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The Journal of Neuroscience, November 1, 1998, 18(21):8637-8647
Localization of Myosin-I
near Both Ends of Tip Links in Frog
Saccular Hair Cells
Jesús A.
García1,
Ann G.
Yee2,
Peter G.
Gillespie3, and
David P.
Corey2, 4
1 Program in Neuroscience and 2 Department
of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, 3 Departments of Physiology and Neuroscience, The Johns
Hopkins University, Baltimore, Maryland 21205, and 4 Howard
Hughes Medical Institute and Neurobiology Department, Massachusetts
General Hospital, Boston, Massachusetts 02114
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ABSTRACT |
Current evidence suggests that the adaptation motor of
mechanoelectrical transduction in vertebrate hair cells is myosin-I
. Previously, confocal and electron microscopy of bullfrog saccular hair
cells using an anti-myosin-I antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to
test whether myosin-I is enriched at or near the side plaques of tip
links, the proposed sites of adaptation, using hair bundles that were
serially sectioned parallel to the macular surface. The highest
particle density occurred at stereocilia bases, close to the cuticular
plate. Also, stereocilia of differing lengths had approximately the
same number of total particles, suggesting equal targeting of
myosin-I to all stereocilia. Finally, particles tended to clump in
clusters of two to five particles in the distal two-thirds of
stereocilia, suggesting a tendency for self-assembly of
myosin-I.
As expected from fluorescence microscopy, particle density was high in
the distal 1 µm of stereocilia. If myosin-I is the adaptation
motor, a difference should exist in particle density between regions
containing the side plaque and those excluding it. Averaging of
particle distributions revealed two regions with approximately twice
the average density: at the upper ends of tip links in a 700-nm-long
region centered ~100 nm above the side plaque, and at the lower ends
of tip links within the tip plaques. Controls demonstrated no such
increase. The shortest stereocilia, which lack side plaques, showed no
concentration rise on their sides. Thus, the specific localization of
myosin-I at both ends of tip links supports its role as the
adaptation motor.
Key words:
hair cell; myosin-I; adaptation; auditory; vestibular; electron microscopy
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INTRODUCTION |
Sensory hair cells adapt to
prolonged deflections of their hair bundles so that the open
probability of the transduction channels returns within tens of
milliseconds to near the resting value of 5-15% (Eatock et al., 1987
;
for review, see Gillespie and Corey, 1997). At least part of adaptation
in vertebrate hair cells results from a mechanical adjustment (Howard
and Hudspeth, 1988; Assad and Corey, 1992) that modulates tension in
the tip links, the 200 nm filaments that connect the tips of
stereocilia to the sides of their taller neighbors (Pickles et al.,
1984). It has been proposed that motor molecules reside in or
near the electron-dense "side plaques" that mark the upper
insertion of each tip link. This structure and the transduction
channel(s) associated with them can move along the stereocilia during
adaptation (Howard and Hudspeth, 1987). Ultrastructural measurements of
side-plaque position before and after adaptation, or before and after
cutting tip links, supports the idea that side plaques can move
(Shepherd et al., 1991).
Current evidence implicates a myosin as the main mechanoenzyme. The
core of a stereocilium is a bundle of cross-linked actin filaments;
myosin molecules are the only motor proteins known to move on
actin. Actin in stereocilia is oriented such that the myosin power
stroke would pull in the direction necessary to provide resting tension
on the tip link (Flock et al., 1981). Unloaded muscle myosin moves
along stereocilia at 1-2 µm/sec, a speed similar to that of the
adaptation motor (Assad and Corey, 1992).
An effort to identify myosins expressed in the bullfrog saccular macula
produced 13 different candidate myosin transcripts from 10 different
genes (Solc et al., 1994). Of these, myosin-I has the properties
necessary for an adaptation motor (Gillespie et al., 1996). First,
myosin-I is found in the stereocilia (Gillespie et al., 1993; Hasson
et al., 1997). Second, it exhibits Ca2+-regulated
actin-activated ATPase activity (Barylko et al., 1992; Burlacu et al.,
1997). Third, the physical compactness of myosin-I would allow
adequate numbers to insert themselves in or near the side plaque (Solc
et al., 1994). Fourth, antibodies directed against cow or frog
myosin-I labeled the tips of frog stereocilia (Gillespie et al.,
1993; Hasson et al., 1997). However, the limited resolution of confocal
microscopy used in those studies (~1 µm) could not adequately
localize myosin-I at or near the ends of tip links.
Preliminary studies using immunoelectron microscopy (Hasson et al.,
1997; Metcalf, 1998) revealed clusters of gold particles near the side
and tip plaques, but these did not present a statistically significant
sample. Here, we extend those findings on the distribution of
myosin-I within the saccular sensory epithelium. Using preembedding immunoelectron microscopy combined with statistical analysis of gold
particle labeling within ultrathin serial sections, we show that
myosin-I distribution is quantitatively enriched in both tip and
side plaques relative to the rest of the stereociliary bundle. The
localization of myosin-I to the side plaque supports its role as the
adaptation motor in bullfrog saccular hair cells.
Some of these results have appeared in preliminary form (García
et al., 1997).
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MATERIALS AND METHODS |
Dissection. Sacculi from adult American bullfrogs
(Rana catesbeiana) were rapidly dissected into an oxygenated
saline solution [containing (in mM): 120 NaCl, 2 KCl, 0.1 CaCl2, 3 dextrose, 5 HEPES, and 0.02% phenol red]
and cleaned of extraneous tissue (Assad and Corey, 1992). The pH was
adjusted to 7.23 with NaOH, and the final osmolarity was 250 mOsm/kg.
