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Cell Motility and the Cytoskeleton 38:146–162 (1997)
Localization of Microtubules Containing
Posttranslationally Modified Tubulin in
Cochlear Epithelial Cells During
Development
Joyce Tannenbaum1,2 and Norma B. Slepecky1,3*
1Department
of Bioengineering and Neuroscience, Institute for Sensory
Research, Syracuse University, Syracuse, New York
2Department of Biology, Syracuse University, Syracuse, New York
3Department of Anatomy and Cell Biology, SUNY Health Science Center,
Syracuse, New York
In the adult gerbil inner ear, hair cell microtubules contain predominantly
tyrosinated tubulin while supporting cell microtubules contain almost exclusively
other isoforms. This cell-type specific segregation of tubulin isoforms is unusual,
and in this respect the sensory and supporting cells in this sensory organ differ from
other cells observed both in vivo and in vitro. Thus, we hypothesized there must be
a shift in the presence and location of tubulin isoforms during development,
directly associated with the onset of specialized functions of the cells. We describe
the appearance and/or disappearance of tubulin isoforms in sensory hair cells and
five different supporting cells (inner and outer pillar cells, Deiters cells, cells of
Kölliker’s organ, and cells of the tympanic covering layer) during development of
the gerbil organ of Corti from birth to 14 days after birth. Tyrosinated tubulin was
initially present in all cells and remained predominant in cells that decrease in
number (Kölliker’s organ and tympanic covering layer) and exhibit active
processes such as secretion and motility (sensory cells). Posttranslational modifications occurred in the supporting cells in a time-dependent manner as the number
and length of microtubules increased and development proceeded, but the
establishment of elongated cell shape and polarity occurred prior to the appearance
of acetylation, detyrosination, and polyglutamylation of tubulin. In the pillar and
Deiters cells, posttranslational modifications progressed from cell apex to base in
the same direction as microtubule elongation. In the pillar cells, posttranslational
modifications occurred first at the apical surfaces. In the pillar cells, the appearance
of acetylated tubulin was rapidly followed by the appearance of detyrosinated
tubulin. In Deiters cells, the appearance of acetylated tubulin preceded the
appearance of detyrosinated tubulin by one or more days. At onset of cochlear
function, detyrosinated tubulin and acetylated tubulin had achieved their adult-like
pattern, but polyglutamylated tubulin had not. Cell Motil. Cytoskeleton 38:146–
162, 1997. r 1997 Wiley-Liss, Inc.
Key words: tubulin; posttranslational modifications; cochlea; organ of Corti; development
INTRODUCTION
Microtubules are prominent structures in the sensory and supporting cells of the organ of Corti in the inner
ear. Based on studies of other cell types, it is thought that
r 1997 Wiley-Liss, Inc.
*Correspondence to: Norma Slepecky, PhD, Dept. of Bioengineering
and Neuroscience, Institute for Sensory Research, Syracuse University,
Syracuse, NY 13244-5290. E-mail Norma_Slepecky@isr.syr.edu
Received 27 December 1996; accepted 30 April 1997
Modified Tubulin in Cochlear Epithelial Cells
their functions include: movement of subcellular organelles [Araki et al., 1993; Ayscough et al., 1993; Baker et
al., 1993], establishment of cell polarity [Hyde and
Hardham, 1993; Glasgow and Daniele, 1994], generating
and maintaining cell shape [Bulinski and Gundersen,
1991; Joshi and Baas, 1993], and provision of structural
support [Deanin et al., 1981; Kirschner and Mitchison,
1986; Bulinski and Gundersen, 1991; Audebert et al.,
1993]. In these cells, microtubules are divided into at
least two subpopulations consisting of tyrosinated tubulin
and posttranslationally modified isoforms (detyrosinated,
acetylated, polyglutamylated, and nontyrosinatable tubulin). They differ with respect to their kinetics of polymerization and depolymerization and their biochemical composition appears to be predictive of their dynamic
properties [Gundersen et al., 1984, 1987; Thompson et
al., 1984; Piperno et al., 1987; Schulze and Kirschner,
1987; Baas and Black, 1990].
The determination of cell polarity and shape and the
permanence of structures containing microtubules are
related to their dynamic properties [Gundersen et al.,
1987; Schulze and Kirschner, 1987]. Microtubules form
at nucleating centers and elongate when newly synthesized tubulin dimers are preferentially added to the free
(non-nucleated) ends. Microtubules shorten by depolymerization, at which time tubulin dimers are removed from
the free ends. Dynamic, or ‘‘short-lived,’’ microtubules
polymerize and depolymerize suddenly and rapidly as
microtubules extend and retract. This is associated with
changes in cell shape and movement. Stable, or ‘‘longlived,’’ microtubules display little or no turnover of
tubulin subunits, resulting in microtubules that are relatively constant in length. These would have the ability to
provide sustained tension and/or support.
Dynamic properties may be determined by the
biochemical composition of tubulin. In microtubules
where depolymerization and polymerization occur rapidly, tubulin is almost entirely in the tyrosinated form
[Schulze et al., 1987; Bulinski and Gundersen, 1991] and
its presence provides a marker for dynamic microtubules.
On the other hand, tubulin present in long-lived microtubules has an increased chance of being modified [Gundersen et al., 1987; Schulze et al., 1987; Bulinski and
Gundersen, 1991]. Thus, posttranslational modifications
are correlated with the presence of stable microtubule
populations and appear in a time-dependent manner
[Kirschner and Mitchison, 1986; Gundersen et al., 1987;
Piperno et al., 1987; Schulze and Kirschner, 1987; Baas
and Black, 1990; Paturle-Lafanechere et al., 1994].
Removal of tyrosine yields detyrosinated tubulin
and this modification may be restricted to tubulin in
microtubules [Arce et al., 1978; Kumar and Flavin, 1981;
Thompson, 1982; Gundersen et al., 1987]. Other modifi-
147
cations that occur primarily to polymerized tubulin include
the addition of an acetyl group to lysine residue 40 of
a-tubulin to form acetylated tubulin [L’Hernault and
Rosenbaum, 1983, 1985] and the addition of one or more
glutamyl residues to the glutamic acid residues at the
carboxy terminal end of a- and b-tubulin to form polyglutamylated tubulin [Eddé et al., 1990; Wolff et al., 1992].
