Striated organelle, a cytoskeletal structure positioned
to modulate hair-cell transduction
Florin Vranceanua, Guy A. Perkinsb, Masako Teradab, Robstein L. Chidavaenzia, Mark H. Ellismanb,
and Anna Lysakowskia,1
aDepartment of Anatomy and Cell Biology, University of Illinois, Chicago, IL 60612; andbNational Center for Microscopy and Imaging Research, Center for
Research in Biological Systems, University of California at San Diego, La Jolla, CA 92093
Edited by Bechara Kachar, National Institutes of Health, Bethesda, MD, and accepted by the Editorial Board January 30, 2012 (received for review January
The striated organelle (SO), a cytoskeletal structure located in the
apical region of cochlear and vestibular hair cells, consists of
alternating, cross-linked, thick and thin filamentous bundles. In
the vestibular periphery, the SO is well developed in both type I
and type II hair cells. We studied the 3D structure of the SO with
intermediate-voltage electron microscopy and electron microscope
tomography. We also used antibodies to α-2 spectrin, one protein
component, to trace development of the SO in vestibular hair cells
open-ended cone attached to the cell membrane along both its up-
contacts with the membrane and adjacent mitochondria, the SO is
connected both directly and indirectly, via microtubules, to some
stereociliary rootlets. The overall architecture of the apical region
in type I hair cells—a striated structure restricting a cluster of large
mitochondria between its filaments, the cuticular plate, and plasma
membrane—suggests that the SO might serve two functions: to
maintain hair-cell shape and to alter transduction by changing the
geometry and mechanical properties of hair bundles.
Much is known about their structure, their mechanical
properties, and their geometric arrangement (1–3). These cells
are embedded in the cuticular plate, a dense structure composed
of an actin gel (4) located at the apical end of the hair cell, which
is thought to act as a rigid platform, helping stereocilia return to
their resting positions after stimulus-evoked displacements (5, 6).
What has never been investigated is how the cuticular plate might
be stabilized by structures underneath it. Do these structures
provide a foundation for the cuticular plate and the hair bundle?
One such structure is the striated organelle (SO), also known
as a laminated (or Friedman’s) body, which is a cytoskeletal
lattice underlying the apicolateral hair-cell membrane and con-
sisting of alternating thick and thin filament bundles. When first
described in vestibular hair cells in the 1960s, this structure was
thought to be a pathological feature (7, 8). It was later found to
be a normal component of mammalian vestibular and cochlear
inner, but not outer, hair cells (9–11). A striated structure,
similar in position but differing in morphological details, has
been described in other vertebrates (12–14).
Slepecky et al. provided a description of the SO (10, 15, 16),
including the periodicity of its filaments, their radial direction,
attachment to the plasma membrane, and association with
microtubules. Previous studies, even those based on conven-
tional transmission electron microscopy (TEM) and deep-etch
freeze fracture (9, 10, 15, 17, 18), did not provide a picture of the
3D structure of the organelle. Electron-microscopic tomography
(EMT) provides a promising approach to study 3D structures.
The approach has been used to study other structural features of
hair cells (19, 20) and is used here to study the SO’s 3D archi-
tecture, localization, shape, size, prevalence, and its connections
with neighboring structures. We also used immunochemistry to
air-cell stereocilia are important for mechanotransduction.
identify and localize one protein component and to trace the
development of the SO over the first postnatal week.
Our findings on SO structure and 3D architecture of the apical
of hair-cell cytoskeletal organization and suggest new hypotheses
about the hair bundle and mechanoelectric transduction.
Material was examined with both conventional TEM and in-
termediate voltage electron microscopy (IVEM, 400 KeV).
Results were similar in both cases. From these data and EMT
software, we produced 3D reconstructions of the SO and the
apical portion of three type I hair cells (cells 1–3) and one type II
cell (cell 4) from the utricular macula. Extrastriolar hair cells
were preferred because they are smaller than those from the
striola, which allowed us to use a magnification high enough
(5,000×) to see details, but low enough to get the whole apical
hair cell in the field of view. Three reconstructions (cells 1, 3, and
4) were partial, and the other (cell 2) encompassed the entire
apical portion of the hair cell. We also obtained tomographic
data, but no reconstructions, from one striolar type I cell (cell 5)
and a second type II cell (cell 6). In each reconstruction, we
modeled the thick and thin filament bundles of the SO, the
stereociliar rootlets (SRs), the cuticular plate, subcuticular mi-
tochondria, and the membranes of the hair cell and surrounding
calyx (the afferent ending innervating a type I hair cell) to obtain
a 3D model of the SO in relation to these other structures.
Structure of the SO. Each SO wascomposed of35–40 thick bundles
of filaments and an equal number of thin bundles (Fig. 1A). These
bundles had thicknesses of 34.6 ± 1.25 nm (mean ± SD), n = 59,
and 11.3 ± 0.45 nm, n = 54, respectively. Individual filaments
within the thick and thin bundles measured ∼10 nm and 6 nm,
respectively. Thick bundles were located immediately subjacent to
the cell membrane (Fig. 1 A and B), and exhibited a periodicity of
128 ± 3.1nm (n = 56); thin bundles were located midway between
Inset) extended radially, like spokes, from the plasma membrane
∼130 nm into the hair cell (Fig. 1B). Along the length of the or-
ganelle, bundles could be oriented vertically (Figs. 1A and 2 A and
B) or spirally (Figs. 1 E and F and 2F). Sometimes both ori-
entations were found in restricted regions of a single organelle.
In type I hair cells, several bundles extended upwards from
their circumferential location below the cuticular plate and
inserted into the apical membrane surrounding the kinocilium
Author contributions: F.V., R.L.C., and A.L. designed research; F.V., G.A.P., R.L.C., and A.L.
performed research; M.H.E.contributed new reagents/analytictools;F.V.,G.A.P., M.T.,R.L.C.,
and A.L. analyzed data; and F.V. and A.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. B.K. is a guest editor invited by the Editorial
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1101003109 PNAS Early Edition
| 1 of 6
(Fig. 1 C and D); the point of insertion is dimpled, as if the bundle
were exerting a pulling force on the apical membrane. Within the
SO, thick bundles often merged with one another (Fig. 1E) or
morphed into thin bundles. Some bundles even appeared to cross
the intercellular cleft between the hair cell and the calyx ending
and insert into the calyx membrane (Fig. 1 E and F). Overall, the
SO is shaped like an inverted conical cage (Fig. 2 and Movies S1
and S2), with the upper, larger opening facing the cuticular plate.