Otolithic membranes overlying saccular epithelia were removed in fresh
saline solution with fine forceps after incubation of the tissue in 75 µg/ml of subtilisin (Type XXVIII; Sigma, St. Louis, MO) for 20 min.
Immunohistochemistry. Tissues were fixed for 18 min in 4%
formaldehyde in 80 mM sodium cacodylate buffer with 4 mM CaCl2, pH 7.35, at room temperature
with gentle agitation. After fixation, tissues were rinsed (one time
for 5 min) in 80 mM sodium cacodylate buffer without
CaCl2 and (three times for 5 min each) in PBS (Life Technologies, Grand Island, NY) diluted to ~250 mOsm, pH 7.4. This and all subsequent steps used PBS adjusted to 250 mOsm. Tissues were permeabilized with 1% Triton X-100 (25 min), washed (one time for
15 min) in PBS, and blocked for 45 min with PBS containing 5% bovine
serum albumin (BSA) (A-6793; Sigma) and 1% normal goat serum (Jackson
ImmunoResearch, West Grove, PA). After the blocking step, tissues were
rinsed briefly with PBS and were incubated for 5 hr in anti-myosin-I
antibody (80 µg/ml) in PBS containing 0.5% BSA and 0.5% normal goat
serum with gentle agitation and washed (three times for 10 min each)
with PBS containing 0.5% BSA (PBS-BSA). The myosin-I antibody was
raised against a fusion protein containing the C-terminal 15 kDa of
frog myosin-I tail, was affinity purified, and was tested for
specificity in immunoblots of frog tissues as described previously
(Hasson et al., 1997). The myosin-VI antibody, used at 5-10 µg/ml,
was raised against the tail of pig myosin-VI (Hasson and Mooseker,
1994); it recognizes frog myosin-VI as well (Hasson et al., 1997).
Control sacculi were processed normally but without primary antibody.
Tissues were incubated overnight at 4°C with protein A coupled to
5-nm-gold particles (J. Slot, University of Utrecht, The Netherlands)
in PBS-BSA.
Additional sacculi were processed in parallel for fluorescence
microscopy using Cy3 goat anti-rabbit (Jackson ImmunoResearch) at a
1:150 dilution to monitor myosin-I immunoreactivity before processing for electron microscopy. Tissues were rinsed (three times
for 10 min each) with PBS-BSA. Cy3-labeled sacculi were mounted on
glass slides in Citifluor (University of Kent, Canterbury, UK) and
viewed with a rhodamine filter on a Zeiss (Oberkochen, Germany)
Axioscope upright microscope.
Individual saccular hair cells were isolated into a saline solution
(Assad and Corey, 1992) over glass slides coated with Cell Tak
(Collaborative Research, Bedford, MA). The slides were incubated at
room temperature (one time for 5 min) to allow attachment of the cells
to the substrate. The cells were fixed for 10 min in 4% formaldehyde
and rinsed (four times for 5 min each) in PBS. The cells were
permeabilized with 1% Triton X-100 (one time for 10 min) and rinsed
(two times for 5 min each) in PBS and then blocked and incubated in
anti-myosin-I antibody (10 µg/ml) as described above for the
entire saccule. The cells were incubated for 1 hr at room temperature
or overnight at 4°C with a FITC-conjugated goat anti-rabbit at 1:150
(Jackson ImmunoResearch). The labeled cells were rinsed (three times
for 5 min each) with PBS and mounted on glass coverslips in
Citifluor to prevent photobleaching. A stack of confocal images
comprising the center 2 µm of a hair bundle were captured using a
BioRad (Hercules, CA) MRC-1000 confocal microscope. The stack of
confocal images was summed using MetaMorph 2.5 (Universal Imaging
Corporation, West Chester, PA).
Electron microscopy. After protein A gold labeling, sacculi
were briefly rinsed in PBS, and the immunohistochemical reaction was
stabilized with 2.5% glutaraldehyde in PBS for 1 hr, followed by
multiple rinses in PBS. This and all subsequent steps were performed at
room temperature. Sacculi were post-fixed with 2% osmium
tetroxide in 1.5% potassium ferrocyanide for 1 hr, rinsed (four
times for 5 min each) with 100 mM sodium cacodylate buffer, and then stained en bloc with 2% uranyl acetate in maleate buffer, pH
6.0, for 1.5 hr. After dehydration in an ethanol series, the tissue was
rinsed briefly in 100% propylene oxide and flat-embedded (1:1) in
EMbed812/Araldite 6005 (Electron Microscopy Sciences, Fort Washington,
PA) and cured for 48 hr at 60°C.
Vertical thin sections were collected from the center of the sensory
epithelium along the axis running parallel to the eighth nerve fibers
and perpendicular to the macular surface using a Reichert
Ultramicrotome E. When vertical sections indicated an optimal region,
sacculi were cut from an epon block and reembedded in the proper
orientation for horizontal serial sections. Thin sections
(silver-gold, ~70 nm) were collected serially, 40-50 at a time,
from near the top of the hair bundle down to the cuticular plate, and
divided into four or five sections per slotted grid. The sections were
suspended within slotted copper grids (Electron Microscopy Sciences)
via a water droplet, and the grids were placed onto floating formvar
sheets. The formvar sheets were collected using a peg loop (Ernest F. Fullam, Inc., Latham, NY) and dried overnight. Each grid was
individually poststained with 2% uranyl acetate and Reynold's lead
citrate. The location of each hair bundle was mapped within a section,
followed serially, and photographed with a JEOL 100CX electron
microscope.
