolation and immunoblotting. Utricular hair
bundle isolation used methods similar to those developed for the frog
saccule (Gillespie and Hudspeth, 1991). After dissection and adhesion
to a Cell-Tak-coated isolation chamber, utricles were incubated for 15 min with 50 µg/ml subtilisin Carlsberg to assist in otolith membrane
removal. To improve recovery of small bundles, we subsequently
incubated utricles for 20 min with 5 mM sulfo-SHPP, using
the conditions described in Gillespie and Hudspeth (1991) for labeling
with N-hydroxysuccinimide derivatives. If the sulfo-SHPP
step was eliminated, bundle recovery was restricted mainly to the large
striolar bundles ("striola-rich bundles"). Utricles then were
immersed in 3% molten methoxylated agarose (Sigma type VIIA) at 34°C
for 3 min and subsequently were cooled at 4°C for 10-15 min. After
the agarose formed a firm gel, the maculae were removed mechanically,
leaving the hair bundles embedded in agarose. Recoveries of isolated
hair bundles were estimated visually (Gillespie and Hudspeth,
1991).
For immunofluorescence the isolated hair bundles, embedded in agarose,
were fixed for 15 min with 1% formaldehyde in 0.1 M phosphate buffer, permeabilized for 15 min with 0.5% Triton-X 100, and
blocked for 30 min with 4 µg/ml hemoglobin, 250 µg/ml donkey
-globulin, and 0.1% Tween 20 in PBS. Hair bundles were incubated
overnight with 20 µg/ml primary antibodies in blocking solution. To
demonstrate specificity, we included a 50-100 µM concentration of the mT2 epitope peptide in the control samples. After
being washed with 0.1% Tween 20 in PBS, the hair bundles were
incubated for 1 hr with 10 µg/ml Cy5-conjugated donkey anti-mouse and
200 nM rhodamine-phalloidin, rinsed in PBS, and mounted
with Aquamount. Then the hair bundles were observed with a Zeiss LSM 410 confocal microscope, using 543 nm and 633 nm HeNe lasers and Zeiss
C-Apo 40× (numerical aperture, 1.2) water-immersion or Zeiss Plan Apo
100× (numerical aperture, 1.4) oil-immersion objectives.
SDS-PAGE and immunoblotting. Dissected agarose blocks
containing hair bundles were heated with SDS-PAGE sample buffer, and samples were separated on 10% acrylamide, low cross-linker SDS gels
(Yamoah and Gillespie, 1996; Hasson et al., 1997). To allow for
estimation of recovery, we ran on the same gels samples of recombinant
frog myosin I, expressed in insect cells, using a baculovirus
expression vector (S. Jean and P. Gillespie, unpublished data).
Proteins were transferred to Immobilon P in a solution containing 5%
methanol and 10 mM 3-(cyclohexylamino)-1-propane sulfonic
acid (CAPS), pH 11 (Hasson et al., 1997). Hemoglobin was included in
samples and in the gel-incubation solution to facilitate the transfer
of small amounts of bundle protein (Gillespie and Gillespie, 1997).
Immunoblots were blocked for 2 hr with 5% Amersham Liquid Block in PBS
and then were incubated for an additional 2 hr with 10 µg/ml mT2 in
the blocking solution. In early experiments we used biotinylated goat
anti-mouse secondary antibodies and streptavidin-alkaline phosphatase,
both diluted 1:1000, to detect bound antibody. To avoid the detection
of endogenously biotinylated macular proteins, we used alkaline
phosphatase-coupled goat anti-mouse secondary antibodies, diluted
1:1000, in later experiments. Chemiluminescence development used CSPD
and methods described previously (Gillespie and Hudspeth, 1991). Myosin
I was quantified by scanning autoradiograms and comparing the
optical density of an unknown with that of purified myosin I
standards.
Transmission electron microscopy. Utricular maculae, fixed
with formaldehyde as described above, were permeabilized for 10 min in
0.1% Triton X-100 in PBS, blocked for 30 min with 5% normal horse
serum in PBS + 1% BSA, and were incubated for 1 hr with the primary
antibody at 10 µg/ml. After being washed, tissue-bound antibodies
were localized by incubating maculae for an additional hour with PBS + 1% BSA containing secondary antibodies conjugated to 3-5 nm gold
particles. Maculae were washed and then post-fixed for 1 hr with 2.5%
glutaraldehyde in PBS and for 1 hr with 1% osmium tetroxide in PBS.
Maculae were dehydrated through an ascending ethanol series and were
passaged through 1:1 ethanol/propylene oxide, 100% propylene oxide,
and 1:1 Eponate 12 resin/polypropylene oxide. After maculae were
infiltrated with three changes of Eponate 12 resin, resin blocks were
polymerized at 60°C, trimmed, and sectioned with an ultramicrotome
(MT2B, DuPont Sorvall, Wilmington, DE). Silver-gold serial sections
(60-70 nm) were collected on colloidin-coated nickel slot grids,
counterstained with uranyl acetate, and examined in a Zeiss 10C/A
transmission electron microscope at accelerating voltages of 60 or 80 kV.
