1 Department of Ophthalmology and Visual Sciences, Washington University School
of Medicine, St Louis, MO 63110, USA
2 Department of Biochemistry and Molecular Biophysics, Washington University
School of Medicine, St Louis, MO 63110, USA
* Author for correspondence (e-mail: andley{at}vision.wustl.edu)
Accepted 17 December 2002
![]() |
Summary |
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Key words: A-Crystallin, Lens, Chaperone, Cell death
![]() |
Introduction |
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A- and
B-knockout mice have been generated to understand the
physiological functions of these proteins
(Brady et al., 1997
;
Brady et al., 2001
).
Disruption of the
A gene causes early-onset cataract in mice
characterized by a central opacity owing to formation of inclusion bodies
comprising
B and HSP25. Lenses of
A/
mice are significantly smaller than normal
(Brady et al., 1997
), and lens
epithelial cells derived from
A/ mice have a
50% slower growth rate in vitro (Andley et
al., 1998
). Disruption of the gene encoding
B does not
result in altered lens morphology or transparency. However, lens epithelial
cells derived from
B/ mice demonstrate
hyperproliferation and genomic instability
(Andley et al., 2001
). These
findings suggest that
A and
B may be essential for optimal
growth of lens epithelial cells.
The lens grows throughout life as fiber cells are added incrementally at
the lens periphery without concomitant loss of any previously formed fibers
(Kuszak et al., 2000). The
lens epithelium also undergoes lifelong growth, but with significant zonal
variation as a function of age. The central epithelium comprises a broad cap
that covers the anterior surface of the lens. The mitotic activity of central
epithelial cells decreases with age
(Mikulicich and Young, 1963
).
The peripheral region of the epithelium contains mitotically active cells
throughout life. As more cells in this region divide, they force the migration
of other cells towards the equator to occupy the differentiating meridional
rows, where they elongate bidirectionally until they become secondary fibers.
Cells of the meridional rows are terminally differentiated and post-mitotic.
Cell division is also absent in the transitional zone just anterior to the
meridional rows (Rafferty and Rafferty,
1981
). Growth and survival factors that influence cell division in
the lens have been extensively studied
(Reddan, 1982
;
McAvoy and Chamberlain, 1989
;
Hyatt and Beebe, 1993; Ishizaki et al.,
1993
; Zelenka et al.,
1997
; Rakic et al.,
1997
; Singh et al.,
2000
). However, regulation of cell division in the lens is not
fully understood.
Several observations suggest that molecular chaperones play important roles
in cell growth and differentiation (Mehlen
et al., 1997; Yokota et al.,
1999
). It has been reported that HSP70, HSP90 and the major
eukaryotic cytoplasmic chaperone TRiC may participate in the quality control
of proteins during the progression of the cell cycle
(Yokota et al., 1999
;
Dunn et al., 2001
). TRiC plays
an important role in cell growth by assisting in the folding of tubulin and
other proteins, and its expression is strongly upregulated during cell growth,
especially from the G1/S transition to early S phase
(Yokota et al., 1999
).
Mutations in TRiC genes cause aberrant chromosome segregation
(Dunn et al., 2001
). Both
HSP70 and HSP90 have been detected in centrosomes of mitotic cells
(Wigley et al., 1999
;
Brown et al., 1996
). The
reported increase in expression of
B in mitotic fibroblasts and its
transient expression in the nucleus during interphase suggests its association
with the cell-cycle machinery (Bhat et
al., 1999
; Djabali et al.,
1999
). In addition, antibodies to phosphorylated
B
recognize midbodies and centrosomes of dividing cells
(Inaguma et al., 2001
). It has
been suggested that chaperones may play a role in quality control of proteins,
particularly in the assembly of microtubules. Although the expression of
A has been shown to enhance the growth of lens epithelial cells
(Andley et al., 1998
), it is
not known whether it directly affects the progression of cells through the
cell cycle.
Recently, mutations in A and
B genes have been shown to be
associated with genetic disorders. In
A, substitution of arginine 116
by cysteine was found to be the cause of one form of autosomal dominant
cataract (Litt et al., 1998
).
In aB, substitution of arginine 120 by glycine was found to be the cause of
another autosomal dominant disease, desmin-related myopathy, and also caused
cataract (Vicart et al.,
1998
). It is not yet known whether these mutations also affect
lens epithelial cell growth in vivo.
We have demonstrated that primary lens epithelial cells derived from
A/ mice grow at a 50% slower rate than
controls (Andley et al., 1998
).
A/ lenses are significantly smaller than
controls (Brady et al., 1997
).
These data suggested that
A might be necessary for lens epithelial
cells to proliferate at a normal rate in vivo.
A protects against
stress-induced apoptosis (Andley et al.,
1998
; Andley et al.,
2000
), and the lack of
A may increase lens epithelial cell
death. We tested the hypothesis that
A prevents cell death at a
specific stage of the cell cycle in vivo and that the smaller size of the
A-knockout mouse lens is owing to the reduced survival of lens
epithelial cells. We labeled the newly synthesized DNA of proliferating lens
cells with 5-bromo-2'-deoxyuridine (BrdU), and followed the BrdU-labeled
cells as they progressed through the cell cycle. We show that
A/ lens cells died as pairs during mitosis.
These studies suggest that
A plays an important role in the regulation
of mitosis in vivo.
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Materials and Methods |
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Labeling of lenses with BrdU
Mice were injected with BrdU intraperitoneally (0.1 ml of a 10 mM solution
of BrdU in sterile PBS) between 9 AM and 10 AM and were sacrificed 1, 3, 8,
16, 24 hours or 2, 3, 5 days later. Previous studies in rodents showed that,
with this dose of BrdU, blood levels peak by 2 hours after injection and
decline substantially thereafter (deFazio
et al., 1987). Whole lenses were dissected under a microscope. In
a 7-day-old lens, blood vessels on the posterior side were used to identify
the orientation of the lens. The lens was placed in tissue culture medium in a
coated tissue culture dish (Falcon 3001, 35 mm). To obtain the
capsule-epithelial wholemounts, the lens was placed posterior-side up, and the
capsule was held with very sharp tweezers and pulled. An opening was made at
the edge of the posterior capsule, and blunt tweezers were used to flatten the
capsule on the culture dish.