Removal of the last two amino acids from the carboxy
terminal end of a-tubulin prevents tyrosine reattachment
and produces tubulin which is nontyrosinatable [Alonso
et al., 1993; Paturle-Lafanechere et al., 1994].
In many studies using tissue-culture, the appearance
of posttranslational modifications to tubulin is correlated
with the loss of dynamic microtubule activity and the
stabilization of cell shape. However, few model systems
exist to study the role of posttranslational modifications,
since most mature cells in vivo, and all cells in culture,
contain mixed populations of microtubules which are
stable and dynamic, as well as being tyrosinated and
posttranslationally modified. The organ of Corti of the
mammalian auditory system presents a unique system in
which to undertake developmental studies of microtubules in vivo because in the adult organ there are cells
which are actively motile (sensory cells) and cells which
provide structural support (pillar and Deiters cells).
Sensory hair cell microtubules are present in loose
networks [Slepecky and Chamberlain, 1985; Steyger et
al., 1989; Furness et al., 1990; Slepecky and Ulfendahl,
1992] and contain predominantly tyrosinated tubulin
[Slepecky et al., 1995]. In contrast, microtubules in the
supporting cells are in discrete, tightly packed bundles
[Angelborg and Engstrom, 1972] containing almost exclusively acetylated, detyrosinated, and polyglutamylated
tubulin [Slepecky et al., 1995].
Because this cell-type specific segregation is so
unusual, we hypothesized that there must be a shift during
development, from newly synthesized tyrosinated tubulin
to posttranslationally modified isoforms, directly associated with the onset of specialized cell functions. Specifically, we were interested in: 1) the relationship between
the appearance of posttranslational modifications to tubulin and the time during which cell polarity is established;
2) the temporal sequence of the posttranslational modifications to tubulin as it relates to changes in cell number,
shape, and size; and 3) how the location of microtubules
in the sensory and supporting cells changes as development proceeds and how this correlates with the onset of
hearing.
In the adult inner ear, the sensory epithelium of the
cochlea spirals around a central core of nerve fibers. In a
cross-section taken through each turn, sensory cells (one
inner and three outer hair cells) and supporting cells
(inner pillar, outer pillar, and three Deiters cells) can be
148
Tannenbaum and Slepecky
seen. Cells in the apical turn code for low-frequency
sounds and those in the middle and basal turns code for
progressively higher frequencies. In the gerbil, as in other
species [Romand, 1987; Lim and Rueda, 1992; Roth and
Bruns, 1992a, 1992b; Pujol et al., 1996], development of
the organ of Corti starts first in the basal turn and
progresses toward the apical turn [Ito et al., 1995; Souter
et al., 1995; Kuhn and Vater, 1996].
Common to all species is the initial presence of
Kölliker’s organ, made up of tall densely packed columnar cells containing irregularly shaped nuclei, and an
undifferentiated mass that will become the sensory and
supporting cells. Development proceeds (see Fig. 1) until
the onset of cochlear function, which coincides with the
disappearance of Kölliker’s organ, the formation of the
inner spiral sulcus, the maturation of the sensory and
supporting cells, the opening of the tunnel of Corti
between the inner pillar and outer pillar and the spaces of
Nuel between the outer hair cells and Deiters cells, the
maturation of the tectorial membrane and basilar membrane, and the formation of synapses with afferent and
efferent nerve terminals.
In the gerbil, these changes take place from 0–14
days after birth [Finck et al., 1972; Harris and Dallos,
1984; Arjmand et al., 1988; Echteler et al., 1989; Harris et
al., 1990; Woolf et al., 1992; Ito et al., 1995; Munyer and
Schulte, 1995; Souter et al., 1995; Kuhn and Vater, 1996;
Schweitzer et al., 1996]. Thus, the experiments described
in this paper were designed to study the appearance
and/or disappearance of the different isoforms of tubulin
during this time.
MATERIALS AND METHODS
Dissection
Following anesthesia by carbon dioxide inhalation,
both temporal bones were removed from gerbils at
several hours, one day (24–36 h), seven days, ten days, or
fourteen days after birth (dab). Prior to 10 dab, when the
middle ear space is not opened and the cochlea not
calcified, each cochlea was exposed and the temporal
bone was dropped directly into fixative. For gerbils 10
dab and older, holes were made in the apex and base of
the cochlea and fixative was perfused through the holes.
Both ears from two gerbils of the same age were
processed and sections were taken from all ears. The care
and use of animals was approved by the Syracuse
University IACUC.
Fixation
Cochleas were fixed in freshly made 3% paraformaldehyde, 0.1% glutaraldehyde, 0.1 M sodium phosphate
buffer (PB), pH 7.5, for approximately three days at room
temperature. Following one day to one week decalcification in 5% EDTA, cochleas were washed in PB, dehy-
Fig. 1. Development of the organ of Corti based on individual tissue
sections taken from the basal turn of the cochlea, selected from
specimens at different ages during development: dab 5 days after birth;
BM 5 basilar membrane; CC 5 Claudius cells; D 5 Deiters cells;
IHC 5 inner hair cells; IP 5 inner pillar cells; ISS 5 inner spiral
sulcus; KO 5 Kölliker’s organ; OHC 5 outer hair cells; OP 5 outer
pillar cells; TM 5 tectorial membrane; TC 5 tunnel of Corti; TCL 5
tympanic covering layer; SV 5 spiral vessel.
drated through increasing concentrations of alcohol and
propylene oxide, infiltrated in Araldite Durcupan ACM,
oriented, and hardened at 45°C for one week.
Light Microscopy
Representative one-micron midmodiolar sections
were cut from all ears. They were placed in drops of water
Modified Tubulin in Cochlear Epithelial Cells
on gelatin/chrome alum subbed slides, dried down at
room temperature, and stained with methylene blue and
Azure II. Sections were examined by light microscopy to
confirm that preservation by this fixation procedure,
which is optimal for immunocytochemistry, was adequate
to allow identification of the cell types and structures, and
that a progression of developmental stages was visible.
Camera lucida drawings from these sections were used to
make the diagrams in Figure 1.
For comparative purposes, most micrographs in this
paper are from the basal turn because it is the only one
present at all stages of development. However, since
development of the organ of Corti proceeds from the
basal to the apical turn, with a lag of 1–2 days in the most
apical portion of the cochlea, observation of cells in the
middle and apical turns provide information regarding
subtle changes in the developmental sequence.