Data analysis. Each section of a bundle was individually
photographed, and the negatives were printed at 8 × 10 inches. An entire hair bundle typically consisted of ~80-90
serial thin sections. In all cases, bundle polarity was unequivocally
determined using two criteria. First, the 9 + 2 microtubule
arrangement of the kinocilium was conspicuous in the majority of
sections. Second, the staircase arrangement of stereocilia from top to
bottom indicated the tallest stereocilia. The tip plaque was readily
apparent in the highest section of each stereocilium. In longitudinal
sections of well fixed bundles, the side plaques are consistently
located ~200 nm (after correcting for shrinkage) above the lower
adjacent stereocilium along the axis of the hair bundle (Shepherd et
al., 1991). In transverse sections of antibody-labeled tissue, we often could detect side plaques. When visible, 64% of these occurred two
sections up from the tip of the shorter adjacent stereocilium. The
remaining 36% of side plaques occurred just one section immediately above (9%) or below (17%) this point. For much of the analysis, therefore, we assumed that the side plaques occurred two sections up
from the adjacent tips, whether or not the plaques were visible.
Each stereocilium was followed serially. For each section, the gold
particles located on its circumference were counted and recorded in two
groups. One sector contained 
of the stereocilium's
circumference on the negative side (opposite the kinocilium), and the
other sector contained the remaining 
. Gold particle counts
from each sector were entered into a spreadsheet program for analysis.
Because the apical surface of a hair cell is not perfectly flat, the
bases of stereocilia were not all in the same section. In mature hair
bundles of the bullfrog saccule, moreover, stereocilia range in height
from ~4 to 9 µm. In the spreadsheet, therefore, we shifted columns
of counts to align the bases of stereocilia before averaging and separately shifted the counts to align the tips. A total of 165,025 gold particles in 10 hair bundles were counted for this study.
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RESULTS |
Immunofluorescent localization of myosin-I
The myosin-I antibody, detected with a fluorescent secondary
antibody, labeled the cytoplasm of frog saccular hair cells, with
reduced labeling in the nucleus and cuticular plate (Fig. 1B). Label was
particularly high in the pericuticular necklace, a vesicle-rich zone
between the cuticular plate and the apical circumferential actin band,
which is thought to be a region of membrane trafficking (Hasson et al.,
1997; Kachar et al., 1997; Richardson et al., 1997). Within a hair
bundle, label was nearly absent from the lowest (proximal) third of
each stereocilium and increased distally, with highest concentration at
the tips of stereocilia (Fig. 1B). Label also
appeared in the bulb of the kinocilium. Specific labeling of the tips
of the stereocilia is similar to that reported previously for this
antibody (Hasson et al., 1997), for a monoclonal antibody raised
against bovine myosin-I (Gillespie et al., 1993), and for
anti-peptide antisera raised against frog myosin-I (Metcalf,
1998).
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Figure 1.
Morphology of bullfrog saccular hair bundles and
labeling with antibody to myosin-I. A, A saccular
bundle typical of those in this study contains ~60 stereocilia in
nine columns. Lengths vary from ~4 µm at the shorter (negative)
edge to ~8 µm at the taller (positive) edge. B,
Labeling of a dissociated hair cell with an antibody to myosin-I.
The secondary antibody was labeled with Cy3; the image is a summation
of a stack of confocal images through the middle 2 µm of the cell.
C, D, Two examples of the tip link
(tl) and the osmiophilic densities marking the
tip-insertion plaque (tp) and side-insertion plaque
(sp). E, Immunogold labeling of the tips
of stereocilia with the myosin-I antibody. Gold particles are
present along the sides of stereocilia but appear more concentrated at
the tip plaques and in a region near the side plaques.
F, Example of the basal tapers of stereocilia and their
insertions into the cuticular plate. Stereocilia are ~150 nm in
diameter where their rootlets insert into the cuticular plate, thicken
to a maximum diameter of ~550 nm ~2 µm above the cuticular plate,
and then narrow to a diameter of 300-400 nm at their distal tips.
G, Immunogold labeling of the bases of stereocilia.
Particles appear around the rootlet at the narrowest part of the taper
where the rootlet enters the cuticular plate (arrow) but
are otherwise scarce in the taper region. Scale bars: A,
B, 2 µm; C-G, 200 nm. (Panel
A was copyrighted by J. A. Assad and D. P. Corey and was reproduced with permission.)
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Immunogold localization of myosin-I
The actin within stereocilia is polarized such that myosins would
tend to move upward, from proximal to distal, if allowed to move freely
on stereocilia. Thus, any myosin isoform might naturally be expected to
accumulate at the tips of stereocilia. If myosin-I has a specific
role in anchoring ion channels to the actin cores of stereocilia and in
maintaining tip-link tension, it should appear more specifically at
either end of the tip links.
For ultrastructural localization of myosin-I, we labeled
formaldehyde-fixed Triton-permeabilized sacculi with the myosin-I antibody, followed by protein A coupled to 5-nm-gold particles. The
tissue was then post-fixed with glutaraldehyde and osmium and sectioned
for electron microscopy. Although the procedure produced good labeling
of stereocilia (Fig. 1E), tip links were not visible,
and the electron dense structures marking tip-link insertions were less
apparent than in bundles rapidly fixed in glutaraldehyde (Fig.
1C,D). In these sections, however, the position of
each side plaque marking the upper end of each tip link can be
inferred from the position of the shorter adjacent stereocilium. Tip
links are uniformly ~200 nm in length (before shrinkage) (Shepherd et
al., 1991) so the side plaque is expected to occur ~200 nm higher
than the tip of the shorter adjacent stereocilium, at least when a
tip link exists between two stereocilia.