BrdU detection
In preparation for BrdU antibody staining, wholemounts were fixed with
ethanol/glycine/water (70:20:10, v/v), pH 2.0 and 0.5% Triton X-100 for 30
minutes, followed by washing three times for 5 minutes each with PBS. Tissues
were stored at 4°C in PBS containing 0.02% sodium azide. To detect BrdU,
wholemounts were treated with a monoclonal antibody from the BrdU labeling kit
(Roche Biochemicals) at a dilution of 1:100 in 1% bovine serum albumin, 0.5%
Tween-20 in PBS for 2 hours, washed extensively in several changes of PBS with
gentle agitation, and exposed to Alexa568-labeled goat anti-mouse
IgG (Molecular Probes) at a concentration of 1:200 diluted in PBS-BSA-Tween
for 2 hours. Tissues were washed three times in PBS. In these studies, the
DNA-binding dye TOTO-1 (Molecular Probes) was also used to determine the total
number of cells. After BrdU labeling, wholemounts were stained with a 1:10 000
dilution of TOTO-1 for 20 minutes. After washing, tissues were mounted on
microscope slides and viewed. BrdU fluorescence was analyzed in the red
channel (excitation 568 nm) of a Zeiss LSM 410 confocal microscope. TOTO-1 was
detected in the green channel (excitation 488 nm). Since the wholemount is not
always completely flat, immunofluorescence detection with confocal microscopy
allowed serial optical sections with a high degree of sensitivity and 3D
images were collapsed into 2D to eliminate optical artifacts. Wholemounts were
sampled by collecting serial images through the lens epithelium in the central
region and in the equatorial region.
Labeling index
To determine the labeling index in different regions of the lens epithelial
wholemounts, tissues were first examined at 10x magnification. The
central region of the wild-type epithelium was identified by its lower degree
of BrdU labeling (as compared with the germinative region), and the area of
the central region was 700-800 µm2. The germinative region near
the periphery of the wholemount had an area of about 180 µm2. In
each wholemount, three 150 µm2 fields of the central lens
epithelial region were examined. BrdU-labeled nuclei were counted in each
field, and the total number of nuclei was determined by counting the
TOTO-1-stained nuclei in the same field. The Scion Image (Scion Corp) program
was used to count the labeled nuclei. For each genotype, six lenses were used.
The labeling index was determined by dividing the number of BrdU-labeled cells
by the total number of cells, multiplied by 100. Statistical analysis was
carried out using the Student's t-test.
In our studies, we compared the labeling index of the peripheral region of
wild-type cells with A/ lens epithelial cells.
Our data showed that a large amount of BrdU labeling was occurring in the
germinative region of the epithelium of 7-day-old mouse lenses. The number of
labeled pairs 24 hours after the BrdU pulse was so high that it was difficult
to assess accurately whether they were members of a pair or not. Thus, the
analysis of the number of BrdU-labeled cells present as pairs is restricted to
the central region of the epithelium.
To follow daughter cells produced after mitosis of BrdU-labeled cells, we
counted the number of pairs of BrdU-labeled nuclei in lens epithelial
wholemounts at 1, 2, 3 or 5 days after labeling. The identification of cell
pairs was carried out as described previously
(Beebe and Masters, 1996). In
these analyses, single-labeled cells at the edge of a field were not counted
as a pair, because it is possible that these could be paired with a nucleus
that was out of the field of view. BrdU-labeled cells were counted as members
of a cell pair when their nuclei were contiguous or separated by one or two
nuclear diameters, and when the staining pattern of their nuclei was
identical. The staining pattern of some pairs of BrdU-labeled nuclei was
uniformly bright, whereas others had faint or diffuse label or a
characteristically punctate appearance. These differences may result from the
availability of BrdU to cells in different phases of the cell cycle at the
time of BrdU injection. Adjacent BrdU-labeled nuclei were only scored as pairs
when both cells had the same labeling pattern.
The proportion of BrdU-labeled cells in pairs was determined at each interval after BrdU injection. In our data, we observed that cell pairs with identical BrdU staining patterns 5 days after labeling were separated by up to four nuclear diameters, either because of migration, or because the cells separating them had undergone cell division.
Analysis of cell death
TUNEL labeling was used to examine cell death in lens epithelial
wholemounts from wild-type and A/ mouse
lenses. Wholemounts were fixed in 4% para-formaldehyde, pH 7.4 for 30 minutes,
permeabilized for 5 minutes in 0.1% Triton X-100/PBS at room temperature, and
apoptotic nuclei were detected using a TUNEL-labeling reaction according to
the manufacturer's instructions (Roche Biochemicals), as described previously
(Andley et al., 1998
). In
explants prepared for double labeling with TUNEL and BrdU, the TUNEL reaction
was carried out first, and then the tissues were treated according to the
protocol described above for BrdU detection. Images were recorded on a
confocal microscope. BrdU was detected in the red channel (568 nm excitation)
and TUNEL in the green channel (488 nm excitation). In other experiments,
TUNEL-labeled tissues were co-labeled with propidium iodide (PI) to visualize
individual nuclei in the wholemount. TUNEL-labeling was detected in the green
channel and PI labeling in the red channel of the confocal microscope.
Labeling of MIP
To examine the cross-sectional profiles of lens fiber cells of wild-type
and A/ lens sections, tissues were treated
with an antibody to MIP (Alpha Diagnostics International). Lenses were mounted
in glycol methacrylate and 3 µm sections were cut in the equatorial plane.
Non-specific binding was blocked by incubation in 10% normal goat serum for 30
minutes. To visualize the distribution of MIP, tissues were incubated
overnight with a 1:100 dilution of a monoclonal antibody to MIP. An
Alexa568-conjugated goat anti-mouse IgG was used as a secondary
antibody (1:200). Immunofluorescence and confocal microscopy were performed as
described above.
Labeling of A
To visualize A in wholemounts of wild-type and
A/ lenses, tissues were incubated overnight
with a 1:50 dilution of a monoclonal antibody to bovine
A (a gift from
P. Fitzgerald, University of California, Davis, CA). An
Alexa568-conjugated goat anti-mouse IgG was used as a secondary
antibody. Wholemounts were mounted on slides and were viewed using a Zeiss LSM
410 confocal microscope equipped with an argon-krypton laser. To visualize
A in mitotic cells, lens epithelial cells were cultured from wild-type
lenses, and primary cells were labeled with the
A antibody.
A
was visualized by Alexa568-conjugated goat anti-mouse IgG as the
secondary antibody, and F-actin was visualized by fluorescein phalloidin
(Molecular Probes).