Connections of the SO to the Kinocilium and Some Stereocilia. The
linkage between the membrane surrounding the kinocilium and
the SO was seen in several cells. On occasion, SO bundles were
observed to connect to SRs. This link was seen in a tomogram
section for cell 1 (Fig. 2A) and in the resulting model (Fig. 2B).
Two neighboring SRs merged, seemingly without interruption,
into separate SO thick bundles. A similar observation was made
in serial TEM photos (Fig. S1). In both instances, the SRs con-
tinuous with SO filaments were located on the circumference of
the hair bundle. The SRs were slightly thicker than their con-
tinuations with the SO (Fig. 2A), suggesting that the two ele-
ments may differ in protein composition, or that, if both are
made up of the same proteins, the latter is bundled differently.
Stereociliar Rootlets. Wemeasuredthediametersofstereociliaand
SRs across all rows of the hair bundle in two hair cells. Stereocilia
nearest the kinocilium (rows 1–3), were significantly thicker than
those in rows 4–10 (stereocilia: rows 1–3, 140.6 ± 3.5, n = 34 vs.
rows 4–10, 116.0 ± 3.4, n = 114, P < 0.05; rootlets: 46.1 ± 1.7, n =
34 vs. 38.0 ± 1.9, n = 114, P < 0.05). In all type I hair cell recon-
structions, the largest SRs, those nearest the kinocilium, bent to
form an angle of 110° within the cuticular plate (cell 2) (Fig. 2C
and Movie S2). A type I cell from the striolar region (Fig. 2C,
Inset), oriented in an appropriate plane, showed several rootlets
bending within the cuticular plate. These long rootlets, on passing
through the cuticular plate (Fig. 2C, Inset), were organized into
two groups (Fig. 2E) that converged toward separate areas on the
hair-cell membrane opposite the kinocilium (the SR insertion
area) (Fig. 2D). The arrangement is best seen by observing the SR
insertion area from different angles, as in the movie (Movie S2).
SRs further from the kinocilium exhibited less and less bending
and most appeared to remain within the cuticular plate (Fig. 2F
and Movie S2).
Subcuticular Mitochondria. Within the confines of the SO, there is
a set of exceptionally large mitochondria compared with those in
the rest of the type I cell or in type II cells. Because mitochondrial
function (Ca2+homeostasis and source of ATP) is related to
overall size, we used our tomograms to measure the volumes and
surface areas of mitochondria from the same portion of the cell
(the subcuticular region, ∼6 μm below the apical cell membrane)
in type I and neighboring type II cells (Table S1). Table S1 indi-
cates that mitochondria inside the SO in type I hair cells are two-
times larger in surface area and three- to four-times larger in
volume than those in type II cells. In one reconstruction (cell 1),
a few SRs emerging from the underside of the cuticular plate
ended on subcuticular mitochondria and appeared to be “teth-
ered” to them (Fig. 3 A and B). In cell 2, other mitochondria were
intimately associated with long stretches of SO bundles (Fig. 3 C
reconstructions: for example, cell 4 (Fig. 4D).
SO in Vestibular Type II Hair Cells. EMT results from the partial
reconstruction of a type II cell (cell 4) are shown in Fig. 4. The SO
is more extensive in the type II hair cell than in type I cells. It is
longer, broader in extent, and the thick bundles are wider (com-
pare Figs. 4 A–C and 5A) and can extend deeper into the hair cell
(∼400 nm, compared with ∼130 nm for typeI haircells). Spherical
structures surround the SO and are more numerous than in type I
modelfroma vantagepointbelow thecuticularplate (Fig.4D),we
is closely associated with four mitochondria along its breadth,
similar to type I cells (Fig. 3 C and D).
Protein Composition of the SO. We have identified one protein in
the SO (Fig. 5). Using an antibody to α-2 spectrin, we labeled the
SO in vestibular hair cells in both confocal (Fig. 5A) and EM
immunogold (Fig. 5 B and C) experiments. Quantification of the
EM label for α-2 spectrin indicated a nonhomogeneous distri-
bution of gold particles, with a tendency for them to be located
immediately adjacent to the thick filament bundles (Fig. 5C) (χ2
test of homogeneity, χ2= 23.1, df = 7, P < 0.002). Western blots
(Fig. S2A) confirmed the presence of α-2 spectrin in the inner
ear. The identities of the bands at 150 kD and 285 kD were
confirmed as α-2 spectrin with mass spectrometry (Fig. S2B).
tomogram showing the structure, periodicity, and location of the SO im-
mediately subjacent to the calyx membrane. (Inset) Thick (numbered) and
thin (arrowhead) filament bundles are composed of several thinner, spiral-
bundled filaments (e.g., 1–4), measuring ∼10 nm and ∼6 nm, respectively.
Note also the cross filaments (small arrows), which EM immunogold studies
indicate are likely spectrin (Fig. 5 B and C, and Insets). (Scale bar, 0.2 μm.) (B)
Cross-section through the neck of a type I cell showing radial distribution of
thick (arrows) and thin (arrowheads) SO filament bundles. They extend from
the hair cell membrane to a depth of ∼130 nm into the cell. (Scale bar, 0.2
μm.) (C and D) Apical insertion of a thick SO filament bundle (arrows) ad-
jacent to the kinocilium. The site of insertion is a dimpled area (arrowhead)
between the kinocilium and nearby SRs. (D) A SO filament bundle angles
downward (arrows), approaching the cell membrane and continuing into
the neck of the type I cell. (Scale bar, 0.5 μm; also applies to C.) (E and F)
Details of hair-cell neck region in cell 2, obtained with SLICER mode in IMOD.
(E) Thick filament bundles of the SO merge together (arrowheads). (Scale
bar, 1 μm.) (F) Higher magnification of the area enclosed in the box in E
shows possible connections (arrows) between thick filament bundles of the
SO and the calyx membrane bridging the intercellular cleft (dashed white
lines show locations of hair-cell and calyx membranes). (Scale bar, 0.25 μm.).
Cal, calyx; CP, cuticular plate; M, mitochondria.
Structure of the SO. (A) Tangential section from a type I hair cell EM
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| www.pnas.org/cgi/doi/10.1073/pnas.1101003109Vranceanu et al.
SO Formation in Relation to Development of the Ear. Using α-2
spectrin as a marker for the SO, we performed confocal im-
munofluorescent (Fig. 5 D–F) and TEM (Fig. 5 G–J) studies on
developing rats during the first two postnatal weeks, a period of
rapid hair-cell differentiation and afferent development (21).