Immunocytochemistry
For immunocytochemistry, Araldite was removed
from additional one-micron midmodiolar sections using a
solution of propylene oxide / absolute ethanol / sodium
hydroxide pellets. Sections were hydrated from ethanol to
water, washed in phosphate buffered saline (PBS) and
processed for immunocytochemistry as described previously [Slepecky and Ulfendahl, 1992]. Specimens were
treated sequentially with goat serum to block nonspecific
activity, primary antibodies diluted 1:100 in PBS, PBS as
a wash, goat serum as a blocker, secondary antibodies
diluted 1:200 in PBS, and PBS as a wash. Slides were
dipped in distilled water to remove salt, air dried, and
stored in the dark at room temperature until viewed. In
some cases, double-labeled sections were treated sequentially with one primary antibody followed by the secondary antibody and then another primary antibody followed
by the secondary antibody. In other cases, simultaneous
incubation in a mixture of both primary antibodies was
followed by simultaneous incubation in a mixture of the
appropriate secondary antibodies (each at the correct final
dilution). Control tissue sections were incubated in a
primary antibody and an inappropriate secondary antibody to show that there was no cross-reactivity of the
secondary antibodies used in the double-labeling experiments. Prior to use, the slides were coverslipped with
glycerol:n-propyl gallate used as mounting medium.
Tissue sections were photographed with a Zeiss Axioskop
microscope using a 1003 oil immersion lens, 100W
fluorescent light source, and Kodak TMax 400 film.
Primary Antibodies
Antibodies for alpha-tubulin (one of the two repeating subunits of tubulin in all microtubules), obtained from
Chemicon (El Segundo, CA, AB935) and Sigma ImmunoChemicals (St. Louis, MO, T9026) were used to localize
149
microtubules regardless of the presence of posttranslational modifications. Antibodies to tyrosinated and acetylated alpha-tubulin were obtained from Sigma ImmunoChemicals (St. Louis, MO, T9028 and T6793). Antibody
to polyglutamylated tubulin was a gift from Dr. Annie
Wolff, College de France, Paris, France [Wolff et al.,
1992]. Antibody to detyrosinated tubulin was a gift from
Dr. J. C. Bulinski, Columbia University, NY, [Gundersen
et al., 1984]. The antibodies were checked by immunoblotting for specificity to tubulin in control tissue (cerebellum) and organ of Corti [Slepecky et al., 1995].
Secondary Antibodies
Appropriate secondary antibodies coupled to fluorescent markers were used to localize primary antibody
binding. They included antibodies against rabbit immunoglobulins coupled to fluorescein (Accurate Chem. and
Sci., Westbury, NY, JGZ1508); antibodies against mouse
immunoglobulins coupled to fluorescein (Chemicon, Temecula, CA, AP130F); antibodies against rabbit immunoglobulins coupled to rhodamine (Accurate Chem. and
Sci., JGZ2508); antibodies against mouse immunoglobulins, coupled to rhodamine (Accurate Chem. and Sci.,
JGM2544).
RESULTS
Type and Location of Microtubules in the
Developing Organ of Corti
As obvious physical changes occurred in cells
during maturation of the organ of Corti, there were
concomitant alterations in microtubules—changes in number, cellular location, and biochemical composition. These
were observed immunocytochemically using isoform
specific antibodies to tubulin on tissue sections of the
developing organ of Corti. Not all results described in this
section can be seen on the few cross-sections through the
organ of Corti presented in the figures because of the
complex configuration of the cells (Fig. 2), especially in
the later stages of development when the cells have
increased in size. If one were to take as a point of
reference the apical surface of one outer hair cell and the
apical surfaces of its adjacent outer pillar and Deiters
cells, one would see that the base of the outer hair cell
slants in one direction (toward the apical turn of the
cochlea), while the basal portions of the pillar and Deiters
cells slant in the opposite direction (toward the base of the
cochlea). Depending on the plane of section, which
changes subtly as one sections through this spiral organ, it
is possible to obtain sections showing the entire length of
the outer hair cells (with only parts of pillar and Deiters
cell processes) or sections showing the entire length of
the outer pillar and Deiters cells (with only parts of outer
150
Tannenbaum and Slepecky
Fig. 2. Diagram showing the complex arrangement of the inner hair
cells (IHC), outer hair cells (OHC), inner pillar cells (IP), outer pillar
cells (OP), and Deiters cells (D) in the mature organ of Corti. OHC1
refers to a first row outer hair cell, and D1 refers to a Deiters cell that
supports a first row outer hair cell. There are three rows of outer hair
cells and three rows of Deiters cells. The microtubule bundles in each
of the supporting cells is identified: the portion of the transcellular
bundle in the apical surface process of the IP; the transcellular and
basal bundles in the pillar process of the IP; the pillar bundle in the OP;
the beam bundle in the OP; the basal bundle in the base of the D cells;
the bundle in the phalangeal process, and the apical process of the D
cells.
hair cells), but never sections showing the entire length of
these cells together.
a-Tubulin
One day after birth, staining with antibodies to
a-tubulin (Fig. 3A-1) showed microtubules throughout
all cells in the developing organ of Corti. There was
diffuse labeling, which is attributed to labeling of individual microtubules, since soluble tubulin should have
leached out of these preparations during processing
(personal communication, Dr. John Tucker). In the developing inner and outer sensory hair cells, networks of
microtubules were seen throughout the cell body, except
in the region of the developing cuticular plate at the apical
surface of these cells. Developing supporting cells were
identified by the position of their nuclei close to the
basilar membrane. While there was strong labeling of
microtubules in all cells, microtubules in the pillar cells
were arranged in parallel bundles rather than in loose
networks seen in the sensory cells and the cells of
Kölliker’s organ. Microtubules were most abundant (based
on the increased intensity of label) in the top half of the
inner pillar. Uniform labeling of microtubules was seen in
the developing Deiters cells, with staining intensity
similar to that seen in the sensory cells. Microtubules
were present in the kinocilia on the apical surface of all
cells in the developing organ of Corti.