Labeling of ß-tubulin
To visualize microtubules in whole mounts of wild-type and
A/ lenses, tissues were incubated overnight
with a 1:100 dilution of a monoclonal antibody to bovine ß-tubulin
(Sigma). An Alexa568-conjugated goat anti-mouse IgG was used as a
secondary antibody. Wholemounts were labeled with TOTO-1 to visualize nuclei.
Wholemounts were mounted on slides and were viewed using a Zeiss LSM 410
confocal microscope equipped with an argon-krypton laser. ß-tubulin was
visualized in the red channel of the confocal microscope (568 nm excitation).
TOTO-1 was detected in the green channel (488 nm excitation).
In other studies, dual labeling of cells was carried out with A and
ß-tubulin antibodies. Cells were first incubated with the
A
antibody and the Alexa488-conjugated goat anti-mouse IgG secondary
antibody (488 nm excitation), fixed again and then treated with an antibody to
ß-tubulin and Alexa568-conjugated secondary antibody (568 nm
excitation). To visualize nuclei, the DNA-binding dye TOPRO-3 was used (647 nm
excitation).
Growth rate
Wet weights of wild-type and A/ lenses were
determined for 7-day-old mice. The neonate lens was too small to be weighed
accurately, but changes in lens size were determined from the diameter, which
could be precisely measured. The equatorial and axial diameters of the lens
were determined by acquiring differential interference contrast images in the
confocal microscope of 4 µm paraffin sections at daily intervals.
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Results |
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|
The wet weight of the wild-type 7-day-old lens was 1.50±0.4 mg,
whereas that of the A/ lens was
0.78±0.10 mg (n=6, P=0.004). We examined whether the
48% smaller lens was due to a decrease in the net production of epithelial
cells or to smaller fiber cells size. We measured the proliferation index and
apoptosis in the lens epithelial wholemount and recorded the cross-sectional
area of secondary fiber cells.
A expression has been shown to enhance lens epithelial growth in
culture, but it is not known whether it affects proliferation in vivo. To test
the hypothesis that
A expression plays a direct role in the regulation
of the cell cycle by preventing cell death at a specific stage of the cell
cycle, the newly synthesized DNA in proliferating cells was labeled in vivo.
A/ and control mice were injected with BrdU to
label cells in the S phase, and the labeled cells were followed as their cell
cycle progressed from the S to the G2 and M phases, and after the
completion of mitosis. Wholemounts of capsule epithelial explants were made,
and labeled nuclei were detected with an antibody to BrdU. Three hours after
BrdU injection, single, labeled nuclei were seen throughout the central
epithelium of the wild-type lens (Fig.
2A-C). In the
A/ lenses, the
majority of the BrdU-labeled nuclei also appeared as single labels, with
distinct staining patterns (Fig.
2D-F). Labeling of the whole mounts with the DNA-binding dye
TOTO-1 stained all nuclei, which could then be counted to determine the
labeling index.
|
The labeling index 3 hours after the pulse was the same (4.5%) in wild-type
and A/ lens epithelial wholemounts
(Fig. 2G). This observation
indicates that the number of cells in the S phase was the same in both
genotypes.
Other mice were killed the next day, by which time the individual cells
labeled in S phase had divided to give pairs of BrdU-labeled cells. Thus, 24
hours after BrdU injection, all the BrdU-labeled nuclei in the lens epithelial
wholemounts were in pairs (Fig.
3A-F). Both members of a cell pair had an identical staining
pattern of their nuclei. The symmetrical pattern of BrdU staining in the
daughter cells significantly assisted their identification as cell pairs. The
labeling index of wild-type and A/ lens
epithelium was determined 24 hours after BrdU injection
(Fig. 3G). The
A/ epithelium had fewer numbers of cell pairs
compared with the wild-type epithelium. The 24-hour labeling index of the
wild-type central epithelium doubled from 4.5±1.5 to 8.6±1.8%
due to mitosis. In the epithelium of
A/
lenses, the 24-hour labeling index increased to 6.4±0.4% (from
4.5±1.3% at 3 hours), about 25% lower than wild-type lenses
(n=18, P=0.008). This result suggests that only some of the
cells were able to complete mitosis. The remainder did not complete mitosis,
and presumably died during or soon after mitosis.
|
Pairs of BrdU-labeled cells remained close to each other for 2-5 days after
the BrdU injection (Fig. 4B).
The wild-type lenses gave us the baseline number of paired BrdU-labeled cells.
The labeling index in the central region of wild-type and
A/ lens epithelial wholemounts at successive
times after the BrdU pulse is shown in Fig.
5. We also divided the 24-hour time period after the BrdU
injection into shorter intervals. The number of BrdU-labeled cells was counted
after 1, 3, 8 and 16 hours in the wild-type and
A/ lens epithelial wholemounts. The labeling
index was the same at 1-8 hours after the BrdU injection but, between 16 hours
and 5 days after the BrdU injection (Fig.
5), the labeling index was significantly lower in the
A/ wholemounts compared with the wild-type.
This result suggests that the lack of
A increased the susceptibility of
both daughter cells to apoptosis.
|
|
We then looked for the number of single-labeled cells in the wild-type and
A/ lens epithelial explants 24 hours after the
BrdU injection. If the number of single-labeled cells were more in the
A/ wholemount, it would mean that one of the
two daughter cells formed in the
A/ epithelial
wholemount had died, and would indicate that cell death occurred after cell
division. The wild-type lenses gave us the baseline level of single-labeled
cells. However, we found that a vast majority of BrdU-labeled cells were
present as pairs in the lenses from both genotypes
(Table 1). This suggests that
the lack of
A increased the susceptibility of both daughter cells to
apoptosis.
|
For each genotype, the labeling index did not change significantly between 1 and 3 days (Fig. 5). This indicates that the BrdU-labeled cells did not undergo a second division during this period. After 5 days, the labeling index in the central region decreased slightly, probably because the nonlabeled cells divided and therefore diluted the labeling index.
In a few cases, tetrads of BrdU-labeled nuclei were also seen 24 hours
after the BrdU injection (Fig.
4A). The cell cycle normally lasts about 24 hours, and the S phase
is about 6 hours, it is therefore unlikely that the tetrads arose from a
second division of a pair of BrdU-labeled daughter cells. These tetrads most
probably resulted from the mitotic division of two precursor daughter cells
that were synchronized from a previous division, consistent with results of
others (Rafferty and Rafferty,
1981).