The SO was not present at birth (postnatal day 0, P0) (Fig. 5D),
but developed rapidly during the first week, making its first ap-
pearance at P2 (Fig. 5 E, G, and H), and appearing to be fully
developed by P6 (Fig. 5 F, I, and J). Because transducer currents
develop between embryonic day (E)16 and E17 in the mouse
(22), and perhaps 1–2 d later in rats, the initial development of
transduction does not require the SO, but it may be required for
Previous studies provided descriptions of the SO (7–11, 15,16, 18)
but were unable to generate a 3D view of the structure. In this
study we did so using EMT. Our reconstructions show that the SO
of type I hair cells has the shape of an inverted, open-ended cone
(a frustum), conforming to the shape of the hair-cell neck. This
cone consists of 35–40 thick filamentous bundles and an equal
number of thin bundles, with an apical extension near the kino-
the lower, constricted neck region, thick filament bundles occa-
sionally merge with each other. The upper ends of at least a few
thick circumferential filament bundles of the SO connect to SRs
and several mitochondria appear tethered to SRs and to SO
bundles. The lower portions of the SO appear to insert into the
plasma membrane. These associations with other elements of the
cell suggest that the SO may be involved in regulation of hair-cell
transduction, as well as contribute to the characteristic shape of
the type I hair cell. In the type II cell, the SO has a more planar
appearance and is not connected to stereocilia or the apical
membrane, suggesting a predominantly structural role.
Our data provide a fresh view of the relationship between the
stereociliar bundle and the cuticular plate. In most accounts, in-
dividual stereocilia have been thought to insert into the cuticular
providing a platform that contributes to the stereocilia returning
to their normal upright positions after bending (6). That SRs ac-
tually traverse the cuticular plate has been noted only occasionally
in the literature (17, 23). Our data are unique in showing that the
SRs reach the plasma membrane opposite the kinocilium. Stabi-
lization of the cuticular plate itself and how it might be anchored
has only been adequately explained in cochlear outer hair cells:
the cuticular plate extends to the apicolateral wall of the cell (23–
25), and stereocilia insert in the lateral cell membrane while still
within the cuticular plate (23). This process is not the case in inner
hair cells nor in vestibular hair cells (26). What provides a foun-
dation for the cuticular plate and thus stabilizes its position?
Jaeger et al. (27) showed that the cuticular plate in bullfrog hair
cells was connected to the axial cytoskeleton through a well-
defined bundle of microtubules. The present data show that the
on the cell membrane: the rootlets bend and extend through the
cuticular plate, inserting into the plasma membrane at two points
opposite the kinocilium, and the apical SO inserts into the apical
cell membrane surrounding the kinocilium. These three attach-
ments potentially form a tripod-like structure, coupling stereocilia
and the kinocilium with subcuticular cytoskeletal elements, pos-
sibly affecting mechanotransduction.
The bending of SRs inside the cuticular plate is an intriguing
finding. The only previous description of such bending is a single
photomicrograph of a bent SR (26). There may be a molecular
gram section of cell 1, a juxtastriolar type I cell, showing the connection
between a SR (arrowhead) and a SO thick filament bundle (arrow). (B) Three-
dimensional reconstruction of the same type I hair cell from a volume
obtained by joining six serial tomograms (3.0 μm total thickness). The apical
part of the SO extends from the lateral aspect of the cell membrane (arrow)
to the apical hair cell membrane near the kinocilium. Two SRs are continuous
with the SO (orange, arrowheads). SO thick (dark blue) and thin (light blue)
filament bundles envelop a group of large mitochondria (transparent light
blue). One possible effect on cell shape and hair bundle tension, based on
filament direction and cell membrane insertions is illustrated: radial con-
traction of the SO elongates the neck of the hair cell (vertical two-headed
arrow), which simultaneously splays the hair bundle through the connection
of the rootlets with the SO (horizontal two-headed arrow). (C–F) Cell 2, an
extrastriolar type I cell, modeled by joining 11 serial tomograms (total vol-
ume thickness = 5.5 μm). (C) One section from this tomogram shows a 110°
bend in two of the longest rootlets (arrowheads). An apical extension of the
SO (arrows) near the kinocilium (not seen in this section), is also shown in the
model (see below, F and F Inset, arrows). (Inset) Section from cell 5, a striolar
type I hair cell, fortuitously illustrating four bent rootlets in a single plane.
(D) The lower of two SRs in C (arrowheads) traverses the cuticular plate and
inserts into the SR insertion area (arrow) on the cell membrane on the op-
posite side of the cell. (E) A view from the top of the hair cell in C and D.
Inside the cuticular plate, long, bent SRs (yellow, arrowheads), originating
near the kinocilium and identical to those in C and D, traverse the cuticular
plate and insert in the plasma membrane; thinner, green stereocilia (located
farther from the kinocilium) do not traverse the cuticular plate. (F) Lateral
view of the 3D model of cell 2. Thick (dark blue) and thin (light blue) SO
filament bundles are distinguished. Arrows point to an extension of the SO
that encircles the kinocilium. Nearby, a kinociliar rootlet (gray, arrowheads)
extends from the kinocilium down toward the neck of the hair cell; another
(arrowheads) is observed extending from the centriole (asterisk). (Inset)
Tomograms and reconstructions of two type I hair cells. (A) Tomo-
Viewed from the kinociliar side, the same model shows apical insertions of
the SO (arrows) on each side of the kinocilium. (Scale bars, 0.5 μm in A–D,
and C Inset); 1 μm in E and F.) Cal, calyx; CP, cuticular plate; KC, kinocilium;
Vranceanu et al.PNAS Early Edition
| 3 of 6
correlate for this bend. Actin is the major component of SRs (16,
to that produced by the actin-related proteins, Arp2/3 (actin-
related protein 2/3 complex) (29–31) and coronin (32). In our
material, only the longest and thickest rootlets, derived from
plate, exhibited this 110° angle; shorter rootlets (those that did not
emerge from the cuticular plate) exhibited less acute angles.
Another prominent feature is the aggregation of large mito-
chondria in the subcuticular region of type I cells. Mitochondrial
functional capacity is related to both overall size and internal
structure (33). Such subcuticular aggregation of mitochondria with
large volumes and surface areas, and “tethering” (34, 35) indicate
the need for tight control of calcium homeostasis or that the SO,
highenergetic requirements. Recent experiments in cochlear outer
hair cells demonstrated that apical mitochondria can act to block
Ca2+diffusion into the hair-cell soma (36). In addition, mito-
chondrial crista structure has recently been related to the high
can provide structural details relevant to the functional status of
surface area, and analysis of crista junction diameter and density).