In longitudinal sections of stereocilia, gold particles appeared along
the sides of all stereocilia (Fig. 1E). Particles
were not observed within the actin cores but occurred between the cores and the membranes. In the hair bundle, they were especially
concentrated in two regions: at the tips of stereocilia, and on the
sides in a region 200-500 nm up from the tip of the shorter adjacent
stereocilium. These are generally the regions expected to contain the
lower and upper insertions of tip links.
As expected from immunofluorescence localization, gold particles were
nearly absent from the basal regions of stereocilia, especially in the
region in which stereocilia taper toward their insertions into the
cuticular plate (Fig. 1G). One striking exception was a ring
of particles around each rootlet at the point where it enters the
apical surface of the hair cell. In this region, particles did appear
to be located within the actin cores; however, the stereocilium
diameter at this point is not much greater than the thickness of a
section so that particles encircling the core might appear to be within
it. Indeed, in cross sections of rootlets (Fig.
2B), it is clear that
particles were around the cores.
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Figure 2.
Immunogold labeling of stereocilia, sectioned
horizontally. A, Myosin-I antibody; section ~2 µm
above the cuticular plate. The orientation is provided by the
conspicuous kinocilium at the top of the image so that
top is positive, bottom is negative, and
tip links would run among stereocilia in a vertical column. Membranes
were removed by detergent before labeling with primary antibody. Gold
particles are visible around the actin cores of stereocilia but not
within the cores. In sections of this height and higher, they often
occur in clusters of two to six particles. B,
Myosin-I antibody; grazing section at the level of the cuticular
plate. Rootlets are apparent as dense cores within stereocilia and are
ringed with gold particles. Particles are concentrated but not
clustered. Particles are not present a bit higher (top
right) or a bit lower (bottom left) than the
level of the apical surface. C, Myosin-VI antibody;
grazing section at the level of the cuticular plate. Particles also
appear concentrated around the stereocilia at the apical surface but do
not ring the rootlet as tightly as for myosin-I. Scale bars, 1 µm.
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At the antibody concentration used (80 µg/ml), nonspecific binding
might produce excessive background. This seems unlikely to be a
problem, because nonspecific sites, if present, would also have to be
absent in the taper region and because we observed a qualitatively
similar distribution of particles with an antibody concentration of 10 µg/ml in preliminary studies.
Longitudinal distribution of myosin-I within stereocilia
Because the gold particles marking myosin-I immunoreactivity
occurred throughout stereocilia, single images, such as Figure 1E, are not sufficient to demonstrate a preferential
concentration of myosin-I at the insertions of tip links. To test
the statistical significance of the concentration at insertions, we
sectioned immunolabeled bundles transversely, perpendicular to the long axis. Serial sections were collected along the lengths of six bundles
labeled with myosin-I antibodies; two bundles labeled with
anti-myosin-VI, and two bundles labeled with no primary
antibody. We then counted the gold particles around each
stereocilium of each cross section and recorded them in a spreadsheet
for analysis. A typical section, taken ~2 µm up from the basal ends
of stereocilia, is shown in Figure 2A. Clusters of
gold particles were observed around most stereocilia and were usually
unambiguously associated with one or another stereocilium. The polarity
of the bundle was identified by the microtubule-containing kinocilium
(Fig. 2A, top) so the positions of
tip links could be inferred, running among vertical columns of
stereocilia. This bundle, for instance, contained nine columns of four
to seven stereocilia each.
A grazing section at the level of the cuticular plate from the same
bundle is shown in Figure 2B. Particles were observed within the cuticular plate and were seen in highest concentration around the rootlets at the point where they emerged from the plate.
To understand the longitudinal distribution of myosin-I in
stereocilia, we averaged the particle counts for all stereocilia in six
bundles (see Materials and Methods). Averages, shown in Figure
3, confirm the qualitative impression of
myosin-I distribution. There is a peak of six to seven particles per
section in the section in which the stereocilia meet the cuticular
plate (Fig. 3A) but otherwise very little label in the lower
taper region (sections 2-10). The number of particles per section
rises to a fairly constant value by the thickest part of the
stereocilium, 20-30 sections from the base, and stays near that level
as the sections approach the tips.
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Figure 3.
Distribution of gold particles along the lengths
of stereocilia with stereocilia aligned by their bases
(A-C) or by their tips
(D). A, Myosin-I antibody.
Filled diamonds indicate the average number of gold
particles per stereocilium in each section, averaged over all the
stereocilia of six bundles. Open symbols indicate the
density of particles per square micrometer of membrane, after
correction for the taper of the stereocilia. The solid
line shows the average diameter of stereocilia as a function of
height, used for the area correction, with five on the
left axis representing 0.5 µm diameter.
B, Myosin-VI antibody. Filled and
open symbols represent particles per section or per
square micrometer for all stereocilia of one bundle. C,
No primary antibody; two bundles. D, Myosin-I
antibody; average of six bundles with all stereocilia aligned by distal
tips. Particle counts on the left half (sections 30-70
from the tip) are not particularly meaningful, because different
regions of tapers were averaged together.
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Are there fewer particles in the taper region simply because the area
per section is less? To determine particles per unit area, we measured
the diameters of 10 stereocilia from three bundles as a function of
height. Most stereocilia are slightly club-shaped, as is apparent in
Figure 1A, rising to a maximum diameter at the top of
the basal taper and then thinning by ~30% near the tips. Assuming
that each section represents ~100 nm of tissue thickness before
shrinkage, we could calculate the particle density per square
micrometer of membrane area. Plotted this way, the concentration at the
lowest section is strikingly higher than elsewhere, and the absence of
particles in the lower taper is still apparent (Fig. 3A).
The concentration per unit area then rises monotonically toward the
tips of stereocilia.