In the peripheral region, the labeling index was 2-3-fold higher as
compared with the central region and many of the pairs of labeled cell nuclei
were immediately adjacent to other labeled pairs
(Fig. 4C). This `overcrowding'
of the BrdU-labeled nuclei made it difficult to assess accurately whether
adjacent BrdU-labeled nuclei were members of a pair or not. However, the
labeling index in the peripheral region was determined to be 30% lower in the
A/ lens epithelial wholemounts than in the
wild-type, confirming that the effects observed in the central region were
also seen in the peripheral region of the epithelium.
During our examination of BrdU labeling in
A/ epithelial wholemounts, we made a
significant observation. We observed cells in the
A/ epithelium that round up and shrink, thus
losing intercellular contacts with neighboring cells (data not shown). These
BrdU-labeled small cell debris resembled apoptotic bodies. The wholemounts
were stained with TUNEL stain to determine whether the smaller nuclei
represented dying cells. The lens epithelial wholemount of the
A/ lenses contained scattered pairs of
apoptotic cells, and these were strongly labeled by the TUNEL assay
(Fig. 6B,C). The labeled nuclei
had a characteristic condensed morphology with small, positively labeled
apoptotic bodies. These apoptotic bodies were often found in close
association, and appeared to be pairs of dying cells. In some cases, they
appeared to be cleared by neighboring cells. The cellular debris or apoptotic
bodies were frequently detected in the
A-knockout wholemounts, but were
rarely detected in the wild-type epithelium
(Fig. 6A,D).
|
To test whether BrdU-labeled cells were dying during mitosis, dual staining
with TUNEL reagents and BrdU antibodies was performed. Mice were injected with
BrdU and, 24 hours later, the wholemounts were treated with TUNEL reagents
first, and then immunostained to detect BrdU.
Fig. 7 shows several closely
spaced, BrdU-positive cells, which were also strongly stained by the TUNEL
assay. These dying cells appeared to be in late stages of mitosis, probably in
anaphase or cytokinesis since, in many cases, they appeared as pairs. We
defined apoptotic cells as those cells whose nuclear diameter was a third or
smaller than the size of a normal nucleus. The BrdU-labeled pairs that stained
positively with the TUNEL reagents were a third or smaller than the size of
the normal nuclei. By contrast, BrdU-labeled pairs that had normal dimensions
were not stained by TUNEL reaction. These data suggest that the lower value of
the labeling index in the A/ lens epithelium
may be due to an increase in apoptotic cell death during cell division.
|
A and
B-crystallins have been shown to protect the
cytoskeleton, and
B and several molecular chaperones are associated
with the mitotic apparatus in dividing cells
(Inaguma et al., 2001
). Since
the integrity of the spindle is essential for accurate cell division to occur,
it is possible that cell death in lens epithelium lacking
A results
from disorganization of the mitotic spindle. To examine the integrity of the
mitotic spindle, wild-type and
A/ lens
epithelial wholemounts were stained with an antibody to ß-tubulin.
Tubulin labeling was visualized by immunofluorescence and confocal microscopy.
Fig. 8A shows that
ß-tubulin was uniformly stained around the nuclei of interphase cells.
During metaphase, chromosome condensation and the bundling of microtubules in
the metaphase spindle was readily detected
(Fig. 8B,C). We examined 20
metaphase spindles each for the wild-type and
A/ lens epithelial wholemounts. There was no
significant difference in the integrity of the metaphase spindle for the two
genotypes.
|
We next examined the anaphase spindle of wild-type and
A/ lens epithelial wholemounts. Anaphase cells
could be readily identified as pairs of cells with condensed chromosomes
(Fig. 8D-F). In anaphase cells
of wild-type wholemounts, well-developed arrays of microtubules nucleated from
both the centrosomes, and were aligned between the separating chromosomes
(Fig. 8D). However, in the
A/ wholemounts, there was a loss of astral
microtubules, and microtubules in the zone between the chromosomes
(Fig. 8E,F). We studied six
wild-type and six
A/ wholemounts immunostained
with the ß-tubulin antibody. The integrity of the spindle was examined in
20 pairs of anaphase cells for each genotype. The results showed that 95% of
the anaphase spindles were well ordered in wild-type epithelium. However, 45%
of the anaphase spindles of the
A/ lens
epithelial wholemounts had a defective phenotype, suggesting that, in the
absence of
A, the anaphase spindle is not well organized and that
A may play a role in maintaining the integrity of the microtubules in
the lens epithelium.
To investigate the cellular distribution of A in mitotic cells,
wild-type mouse lens epithelial cells were cultured, and the primary cells
were immunostained with antibody to
A. The cytoskeletal protein F-actin
was stained with fluorescein phalloidin, and cellular morphology was imaged by
differential interference contrast. These studies showed that
A was
excluded from the chromosomes, but was highly concentrated in the middle of
dividing cells (Fig. 9A,B).
During interphase,
A was distributed uniformly throughout the cytoplasm
(Fig. 9A).
|
To examine further whether A may play a role in the integrity of the
mitotic spindle, wild-type mouse lens epithelial cells were also double
immunostained with
A and ß-tubulin antibodies, to determine the
distribution of
A in relation to the mitotic spindle.
Fig. 10 shows the distribution
of
A (green) and ß-tubulin (red) in metaphase, anaphase and
cytokinesis. These stages of mitosis were recognized by chromosome
condensation using TOPRO-3 fluorescence (shown in blue). As can be seen in
Fig. 10,
A
immunofluorescence was excluded from the chromosomes of the dividing cells at
all stages of mitosis. During metaphase,
A appears to be concentrated
in the centrosomes of the mitotic spindle
(Fig. 10A-C). However, the
punctate staining pattern observed suggest that the microtubules are not
necessarily the primary target of
A during early mitosis. During
anaphase,
A was highly concentrated in the region between the
chromosomes. However, significant punctate immunostaining for
A was
also observed (Fig. 10E,F).
This staining pattern suggests that
A associates with microtubules as
well as with other cytoskeletal elements during anaphase. At cytokinesis, the
intercellular bridge microtubules were labeled by the
A antibodies
(Fig. 10H,I). At this stage,
there appears to be a strong association with the microtubule cytoskeleton,
corroborating the results shown in Fig.