Seven days after birth, all antibodies to tubulin and
tubulin isoforms used in this study gave weaker labeling
of sensory cells at this stage of development than at 1 dab
and 10 dab. The decreased intensity was consistently
found on tissue sections from all specimens, even though
they were fixed, processed. and stained at different times.
Antibodies for a-tubulin (Fig. 3A-7) weakly labeled
microtubules in most cells. The pillar cells appeared the
most obviously changed at this time. The inner pillar cell
was extending a process, so that the apical portion of the
inner pillar was slightly bent over the outer pillar. A
similar process was being extended by the outer pillar, to
eventually interdigitate between the outer hair cells in the
first row. The microtubule bundle in the inner pillar cell
was more extensively labeled than at 1 dab, suggesting an
increase in the number and length of the microtubules.
While the inner pillar microtubules displayed prominent
labeling along their length, the outer pillar cells showed
only weak labeling. The basal regions of the inner and
outer pillars were enlarging and the basal portions of both
showed only diffuse labeling. However, microtubules in
these regions and throughout the outer pillar bundle were
parallel to each other, unlike the loose networks seen
previously. Microtubules in the phalangeal processes of
the Deiters cells were labeled, but networks of microtubules in the base of the Deiters cells could not be
differentiated from the microtubules in the outer hair
cells. Kinocilia could no longer be detected.
At 10 dab, antibodies to a–tubulin (Fig. 3A-10)
showed that the organ of Corti is approaching adult-like
shape. Networks of microtubules remained in what was
left of Kölliker’s organ and the tympanic covering layer.
The inner and outer hair cells displayed discrete networks
of microtubules. Strongly labeled microtubule bundles
were found throughout the inner pillar cells (transcellular
and basal bundles) and outer pillar cells (beam and pillar
bundles). Distinct bundles of microtubules were observed
in the phalangeal processes and basal portions of the
Deiters cells.
At 14 dab, labeling with antibodies to a-tubulin
(Fig. 3A-14) showed that the organ of Corti was almost
fully mature. Microtubules were present in very diffuse
networks in what little remained of Kölliker’s organ and
the tympanic covering layer. Hair cells contained networks of microtubules, diffuse throughout the central
Modified Tubulin in Cochlear Epithelial Cells
Fig. 3. Immunocytochemical localization of tubulin isoforms in the organ of Corti as seen in tissue
sections taken from the basal turn of the cochlea at different ages (dab 5 days after birth). (A) Antibodies to
a-tubulin; (B) antibodies to tyrosinated tubulin; (C) antibodies to detyrosinated tubulin; (D) antibodies to
acetylated tubulin; (E) antibodies to polyglutamylated tubulin. Distribution of the different isoforms shifts
as development proceeds on days 1, 7, 10, 14, and 55 after birth. Long arrow, inner pillar cell; short arrow,
outer pillar cell; double arrows, Deiters cells; horizontal arrow, kinocilium; * 5 nerves.
151
152
Tannenbaum and Slepecky
Fig. 3. (Continued.)
Modified Tubulin in Cochlear Epithelial Cells
153
regions and dense just below the cuticular plates and near
the synapses. Microtubules were prominent in both
bundles in each of the pillar cells, displaying equal
intensity labeling in both cells.
Tyrosinated Tubulin
At 1 dab, antibodies specific for tyrosinated tubulin
(Fig. 3B-1) gave a labeling pattern that was almost
similar to that seen with antibodies to a-tubulin in most
cells of the organ of Corti. However, unlike the labeling
pattern seen with antibodies to a-tubulin where the
intensity was greater in the apical half of the cell, the
labeling for tyrosinated tubulin was intense and uniform
along the entire length of the inner pillar cell.
At 7 dab, antibodies to tyrosinated tubulin (Fig.
3B-7) also displayed decreased labeling in the sensory
cells and nerve fibers. In the base of the inner pillar and
throughout the outer pillar, the microtubules labeling for
tyrosinated tubulin appeared more in networks than in
discrete bundles. The intensity of labeling for tyrosinated
tubulin in the apical half of the inner pillar was much less
than that seen in the basal half of the cell. Labeling was
seen in the phalangeal processes of the Deiters cells near
its apical surface.
At 10 dab, antibodies to tyrosinated tubulin (Fig.
3B-10) showed diffuse labeling of microtubules in the
remains of Kölliker’s organ and the tympanic covering
layer, especially along the apical surfaces of the cells. The
pillar cells, while larger, were not yet mature. This is
based on the presence of tyrosinated tubulin in the basal
region of the pillar cells, where microtubules were not yet
organized into discrete bundles. Microtubules in Deiters
cells could not be differentiated from those in outer hair
cells.
At 14 dab, antibodies for tyrosinated tubulin (Fig.
3B-14) weakly labeled the microtubules in the few cells
remaining in Kölliker’s organ and the tympanic covering
layer. Antibodies diffusely labeled the sensory cells and
portions of the Deiters cells in the phalangeal processes
and basal regions. While there was weak diffuse labeling
of tyrosinated tubulin in the inner and outer pillar cells,
tyrosinated tubulin was conspicuously lacking in the
microtubules forming the bundles.
Detyrosinated Tubulin
Fig. 3. (Continued.)
At 1 dab, antibodies to detyrosinated tubulin (Figs.
3C-1, 4A8-1, 4B8-1, 5A8-1, 5B8-1) weakly labeled all
cells in the developing organ of Corti. In Kölliker’s organ,
microtubules were present along the lateral edges of the
columnar cells. In the developing sensory cells, detyrosinated tubulin weakly labeled microtubules throughout the
hair cells and especially the region directly below the
cuticular plate. In the developing pillar cells, detyrosinated tubulin was present in microtubules primarily at the
154
Tannenbaum and Slepecky
Figs. 4 and 5. In the inner pillar cell (vertical arrow) at 1 dab, the appearance of acetylated tubulin is
rapidly followed by the appearance of detyrosinated tubulin. In the Deiters cell (angled arrows), acetylated
tubulin precedes detyrosinated tubulin by several days. Using double labeling techniques on individual
sections, this pattern can be seen in the middle (Figs. 4A,A8, Figs. 5A,A8) and basal (Figs. 4B,B8; Figs. 5B,
B8) turns of the cochlea.
apical surface of the inner pillar. The region most
intensely labeled was increased in length from that seen
several hours after birth, and the microtubules appeared
to be more tightly packed. This correlated with the
decrease in labeling seen in the inner pillar cells with
antibodies to tyrosinated tubulin. Labeling of microtubules in the outer pillar and Deiters cells was not greater
than that seen in the outer hair cells at this time.