Control organs were treated during the primary antibody incubation step
with blocking solution containing (1) no primary antibody, (2)
nonimmune mouse serum, or (3) mT2 preadsorbed with the mT2-epitope peptide. For preadsorption, 500 µM peptide in PBS was
mixed for 30 min with 100 µg/ml mT2; then this solution was diluted
1:10 before use.
Morphometric analyses. Photographic negatives of individual
stereocilia containing both tip and side insertional plaques were enlarged to a total magnification of 100,000×. The tip and side plaques were defined, respectively, as the electron-dense regions located at the stereociliary tip and adjacent to a distinct notch in
the nonkinociliary side of the stereociliary membrane. Although these
structures anchor tip links, because of the harsh conditions required
for preembedding immunoelectron microscopy, tip links were observed
only rarely. Quantitative analysis of gold particle labeling was
performed only on negatives of stereocilia possessing both tip and side
plaques. After identifying on a section a stereocilium that contained
tip and side plaques, we ensured that this stereocilium was not
analyzed on adjacent sections also. Using a 10× loupe eyepiece, we
then verified the shape and diameter of presumptive gold particles on
photographic prints and, using transparent overlays, placed a dot over
each identified gold particle. Clusters of gold particles were included
in our analysis only when individual particles within a cluster could
be discerned and counted. For publication, photographic negatives were
scanned digitally, and image brightness and contrast were adjusted to
optimize the visualization of gold particles and stereociliary
structures.
The distribution of gold particles was measured after each stereocilium
was divided with spatial bins. To create these bins, we bisected the
stereocilium along its longitudinal midline into kinociliary and
nonkinociliary halves. Starting at the stereociliary tip, pairs of bins
in spatial register, extending 200 nm along the length of a
stereocilium, were placed adjacent to each other across the midline.
Gold particles therefore were counted within bins that each encompassed
one-half of a stereocilium horizontally and 200 nm longitudinally.
Our initial analysis suggested that gold particles were located
preferentially in bins containing the tip or side insertional plaques.
To test this hypothesis further, we counted the number of gold
particles located within a series of 75 nm square bins, centered on the
side plaque and continuing apically and basally on the nonkinociliary
side of the stereociliary shaft. A control analysis was performed on
the kinociliary side of the stereociliary shaft, using bins in spatial
register with those on the nonkinociliary side. Paired bins extended to
a point ~200 nm below the tip plaque. In addition, to compare the
amount of gold immunoreactivity immediately surrounding the tip and
side plaques, we counted the number of gold particles located within a
100 nm diameter arc centered on these structures. We then determined
the average gold particle density by dividing the number of gold
particles by the area of each arc contained within the
stereocilium.
Statistical procedures. Unless otherwise stated, statistical
comparisons of morphometric data were based on a one-way ANOVA. Where
appropriate, post hoc pairwise multiple comparisons were performed with the Tukey multiple comparison test, adjusting, when
necessary, for unequal group sizes.
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RESULTS |
Phalloidin labeling of utricular hair bundles
We examined the distribution of myosin I in hair bundles of the
bullfrog's utricle. To visualize utricular hair bundles, we labeled
bullfrog utricles with rhodamine-phalloidin. At low magnification,
phalloidin labeling clearly revealed the shape of the sensory
epithelium and the presence of the striola, a characteristic curved
band of larger hair bundles, near the abneural edge of the macula (Fig.
1A). At higher
magnifications (Fig. 1B) we could visualize the shape
and size of individual hair bundles and could observe circumferential
actin belts, which circumscribe hair cells at the level of the adherens
junctions. As previously described (Baird, 1994a,b), cells within the
striola generally had larger bundles and were packed less densely than
hair cells in the surrounding medial and lateral extrastriolar regions.
Within the striola, hair cells abruptly reversed their bundle
polarization about a line located near the lateral edge of the striolar
region (dashed line, Fig. 1B). Hair cells
in the inner striolar rows immediately adjacent to this line were
smaller and less densely packed than those in the adjacent outer
striolar rows. This pattern of hair bundles coincides with the known
distribution of hair cells with differing adaptation kinetics: Type C
and Type F hair cells in the outer striolar rows adapt rapidly, Type C
and Type E hair cells in the inner striolar rows adapt more slowly, and
Type B hair cells in the medial and lateral extrastriolar regions
display little or no adaptation over a time scale of several seconds
(Baird, 1992, 1994b).
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Figure 1.