9B. These observations suggest that the relative distribution of
A is dependent upon the relative position of a cell during the cell
cycle.
|
The A-knockout mice had 40-50% smaller lenses in comparison with
control mice, indicating that deletion of the gene encoding
A resulted
in a lens growth deficiency. To determine when the growth deficiency became
apparent, the size of the lenses was recorded as a function of age. The lens
at postnatal day 0 was 40% smaller, and the growth lagged that of the control
mice throughout the observation period
(Fig. 11). The number of fiber
cells could not be accurately determined because the 7-day-old
A-knockout lenses had disorganized fiber cell morphology in the central
region of the lens.
|
To assess further if the smaller size of the
A/ lenses was due to smaller fiber cells, we
examined the cross-sectional profiles in the periphery of the lenses using MIP
immunofluorescence to visualize the fiber membranes. Lens slices (3 µm)
were cut in the equatorial plane. Light microscopic analysis of the lenses
indicated that the mean cross-sectional area of the secondary fiber cells in
the cortex of the lens was 9.5±4.8 µm2 for wild-type and
9.1±3.8 µm2 for the
A/
(n=20, P=0.38). Although the cross-sectional profiles of the
fiber cells appears to be indistinguishable for the wild-type and
A/ cells, since the wild-type lens is larger,
its fiber cells are probably longer. However, the fiber cells of the wild-type
and
A/ lenses had a distinctly altered
organization, with fiber cell membranes being significantly smoother in the
wild-type lens (Fig. 12).
A has been shown to associate with membranes
(Boyle and Takemoto, 1996
;
Cobb and Petrash, 2000
) and
our studies suggest that it might affect the organization of the fiber cells.
Taken together, these observations suggest that the reduced size of the
A/ lens is due to a reduction in the net
production of epithelial cells, and hence the formation of fewer fiber
cells.
|
![]() |
Discussion |
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It is also possible that only one of the two daughter cells died soon after
mitosis in the A/ wholemount. In this case, we
would have observed single BrdU-labeled cells 24 hours after the BrdU pulse.
However, the absence of single BrdU-labeled cells 24 hours after the pulse in
the
A-knockout wholemounts argues against this possibility. Overall,
the data suggest that the lack of
A increased the susceptibility of
both daughter cells to apoptosis.
Although the emphasis in this study has been on the central region of the
7-day-old lens epithelial wholemounts, similar effects of the lack of A
were seen in the germinative and central region. The labeling index in the
peripheral (germinative) region was also determined to be 30% lower in the
A/ lens epithelial wholemounts than in the
wild-type, confirming that the effects observed in the central region were
also seen in the peripheral region of the epithelium. However, due to
overcrowding of the BrdU-labeled nuclei in this region, we were unable to
ascertain whether the labeled nuclei were members of a pair.
Deletion of the gene encoding A results in an increased light
scattering in the fiber cells in the central region of 7-week-old mouse lenses
(Brady et al., 1997
). In older
mice, the entire lens becomes opaque. The increased light scattering is due to
the formation of inclusion bodies within the interior fiber cells of the
A-knockout lens. The inclusion bodies primarily comprise
B and
HSP25, two other members of the small HSP family that can form co-aggregates
with
A. The presence of these inclusion bodies has been taken as an
indication that
A is necessary for maintaining the solubility of
B and HSP25. Others have also shown that the 7-week-old
A/ lens is smaller than the wild-type
(Brady et al., 1997
). We
confirmed and extended this finding in this study and showed that the growth
defect in the
A/ lenses occurred early in
life. Lenses of newborn mice were 40% smaller and continued to lag in growth
throughout early postnatal life. It had not been investigated in earlier
studies whether the lens is smaller because of fewer fiber cells or smaller
fiber cells (due to loss of a major cytoplasmic protein). The current work
suggests that, during cell division in vivo, only some of the
A/ cells were able to produce daughter
cells, and the others died during or soon after completing mitosis.
The hypothesis that cells were dying during mitosis is supported by our
observations that there were more TUNEL-positive cells in the
A/ lens epithelial wholemounts, and these
TUNEL-positive cells often appeared as pairs. These dying cells were
considerably reduced in size. Furthermore, our studies revealed that
BrdU-labeled cells similar in appearance to apoptotic bodies were detected
more frequently in the
A/ lens epithelium than
in the controls. These apoptotic bodies also strongly stained with the TUNEL
stain, and were almost always found in pairs, suggesting that the lack of
A increased the susceptibility of both daughter cells to apoptosis.
The present results suggest that A may act at the checkpoint that
controls the exit of cells from mitosis. The exit of cells from mitosis is
controlled by checkpoints that monitor the assembly and position of the
mitotic spindle. Cells defective in assembly of the mitotic spindle produce
abnormal cells that die by apoptosis
(Paulovich et al., 1997
;
Karsenti and Vernos, 2001
).
Cell death associated with mitosis has been observed in a few experimental
systems (Chen et al., 2002
).
Cells can also arrest in mitosis when the microtubule spindle assembly is
disrupted, with drugs such as nocodazole
(Lanni and Jacks, 1998
). The
mitotic spindle performs several well-documented functions, including the
capture and segregation of the duplicated parental chromosomes to daughter
cells (Shimoda and Soloman,
2002
; Heald,
2000
). The central spindle is required for cell cleavage
(Glotzer, 1997
) and the
essential components of the central spindle required for cytokinesis have been
identified (Mishima et al.,
2002
). Our studies showed that the anaphase spindle of
A/ cells was not properly assembled in a
significant proportion of mitotic cells, and suggest that
A/ cells died at mitosis in anaphase or
cytokinesis. This points to a role of the spindle assembly in the apoptotic
cell death in this study.
The current work showed that, during mitosis in mouse lens epithelial
cells, the distribution of A increased around the centrosomes during
metaphase. However, additional punctate staining patterns observed
(Fig. 10B,C) suggest that the
microtubules are not necessarily a main target of
A during early
mitosis.
A was excluded from the chromosomes of dividing cells, but
continued to be expressed at a high level around the chromosomes, and in the
middle of the cell during anaphase. This staining pattern suggests that
A probably associates with the microtubules as well as other
cytoskeletal elements. At cytokinesis, the intercellular bridge microtubules
were strongly labeled by the
A antibodies. At this stage, there
appeared to be a strong association with the microtubule cytoskeleton
(Fig. 10H,I).