At 7 dab, antibodies to detyrosinated tubulin (Fig.
3C-7) showed weak labeling along the apical surface of
Modified Tubulin in Cochlear Epithelial Cells
cells in Kölliker’s organ and little labeling of microtubules in the sensory hair cells. However, the microtubules
in the inner pillar appeared more numerous than at 1 dab,
based on the increased intensity of labeling. Microtubules
were present both in the bundle along the apical surface
and the bundle in the pillar process. Labeling was
uniform along most of the inner pillar length, where little
labeling for tyrosinated tubulin was found. Labeling for
detyrosinated tubulin was strong but more patchy than
that seen with antibodies to acetylated tubulin. The outer
pillar showed weak and patchy labeling for detyrosinated
tubulin, and the phalangeal processes of the Deiters cells
were labeled.
At 10 dab, antibodies to detyrosinated tubulin (Fig.
3C-10) labeled predominantly microtubule bundles in the
pillar and Deiters cells. Labeling of the inner pillar cells
was more intense than labeling of the outer pillar cells.
However, labeling was more intense in the mid-portion of
the inner pillar cell than it was in the apical process, while
in the outer pillar cell it was more intense in the beam
bundle than the pillar bundle. Deiters cell microtubule
bundles were intensely labeled in the phalangeal processes, with some labeling of the basal bundles under the
outer hair cells.
At 14 dab, antibodies to detyrosinated tubulin (Fig.
3C-14) labeled predominantly microtubules in the pillar
and Deiters cells. Labeling was equally intense and
distributed uniformly along the entire length of the
microtubules in the pillar processes in these cells; however, the beam bundle in the outer pillar was still more
intensely labeled than the portion of the transcellular
bundle at the apical surface of the inner pillar cells.
Acetylated Tubulin
At 1 dab, antibodies to acetylated tubulin (Figs.
3-D1, 4A-1, 4B-1, 5A-1, 5B-1) labeled only a limited
number of microtubules present. Microtubules were seen
in the columnar cells of Kölliker’s organ, where staining
appeared bright only along the lateral margins. In the
sensory cells, there was little if any labeling. There was
strong labeling of the kinocilia on the apical surfaces of
all cells. Bundles of microtubules containing acetylated
tubulin appeared prominently in the upper third of the
inner pillar cells; there was no acetylated tubulin in the
lower portion of the inner pillar cells or in any region of
the outer pillar. Although detyrosinated and acetylated
tubulin appeared almost simultaneously in the inner
pillar, close examination of double-labeled sections (Figs.
4, 5) showed that the extent of labeling of detyrosinated
tubulin was always less than that for acetylated tubulin.
Acetylated tubulin appeared along the length of the
Deiters cells and in the processes interdigitated between
the apical surfaces of the outer hair cells, at a time where
155
there was only weak, diffuse labeling for detyrosinated
tubulin (Figs. 4, 5).
At 7 dab, antibodies to acetylated tubulin (Fig.
3D-7) labeled a few microtubules along the apical surface
of the remaining cells of Kölliker’s organ. Little if any
labeling was seen in the sensory cells. In the inner pillar,
labeling for acetylated tubulin was most intense in the
transcellular bundle along the apical surface of the cell,
and strong continuous labeling was found along most of
the length of the pillar process. There was labeling in the
apical region of the outer pillar, especially throughout the
beam bundle interdigitating between the outer hair cells,
but labeling in the pillar bundle was patchy and similar to
that seen for detyrosinated tubulin. Labeling for acetylated tubulin was seen in the apical processes of the
Deiters cells, as well as along the phalangeal processes.
At 10 dab, antibodies to acetylated tubulin (Fig.
3D-10) continued to weakly label the apical surface of the
few remaining cells in Kölliker’s organ, but showed no
labeling of the cells in the tympanic covering layer. The
inner and outer hair cells lacked acetylated microtubules.
In sections where both inner and outer pillar cells were
cut through the middle of the microtubule bundles,
labeling for acetylated tubulin was equally intense in both
the inner and outer pillar cells. It was also of equal
intensity in the transcellular and basal bundles in the inner
pillar and the beam and pillar bundles in the outer pillar
cells, unlike the labeling seen for detyrosinated tubulin.
Microtubules throughout the Deiters cells were also
prominent, and labeling was more intense than that seen
for detyrosinated tubulin.
At 14 dab, antibodies to acetylated tubulin (Fig.
3D-14) labeled the organ of Corti with a pattern similar to
that seen at 10 dab. There was weak labeling in the few
cells remaining in Kölliker’s organ (which become the
inner hair cell supporting cells) and no labeling in the few
cells remaining in the tympanic covering layer. Sensory
cells also lacked labeling for acetylated microtubules. In
both the inner and outer pillar, labeling for acetylated
tubulin was equally intense and distributed along the
entire length of all the microtubule bundles. Labeling for
acetylated tubulin was also intense in the basal bundle
and phalangeal process bundle of the Deiters cells, but
intensity of labeling for acetylated tubulin remained
greater than that seen for antibodies to detyrosinated
tubulin.
Polyglutamylated Tubulin
At 1 dab, antibodies specific for polyglutamylated
tubulin (Fig. 3E-1) showed weak but diffuse labeling in
most cells and kinocilia of the developing organ of Corti.
Both pillar cells were diffusely labeled, with intense
labeling in the upper quarter of the inner pillar. When
single sections were double-labeled, even at this early
156
Tannenbaum and Slepecky
stage labeling for polyglutamylated tubulin was less
intensive along the length of the pillar cell than labeling
for acetylated tubulin.
At 7 dab, antibodies to polyglutamylated tubulin
(Fig. 3E-7) showed no apparent labeling in the cells of
Kölliker’s organ and the tympanic covering layer, although there was weak, diffuse labeling in the apical
portion of the sensory cells. The antibodies intensely
labeled microtubule bundles in the apical region of the
inner pillars. No labeling was seen in the outer pillars and
little labeling was seen in the Deiters cell processes.