Isolation and protein analysis of utricular hair
bundles. A, Rhodamine-phalloidin labeling of hair
bundles in the whole-mount utricle. B,
Rhodamine-phalloidin labeling, at higher magnification, of the striola
and surrounding medial and lateral extrastriolar regions. The
dotted line marks the reversal of hair bundle
polarization, dividing the striola into the medial and the lateral
zones. MES, Medial extrastriola; MS,
medial striola; LS, lateral striola; LES,
lateral extrastriola; O, outer striolar rows;
I, inner striolar rows. C,
Rhodamine-phalloidin labeling of hair bundles isolated with
sulfo-SHPP. D, Protein immunoblot labeled with mT2, a
monoclonal antibody against myosin I. mT2 labeled bands at the
expected molecular mass of myosin I. Utricular
bundles, Hair bundles from ~14 utricular equivalents;
Utricular macula, total protein from one utricular
macula; Saccular macula, total protein from one saccular
macula; rMI, 50 pg of recombinant myosin.
E, Rhodamine-phalloidin labeling of hair bundles
isolated without sulfo-SHPP treatment. Note the relative paucity of
extrastriolar hair bundles. F, Protein immunoblot
labeled with mT2 of bundles from striola-rich (without sulfo-SHPP) and
whole utricle (with sulfo-SHPP) preparations. Striola-rich
bundles, Hair bundles extracted without sulfo-SHPP treatment
from ~15 utricular equivalents; Utricular bundles,
hair bundles extracted with sulfo-SHPP treatment from ~7 utricular
equivalents. Scale bars: 150 µm in A,
C, E; 20 µm in B.
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Hair bundle isolation and myosin I immunoblotting
To identify and localize myosin I within utricular hair
bundles, we used the monoclonal antibody mT2 (also known as M2; Wagner et al., 1992). The specificity of mT2 for myosin I has been
confirmed by epitope-mapping studies, which localized high-affinity
antibody binding to a 12-amino-acid region with no more than four
identical amino acids in any other known myosin I (Metcalf, 1996). In
some experiments described below, we used a synthetic peptide
containing this region to demonstrate the specificity of mT2
labeling.
To isolate utricular hair bundles and confirm that these bundles
contain myosin I, we adapted the "twist-off" isolation used to
purify hair bundles from the bullfrog's saccule (Gillespie and
Hudspeth, 1991). The undulating shape of the utricle and the large
number of small hair bundles introduced challenges that had not been
faced with saccular hair bundle isolation. Because utricles did not
adhere well to the Cell-Tak-coated experimental chamber, we used an
agarose that remained molten at 34°C for an extended period of time;
this extra time allowed us to reposition utricles that had detached
from the bottom of the chamber during agarose irrigation. In addition,
to isolate the small extrastriolar hair bundles that constituted the
majority of utricular bundles, we found it necessary to modify surface
amino groups with the water-soluble N-hydroxysuccinimide
sulfo-SHPP. We also took advantage of the poor recovery of
extrastriolar hair bundles when we were not using sulfo-SHPP to isolate
hair bundles preferentially from the striolar region. Although the
mechanism of action of sulfo-SHPP modification is unknown, this reagent
also enhances the recovery of saccular hair bundles (P. Gillespie,
unpublished data). When sulfo-SHPP was used, recoveries of utricular
hair bundles ranged from 50 to 100%, comparable to recoveries of
saccular hair bundles.
Phalloidin labeling of isolated utricular hair bundles in agarose with
(Fig. 1C) or without (Fig. 1E) sulfo-SHPP
treatment revealed that the distinctive distribution patterns of
striolar and extrastriolar hair bundles were retained after isolation
and closely corresponded to those seen in intact maculae (Fig.
1A). In particular, the striolar and extrastriolar
regions were identified easily, and the smaller, more dispersed hair
bundles of the inner striola rows were distinguished readily from the
larger hair bundles of the outer striolar rows (Fig.
1B,C).
Using mT2, we found that purified utricular hair bundles contained
myosin I in substantial amounts (Fig. 1D). Hair
bundle myosin I comigrated with myosin I from whole saccular
macula and from whole utricular macula at ~105 kDa, the migration
position for myosin I in low cross-linker polyacrylamide gels (Fig.
1D). Other bands, seen in addition to the myosin I
monomer, were observed only sporadically. Utricular hair bundle myosin
I migrated slightly faster than recombinant frog myosin I, which
was ~3 kDa larger because of its N-terminal fusion partner. By
quantitative immunoblotting, we estimated that the hair bundles of one
utricle contained 22 ± 10 pg of myosin I. Although this value
is considerably larger than that for saccular hair bundles (3 pg;
Gillespie et al., 1993), conversion by the relative number of bundles
per organ (Baird and Schuff, 1994) and the average number of
stereocilia per bundle (Baird, 1994a) indicates that the amount of
myosin I per stereocilium is only approximately fourfold greater in
the utricle. There was approximately fourfold more myosin I per
utricular equivalent of bundles in the entire utricle preparation than
in the striola-rich preparation (Fig. 1F) (data not
shown). These results demonstrate that mT2 detects myosin I in
utricular hair bundles.