Previous immunohistochemical studies have provided data on
proteins in this part of the cell. The cuticular plate of vestibular
hair cells is immunoreactive for actin, tropomyosin, and myosin
(16, 37). Demêmes and Scarfone (38) demonstrated fodrin (α-2
spectrin) immunoreactivity in the SO and cuticular plate and
suggested that fodrin participates in a Ca2+-dependent cross-
linking of actin filaments. Although we have not yet been able to
confirm actin, we have confirmed by EM immunogold, Western
blot, and mass spectrometry that α-2 spectrin is an integral,
major component of the SO.
Physiological studies support a contractile hypothesis for
something in the neck of the type I, but not type II, hair cell. K+
depolarization induces not only reversible shape changes in type I
hair cells (39–41), but also a rise in cytosolic Ca2+concentration
(42) that could trigger such changes. Rising Ca2+levels have been
suggested as a potential substrate for mechanotransduction (3),
with the large SO-associated mitochondria potentially playing
a role (36, 43) by promoting reuptake of Ca2+after mechano-
transduction. A prior study (44) has shown that in the presence of
local elevated calcium levels, fodrin (α-2 spectrin) can change
from a diffuse distribution within the cytoplasm to a submem-
branous position, or a patch. If this were also the case in hair cells,
it is conceivable that the SO could change its dimensions and ri-
gidity depending on variations in intracellular Ca2+concentration
(with the latter regulated, in part, by the large subcuticular mito-
chondria), and thus influence hair bundle or cuticular plate
movements, or tilting of the cell neck. Any of these actions could
alter the sensitivity of the transduction apparatus, and thus,
mechanotransduction (45). Rüsch and Thurm (46) showed that
hair bundles at different sites on the sensory epithelium exhibit
differences in amplitude and in the time course of deflection. Our
data suggest that some of these differences could be because of
variationsincuticular plate size,hair-bundlesize,SOshape,cross-
linking, or perhaps even mitochondrial size.
Our findings suggest a dual function for the SO in type I hair
cells. One structural role is as a cytoskeletal specialization
aments. (A) Cell 1: SRs can terminate on subcuticular mitochondria (arrow-
heads); left arrowhead points to a mitochondrion that receives two distinct
rootlets. (B) Model view of area enclosed in white box in A shows contacts
between the distal end of several rootlets and subcuticular mitochondria.
Two black arrowheads are identical to those in A; two white arrowheads
point to additional mitochondrial-rootlet connections. (C and D) Cell 2: SO
filaments are closely associated (arrowheads) with subcuticular mitochon-
dria, as seen in a tomogram section (C) and in a model view (D) that high-
lights linkages (red) between the SO and subcuticular mitochondria. (Scale
bars, 0.25 μm.) CP, cuticular plate; M, mitochondria.
Connections between subcuticular mitochondria and SRs or SO fil-
modeled by joining nine serial tomograms (total volume thickness = 4.5 μm).
(A) A large portion of the SO is visualized in a single plane in SLICER mode,
illustrating the planar nature of the SO in type II cells. Morphing of a thick
bundle into a thin one can be observed (arrowhead). (B) A partial 3D re-
construction of the same hair cell. “D” in B refers to the view in D (i.e., from
under the cuticular plate). (C) In another section from the same hair cell,
note the dense spherical objects (arrowheads, depicted as pink spheres in B
and D) aligned with many of the thin filaments along the margins of the SO.
(D) View from below the cuticular plate: SRs are not connected to the SO,
but may be associated with the dense spherical objects (pink spheres). Most
mitochondria, except for those in close association with the SO, have been
removed for clarity. (Scale bars, 0.5 μm.) CP, cuticular plate; KC, kinocilium;
M, mitochondria; *, centriole.
The striated organelle in cell 4, an extrastriolar type II hair cell,
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| www.pnas.org/cgi/doi/10.1073/pnas.1101003109Vranceanu et al.
responsible for the constricted neck characteristic of these hair cells
(10, 15, 18). The architecture of the SO in type I and type II cells
is one of many differences, besides IK,L(the outward potassium
current present in type I hair cells), of a functional difference be-
tween type I and type II cells (47). Another potential structural role
is to provide a tripartite foundation for the cuticular plate. Neither
structuralrequirementisobviouslypresent intype IIhaircells, nor
was a tripartite arrangement present in the one type II hair cell
we reconstructed. Its columnar shape is similar to many columnar
epithelial cells lacking an SO. Concerning a dynamic role, the
overallarchitectureoftheapical regionintype I cells—a clusterof
large mitochondria constrained between the SO and the cuticular
plate, the plasma membrane and the adjacent calyx, the dense-
cored vesicles—suggests that the SO is part of a larger functional
complex regulating mechanotransduction or other physiological
functions. Further analysis of the morphology, composition, and
location, in combination with physiological studies, should shed
light on its function.
Materials and Methods
Animals. Normal adult chinchillas (Chinchilla lanigera) were used for tomo-
graphic studies. Adult Long-Evans rats (Rattus norvegicus), weighing 230–
330 g, were used for immunochemistry studies. For the development studies,
14 Long-Evans rat pups were studied. Two pups were killed on each day
(beginning at birth, P0 to P6), one for immunofluorescent studies and the
other for TEM. The Institutional Animal Care and Use Committee at the
University of Illinois approved procedures involving animals.
and for IVEM. Our TEM methods were described previously (45). We analyzed
a total of 41 tomograms using research facilities at the National Center for
Microscopy and Imaging Research. For each reconstruction, a series of
images at regular tilt increments was collected as described previously (30).
Three-Dimensional Reconstruction. Pixel sizes in our reconstructions were
2.8 nm and 1.96 nm. IMOD software (46) was used for alignment. Warped
reconstructions were processed with TxBR software (47). Measurements
were made with 3dmod (“Object info” feature). Stereocilia and rootlet
perimeters were measured at the same distance above the cell membrane
(5.6 nm). Volumes and surface areas of mitochondria below the cuticular
plate and within 6 μm of the apical surface of the cell were also measured.
Movies were made with Amira software (version 5.2.1, Mercury/TGS).
anticalretinin (Chemicon). Secondary antibodies for confocal experiments
component of the SO. (A) Confocal mi-
croscopy shows cuticular plate (arrows)
and SO (arrowheads) immunoreactivity
for α-2 spectrin (red). Calretinin antibody
(green) distinguishes type II (II) from
type I (I) hair cells. (Inset) Higher mag-
nification Volocity reconstruction of α-2
spectrin label (red) in a type II hair cell
shows the SO, which appears to hang
down in two large flaps (arrowheads)
from the cuticular plate (CP). (Scale bars,
2 μm.) (B) EM immunogold with an an-
tibody to α-2 spectrin (gold particles)
localizes the protein to the cuticular
plate (arrowheads) in a type I cell (I) and
to the SO (black box) in a type II cell (II).