At 10 dab, antibodies to polyglutamylated tubulin
(Fig. 3E-10) showed only weak labeling of microtubules
in the remains of Kölliker’s organ and the tympanic
covering layer. Weak diffuse labeling for polyglutamylated tubulin was seen in the networks of microtubules
in the inner and outer hair cells, their supporting cells, and
in nerve fibers. In the supporting cells, the appearance of
polyglutamylated tubulin lagged behind acetylated tubulin; it was present only in the upper third of the inner
pillar, upper quarter of the outer pillar, and small spots at
the apical surfaces of the Deiters cells.
At 14 dab, antibodies to polyglutamylated tubulin
(Fig. 3E-14) weakly labeled the few remaining cells of
Kölliker’s organ and the tympanic covering layer. There
was still diffuse labeling of the sensory cells and supporting cells, and stronger labeling along the entire length of
the microtubule bundles in the pillar and Deiters cells.
While the labeling of the microtubule bundles in the
supporting cells had attained its adult-like pattern, diffuse
labeling was increased in all these cells when compared
to that seen in the adult at 55 dab (Fig. 3E-55).
DISCUSSION
Summary of Results
Figure 6 presents a diagram summarizing our findings.
A few hours after birth, the organ of Corti appears
as a mass of tightly packed, undifferentiated cells in a
small region of the basal turn of the cochlea. Microtubules, arranged in networks parallel to the long axis of the
cells, are present in Kölliker’s organ, the tympanic
covering layer, and all sensory and supporting cells. Our
results differ from those seen by other investigators using
transmission electron microscopy [Ito et al., 1995] in that
we see many more microtubules in the sensory and supporting cells at this early stage of development. However, at
the later stages our results confirm the sequence of
microtubule assembly and bundle formation seen in the
gerbil [Ito et al., 1995] and mouse [Tucker et al., 1992,
1995; Henderson et al., 1994; Henderson et al., 1995].
While the adult organ of Corti contains sensory and
supporting cells which display cell-type specific localization of modified and unmodified tubulins, this is not the
Fig. 6. Diagram summarizing the temporal and spatial pattern of the
appearance of tyrosinated, acetylated, and detyrosinated tubulin during
development of the organ of Corti.
case early in development. Several hours after birth,
labeling with antibodies to tyrosinated tubulin displays a
pattern similar to that for antibodies to a-tubulin; strong
labeling of all cells with increased intensity of labeling in
the apical third of the inner pillar cell. Labeling for
posttranslationally modified tubulin is restricted to small
areas along the apical surface of all cells, and a larger
region near the apical surface of the inner pillar cell. One
explanation for the few microtubules seen by transmission electron microscopy at this time might be that the
more dynamic microtubules, composed of tyrosinated
tubulin, are not preserved during processing. Some of
these steps (including incubation with cold solutions)
could induce dynamic microtubules to depolymerize. As
the microtubules are stabilized, reflected in the increased
labeling for posttranslational modifications to tubulin, the
microtubules remain and the results seen at the ultrastructural level are more similar to the immunofluorescent
Modified Tubulin in Cochlear Epithelial Cells
results presented here. Another is that tissue sections for
light microscopy are thicker than those used for electron
microscopy, so that microtubules are easier to detect.
At 7 dab, we consistently see a general decrease in
the labeling for tubulin (a-tubulin as well as tyrosinated
and posttranslationally modified tubulins). A similar
decrease in intensity of labeling for tubulin has been seen
at a comparable stage of development in isolated outer
hair cells in the rat [Vago et al., 1996]. This decrease
might reflect depolymerization of microtubules, and the
decrease in the number or length of microtubules could
destabilize the rigid cytoskeleton and allow the remodeling necessary for cells to attain their mature location,
position, shape, and size. In supporting cells, the depolymerization could be associated with the reorganization of
microtubules into specific bundles; there is evidence for
the loss of almost 2,000 microtubules in the transcellular
bundle of the inner pillar cell prior to the formation of the
basal bundle [Tucker et al., 1993]. In sensory cells,
depolymerization could be associated with the release of
microtubule-associated proteins; these could become available for interaction with actin and thus provide a mechanism by which microtubules could help align actin
filaments in the cuticular plate [Sobkowicz et al., 1995].
However, the decreased labeling could instead reflect
alterations to, rather than depolymerization of, microtubules; interactions of microtubules with proteins which
may block antigenic sites would prevent antibody binding. Alternatively, microtubules may remain unaltered but
cells increase in size so rapidly that microtubule formation cannot keep up. This would result in a temporary
decrease in microtubule density, which recovers as synthesis and polymerization continue.
Tyrosinated Tubulin in Cells That Decrease in
Number or That Will be Involved With Active
Processes Such as Secretion and Motility
In the developing organ of Corti, Kölliker’s organ
and the tympanic covering layer are initially large, with
numerous cells that play a role in the synthesis and
secretion of the tectorial and basilar membranes, respectively. While they initially contain microtubules composed of tyrosinated, detyrosinated, acetylated, and polyglutamylated tubulin, at later stages they are enriched
primarily with tyrosinated tubulin. Concomitant with the
decrease in labeling for microtubules containing modified
isoforms of tubulin, the cells diminish in size and
number; by 14 dab, Kölliker’s organ and the tympanic
covering layer have almost completely disappeared.
The presence of posttranslationally modified tubulin in these cells during the early stages of development
might provide the initial mechanism for stabilizing shape
as the cells in the organ of Corti differentiate. In the later
stages, when the levels of the posttranslational modifica-
157
tions change, the continued presence of predominantly
tyrosinated tubulin in Kölliker’s organ and the tympanic
covering layer might be related to secretion, based on the
role of microtubules in organelle movement in other cell
types [Araki et al., 1993; Ayscough et al., 1993]. It may
also be related to the decrease in cell size and number in
both structures, since microtubules containing tyrosinated
tubulin play important roles in cells undergoing rapid
changes in morphology [Robson and Burgoyne, 1989;
Alfa and Hyams, 1991; Pagh-Roehl et al., 1991]. However, the continued presence of tyrosinated tubulin cannot
be totally responsible for the dramatic change in the cells
of these accessory structures. The sensory hair cells also
lack detyrosinated, acetylated, and polyglutamylated tubulin in the developing and adult organ of Corti [Slepecky et
al., 1995], yet sensory cells do not decrease in size or
number.