Confirming that myosin molecules were present in utricular hair
bundles, photoaffinity labeling of utricular bundle proteins with
[
-32P]UTP and vanadate (Gillespie et al., 1993)
identified proteins of 105 and 230 kDa, probably myosin I and myosin
VIIa (Hasson et al., 1997) (data not shown).
Fluorescence microscopy
When we used mT2 to label intact utricular maculae, we found that
immunofluorescence corresponding to myosin I was present in
utricular hair bundles throughout the striola (Fig.
2B) and the surrounding
medial (Fig. 2A) and lateral (Fig. 2C)
extrastriolar regions. Myosin I was also present in supporting cell
apices (data not shown), as previously reported for the saccule
(Gillespie et al., 1993). Close examination of whole-mount utricles and
resin-embedded cross sections (Fig. 2D) indicated
that striolar and extrastriolar hair bundles were labeled in their
entirety. Hair bundles in the inner striolar rows, primarily belonging
to cells with slow adaptation kinetics (Baird, 1992, 1994b), were
labeled less intensely than bundles of nonadapting Type B hair cells in
extrastriolar regions (Fig. 2A,C,D) and bundles of
rapidly adapting hair cells in outer striolar rows (Fig.
2B,D). In control experiments in which primary antibodies were omitted (data not shown), replaced with nonimmune mouse
serum (Fig. 2E), or preadsorbed with mT2-epitope
peptide (Fig. 2G), no immunoreactivity was visible in
utricular hair bundles (compare Fig. 2, E and F;
G and H). Under each of these control conditions, only weak, nonspecific reactivity was visible within the
macula, usually within blood vessels.
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Figure 2.
Immunofluorescence localization of myosin I in
utricular hair cells. A-C, mT2 labeling in whole-mount
utricles. A, Medial extrastriolar region
(MES). B, Striola. Arrows
mark the borders between the inner striolar (IS) rows
and the medial (left) and lateral (right)
outer striolar (OS) rows. Note that hair bundles in the
inner striolar rows are labeled less intensely than those in the
extrastriolar regions and outer striolar rows. C,
Lateral extrastriolar region (LES). D,
mT2 labeling in a cross section of the utricular macula. Large
arrows mark the medial (left) and lateral
(right) edges of the striola region; small
arrows mark the borders between the inner striolar rows and the
medial (left) and lateral (right) outer
striolar rows. Note that immunoreactivity occurs throughout
extrastriolar and outer striolar hair bundles and that inner striolar
hair bundles (asterisks) are immunolabeled less
intensely than hair bundles in the extrastriolar and outer striolar
rows. E-H, Control experiments. Fluorescence
(E, G) and differential interference
contrast (DIC; F, H) images of
medial extrastriolar hair bundles were obtained after mT2 was replaced
with nonimmune mouse serum (E, F)
or preadsorbed with a synthetic peptide derived from the tail region of
bullfrog myosin I (G, H). Note
that fluorescence was abolished under both control conditions. Scale
bars: 50 µm in A-D; 25 µm in
E-H.
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We also examined the distribution of myosin I in hair bundles
isolated from the utricular macula. When agarose-embedded utricular hair bundles were double-labeled for filamentous actin (Fig.
3A) and myosin I (Fig.
3B), generally similar labeling patterns were observed with
the two reagents. One exception, however, was that the inner striolar
bundles of slowly adapting hair cells contained less myosin I than
bundles in the outer striolar rows, derived primarily from rapidly
adapting hair cells (Fig. 3B). Z-series projections taken at different focal planes indicated that myosin I
immunoreactivity was present throughout the length of all hair bundles.
Confirming that the antibody recognized myosin I, the synthetic mT2
peptide reduced nearly completely the mT2 labeling (Fig.
3C,D). When higher magnification pseudo-color images of rhodamine-phalloidin (red) and mT2 (green) labeling were combined digitally, hair bundles with both filamentous actin and myosin I
labeling appeared yellow-brown (Fig. 3E). Hair bundles in
the inner striola, with strong filamentous actin labeling and little myosin I immunoreactivity, appeared red. Modest amounts of Cy5 fluorescence not associated with hair bundles were also present; this
signal (green in Fig. 3E) corresponded to
mT2 binding to the agarose gel or structures trapped within it.
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Figure 3.
Immunofluorescence localization of myosin I in
isolated utricular hair bundles. A-D, Isolated
agarose-embedded utricular hair bundles were double-labeled with
rhodamine-phalloidin (A, C) and mT2
(B, D). Hair bundles in all regions
contain filamentous actin; hair bundles in the inner striolar contain
less myosin I than do bundles in the outer striolar rows and
surrounding extrastriolar regions (B). When mT2
was preadsorbed with a synthetic peptide derived from the tail region
of bullfrog myosin I, negligible immunoreactivity was seen in
utricular hair bundles. D, E,
Superimposed high power pseudo-color images of hair bundles labeled
with rhodamine-phalloidin (red) and mT2
(green). Hair bundles with both filamentous actin
and myosin I labeling appear yellow-brown; hair
bundles in the inner striolar rows with strong actin labeling and weak
myosin I labeling appear red; antibody reactivity
unassociated with hair bundles appears green. Scale
bars: 50 µm in A-D; 10 µm in
E.