We performed a similar analysis on stereocilia aligned by their bases
for an antibody raised against myosin-VI (Hasson and Mooseker, 1994)
and for no primary antibody. The no primary control should reflect
nonspecific binding of the protein A gold (Fig. 3C).
Myosin-VI and myosin-VIIa are also present in stereocilia of the frog
saccule (Hasson et al., 1997) but should have different distributions
from myosin-I if they have different functions. Myosin-VI also
appeared highest at the level of the cuticular plate (Fig.
3B) but is not so tightly grouped around each rootlet (Fig.
2C) as is myosin-I (Fig. 2B). The
elevated concentration at this site is consistent with
immunofluorescence observations but may be a consequence of the higher
concentration of myosin-VI throughout the cuticular plate (Hasson et
al., 1997). Elsewhere in the stereocilia, however, myosin-VI is at
nearly uniform concentration when normalized for surface area.
When the stereocilia are aligned by their tips (Fig. 3D), it
is apparent that the particle density was highest in the top 0.6-0.8
µm, as expected from fluorescence images such as Figure 1B. In the shaft region, 1-3 µm down from the tip,
the particle density was fairly constant at ~10 particles per
section. The drop in concentration further down (3-7 µm from the
tip) probably reflects the low concentration in the tapered regions,
because stereocilia of different lengths were averaged together. Thus, the meaningful region of this plot is restricted to the top 30 sections
in which the diameters are nearly constant. The marked increase in
myosin-I immunoreactivity in the tips of stereocilia was not
observed for myosin-VI or for the no primary control (see below). Thus,
the high myosin-I immunoreactivity at the tips of stereocilia is
most likely related to its function within stereocilia.
Distribution of myosin-I among stereocilia
We noticed that the density of particles in the shorter
stereocilia seemed to be higher than in longer stereocilia. If
myosin-I is regulated to maintain a certain concentration in
stereocilia, we would expect the density at equivalent points in
different stereocilia to be similar. On the other hand, if each
stereocilium has a certain amount of myosin-I, then the
concentration should be higher for shorter stereocilia. To test these
hypotheses, we counted the total number of particles in each
stereocilium and plotted it against the overall length of the
stereocilium, measured in sections (Fig.
4). Although there is considerable
variability among stereocilia, there is no tendency for longer
stereocilia to have more myosin-I immunoreactivity (Fig.
4A). Thus, the mechanism for targeting myosin-I to
stereocilia seems to put approximately the same amount in each
stereocilium. Because all stereocilia have few particles in the basal
taper region, the shortest stereocilia (with only ~2 µm in length
above the taper region) tend to have a much higher concentration in the
upper part than do longer stereocilia.
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Figure 4.
Number of particles in each stereocilium as a
function of stereocilium length. A, Myosin-I
antibody. The line is a least-squares fit to the data
and indicates no significant correlation of particle number with
length; long stereocilia do not seem to have more myosin-I than
short stereocilia. B, Myosin-VI antibody. A
least-squares fit indicates a significant correlation with length
at the p < 10 15 level.
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A different result was seen for myosin-VI. Longer stereocilia tended to
have proportionately more myosin-VI immunoreactivity than shorter
stereocilia (Fig. 4B), suggesting that myosin-VI is
distributed to maintain a certain concentration.
Clustering of myosin-I
In Figure 2A, which shows a section taken ~2
µm from the base of the bundle, gold particles often appear to occur
in clusters. Clustering might result from clumping of protein A gold,
from clumping of the primary antibody, from multiple antibodies in the
antiserum binding to different epitopes on a single myosin-I molecule, or from clustering of myosin-I molecules themselves. At
the level of the cuticular plate, however, gold particles occurred in
clusters less frequently (Fig. 2B), suggesting that
the clustering is not caused by clumping of protein A, clumping
of the primary antibody, or by multiple epitopes. To determine whether
the clustering was statistically significant, we counted the number of
particles in a cluster at different heights: for 21 stereocilia labeled with myosin-I antibody, for 27 stereocilia labeled with myosin-VI antibody, and for 21 stereocilia with no primary antibody. Particles were considered to be in a cluster if they were separated by less than
two particle diameters (~10 nm). For statistical analysis, we
combined data from ten sections so that data were placed into seven
groups, each representing increments in height of ~1 µm.
Figure 5 shows histograms for three
representative groups containing data from the first, third, and fifth
micrometer of stereocilium height and a histogram derived from a theory
that assumes random distribution of particles. Each histogram in Figure
5 shows the clustering in two ways: the striped bar is
the proportion of clusters of a certain size, and the solid
bar is the proportion of particles that occurs in a cluster
of a certain size. The second method tends to reveal large clusters,
because a cluster of six particles, for instance, would be counted once
by the first method and six times by the second.
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Figure 5.
Clustering of gold particles at different heights.
In each panel, the striped bar indicates
the proportion of clusters containing the indicated numbers of
particles, and the solid bar indicates the proportion of
particles in a cluster of a certain size. A, Myosin-I
antibody; sections 1-10, counting from stereocilia bases.
B, Sections 21-30. C, Sections 41-50.
D, Apparent clustering assuming random juxtaposition of
particles expected from the average density in sections 41-50 (from
Fig. 3A). The clustering observed in B
and C is apparently not random. E-H,
Myosin-VI antibody; clusters scored at the same height. Clustering
observed in F and G is not substantially
different from that occurring at random.
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The histograms confirm the impression of clustering higher in the
bundle. In the first micrometer, more than two-thirds of particles
occurred as isolated particles (Fig. 5A), whereas fewer than
a third were isolated particles in the third or fifth micrometers (Fig.