Posttranslational Modifications to Tubulin Do Not
Correlate With the Development of Cell Polarity
and Shape
From study of cells in culture, it has been suggested
that the appearance of posttranslationally modified tubulin correlates with stabilization of microtubules. Selective
stabilization would provide the basis for asymmetry,
resulting in the establishment of cell polarity and shape
during morphogenesis [Kirschner and Mitchison, 1986].
Our study of posttranslational modifications to tubulin in
the developing organ of Corti does not support this
mechanism. At 1 dab, sensory and most supporting cells
contain microtubules composed of predominantly tyrosinated tubulin, with labeling for posttranslationally modified tubulins found only in restricted regions near the
apical surface. The outer pillar cells lack detectable
microtubules at this stage. Yet all these cells are already
asymmetric and elongated. One interpretation of our
results is that microtubules are stabilized prior to the
onset of posttranslational modifications, supporting observations by others that the appearance of posttranslational
modifications to tubulin are neither associated with nor
required for the onset of microtubule stability [Gundersen
and Bulinski, 1986; Schulze et al., 1987; Khawaja et al.,
1988; Pepperkok et al., 1990; Xiang and MacRae, 1995].
Another is that the establishment of cell polarity does not
require the presence of stable microtubules, or even any
microtubules at all [Alfa and Hyams, 1991; Bre et al.,
1991; Euteneuer and Schliwa, 1992].
Posttranslational Modifications to Tubulin in Cells
That Provide Mechanical Support to the Organ of
Corti
While our results do not suggest a correlation
between the appearance of posttranslationally modified
microtubules and the establishment of cell polarity, they
158
Tannenbaum and Slepecky
do suggest that the appearance of posttranslationally
modified microtubules is correlated with the elongation
of supporting cell microtubules, seen first in the inner
pillar and Deiters cells and then in the outer pillar cells
[Tucker et al., 1992, 1995; Ito et al., 1995] Over time, the
posttranslational modifications to tubulin may provide
sites for binding of specific microtubule-associated proteins that further stabilize the cell and cytoskeleton. Actin
and intermediate filaments have been shown to associate
with stable microtubules [Griffith and Pollard, 1978;
Gurland and Gundersen, 1995] and both actin and
intermediate filaments are prominent components of the
pillar cells, associated with microtubules in the mature
cochlea [Slepecky and Chamberlain, 1983; Raphael et al.,
1987; Schulte and Adams, 1989]. While nothing is known
about the appearance of intermediate filaments during
development of the gerbil cochlea, actin is recruited into
the microtubule bundles of the pillar and Deiters cells at a
stage late in development [Kuhn and Vater, 1996] when
we find detyrosination and acetylation of tubulin have
already occurred. The abundance of all three of these
cytoskeletal elements, as well as the highly ordered
organization of the actin filaments with microtubules,
suggests they play a role in providing mechanical support
to the organ of Corti, required for proper amplitude and
frequency of vibration in response to sound stimulus.
Pillar cells. In the pillar cell microtubules, a
decrease in tyrosinated tubulin labeling is paralleled by
an almost simultaneous increase in acetylated and detyrosinated tubulin. The timing and pattern of appearance
of these posttranslational modifications correlates with an
increase in microtubule number and length. This sequence is commonly found in developing cells [Thompson, 1982; Cummings et al., 1984; Alonso et al., 1988;
Beltramo et al., 1989; Gundersen et al., 1989; Bre et al.,
1991; Bulinski and Gundersen, 1991] and is similar to the
pattern seen in fibroblasts, following cell division or
recovery from drug treatment [Bulinski et al., 1988].
The difference between the intensity of labeling in
the inner and outer pillar cells at each of the early stages
studied can be explained if the developmental changes in
the number and location of gerbil pillar cell microtubules
are similar to those seen in the mouse [Tucker et al., 1992;
Henderson et al., 1995; Mogensen et al., 1997]. Ultrastructural analysis has shown that microtubule nucleation
occurs first in the inner pillar cell, near the centrioles at
the apical surface. Pericentriolar material migrates to the
periphery of the cell and the newly formed microtubules
also migrate and are anchored there. Microtubules begin
to elongate toward the base of the columnar cell, and
additional microtubules are added to the forming bundle.
As development proceeds, the cell increases in size and
changes in shape. The pericentriolar nucleating and
anchoring site migrates laterally, forming a thin apical
surface process, filled with microtubules, which extends
over the outer pillar cell (see Fig. 2). The microtubules
continue to elongate at their free ends, bend toward the
base of the cell, and elongate further so that many long
microtubules span the distance between the apical surface
and the base of the cell, forming the transcellular bundle.
At the same time that this bundle is being formed, a large
number of shorter microtubules (2,000 out of the 3,000
present in each inner pillar cell) are released from their
apical anchoring site and migrate to the base of the cell,
where their ends are captured at a basal anchoring site. At
this location, the microtubules elongate toward the midregion of the cell to form the basal bundle.
Our labeling of microtubules with antibodies to
tubulin during development reflects this pattern. The
inner pillar microtubules accumulate first in large numbers at the apical surface. As these microtubules elongate
toward the base of the cell, they contribute to the
transcellular bundle. The inner pillar labels first in the
apical third of the cell with antibodies to a-tubulin and
tyrosinated tubulin. As the microtubules in the apical
surface bundle increase in number and length, posttranslationally modified isoforms appear. The addition and
elongation of microtubules is correlated with the appearance of acetylated and detyrosinated tubulin, with labeling increasing in width, length, and intensity. Early in
development, labeling for posttranslationally modified
isoforms of tubulin shows that microtubules in the
transcellular bundle span the distance between the apical
and basal surfaces of the cell. The appearance of detyrosinated, acetylated, or polyglutamylated tubulin is not
directly responsible for the bending of the transcellular
bundle because: 1) the posttranslational modifications
precede bending, and 2) a similar pattern of posttranslational modifications is observed at the apical surface of
outer pillar cells where the microtubule bundles do not
bend, but remain short and parallel to the apical surface of
the cell [Tucker et al., 1995].