A is
redistributed into the cytoplasm during interphase
(Fig. 9A). These observations
suggest that the relative distribution of
A is dependent upon the
relative position of a cell in the cycle. Interestingly, a recent study showed
that a phosphorylated form of
B is associated with centrosomes and
midbodies during cytokinesis (Inaguma et
al., 2001
). It is possible that
A, a closely related
protein, may also be involved with the quality control of proteins such as
tubulin, a major component of the mitotic spindle. Such a role for
A
would be consistent with the importance of small HSPs in organization of
different elements of the cytoskeleton such as the intermediate filaments and
actin (Nicholl and Quinlan,
1994
; Perng et al.,
1999
; Head and Goldman,
2000
), and by its association with actin
(Gopalakrishnan and Takemoto,
1992
; Fitzgerald and Graham,
1991
).
B has also been suggested to be a chaperone for
tubulin (Arai and Atomi, 1997
;
Liang and MacRae, 1997
;
Leroux et al., 1997
). The
hypothesis that
A may play a role in cell division in the lens
epithelium is supported by findings that molecular chaperones are involved in
various aspects of cell growth and differentiation
(Yokota et al., 1999
;
Wigley et al., 1999
;
Inaguma et al., 2001
;
Brown et al., 1996
;
Dunn et al., 2001
). Studies on
synchronized cells in primary cultures are necessary to identify further the
cell-cycle stage at which
A is involved.
A is expressed very early during lens development
(Robinson and Overbeek, 1996
).
Our studies suggest that
A may play a role in preventing apoptosis in
vivo. During eye development, apoptosis is morphologically documented in the
very early stages of lens vesicle formation
(Garcia-Porrero et al., 1984
;
Ishizaki et al., 1993
), is
thought to play a role in primary or secondary fiber cell maturation and
denucleation (Bassnett and Mataic,
1997
; Bassnett,
2002
), and may be associated with differentiation of lens
epithelial cells into fibers in the rat lens
(Ishizaki et al., 1998
). A
recent study suggests that the addition of
A to lens epithelial cells
induces differentiation (Boyle and
Takemoto, 2000
). Aberrant proliferation and apoptosis occurs in
vivo in transgenic mouse lenses expressing the polyoma large T antigen, and in
the developing lenses of Rb-deficient mice
(Griep et al., 1993
;
Morgenbesser et al., 1994
).
Cell-cycle entry and cell death has been reported in post-mitotic lens fiber
cells by overexpression of E2F (Chen et
al., 2000
). Apoptosis does not seem to play a major role in
age-related cataract formation (Harocopos
et al., 1998
). Small HSPs regulate programmed cell death
(Arrigo, 2000
;
Kamradt et al., 2001
;
Paul et al., 2002
). It is not
yet known whether the cell death that we have observed occurs by p53-dependent
mechanisms, although p53 expression has been documented in the murine lens
epithelium (Pokroy et al.,
2002
). Additional studies will be necessary to investigate the
possible role of
A during embryonic growth of the lens.
The present study raises new and important questions about the function of
A in the lens and in other tissues. The expression of
A and
B in nonlenticular tissues, their in vitro properties such as their
autokinase activity (Kantorow and
Piatigorsky, 1994
), interaction with cytoskeletal elements
(Quinlan, 2002
), and the
ability to protect cells from stress-induced apoptosis
(Andley et al., 1998
), suggest
that these proteins have general cellular functions. Further studies in
cultured cells are needed to ascertain whether the expression of
A
directly alters the cell cycle.
In summary, we have demonstrated that A/
lenses undergo slower proliferation and higher apoptosis in vivo than the
wild-type controls. Moreover, our data suggest that
A expression
protects lens epithelial cells in vivo from apoptotic cell death associated
with mitosis. These results are consistent with the observed protective
phenotype conferred by
A expression in vitro in cultured lens
epithelial cells (Andley et al.,
1998
; Andley et al.,
2000
). It is interesting to note that, in contrast to the slower
growth observed in
A/ lens epithelial cells,
the absence of
B, the aggregation partner of
A in the lens,
enhances the frequency of hyperproliferation and tetraploidy in cultured lens
epithelial cells, suggesting a cell-cycle-associated role of
B
(Andley et al., 2001
). Further
studies are necessary to investigate whether the mitosis-associated cell death
that we have observed in the
A/ lens
epithelium occurs by a p53-dependent mechanism in vivo. The system used here
can serve as a useful model for future studies.
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Acknowledgments |
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References |
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---|
Andley, U. P., Song, Z., Wawrousek, E. F. and Bassnett, S.
(1998). The molecular chaperone A-crystallin enhances lens
epithelial cell growth and resistance to UVA stress. J. Biol.
Chem. 273,31252
-31261.
Andley, U. P., Song, Z., Wawrousek, E. F., Fleming, T. P. and
Bassnett, S. (2000). Differential protective activity of
A and
B-crystallins in lens epithelial cells. J.
Biol. Chem. 275,36823
-36831.
Andley, U. P., Song, Z., Wawrousek, E. F., Brady, J. P.,
Bassnett, S. and Fleming, T. P. (2001). Lens epithelial cells
derived from B-crystallin knockout mice demonstrate hyperproliferation
and genomic instability. FASEB J.
15,221
-229.
Arai, H. and Atomi, Y. (1997). Chaperone activity of alpha B-crystallin suppresses tubulin aggregation through complex formation. Cell Struct. Funct. 22,539 -544.[Medline]
Arrigo, A. P. (2000). sHsp as novel regulators of programmed cell death and tumorigenicity. Pathol. Biol. 48,280 -288.[Medline]
Bassnett, S. (2002). Lens organelle degradation. Exp. Eye Res. 74, 1-6.[CrossRef][Medline]
Bassnett, S. and Mataic, D. (1997). Chromatin
degradation in differentiating fiber cells of the eye lens. J. Cell
Biol. 137,27
-49.
Beebe, D. C. and Masters, B. R. (1996). Cell lineage and the differentiation of corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 37,1815 -1825.[Abstract]
Bhat, S. P. and Nagineni, C. N. (1989).
B subunit of lens-specific protein
-crystallin is present in
other ocular and non-ocular tissues. Biochem. Biophys. Res.
Commun. 158,319
-325.[Medline]
Bhat, S. P., Hale, I. L., Matsumoto, B. and Elghanayan, D.
(1999). Ectopic expression of B-crystallin in Chinese
hamster ovary cells suggests a nuclear role for this protein. Eur.