(Inset) Higher magnification of the area
enclosed within the box in B. Gold par-
ticles (arrows) had a tendency to be lo-
cated over the cross-links between the
thick and thin filament bundles. (Scale
bars, 0.5 μm.) (C) Quantification of the
α-2 spectrin EM immunogold results. In
14 SO profiles, such as this example (SO)
from a type II hair cell (II), gold particles
were identified within an area delin-
eated by a white line circumscribing
the SO profile. Starting with each thick
filament bundle, intervals between thick
bundles were divided into eight equally
spaced samples running parallel to the
thick bundles, and the interval into
which each particle fell was determined.
(Inset) For 363 particles, sums in the
eight sample bins are illustrated, super-
imposed upon a schematic of the SO. A
χ2test of homogeneity (χ2= 23.1, df = 7,
P < 0.002) indicated a preference for
intervals immediately adjacent to the
thick filament bundles. (D–F) Confocal
microscopy of α-2 spectrin antibody in
developing rat crista in the first post-
natal week. (D) At birth (P0), there is no
label in the cuticular plate (CP, short ar-
row), and only weak immunoreactivity in the upper half of the hair cell. (E) At P2, α-2 spectrin antibody labels the cuticular plate less intensely than at P6 (F),
when it also labels parts of the lateral membrane in the region of the SO. (Scale bars, 5 μm.) (G–J) Normal TEM of developing rat hair cells. At P2 (G and H), the
SO is just beginning to form in a hair cell of indeterminate type. The calyx ending, which defines type I hair cells, typically does not begin to form until P4 (21).
(Scale bars in G and H, 0.5 μm.) At P6 (I and J), both the calyx (Cal) surrounding the type I cell and the SO (arrowheads) are well developed. Several stereociliar
rootlets can also be observed within the cuticular plate in I. (Scale bars, 0.5 μm in I and 2 μm in J.)
The α-2 spectrin is one protein
Vranceanu et al.PNAS Early Edition
| 5 of 6
(Chemicon) were: Alexa 488-conjugated donkey anti-goat; Alexa 594-conju-
gated donkey anti-mouse. Secondary antibody for EM immunogold experi-
ments (Aurion, EM Sciences) were: gold-conjugated rabbit anti-mouse. We
used calretinin antibody as a marker of hair-cell type (48).
Immunohistochemistry. Three rats were used for immunohistochemical
studies and three for Western blots. Fixation, confocal microscopy, and EM
immunogold procedures were identical to those described in a recent study
(48), which can be consulted for details.
EM Immunogold Quantification. To test if there were a nonuniform distri-
bution of particles, we divided SO profiles into intervals starting with each
thickfilamentbundle,dividedeachinterval intoeight equally spacedsamples
parallel to the filament bundles, and determined the interval into which each
particle fell. A χ2test of homogeneity was performed.
More detailed descriptions of EM tomography, immunogold quantifica-
tion, mass spectrometry, and Western blot analysis are given in SI Materials
ACKNOWLEDGMENTS. We thank Mr. Steven D. Price, Mr. John Crum, and Mr.
Biomedical Consortium-University of Illinois at Chicago Proteomics, Metabolo-
established in part by a grant from The Searle Funds at the Chicago Community
spectrometry data; and Dr. Jay M. Goldberg and the anonymous reviewers for
providing critical comments on a previous version of the manuscript. This work
was supported by National Institutes on Deafness and other Communication
Disorders Grant DC-02521 and a grant from the American Hearing Research
Foundation (to A.L.); the 2008 Tallu Rosen Grant in Auditory Science from the
National Organization for Hearing Research Foundation (to A.L. and F.V.); and
National Center for Recearch Resources Grant P41 RR004050 (to M.H.E.).
1. Dierkes K, Lindner B, Jülicher F (2008) Enhancement of sensitivity gain and fre-
quency tuning by coupling of active hair bundles. Proc Natl Acad Sci USA 105:
2. Fettiplace R, Hackney CM (2006) The sensory and motor roles of auditory hair cells.
Nat Rev Neurosci 7:19–29.
3. Hudspeth AJ (2005) How the ear’s works work: Mechanoelectrical transduction and
amplification by hair cells. C R Biol 328:155–162.
4. DeRosier DJ, Tilney LG (1989) The structure of the cuticular plate, an in vivo actin gel. J
Cell Biol 109:2853–2867.
5. Forge A, Wright T (2002) The molecular architecture of the inner ear. Br Med Bull 63:
6. Tilney MS, et al. (1989) Preliminary biochemical characterization of the ste-
reocilia and cuticular plate of hair cells of the chick cochlea. J Cell Biol 109:1711–
7. Friedmann I, Cawthorne T, McLay K, Bird ES (1963) Electron microscopic observations
on the human membranous labyrinth with particular reference to Méniére’s Disease.
J Ultrastruct Res 49:123–138.
8. Friedmann I, Cawthorne T, Bird ES (1965) Broad-banded striated bodies in the sensory
epithelium of the human macula and in neurinoma. Nature 207:171–174.
9. Ross MD, Bourne C (1983) Interrelated striated elements in vestibular hair cells of the
rat. Science 220:622–624.
10. Slepecky N, Hamernik R, Henderson D (1981) The consistent occurrence of a striated
organelle (Friedmann body) in the inner hair cells of the normal chinchilla. Acta
11. Spoendlin H (1966) The organization of the cochlear receptor. Adv Otorhinolaryngol
12. Hoshino T (1975) An electron microscopic study of the otolithic maculae of the lam-
prey (Entosphenus japonicus). Acta Otolaryngol 80:43–53.
13. Mørup Jørgensen J (1982) Microtubules and laminated structures in inner ear hair
cells. Acta Otolaryngol 94:241–248.
14. Peusner KD, Lindberg NH, Mansfield PF (1988) Ultrastructural study of calycine syn-
aptic endings of colossal vestibular fibers in the cristae ampullares of the developing
chick. Int J Dev Neurosci 6:267–283.
15. Slepecky N, Hamernik RP, Henderson D (1980) A re-examination of a hair cell or-
ganelle in the cuticular plate region and its possible relation to active processes in the
cochlea. Hear Res 2:413–421.
16. Slepecky N, Chamberlain SC (1985) Immunoelectron microscopic and immunofluo-
rescent localization of cytoskeletal and muscle-like contractile proteins in inner ear
sensory hair cells. Hear Res 20:245–260.