5B,C). Conversely, in the first
micrometer, <7% occurred in clusters of four or more, whereas 24 and
32% were in clusters of four or more in the third or fifth
micrometers, respectively. Groups above the second micrometer
appeared similar in their extent of clustering.
Is the apparent clustering a consequence of higher number of particles
in the upper regions of stereocilia so that particles are more likely
to fall randomly near another particle? We compared the results to a
simple theory in which 10 particles (the average density in upper
sections) fall randomly on the perimeter of a stereocilium and are
scored for clustering by our criteria. The probabilities are shown in
Figure 5D. The theory predicts that nearly two-thirds of
particles would be scored as singles if they were truly independent,
and <1% would randomly occur in clusters of four or more. We also
looked for clustering of myosin-VI immunoreactivity by this method
(Fig. 5E-H). In contrast to myosin-I, myosin-VI antibodies did not seem to show significant clustering above that expected from random distribution at any height in the stereocilia. Thus, particles associated with the myosin-I antibody cluster significantly in the upper part but not the lower part of stereocilia, suggesting that myosin-I molecules are more aggregated above the
taper region.
Localization at tip-link insertions
The marked increase in immunogold label we observed in the distal
0.8 µm of the stereocilia is not sufficient to identify myosin-I
as the adaptation motor. If myosin-I anchors transduction channels
to the actin cores of stereocilia and serves to regulate tip-link
tension, it should be located specifically near either end of tip
links. We would then expect a difference in myosin-I density between
the positive side of a stereocilium, from which a tip link rises to the
next taller stereocilium, and the negative side, which receives a tip
link from the next shorter stereocilium, somewhat down from the
tip.
To detect such a difference, each stereocilium was divided into two
sectors: one encompassing of the diameter on the negative
side, which would contain the side plaque, and one encompassing the
remaining of the diameter, which would contain the tip
plaque. Particles were counted separately for the two sectors and
normalized for surface area. Because we could reliably infer the
position of the side plaque as approximately two sections from the tip
of the shorter adjacent stereocilium (see Materials and Methods),
sections could be aligned by either tips or side plaques. These
alignments are shown in Figure 6.
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Figure 6.
Distribution of gold particles along the lengths
of stereocilia, with stereocilia aligned by the inferred position of
the side-insertion plaques (A-D) or by the tips
(E-H). Data for the negative side section
() are connected by dashed lines; those for
the positive side section () are connected by solid
lines. Sections are illustrated in cross-section in
A (left). Particles per stereocilium were
calculated for each sector of each section, and sections were
normalized to full circumference; thus, 10 particles per stereocilia
per section corresponds to a surface density of ~90
particles/µm2. A, Illustration of
alignment by side plaques. Stereocilia ended about six sections above
the side plaque, on average. B, Antibody to myosin-I.
At each height, the density of particles in the negative side section
was 60-90% higher than in the remaining circumference, over a length
of ~700 nm around the side plaques. C, Antibody to
myosin-VI. D, No primary antibody. Distribution of
protein A gold apparently reflects surface area. E,
Illustration of alignment by tips. Inferred positions of side plaques
are plotted in F and indicated by the filled
circles. F, Antibody to myosin-I. The density
on the positive side rose abruptly in the most distal two sections and
fell on the negative side. G, Antibody to myosin-VI.
H, No primary antibody. No such dichotomy was observed
for myosin-VI. Error bars represent the maximum mean ± SE
for each sector.
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When the sections were aligned by side plaques for averaging (Fig.
6A), the density of particles was generally constant
and similar for both sectors until the distal micrometer of the
stereocilia. Then, for a 700 nm region centered approximately one
section above the inferred side-plaque position, the density on the
negative rose to nearly twice that of the positive (Fig. 6B). No such rise was observed for stereocilia labeled with myosin-VI antiserum (Fig. 6C) or
with no primary antibody (Fig. 6D). Closer to the
tips, the concentration on the negative side fell, whereas that on the
positive side rose.
Because the distance from the side plaques to the tip of a stereocilium
is somewhat variable, alignment by side plaques does not properly
illustrate the concentrations at the very tips. Thus, we also aligned
the particle counts by the tips of stereocilia for averaging (Fig.
6E). Again, counts were similar between sectors until
the distal micrometer. In this plot (Fig. 6F), the
particle density in the positive sector is seen to rise most
dramatically in the final two sections (~200 nm), and the density in
the negative sector is seen to fall in the same region. The inferred
positions of side plaques are shown in Figure 6F;
they occur, on average, six sections down from the tip. Again, no such
rise was observed for stereocilia labeled with myosin-VI antiserum
(Fig. 6G) or with no primary antibody (Fig.
6H).
We have also analyzed two bundles in which the stereocilia were divided
into six sectors of each. In these, the variability in
density was larger, because we had fewer bundles divided into more
sectors, but the trend was the same. In particular, the density at the
very tip was highest in the sixth at the positive edge (where the tip
link inserts) and less in the sixths at the sides (data not shown).
[The shape of stereocilia tips creates a possible artifact for one
point in Figure 6, right plots. Although some tips are relatively square (Fig. 1C), others are slanted or rounded
such that the positive side is taller than the negative side (Fig. 1D). The slant is not caused simply by the nipple of
membrane seen at the lower end of tight tip links but includes a
slanted actin core as well. Thus, a section passing through the very
tip might include material in the positive sector but
nothing in the negative . A lack of particles in the negative
could reflect a lack of stereocilium rather than a lack of
myosin-I. Thus, the top-most point on the negative side is less
reliable than others and was not plotted. On the other hand,
longitudinal sections (Fig. 1E) also showed a similar
lack of particles at the very tip of the negative side.]