J. Cell Biol. 78,143
-150.[Medline]
Boyle, D. L. and Takemoto, L. J. (1996). EM immunolocalization of alpha-crystallins: association with the plasma membrane from normal and cataractous human lenses. Curr. Eye Res. 15,577 -582.[Medline]
Boyle, D. L. and Takemoto, L. (2000). A
possible role for -crystallins in lens epithelial cell differentiation.
Mol. Vis. 6,63
-71.[Medline]
Brady, J. P., Garland, D., Douglas-Tabor, Y., Robison, W. G.,
Jr, Groome, A. and Wawrousek, E. F. (1997). Targeted
disruption of the mouse A-crystallin gene induces cataract and
cytoplasmic inclusion bodies. Proc. Natl. Acad. Sci.
USA 94,884
-889.
Brady, J. P., Garland, D., Green, D. E., Tamm, E. R., Giblin, F.
J. and Wawrousek, E. F. (2001). B-crystallin in lens
development and muscle integrity: a gene knockout approach. Invest.
Ophthalmol. Vis. Sci. 42,2924
-2934.
Brown, C. R., Doxey, S. J., Hong-Brown, L. O., Martin, R. L. and
Welch, W. J. (1996). Molecular chaperones and the centrosome.
A role for TCP-1 in microtubule nucleation. J. Biol.
Chem. 271,824
-832.
Chen, Q., Huang, F. C., Fromm, L. and Overbeek, P. A.
(2000). Induction of cell cycle entry and cell death in
postmitotic lens fiber cells by overexpresion of E2F1 or E2F2.
Invest. Ophthalmol. Vis. Sci.
41,4223
-4231.
Chen, Q. M., Merrett, J. B., Dilley, T. and Purdom, S. (2002). Down-regulation of p53 with HPV E6 delays and modifies cell death in oxidant response of human diploid fibroblasts: an apoptosis-like cell death associated with mitosis. Oncogene 21,5313 -5324.[CrossRef][Medline]
Cobb, B. A. and Petrash, J. M. (2000).
Characterization of alpha-crystallin-plasma membrane binding. J.
Biol. Chem. 275,6664
-6672.
deFazio, A., Leary, J., Hedley, D. and Tattersall, M. (1987). Immunohistochemical detection of proliferating cells in vivo. J. Histochem. Cytochem. 35,571 -577.[Abstract]
de Jong, W. W., Leunissen, J. A. M. and Voorter, C. E. M.
(1993). Evolution of the -crystallin/small heat shock
protein family. Mol. Biol. Evol.
10,103
-126.[Abstract]
Djabali, K., Piron, G., de Nechaud, B. and Portier, M. M.
(1999). B-crystallin interacts with cytoplasmic
intermediate filament bundles during mitosis. Exp. Cell
Res. 253,649
-662.[CrossRef][Medline]
Dunn, A. Y., Melville, M. W. and Frydman, J. (2001). Review: cellular substrates of the eukaryotic chaperonin TRiC/CCT. J. Struct. Biol. 135,176 -184.[CrossRef][Medline]
Fitzgerald, P. and Graham, D. (1991). Ultrastructural localization of alpha A-crystallin to the bovine lens fiber cytoskeleton. Curr. Eye Res. 10,417 -436.[Medline]
Garcia-Porrero, J. A., Colvee, E. and Ojeda, J. L. (1984). The mechanism of cell death and phagocytosis in early chick lens morphogenesis: a scanning electron microscopy and cytochemical approach. Anat. Rec. 208,123 -136.[Medline]
Glotzer, M. (1997). The mechanism and control of cytokinesis. Curr. Opin. Cell Biol. 9, 815-823.[CrossRef][Medline]
Gopalakrishnan, S. and Takemoto, L. (1992). Binding of actin to lens alpha-crystallins. Curr. Eye Res. 11,929 -933.[Medline]
Griep, A. E., Herbert, R., Jeon, S., Lohse, J. K., Dubielzig, R. R. and Lambert, P. F. (1993). Tumorigenicity by human papilloma virus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation. J. Virol. 67,1373 -1384.[Abstract]
Harocopos, G. J., Alvares, K. M., Kolker, A. E. and Beebe, D. C. (1998). Human age-related cataract and lens epithelial cell death. Invest. Ophthalmol. Vis. Sci. 39,2696 -2706.[Abstract]
Head, M. W. and Goldman, J. E. (2000). Small heat shock proteins, the cytoskeleton, and inclusion body formation. Neuropathol. Appl. Neurobiol. 26,304 -312.[CrossRef][Medline]
Heald, R. (2000). Motor functions in the mitotic spindle. Cell 102,399 -402.[Medline]
Horwitz, J. (1992). The function of
-crystallins. Proc. Natl. Acad. Sci. USA
89,10449
-10453.[Abstract]
Horwitz, J. (2000). The function of alpha-crystallin in vision. Semin. Cell Dev. Biol. 11, 53-60.[CrossRef][Medline]
Hyatt, G. A. and Beebe, D. C. (1985).
Regulation of lens cell growth and polarity by an embryo-specific growth
factor and by inhibitors of lens cell proliferation and differentiation.
Development 117,701
-709.
Inaguma, Y., Ito, H., Iwamoto, I., Saga, S. and Kato, K.
(2001). B-crystallin phosphorylated at Ser-59 is located
in centrosomes and midbodies during mitosis. Eur. J. Cell
Biol. 80,741
-748.[Medline]
Ishizaki, Y., Voyvodic, J. T., Burne, J. F. and Raff, M. C. (1993). Control of lens epithelial cell survival. J. Cell Biol. 121,899 -908.[Abstract]
Ishizaki, Y., Jacobson, M. D. and Raff, M.
(1998). A role for caspases in lens differentiation.
J. Cell Biol. 140,153
-158.
Kamradt, M. C., Chen, F. and Cryns, V. L.
(2001). The small heat shock protein B-crystallin
negatively regulates cytochrome c- and caspase-8-dependent activation of
caspase-3 by inhibiting its autoproteolytic maturation. J. Biol.
Chem. 276,16059
-16063.
Kantorow, M. and Piatigorsky, J. (1994).
-Crystallin/small heat shock protein has autokinase activity.
Proc. Natl. Acad. Sci. USA
91,3112
-3116.[Abstract]
Karsenti, E. and Vernos, I. (2001). The mitotic
spindle: A self-made machine. Science
294,543
-547.