17. Takasaka T, Shinkawa H, Hashimoto S, Watanuki K, Kawamoto K (1983) High-voltage
electron microscopic study of the inner ear. Technique and preliminary results. Ann
Otol Rhinol Laryngol Suppl 101:1–12.
18. Sans A (1989) Ultrastructural study of striated organelles in vestibular sensory cells of
human fetuses. Anat Embryol (Berl) 179:457–463.
19. Auer M, et al. (2008) Three-dimensional architecture of hair-bundle linkages revealed
by electron-microscopic tomography. J Assoc Res Otolaryngol 9:215–224.
20. Lenzi D, Runyeon JW, Crum J, Ellisman MH, Roberts WM (1999) Synaptic vesicle
populations in saccular hair cells reconstructed by electron tomography. J Neurosci 19:
21. Rüsch A, Lysakowski A, Eatock RA (1998) Postnatal development of type I and type II
hair cells in the mouse utricle: acquisition of voltage-gated conductances and dif-
ferentiated morphology. J Neurosci 18:7487–7501.
22. Géléoc GS, Holt JR (2003) Developmental acquisition of sensory transduction in hair
cells of the mouse inner ear. Nat Neurosci 6:1019–1020.
23. Furness DN, Mahendrasingam S, Ohashi M, Fettiplace R, Hackney CM (2008) The di-
mensions and composition of stereociliary rootlets in mammalian cochlear hair cells:
Comparison between high- and low-frequency cells and evidence for a connection to
the lateral membrane. J Neurosci 28:6342–6353.
24. Liberman MC, Dodds LW (1987) Acute ultrastructural changes in acoustic trauma:
Serial-section reconstruction of stereocilia and cuticular plates. Hear Res 26:45–64.
25. Nunes FD, et al. (2006) Distinct subdomain organization and molecular composition
of a tight junction with adherens junction features. J Cell Sci 119:4819–4827.
26. Liberman MC (1987) Chronic ultrastructural changes in acoustic trauma: Serial-section
reconstruction of stereocilia and cuticular plates. Hear Res 26:65–88.
27. Jaeger RG, Fex J, Kachar B (1994) Structural basis for mechanical transduction in the
frog vestibular sensory apparatus: II. The role of microtubules in the organization of
the cuticular plate. Hear Res 77:207–215.
28. Tilney LG, DeRosier DJ (1986) Actin filaments, stereocilia, and hair cells of the bird
cochlea. IV. How the actin filaments become organized in developing stereocilia and
in the cuticular plate. Dev Biol 116:119–129.
29. Maiti S, Bamburg J (2004) Actin-capping and -severing proteins. Encylopedia of Bi-
ological Chemistry, eds Lennarz WJ, Lane MD, pp 19–26.
30. Mullins RD, Heuser JA, Pollard TD (1998) The interaction of Arp2/3 complex with actin:
Nucleation, high affinity pointed end capping, and formation of branching networks
of filaments. Proc Natl Acad Sci USA 95:6181–6186.
31. Saarikangas J, Zhao H, Lappalainen P (2010) Regulation of the actin cytoskeleton-
plasma membrane interplay by phosphoinositides. Physiol Rev 90:259–289.
32. Cai L, Makhov AM, Schafer DA, Bear JE (2008) Coronin 1B antagonizes cortactin and
remodels Arp2/3-containing actin branches in lamellipodia. Cell 134:828–842.
33. Perkins GA, et al. (2010) The micro-architecture of mitochondria at active zones:
Electron tomography reveals novel anchoring scaffolds and cristae structured for
high-rate metabolism. J Neurosci 30:1015–1026.
34. Boncompagni S, et al. (2009) Mitochondria are linked to calcium stores in striated
muscle by developmentally regulated tethering structures. Mol Biol Cell 20:
35. Csordás G, et al. (2010) Imaging interorganelle contacts and local calcium dynamics at
the ER-mitochondrial interface. Mol Cell 39:121–132.
36. Beurg M, Nam JH, Chen Q, Fettiplace R (2010) Calcium balance and mechano-
transduction in rat cochlear hair cells. J Neurophysiol 104:18–34.
37. Hasson T, et al. (1997) Unconventional myosins in inner-ear sensory epithelia. J Cell
38. Demêmes D, Scarfone E (1992) Fodrin immunocytochemical localization in the stri-
ated organelles of the rat vestibular hair cells. Hear Res 61:155–160.
39. Zenner HP (1986) Motile responses in outer hair cells. Hear Res 22:83–90.
40. Didier A, Decory L, Cazals Y (1990) Evidence for potassium-induced motility in type I
vestibular hair cells in the guinea pig. Hear Res 46:171–176.
41. Tanigawa T (1997) Motility of the vestibular hair cell of the guinea pig and bull frog.
Nippon Jibiinkoka Gakkai Kaiho, 100:264.275. Japanese.
42. Chabbert C, Devau G, Sladeczek F, Lehouelleur J, Sans A (1991) Intracellular free
calcium in isolated vestibular hair cells and potassium iontophoresis. Neuroreport 2:
43. Szabadkai G, Duchen MR (2008) Mitochondria: The hub of cellular Ca2+signaling.
Physiology (Bethesda) 23:84–94.
44. Perrin D, Aunis D (1985) Reorganization of alpha-fodrin induced by stimulation in
secretory cells. Nature 315:589–592.
45. Rabbitt RD, Boyle R, Highstein SM (2010) Mechanical amplification by hair cells in the
semicircular canals. Proc Natl Acad Sci USA 107:3864–3869.
46. Rüsch A, Thurm U (1989) Cupula displacement, hair bundle deflection, and physio-
logical responses in the transparent semicircular canal of young eel. Pflugers Arch
47. Rüsch A, Eatock RA (1996) A delayed rectifier conductance in type I hair cells of the
mouse utricle. J Neurophysiol 76:995–1004.
48. Lysakowski A, et al. (2011) Molecular microdomains in a sensory terminal, the ves-
tibular calyx ending. J Neurosci 31:10101–10114.
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| www.pnas.org/cgi/doi/10.1073/pnas.1101003109Vranceanu et al.
Vranceanu et al. 10.1073/pnas.1101003109
SI Materials and Methods
Animals. Normal adult chinchillas (Chinchilla lanigera) were used
for tomographic studies. Adult Long-Evans rats (Rattus norvegi-
For the development studies, 14 Long-Evans rat pups were
studied. Two pups were killed on each day [from birth (postnatal
for transmission electron microscopy (TEM). Procedures in-
volving animals were approved by the Institutional Animal Care
and Use Committee at the University of Illinois at Chicago.