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Transmission electron microscopy
To confirm the localization of myosin I obtained from
immunofluorescence experiments and to refine its localization by using the higher resolution afforded by immunoelectron microscopy, we used a
preembedding procedure, using secondary antibodies conjugated with 3-5
nm gold particles. Control experiments, including omission of the
primary antibody, substitution of the primary antibody with nonimmune
mouse serum, or preadsorption of the primary antibody with synthetic
myosin peptides, showed that nonspecific immunoreactivity was
negligible (Fig. 4).
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Figure 4.
Immunoelectron microscopy controls. Few gold
particles were observed when mT2 was preadsorbed with a synthetic
peptide derived from the tail region of bullfrog myosin I (A,
B) or replaced with nonimmune serum (C) or blocking
serum (D). Scale bars: 100 nm in A-D.
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mT2 gave a robust labeling of utricular cells, particularly hair cells.
Gold particles corresponding to myosin I were observed in the
microvilli of hair cells and supporting cells (data not shown) and in
the cell apices and stereocilia of extrastriolar (arrowheads, Fig. 5) and
striolar (arrowheads, Fig. 6)
hair cells. In stereocilia, gold particles were located preferentially
close to the cell membrane, throughout the length of each stereocilium (Figs. 5B-D, 6B,C). Gold particles were
observed at the ankle region at the stereociliary base, particularly at
the junction where the stereociliary membrane meets with the apical
membrane of the hair cell (arrowheads, Fig. 5B).
Gold particles also were found along the stereociliary rootlet within
the cuticular plate (arrow, Fig. 5B).
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Figure 5.
Immunoelectron microscopic localization of myosin
I in extrastriolar hair cells. Shown are the locations of side
insertional plaques (A, C,
D, arrows) and gold immunoreactivity
(B-D) in medial extrastriolar Type B hair
bundles. Note the presence of gold particles in the stereociliary
rootlet (B, small arrow), ankle region
(B, arrowheads), side plaques
(C, D, arrowheads), and
stereociliary tips (C, D,
arrowheads). In D, there are 40 counted
gold particles, but only three are indicated at the tips
of the arrowheads. As an example, the
arrowhead on the top right of the panel
is pointing to a single gold particle; in addition, there are two
clearly identifiable particles immediately below this one. To the
right of those two particles is a cluster that would not
be included in the particle count because of the difficulty in
discerning the number of particles present. Images were obtained at
accelerating voltages of 60 kV (A-C) or 80 kV
(D). Scale bars: 500 nm in A; 200 nm in B; 100 nm in C,
D.
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Figure 6.
Immunoelectron microscopic localization of myosin
I in striolar hair cells. Shown are the locations of side
insertional plaques (large arrows, A-C)
and gold immunoreactivity (B, C) in
stereocilia from an outer striolar Type C hair bundle. Note the
presence of gold particles at the tip and side plaques
(arrowheads, B, C). All
images were obtained at an accelerating voltage of 60 kV. Scale bars:
500 nm in A; 100 nm in B,
C.
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If myosin I is the adaptation motor of the hair cell, then this
myosin should be associated with the osmiophilic structure at the
upper end of the tip link, the side insertional plaque, which is
thought to house the motor molecules. We observed gold particles in and
around the side plaques in both extrastriolar (arrowheads,
Fig. 5C,D) and striolar (arrowheads, Fig.
6B,C) hair cells. In addition, gold particles
frequently were found on the second osmiophilic structure associated
with tip links, the tip insertional plaque. This structure is located
on the kinociliary side of each stereociliary tip, at the base of a tip
link; like the side plaque, the tip plaque may contain structures that
are important for transduction and adaptation. Gold particles were observed more frequently around the interior edges of the plaques rather than within them and also frequently were observed immediately above and below the side plaques.
The small gold particles used in this study were difficult to see on
negatives of stereociliary sections without further magnification. When
higher accelerating voltages were used (80 kV), gold particles were
easier to distinguish, but the opacity of the tip and side plaques
decreased (Fig. 5D). Larger gold particle sizes did not work
with our protocol; this protocol nonetheless was preferred because of
the greater ultrastructural preservation as compared with that obtained
with protocols that used greater concentrations of or longer exposure
to detergent.
Morphometric analysis
To confirm that myosin I was located in the side and tip
insertional plaques at levels significantly higher than adjacent areas,
we sectioned stereocilia parallel to the plane of symmetry of the
bundle and counted gold particles along the stereociliary shaft. We
only examined sections that contained both a side and a tip plaque.
Within the 64 individual stereocilia from 19 hair bundles that met this
criterion, we counted the number of gold particles in rectangular bins
paired across the midline of each stereocilium (Fig.