In general, the quantitative differences between locations in
stereocilia expected to contain upper and lower tip-link insertions are
consistent with a specific localization of myosin-I at either end of
the tip link. A graphical summary of the data are shown in Figure
7 in which the concentration of
myosin-I immunoreactivity is shown as density of shading. A
transmission electron micrograph is reproduced at the same scale for
comparison. The concentration is highest in two locations: on the
negative side in a fairly broad region centered around the side plaque,
and on the positive side at the very tip, near the tip plaque.
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Figure 7.
Summary of the data from Figure 6 for myosin-I
immunoreactivity. The intensity scale indicates
particles per square micrometer.
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Testing the localization of myosin 1 at side plaques
Because the electron-dense structure marking the side insertion is
typically 30-50 nm across, it is surprising to find myosin-I immunoreactivity in a band extending almost 700 nm along the negative side of each stereocilium. It is possible that our estimation of side
insertion position is inaccurate and that the alignment by insertion
smeared the true distribution. However, when plaques were clearly
visible, they always occurred within one section of the inferred
position. It is also possible that only some tip links are intact and
that motors broken loose from a transduction complex tend to accumulate
several hundred nanometers closer to the tips. Finally, it is possible
that the adaptation motor is a more broadly distributed structure and
that the specific electron density only reflects one element of the
motor. In any case, we sought to test further the association of
myosin-I immunoreactivity with motor function.
The data in Figure 6 are averages of stereocilia both with and without
visible side plaques, and perhaps some of those not clearly visible
were missing altogether. We therefore asked whether the particle
density would be higher at clearly identified side plaques. Comparisons
were made in two ways. In one test, we compared negative and positive
sectors for three bundles (Fig.
8A) and found that the
density of particles at visible side plaques (negative sectors) was
almost 300% higher than positive sectors of the same sections. In the
averages of all stereocilia (Fig. 6B), the density at
inferred side plaques was only 70% higher. In another test, we
compared negative sectors for stereocilia with and without visible side
plaques for two bundles. Stereocilia that contained a visible side
plaque had a 71 ± 14% higher density of particles within 150 nm
of the side plaque than at the equivalent location in stereocilia
without a visible plaque (data not shown).
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Figure 8.
Tests of the specificity of myosin-I
immunoreactivity. A, Density of particles at
morphologically identified side-insertion plaques. Filled
bars represent the negative sector; open
bars represent the positive sector. Particles were
counted in one section each of 19 stereocilia of three bundles. The
particle density at identified side plaques is higher than for all
stereocilia averaged. B, Particle density for the
shortest stereocilia, aligned by tips. The shortest stereocilium of
each column (n = 53 from about nine columns in each
of six bundles) was selected for averaging and analyzed as for Figure
6. Whereas the positive sector showed a rise in density at the very
tips that matched that of all stereocilia, the negative sector (which
would not contain a side plaque) was not significantly different from
the positive sector over most of the length. Both sectors showed a
decline in the taper region, as expected from Figure 3. Error bars
represent the maximum mean ± SE for each sector.
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Finally, we reasoned that the shortest stereocilia of each bundle
should differ from all others, because they have no tip link inserting
into a side density and might have no special concentration of
particles along the negative side. Thus, we identified all the shortest
stereocilia in six bundles and averaged their distributions separately.
Figure 8B shows the concentrations, with the sections aligned by tips. (These stereocilia, of course, could not be aligned by
side inserts.) Two features of the distribution are striking. Like all
other stereocilia, the concentration on the positive side rises at the
very tips where we would expect the lower insertion of a tip link
extending to the next taller stereocilium. On the other hand, the
negative side shows a concentration not significantly different from
the positive side, in the region in which other stereocilia have their
highest concentration and most conspicuous difference. Thus, the high
concentration of myosin-I immunoreactivity is associated with a
tip-link insertion.
| |
DISCUSSION |
Myosin-I immunoreactivity is high at both ends of tip links
Where exactly should we expect to find myosin-I? A
myosin-mediating adaptation may serve two functions. First, it would be the force-producing protein of the adaptation motor. If the major constituents of the motor are incorporated within the side plaque, myosin-I should be in or near the plaque. Second, and more
generally, myosin-I should help anchor the transduction channels to
the actin cores of stereocilia to prevent tip-link tension from pulling channels out of the plasma membrane. Transduction channels are likely
to be placed at both ends of tip links (Denk et al., 1995), so we might
expect myosin-I to anchor the channels at the lower ends of the tip
links where they insert into the tips of stereocilia, and thus to be
present in the tip plaques as well.
We found that the density of myosin-I immunoreactivity is highest at
the distal 0.8 µm of stereocilia, consistent with studies using
fluorescence microscopy and a variety of antibodies to myosin-I (Gillespie et al., 1993; Hasson et al., 1997; Metcalf, 1998). Electron
microscopy revealed that the density is not homogeneous around the
circumference of the tips. On the positive side of each stereocilium,
the density is highest in the top 0.2 µm, near the lower insertion of
a tip link (Fig. 7). On the negative side, the highest density occurs
in a band of about seven sections (~700 nm), with the center of the
band approximately coincident with the inferred positions of the side
plaques marking the upper insertion of tip links. Additional tests
support the idea that myosin-I immunoreactivity is specifically
associated with the side plaques, which is consistent with the proposal
that myosin-I is the motor protein of the adaptation apparatus.