Kuszak, J. R., Clark, J. I., Cooper, K. E. and Rae, J. L. (2000). Biology of the lens: Lens transparency as a function of embryology, anatomy, and pathology. In Principles and Practice of Ophthalmology, Vol. 2, 2nd edn (ed. D. M. Albert and F. A. Jakobiec), Chapter 100, pp.1355 -14085. Philadelphia: W. B. Saunders.
Lanni, J. S. and Jacks, T. (1998).
Characterization of the p53-dependent post-mitotic checkpoint following
spindle disruption. Mol. Cell. Biol.
18,1055
-1064.
Leroux, M. R., Melki, R., Gordon, B., Batelier, G. and Candido,
P. M. (1997). Structure-function studies on small heat shock
protein oligomeric assembly and interaction with unfolded peptides.
J. Biol. Chem. 272,24646
-24656.
Liang, P. and MacRae, T. H. (1997). Molecular
chaperones and the cytoskeleton. J. Cell Sci.
110,1431
-1440.
Litt, M., Kramer, R., LaMorticella, D. M., Murphey, W.,
Louvrien, E. W. and Weleber, R. G. (1998). Autosomal dominant
congenital cataract associated with a missense mutation in the human alpha
crystalline gene CRYAA. Hum. Mol. Genet.
7, 471-474.
McAvoy, J. W. and Chamberlain, C. G. (1989). Fibroblast growth factor induces different responses in lens epithelial cells depending on its concentration. Development 107,221 -228.[Abstract]
Mehlen, P., Mehlen, A., Godet, J. and Arrigo, A. P.
(1997). hsp27 as a switch between differentiation and apoptosis
in murine embryonic fibroblasts. J. Biol. Chem.
272,31657
-31665.
Mikulicich, A. G. and Young, R. W. (1963). Cell proliferation and displacement in the lens epithelium of young rats injected with tritiated thymidine. Invest. Ophthalmol. 2, 344-354.
Mishima, M., Kaitna, S. and Glotzer, M. (2002). Central spindle assembly and cytokinesis requires a kinesin-like protein/RhoGAP complex with microtubule bundle activity. Dev. Cell 2,41 -54.[Medline]
Morgenbesser, S. D., Williams, B. O., Jacks, T. and DePinho, R. A. (1994). p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature 371, 72-74.[CrossRef][Medline]
Nicholl, I. D. and Quinlan, R. A. (1994). Chaperone activity of alpha-crystallins modulates intermediate filament assembly. EMBO J. 13,945 -953.[Abstract]
Paul, C., Manero, F., Gonin, S., Kretz-Remy, C., Virot, S. and
Arrigo, A. P. (2002). Hsp27 as a negative regulator of
cytochrome c release. Mol. Cell. Biol.
22,816
-834.
Paulovich, A. G., Toczyski, D. P. and Hartwell, L. H. (1997). When checkpoints fail. Cell 88,315 -321.[Medline]
Perng, M. D., Cairns, L., van den Ijssel, P., Prescott, A.,
Hutcheson, A. M. and Quinlan, R. A. (1999). Intermediate
filament interactions can be altered by HSP27 and B-crystallin.
J. Cell Sci. 112,2099
-2112.
Pokroy, R., Tendler, Y., Pollack, A., Zinder, O. and Weisinger,
G. (2002). p53 expression in the normal murine eye.
Invest. Ophthalmol. Vis. Sci.
43,1736
-1741.
Quinlan, R. (2002). Cytoskeletal competence requires molecular chaperones. Prog. Mol. Biol. Subcellular Biol. 28,219 -233.
Rafferty, N. S. and Rafferty, K. A. (1981). Cell population kinetics of the mouse lens epithelium. J. Cell. Physiol. 107,309 -315.[Medline]
Rakic, J. M., Galand, A. and Vrensen, G. F. (1997). Separation of fibers from the capsule enhances mitotic activity of human lens epithelium. Exp. Eye Res. 64, 67-72.[CrossRef][Medline]
Reddan, J. R. (1982). Control of cell division in the ocular lens, retina and vitreous. In Cell Biology of the Eye (ed. D. McDevitt), pp. 299-275. New York: Academic Press.
Robinson, M. L. and Overbeek, P. A. (1996). Differential expression of alpha A- and alpha B-crystallin during murine ocular development. Invest. Ophthalmol. Vis. Sci. 37,2276 -2284.[Abstract]
Sax, C. and Piatigorsky, J. (1994). Expression
of the -crystallin/small heat-shock protein/molecular chaperone gene in
the lens and other tissues. Adv. Enzymol. Relat. Areas Mol.
Biol. 69,155
-201.[Medline]
Shimoda, S. L. and Soloman, F. (2002). Integrating functions at the kinetochore. Cell 109, 9-12.[CrossRef][Medline]
Singh, D. P., Ohguro, N., Kikuchi, T., Sueno, T., Reddy, V. N., Yuge, K., Chylack, L. T. and Shinohara, T. (2000). Lens epithelium derived growth factor LEDGF: Effects on growth and survival of lens epithelial cells, keratinocytes and fibroblasts. Biochem. Biophys. Res. Commun. 267,371 -381.[CrossRef]
Srinivasan, A. N., Nagineni, C. N. and Bhat, S. P.
(1992). alpha A-crystallin is expressed in non-ocular tissues.
J. Biol. Chem. 267,23337
-23341.
Vicart, P., Caron, A., Guicheney, P., Li, Z., Prevost, M.-C.,
Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M. et al.
(1998). A missense mutation in the B-crystallin chaperone
gene causes a desminrelated myopathy. Nat. Gen.
20, 92-95.[CrossRef][Medline]
Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R.,
Muallem, S., DeMartino, G. N. and Thomas, P. J. (1999).
Dynamic association of proteasomal machinery with the centrosomes.
J. Cell Biol. 145,481
-490.
Yokota, S., Yanagi, H., Yura, T. and Kubota, H.
(1999). Cytosolic chaperonin is up-regulated during cell growth.
Preferential expression and binding to tubulin at G1/S transition
through early S phase. J. Biol. Chem.
274,37070
-37078.
Zelenka, P. S., Gao, C. Y., Rampalli, A., Aurora, J., Chauthwale, V. and He, H. Y. (1997). Cell cycle regulation in the lens: proliferation, quiescence, apoptosis and differentiation. Prog. Retinal Eye Res. 16,303 -322.[CrossRef]