7A). In our initial analysis
we used pairs of bins that began at stereociliary tips and continued in
200 nm increments basally toward the stereociliary rootlet. Each pair of bins included one from the kinociliary side of a stereocilium and
one from the nonkinociliary side.
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Figure 7.
Morphometric analysis of myosin I localization,
using coarse spatial bins. A, Schematic diagram of
rectangular sampling grid with typical location of tip and side
insertional plaques. B, Percentage of stereocilia with
tip plaques located in the indicated bins. The tip plaque was always
located in the apical-most kinociliary bin. C, Mean
number and SD of gold particles found in each 200 nm kinociliary bin.
D, Percentage of stereocilia with side plaques located
in the indicated bins. The location of the side plaque on the
nonkinociliary side was variable. E, Mean number and SD
of gold particles found in each 200 nm nonkinociliary bin.
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The mean number of gold particles per bin was greatest near the
stereociliary tip and decreased significantly in more basal locations
(Fig. 7C,E). This trend was most apparent on the kinociliary side of stereocilia, the side at which the tip plaque was always found
(Fig. 7B).
On the nonkinociliary side of the stereocilium, the mean number of gold
particles was greatest in the second bin from the tip and decreased for
successively basal bins (Fig. 7E). The less striking
concentration of gold particles in any particular nonkinociliary bin
appeared to be attributable to the variable location of the side plaque
relative to the tips of the stereocilia. The side plaque was located in
the second bin from the tip in only 50% of stereocilia; the remaining
side plaques were located mainly in more basal locations (Fig.
7D). This was particularly true for inner striolar hair
bundles, in which >75% of stereocilia had side plaques located 400 nm
or more from the stereociliary tip.
For each stereocilium we compared the number of gold particles in bins
containing tip plaques, bins containing side plaques, and bins without
a plaque (Table 1). The overall gold
immunoreactivity in Type F hair bundles was significantly greater than
in other hair bundle types (p < 0.001). In
addition, the number of gold particles in extrastriolar and outer
striolar hair bundles was significantly greater than in inner striolar
hair bundles (p < 0.05), confirming our
immunofluorescence observations.
The number of gold particles located in bins containing either a
tip or a side plaque, regardless of hair bundle location or morphology,
was significantly higher than in bins not containing a plaque
(p < 0.0001) (Table 1). With the exception of
inner striolar hair bundles (p > 0.10), this
observation was true for hair bundles in different macular regions and
for different hair bundle types (p < 0.001).
There were no significant differences in the number of gold particles
between membrane-only bins at the same level of the stereocilium on
kinociliary and nonkinociliary sides (p > 0.40).
To test more directly the hypothesis that gold immunoreactivity was
associated with the side insertional plaque, we examined the
distribution of gold particles in and around the plaque, using small
square bins (Fig. 8A).
These bins, 75 × 75 nm, were similar in size to the side plaque,
which had a mean diameter of 76 ± 13 nm [n = 64 bins]. As a control, similar bins were used to examine gold particles
on the kinociliary side; the number of gold particles per bin on the
kinociliary side was essentially constant (p > 0.10 for all pairwise combinations; Table
2). There was also no significant
difference between the number of gold particles in kinociliary bins and
the outermost basal (p > 0.10) or apical (p > 0.05) nonkinociliary bin. Regardless of
hair bundle location or morphology, the number of gold particles on the
nonkinociliary side was greatest in the bin containing the side plaque
and systematically decreased in bins located basally or apically to the
density (Fig. 8C). The number of gold particles in the bin
containing the side plaque was significantly greater than in the
outermost basal (p < 0.05) and apical
(p < 0.002) bins. This significance was
increased greatly when the centralmost bin was pooled with the bins
located above and below it and the pooled bins were compared with the apical or basal outermost three bins (p < 0.0001), confirming our initial observation that gold immunoreactivity
was located immediately adjacent to as well as within the side
osmiophilic density.
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Figure 8.
Morphometric analysis of myosin I localization,
using fine spatial bins. A, Schematic diagram of 75 nm
square sampling grid with typical location of tip and side insertional
plaques. B, Mean number and SD of gold particles located
in each bin on the kinociliary side. There was no significant
difference in the number of gold particles between any two pairs of
bins on the kinociliary side of the stereocilium
(p > 0.10). C, Mean number
and SD of gold particles located in each bin on the nonkinociliary
side. On this side the number of gold particles in the bin containing
the side plaque was significantly greater than that in basal or apical
bins located >200 nm from the side plaque
(p < 0.05).