If myosin-I is associated with the motor, why is its distribution
broader than the size of the side plaque? It may be that the motor
apparatus is somewhat diffuse; myosin-I might attach to a tether or
harness protein, and these proteins might attach to the osmiophilic
plaque. Thus, the plaque might represent only the most condensed part
of the motor. If all myosin molecules in the side-plaque region are
active components of the motor, we might expect them to be mostly above
the side plaque, because they would all be attempting to climb upward
and would be restrained only by the tethers. The observed distribution
is consistent with this idea. Alternatively, myosin-I might collect
in the vicinity of the motor and there might be some equilibrium
association with the active motor that is contained entirely within the
plaque. Previous experiments indicating that only about half of the tip links are intact in this preparation of bullfrog saccule (Assad et al.,
1991; Denk et al., 1995) and that intentionally cut tip links of
chicken hair cells can regenerate in 10-15 hr (Zhao et al., 1996)
suggest a dynamic assembly and disassembly of the channel-motor complex. Similarly, myosin-I molecules above the side plaque might
have been part of a motor complex that climbed and dispersed after loss
of its tip link.
Myosin-I is also concentrated at the rootlet
Although our focus was primarily directed at the tips of
stereocilia, we found also a particular concentration of gold particles at the very bases of stereocilia, exactly where the narrow rootlet enters the apical surface of the hair cell. At one section above or
below this point, the concentration dropped to near zero. Adjusted for
surface area, the concentration at this point was as high or higher
than at the tip-link insertion plaques. We can only speculate about the
function of this small group of myosin-I molecules. They may
constitute a barrier that prevents excessive myosin-I or other
myosin isozymes in the cell body from ascending the actin filaments of
the stereocilia. They may provide structural support, linking the
rootlets to the actin of the cuticular plates. Finally, they may
represent a reserve of myosin-I involved in normal turnover of the
adaptation complex.
Myosin-I clusters in more distal regions of stereocilia
In cross-sections of hair bundles above the taper region, gold
particles labeling the myosin-I antibody clearly tended to cluster
(Fig. 2). The clustering was statistically significant above the taper
region but not significantly different from random in the lowest 1 µm
of the stereocilia. This argues against clustering that results from
clumping of protein A gold, from clumping of the myosin-I antibody,
or from the polyclonal antibodies binding to multiple epitopes of a
single myosin-I molecule. Instead, it suggests that myosin-I
itself tends to self-associate and does so as it ascends a
stereocilium. Indeed, the recombinant tail of myosin-I has been
shown to aggregate in vitro (R.A. Dumont and P.G. Gillespie,
unpublished data). Such aggregation might play a role in assembly of
the motor complex.
Myosin-I is uniformly allocated among stereocilia but
inhomogeneously distributed within them
The number of gold particles per stereocilium varied considerably
among stereocilia but did not demonstrate any significant correlation
with length; that is, short stereocilia had as much total myosin-I
immunoreactivity as long stereocilia, on average. If stereocilia have a
certain number of myosin-I binding sites per unit length (for
instance, actin monomers) and the cell inserts enough myosin to fill
those sites, taller stereocilia should have had more myosin-I. In
contrast to myosin-I, total myosin-VI immunoreactivity did show a
correlation with length. It appears, therefore, that a cell puts a
certain amount of myosin-I into each stereocilium, and the
distribution within the stereocilium then depends on factors secondary
to the allocation mechanism. Allocation of myosin-VI may follow a
different mechanism.
Within a stereocilium, the myosin-I immunoreactivity (normalized for
surface area) rises uniformly from the taper region to a height of 4-5
µm and then plateaus. Even apart from the density at insertion
plaques, myosin-I is not randomly distributed within a stereocilium.
It may be that the normal motor activity of myosin-I and the
polarity of the actin causes accumulation toward the tips. Alternatively, binding sites on actin filaments may be more exposed toward the tips; for instance, myosin-I might not bind the pointed ends of actin in the taper region but might bind or accumulate at the
barbed ends of actin filaments that occur as stereocilia decrease in
diameter distally. Finally, the distribution of myosin-I may not
represent static binding sites so much as its dynamic turnover in the
stereocilium.
Insertion plaques are associated with dozens of
myosin-I molecules
The amount of myosin-I has been estimated by biochemical
methods at ~500 molecules per stereocilium (Gillespie et al., 1993). The particle counts described here suggest that the amount may be
higher. We observed ~400 particles per stereocilium. The cluster analysis, specifically the lack of clustering in the taper region, provides evidence that an individual myosin-I molecule is not associated with more than one protein A gold particle. Because it is
unlikely that every myosin-I molecule had an antibody bound to it,
the number of myosin-I molecules per stereocilium is probably several-fold higher than 400. Of these particles, ~40 were broadly associated with the insertion densities. With adjustment for labeling efficiency, it appears that there are at least several dozen
myosin-I molecules associated with the adaptation motor. This
estimate fits well with those in previous studies (Hudspeth and
Gillespie, 1994).
| |
FOOTNOTES |
Received May 27, 1998; revised Aug. 12, 1998; accepted Aug. 14, 1998.
This work was supported by a National Institute of Neurological
Disorders and Stroke Minority Predoctoral National Research Service
Award (to J.A.G.) and by National Institutes of Health Grants DC00304
(to D.P.C.) and DC02368 (to P.G.G.). D.P.C. is an Investigator of the
Howard Hughes Medical Institute. We especially thank Abigail Peterson
for counting gold particles and appreciate comments on this manuscript
from Eunice Cheung and Drs. Jaime García-Añoveros,
Jeffrey R. Holt, and Emily R. Liman. We thank Dr. Tama Hasson (Yale
University) for the gift of the myosin-VI antibody.
Correspondence should be addressed to David P. Corey, WEL414,
Massachusetts General Hospital, Boston, MA 02114.
| |
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Top
Abstract
Introduction