At the early stages of development, the outer pillar
cell lacks detectable microtubules even when large numbers of long and posttranslationally modified microtubules are present in the inner pillar cell. Only at several
days after birth are outer pillar cell microtubules observed, nucleated and anchored near the centriole at the
apical surface of the columnar cell. As development
proceeds, the pericentriolar material divides in two and
each portion moves laterally, one toward the inner pillar
cells and one toward the outer hair cells. As the outer
pillar cell apical process extends to interdigitate between
the first row of outer hair cells, the nucleating site and
anchoring site (centrioles and pericentriolar material)
moves to the lateral-most edge of this process (see Fig. 2)
near the outer hair cell [Tucker et al., 1995]. Unlike
microtubules in the apical process of the inner pillar cell,
Modified Tubulin in Cochlear Epithelial Cells
the microtubules nucleated and anchored in the apical
processes of the outer pillar cell do not continue to
elongate and bend. Most remain relatively short (around
10 µm in the mouse) and run parallel to the apical surface
of the outer pillar cell, forming the beam bundle. This is
the first region of the outer pillar cell where we found
intense labeling of posttranslationally modified microtubules.
Other short microtubules are released from the
apical anchoring site and migrate down toward the basal
surface of the outer pillar cell, where their ends are
captured. They then elongate upward to form the pillar
bundle, which becomes anchored at the pericentriolar
material that was established at the apical surface of the
outer pillar, near the inner pillar cell, earlier in development. Our immunocytochemical results confirm this
sequence. Since most of the microtubules that eventually
comprise the pillar bundle in the outer pillar cell do not
accumulate at the apical surface of the cell or elongate as
a group, labeling with antibodies to a-tubulin is initially
weak and remains weak in this region. Moreover, since
all the microtubules at the apical surface are newly
formed, most tubulin in the microtubules is tyrosinated.
While the appearance of acetylated tubulin is rapidly
followed by detyrosinated tubulin in the outer pillar cell,
as it is in the inner pillar cell, our results suggest that
posttranslational modifications occur as the microtubules
are migrating, as reflected in the relatively weak and
patchy labeling for acetylated and detyrosinated tubulin
seen along the length of the pillar bundle even up to 10
dab.
Deiters cells. Labeling for acetylated tubulin allows
us to detect the Deiters cells between the developing outer
hair cells even at 1 dab, and indicates that microtubules
are present in these cells several days before they can be
detected by transmission microscopy [Ito et al., 1995].
Unlike the sequence of appearance of the posttranslational modifications in the pillar cells, acetylated tubulin
precedes detyrosinated tubulin by several days. A similar
sequence has been observed in developing Drosophila
embryos, where acetylated tubulin appears first and
detyrosinated tubulin appears at a later stage [Warn et al.,
1990]. It should be noted that when there is a difference
between the timing of appearance of these tubulin
isoforms, acetylation does not always precede detyrosination. In developing myoblasts, detyrosinated tubulin is
detected first in elongating portions of the cell; acetylated
tubulin increases occur much later [Gundersen et al.,
1989].
SUMMARY
Our results show that during development:
159
1) Tyrosinated tubulin is initially the predominant
form of tubulin in all cells.
2) Posttranslational modifications occur in the supporting cells in a time-dependent manner as
development proceeds; progression from cell
apex to base is in the same direction as microtubule elongation.
3) Establishment of cell polarity appears to precede
posttranslational modifications to tubulin, especially as shown in the development of the outer
pillar cells.
4) Tyrosinated tubulin remains the predominant
form of tubulin in cells that decrease in number
and that retain active processes such as secretion
and motility.
5) The appearance of acetylated tubulin is rapidly
followed by the appearance of detyrosinated
tubulin in the pillar cells, but the appearance of
acetylated tubulin precedes the appearance of
detyrosinated tubulin in the Deiters cells by
several days.
6) The cell-type specific pattern of posttranslational modifications appears to be established at
the time of onset of cochlear function (12 dab);
however, the intensity of labeling for polyglutamylated tubulin does not appear adult-like until
cochlear function is also mature (30 dab).
In the cochlea, sensory and supporting cells play important but differing functional roles. In ears from adult
animals, microtubules in the inner and outer sensory hair
cells are arranged in loose networks found throughout the
cell, while those in the supporting cells are arranged
almost exclusively in tightly packed bundles which run
the length of each cell [Angelborg and Engstrom, 1972;
Steyger et al., 1989; Furness et al., 1990; Slepecky and
Ulfendahl, 1992; Raphael et al., 1994; Kuhn and Vater,
1995]. In outer hair cells, microtubules may be involved
in remodeling of the cytoskeleton following shape changes
related to outer hair cell motility. In both inner and outer
hair cells, microtubules are found in areas where organelles are being transported; in the apex of the cell adjacent
to the cuticular plate and in the base of the cell near the
synaptic vesicles. Thus, microtubules in sensory cells are
thought to be dynamic and involved in active processes.
The pillar cells possess microtubules in bundles, larger
than any others described so far for mammalian cells
[Tucker et al., 1992]. It has been assumed that these
microtubules provide rigidity and mechanical support to
the organ of Corti.
Microtubules in supporting cells of the cochlea also
differ biochemically and structurally from those found in
the sensory cells. They are enriched with microtubules
containing posttranslationally modified isoforms of tubulin, suggesting that they are stable and long-lived; micro-
160
Tannenbaum and Slepecky
tubules in the sensory cells are predominantly tyrosinated, suggesting that they are dynamic and short-lived
[Slepecky et al., 1995]. The supporting cell microtubules
differ from those in the sensory cells in that they are
composed of 15 protofilaments [Saito and Hama, 1982;
Kikuchi et al., 1991a], instead of the more commonly
found 13 protofilaments. It is interesting that supporting
cells in the sensory epithelia of the vestibular system
contain microtubules with 13 protofilaments and both
tyrosinated and detyrosinated tubulin [Kikuchi et al.,
1991b; Ogata and Slepecky, 1995]. Since the organ of
Corti vibrates during stimulation while the vestibular
sensory epithelium is stationary, the differences in the
microtubules may be correlated with the mechanical
properties of the supporting cells. One can further speculate that alterations in the pattern and location of posttranslationally modified tubulin isoforms might change the
mechanics of this vibratory organ and subsequently alter
the frequency selectivity and sensitivity of this sensory
organ.
ACKNOWLEDGMENTS
We thank Molly Watts for help with the diagrams
based on the tissue sections, Tammy Stage for help with
photography, and the Deafness Research Foundation for
funding.
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