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To compare the overall gold immunoreactivity in the immediate vicinity
of the tip and side insertional plaques, we determined the density of
gold particles located within arcs of 100 nm diameter, centered on the
tip and side plaques. In the 64 stereocilia that were examined in this
study, the gold particle density within 100 nm of the center of the
side plaque (1.7 ± 0.5 gold particles per 1000 square nanometers)
was significantly greater than the density within 100 nm of the center
of the tip plaque (0.9 ± 0.4 particles per 1000 square
nanometers) (p < 0.05) and greater than the
control value density (0.7 ± 0.4 per 1000 square nanometers) (p < 0.01). The gold particle density of the
tip plaque was not significantly different from the control arc.
| |
DISCUSSION |
Morphologically distinct hair cells in the bullfrog utricle differ
in their rates and extent of adaptation (Baird, 1994a,b). Recent
biochemical, pharmacological, and immunocytochemical studies suggest
that myosin I is responsible for the adaptation of mechanoelectrical transduction current in vestibular otolith hair cells (Gillespie et
al., 1993; Walker and Hudspeth, 1996; Yamoah and Gillespie, 1996;
Burlacu et al., 1997; Garcia et al., 1997; Hasson et al., 1997). Here,
we demonstrate that utricular hair cells of widely varying adaptation
kinetics contain myosin I throughout their hair bundles. In
addition, we demonstrate significant concentrations of myosin I
within the insertional plaques that anchor the tip links, providing
more compelling evidence that myosin I is the adaptation motor.
Although the the rate of adaptation in utricular hair cells ranges from
milliseconds to more than several seconds, all utricular hair cells
nevertheless contain myosin I molecules properly placed to perform
adaptation.
Myosin I in utricular stereocilia
To determine the specificity of mT2 for myosin I in utricular
hair bundles, we adapted previously published methods for bundle isolation (Gillespie and Hudspeth, 1991). By adding sulfo-SHPP, a
water-soluble primary amine-reactive labeling reagent, we improved substantially the recovery of utricular hair bundles. Protein immunoblotting of purified hair bundles with mT2 demonstrated that the
antigen recognized by this antibody is similar in mass to myosin I
in other frog tissues or expressed in insect cells. These results show
that the antibody labels myosin I selectively, justifying its use in
immunolabeling experiments.
Myosin I in the side insertional plaque
In the distal region of each stereocilium, regardless of hair
bundle morphology and, hence, adaptation kinetics, myosin I immunoreactivity was found within and adjacent to the insertional plaques at the stereociliary tip and on the side of the stereocilium near the tip of the adjacent, shorter stereocilium (see Figs. 5-8).
These structures correspond with the insertion points of the tip links
(Furness and Hackney, 1985). Gold particles were found at higher
density within 200 nm of each tip insertional plaque and within 75 nm
of each side insertional plaque, as compared with adjacent membrane
areas.
Localization at the side insertional plaque lends further support to
the hypothesis that myosin I is the myosin isozyme responsible for
adaptation. Myosin molecules are likely to constitute the adaptation
motor of the hair cell, which is thought to reside in or near the side
plaque (Corey and Assad, 1992; Hudspeth and Gillespie, 1994; Gillespie
and Corey, 1997). Our results coincide well with localization, using a
different antibody against myosin I, both with preliminary results
(Hasson et al., 1997) and with a statistical analysis of gold particle
location within serial section reconstructions of several hair bundles
(Garcia et al., 1997; D. Corey, personal communication). Although
another immunoelectron microscopic investigation failed to find a
correlation between myosin I location and the side plaque (Metcalf,
1998), the labeling density encountered in that study appeared too low
to draw meaningful statistical conclusions. The immunoelectron
microscopy data obtained so far, therefore, show that myosin I
is concentrated where the adaptation motor is thought to be located
and, hence, support its proposed role as the active component of the
adaptation motor.
Gold immunoreactivity corresponding to myosin I near the side
insertional plaque was observed most frequently around the plaque,
rather than within it. One view of the side plaque is that it contains
50-100 tightly packed myosin molecules (Hudspeth and Gillespie, 1994;
Gillespie and Corey, 1997); with such a structure, antibodies may be
excluded sterically from the central regions of aldehyde-fixed plaques.
Alternatively, myosin I may not form the side plaque as previously
suggested. Instead, the central core of the plaque could be constituted
of other, unknown molecules, with myosin I hovering around the
periphery of the plaque.
Myosin I within the stereocilia
In addition to its concentration in insertional plaques, we also
found myosin I at the membrane along the stereociliary shaft and
adjacent to the apical membrane over the cuticular plate. Myosin I
also was observed in the ankle region, where each stereocilium tapers
as its rootlet enters the cuticular plate. Occasional myosin I
immunoreactivity was associated with the rootlet within the cuticular
plate. The rootlets might serve as a track for initiating myosin I
movement into stereocilia; alternatively, myosin I might participate
in cross-linking rootlet actin filaments to the cuticular plate, as
hypothesized for myosin VI (Hasson et al., 1997).
The majority of myosin I molecules at stereociliary tips was
associated with the tip insertional plaque, at the lower end of the tip
link. If the transduction apparatus is symmetric, with channels and
motors at either end of a tip link (Denk et al., 1995), myosin I
would be expected at this position, although it should not play an
active role in adaptation. Other myosin I molecules may be found at
stereociliary tips because of the dynamic nature of the transduction
apparatus (Zhao et al., 1996); recycled or unused adaptation
motor myosin molecules may accumulate at the ends of their actin
tracks. Not all myosin I is at distal regions of the stereocilia,
however; some mechanism must exist to shuttle motor molecules back to
the soma or to degrade them at stereociliary tips.