FixationforTomographicStudies.Five adult chinchillas were deeply
anesthetized with Nembutal (80 mg/kg) and perfused trans-
cardially with 200 mL physiological saline containing heparin
(2,000 IU) followed by 8 mL/g body weight of a trialdehyde
fixative, as described previously (1). After perfusion, vestibular
epithelia were dissected in 0.1 M PB at 4 8C.
Electron Microscope Tomography. We used serial sections of
chinchilla utricular macula for conventional TEM and for in-
termediate-voltage electron microscopy (IVEM). Our TEM
methods have been described previously (1).
Semithin sections (0.5 μm) for IVEM were sectioned with
a diamond Histoknife (Diatome) and serial sections were col-
lected onto Formvar-coated or Formvar/Luxel-coated, carbon-
coated, 1 × 2-mm single slot grids (Ted Pella). All sections were
stained with 2% (wt/vol) uranyl acetate (10 min) and Sato lead
stain (10 min), and then carbon-coated again. Fiducial cues
consisting of 20-nm colloidal gold particles were deposited on
one side of each section.
We analyzed a total of 41 tomograms using researchfacilities at
the National Center for Microscopy and Imaging Research. For
each reconstruction, a series of images at regular tilt increments
was collected as described previously (2). Tilt series were re-
corded with a Gatan 4 K × 4 K camera at 5,000× magnification.
We used a magnification of 5,000× to comfortably include the
entire circumference of the apical part of type I hair cell, the
proximal part of the hair bundle, and most of the type I hair cell
constriction (neck) in the field of interest. The z-resolution of the
tomographic volumes was 8 nm, compared with the 70-nm
thickness of ultrathin sections, which allowed us to visualize
objects at a much higher effective z-depth resolution (8 nm vs. 70
nm) compared with conventional EM. Some elements were re-
examined at high magnification (20,000–25,000×).
Three-Dimensional Reconstruction. The pixel sizes in our recon-
structions were 2.8 nm and 1.96 nm. The IMOD software package
(3) was used for rough alignment, and in some cases for fine
alignment and reconstruction. Warped reconstructions were
processed (fine alignment and reconstruction) using the TxBR
software package (4). Serial tomograms of hair cells were joined
using Etomo (IMOD) and four final volumes were obtained:
three type I hair cells, (cell 1 from 6 serial tomograms, cell 2
from 11 serial tomograms, and cell 3 from 5 serial tomograms),
and one type II hair cell (cell 4, from 9 serial tomograms). For
each final volume (tomogram), segmentation was performed by
manual tracing in the planes of highest resolution and by auto-
mated isosurface rendering with the program 3dmod (IMOD).
Reconstructions were visualized using 3dmod. Cell 5, a striolar
type I hair cell, and cell 6, an extrastriolar type II hair cell, were
visualized with 3dmod as eight and three serial tomograms, re-
spectively, but not segmented. The program allows stepping
through slices of the reconstruction in any orientation (SLICER
option) and tracking or modeling features of interest in any of
the three dimensions. For quantification, measurements were
made using 3dmod (“Object info” feature). Stereocilia and
rootlet perimeters were each measured at the same distance
above the cell membrane (5.6 nm), and the volumes and surface
areas of mitochondria below the cuticular plate within 6 μm of
the apical surface of hair cell were also measured. Movies of
surface-rendered volumes and slices through the reconstructions
were made using Amira software (version 5.2.1, Mercury/TGS).
EM Immunogold Quantification. To test if there were a nonuniform
distribution of particles, we examined 14 striated organelles (SO)
profiles from 12 hair cells (five type I and seven type II) and
divided them into intervals. Starting with each thick filament, an
interval was divided into eight equally spaced samples running
which each particle fell. A χ2test of homogeneity using “a single
classification with equal expectations” (χ2= 23.1, df = 7, P <
0.002) indicated a preference for the intervals immediately ad-
jacent to the thick filament.
Antibodies. Primary antibodies used were: mouse anti α-2 spectrin
(Chemicon), goat anti-calretinin (Chemicon). Secondary anti-
bodies for confocal experiments (Chemicon) were: Alexa 488-
conjugated donkey anti-goat; Alexa-594-conjugated donkey anti-
mouse. Secondary antibody for EM immunogold experiments
We used calretinin antibody as a marker of hair-cell type (5).
Immunohistochemistry. Three animals were used for immunohis-
tochemical studies andthree forWestern blots. Fixation, confocal
microscopy, and EM immunogold procedures were identical to
those described in a recent study (5), which can be consulted
for details. Western blots were done to verify that the hair-cell
staining found in rat endorgans was α-2 spectrin. For Western
blots, animals were anesthetized (Nembutal, 80 mg/kg, i.p.) and
decapitated. Vestibular endorgans, cochleae, vestibular ganglion,
brain (positive control), and vibrissae (negative control) were
harvested from three adult rats within 10 min of killing. Methods
were identical to those published previously (5), except that the
ECL detection kit was obtained from GE Healthcare Life Sci-
ences. Antibody incubation conditions were mouse anti–α-2
spectrin antibody (diluted 1:2,000) for 4 h at room temperature,
washed 3 × 5 min in PBS-Tween, and HRP-conjugated goat anti-
mouse IgG (diluted 1:30,000) for 1 h at room temperature. The
identities of bands from Western blots were confirmed with mass
spectrometry in the University of Illinois at Chicago Proteomics
Laboratory. Scaffold software (v.22.214.171.124, Proteome Software) was
used to validate mass spectrometry-based peptide and protein
Mass Spectrometry. Analysis was performed by the University of
Illinois at Chicago Research Resources Center’s Mass Spectrom-
etry, Metabolomics, and Proteomics Facility. The in-gel tryptic
digestion was performed according to the protocol described by
and alkylated with iodoacetamide in the dark, before overnight
digestion with trypsin at 37 8C in 50 mM ammonium bicarbonate.
Peptides were concentrated and analyzed with a Thermo Orbitrap
Velos mass spectrometer using a chip-based HPLC system (Agi-
Vranceanu et al. www.pnas.org/cgi/content/short/1101003109 1 of 4
lent Chip Cube) adapted to run on the Orbitrap Velos (7) using
collision-induced dissociation fragmentation.
exe (version 4.0.2, Institute for Systems Biology), converted to the
Mascot generic format using MzXML2Search and then submitted
to a Mascot search engine (version 2.2.04). Charge state decon-
assuming the digestion enzyme was trypsin. Mascot was searched
with a fragment ion mass tolerance of 0.60 Da and a parent ion
tolerance of 10.0 ppm. Oxidation of methionine, acetylation of
lysine, and the N terminus and iodoacetamide derivative of cyste-
ine were specified in Mascot as variable modifications.