Modification of adaptation rate in utricular hair cells
Our experiments indicate that gross inequities in myosin I
distribution cannot account for the range of adaptation kinetics seen
in utricular hair cells. If hair cells have similar amounts of myosin
I within their adaptation motors, despite differing adaptation
kinetics, how do these different cell types regulate their adaptation
rates? Several explanations can account for these differences.
The rate of adaptation depends on both the force applied to the
adaptation motor and on the concentration of Ca2+
felt by the motor (Assad and Corey, 1992), as well as on the number of
myosin molecules in an adaptation motor and on their kinetic and
mechanical properties (Gillespie and Corey, 1997). Although increasing
the number of myosin molecules per adaptation motor could reduce the
rate of slipping adaptation, we saw no correlation between myosin I
immunoreactivity in side insertional plaques and the adaptation rate of
the utricular hair cell types.
Differences in adaptation rate also could arise from variations in the
concentration or association kinetics of mobile Ca2+
buffers (Ricci and Fettiplace, 1997). Utricular hair cells contain the
mobile Ca2+ buffers calbindin, calretinin,
calmodulin, parvalbumin, and S-100 (Baird et al., 1997). Cells with
high levels of Ca2+ buffers should adapt more slowly
than those with lower amounts. Although variations in
Ca2+ buffer distribution are seen within the
bullfrog utricle (Baird et al., 1997), it seems unlikely that these
variations could account for the differences seen in the adaptation
rate.
Adaptation kinetics also might be affected by the concentration of
calmodulin, a critical regulator of myosin I (Barylko et al., 1992;
Zhu et al., 1996), including myosin I of the hair bundle (Walker and
Hudspeth, 1996). Detectable calmodulin immunoreactivity is absent from
nonadapting extrastriolar hair cells within the utricle (Baird et al.,
1997). Although myosin I is present in these hair cells, the
diminished calmodulin concentration in these cells may limit the myosin
activity. Different permutations of myosin I and
Ca2+ binding proteins could be responsible for the
range of adaptation kinetics seen in different utricular hair cell
types. Because additional mechanisms may account for the variability of
adaptation, however, further investigation certainly will be required
to establish definitively why adaptation rates vary in these cell
types.
Universality of the adaptation motor
Our results confirm that myosin I is present in all utricular
hair cells, even those that do not adapt appreciably to sustained displacements. This apparent discrepancy may reflect different time
scales of adaptation in different hair cell types. The central role of
adaptation is to ensure that the tension applied to gating springs is
optimal so that transduction channels can respond sensitively to
displacements (Howard and Hudspeth, 1987b; Howard et al., 1988). For
example, although type B hair cells do not adapt to stimuli as long as
2 sec (Baird, 1994b), myosin I is present at the side insertional
plaque of type B hair cells in amounts comparable to amounts in other
hair cell types. Instead, the presence of an active motor molecule may
account for type B cells being on their displacement-response relation
at rest near the steepest and, hence, most sensitive point (Baird,
1994b), a position that should require the substantial application of
force to gating springs (Hudspeth, 1992). Rather than being used to
reset the transduction apparatus on a time scale of tens of
milliseconds, myosin I (and the adaptation motor) may operate on a
much slower time scale in type B cells, presumably with similar
Ca2+ feedback. Although precluding rapid adaptation,
a slow time scale for the adaptation motor would ensure that these
cells nevertheless could detect small stimuli.
Nonadapting hair cells of the bullfrog utriculus are unlikely to be
exceptional in possessing the motor molecule responsible for adaptation
without also exhibiting rapid adaptation. Setting resting gating spring
tension should be a universal requirement for hair cells, and the
adaptation motor is well suited to perform this role. We suspect that
even hair cells of the mammalian cochlea, which exhibit little or no
adaptation (Géléoc et al., 1997) (but see also Kros et al.,
1992), also will use such a motor and will be found to have myosin I
located near their tip link anchors.
| |
FOOTNOTES |
Received Feb. 10, 1998; revised April 2, 1998; accepted April 7, 1998.
This work was supported by National Institute on Deafness and Other
Communication Disorders Grants DC03028 (P.S.S.), DC02368 (P.G.G.), and
DC02040 (R.A.B.) and funds from the Oregon Lions Sight and Hearing
Foundation (R.A.B.). P.G.G. is a Pew Scholar in the Biomedical
Sciences.
Correspondence should be addressed to Dr. Peter S. Steyger, Oregon
Hearing Research Center, Oregon Health Sciences University, 3181 Sam
Jackson Park Road, Portland, OR 97201.
| |
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Abstract
Introduction
Compound via MeSH