Criteria for Protein Identification. Scaffold software (v. 126.96.36.199,
Proteome Software) was used to validate MS/MS-based peptide
and protein identifications. Peptide identifications were accepted
if they could be established at greater than 95.0% probability as
specified by the Peptide Prophet algorithm (8) and if they con-
tained at least two identified peptides. Protein probabilities were
assigned by the Protein Prophet algorithm (9). Proteins that
contained similar peptides and could not be differentiated based
on MS/MS analysis alone were grouped to satisfy the principles
1. Lysakowski A, Goldberg JM (1997) A regional ultrastructural analysis of the cellular and
synaptic architecture in the chinchilla cristae ampullares. J Comp Neurol 389:419–443.
2. Perkins GA, et al. (2010) The micro-architecture of mitochondria at active zones:
Electron tomography reveals novel anchoring scaffolds and cristae structured for high-
rate metabolism. J Neurosci 30:1015–1026.
3. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-
dimensional image data using IMOD. J Struct Biol 116:71–76.
4. Lawrence A, Bouwer JC, Perkins G, Ellisman MH (2006) Transform-based backprojection
for volume reconstruction of large format electron microscope tilt series. J Struct Biol
5. Lysakowski A, et al. (2011) Molecular microdomains in a sensory terminal, the vestibular
calyx ending. J Neurosci 31:10101–10114.
6. Kinter M, Sherman NE (2000) Protein Sequencing and Identification Using Tandem
Mass Spectrometry (Wiley-Interscience, New York), pp 152–160.
7. Schilling AB, Crot C, Helseth DL, Xu H, Davis RG (2010) Conversion of an Agilent Chip
Cube system for the analysis of proteomics samples using a LTQ-FT Ultra mass
spectrometer. 58th ASMS Conference on Mass Spectrometry, Salt Lake City, UT May
24–27, 2010, Poster # MP612.
8. Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to
estimate the accuracy of peptide identifications made by MS/MS and database search.
Anal Chem 74:5383–5392.
9. Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying
proteins by tandem mass spectrometry. Anal Chem 75:4646–4658.
rootlets (SR) in a striolar type I hair cell are directly continuous with stereocilia (arrowheads) and enter the SO (SO, short arrows). In the lower part of the neck,
long arrows show insertions of the thick SO filaments into the cell membrane. Some microtubules (Mt, long, thin arrows) in A and C connect SRs to the SO
below the central portion of the cuticular plate. Vesicular structures (asterisks in B and C) line up in parallel with microtubules (Mt) and the SO. (D) Section
through the median plane of a type I hair cell, showing gray bands (short arrows) that lie between the thick filaments of the SO forming a narrow band along
the cell membrane. Our EM immunogold results indicate this gray band is α-2 spectrin. (Scale bar in D, 1 μm; applies to all panels.) CP, cuticular plate; M,
Serial section TEM of SOs. Ultrathin sections from a serial section study of type I vestibular hair cells in chinchilla crista. (A and B) Actin stereociliar
Vranceanu et al. www.pnas.org/cgi/content/short/11010031092 of 4
ganglia (VG), and rat cochleae labeled with an anti–α-2 spectrin antibody. The blot shows two spectrin bands. The upper band corresponds to the full-length
α-2 spectrin protein (285 kDa), the lower band to a well-characterized proteolytic fragment at ∼150 kDa (1). (B) Mass spectrometry results. (Upper) Results of
our mass spectrometry runs on the upper (285 kDa) band in our immunoprecipitation gels. With 46% coverage and 118 unique peptides, there is 95–100%
confidence that the band is α-2 spectrin. (Lower) Results of mass spec runs on the lower, 150-kDa band in our gels. Again, with 43% coverage and 108 unique
peptides, there is a similar level of confidence that this band is also α-2 spectrin.
Antibody validation. (A) Immunoblot of rat brain (positive control for α-2 spectrin), vibrissae (negative control), vestibular end organs (VO), vestibular
1. Harris AS, Croall DE, Morrow JS (1988) The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site. J Biol Chem 263:15754–15761.
Vranceanu et al. www.pnas.org/cgi/content/short/11010031093 of 4
Table S1. Subcuticular mitochondrial dimensions Download full-text
Type I hair cells Neighboring type II hair cells
n SA (106nm2) Vol (108nm3) SA/Vol ratioN SA (106nm2) Vol (108nm3) SA/Vol ratio
Cell 1 juxtastriolar
Cell 2 extrastriolar
Cell 3 extrastriolar
1.74 ± 0.79
1.58 ± 0.73
1.61 ± 0.81
1.64 ± 0.09
1.46 ± 0.61
1.61 ± 1.05
1.39 ± 0.92
1.49 ± 0.11
0.90 ± 0.56
0.75 ± 0.36
0.95 ± 0.56
0.87 ± 0.10
0.45 ± 0.29
0.38 ± 0.19
0.40 ± 0.21
0.41 ± 0.04
Samples, region and cell number from which samples were taken; n, number of mitochondria measured for each type of cell; Values, mean ± SD. Only
complete mitochondria from the subcuticular region (∼6 μm below the apical cell membrane) in type I and neighboring type II hair cells were reconstructed. As
mitochondrial function is related to overall size (in particular to surface area), mean volumes (Vol) and surface areas (SA) were measured for each organelle,
and surface to volume ratios (SA/Vol) were calculated. Student t test was used to compute significance levels for type I vs. type II mitochondrial surface areas
(P < 0.0001) and volumes (P < 0.0001).
presentation of modeled apical structures from cell 1 (a juxtastriolar type I hair cell): hair-cell membrane, cuticular plate, subcuticular mitochondria, kinocilium,
SRs, SO—thick and thin filaments—and connections between SRs and SO. Extracellular structures: calyx terminal, containing dense core vesicles. All labels have
been colored to match the color-coding of each modeled structure.
Movie showing digital sections through the tomogram (six serial semithin sections at 0.5 μm per section, a total of 3 μm joined volume) and
presentation of apical structures modeled from cell 2 (an extrastriolar type I hair cell): hair cell membrane, cuticular plate, subcuticular mitochondria, kino-
cilium, SRs, centriole and kinociliar rootlets, SO—thick and thin filaments—and SRIA (SR inserting areas). Extracellular structures: calyx terminal, containing
dense core vesicles. Labels have been colored to match the color-coding of each modeled structure.
Movie showing digital sections through the tomogram (11 serial semithin sections at 0.5 μm per section, a total of 5.5 μm of joined volume) and
Vranceanu et al. www.pnas.org/cgi/content/short/11010031094 of 4