Download Heat stress-induced localization of small heat shock

Experimental Cell Research 299 (2004) 393 – 403
www.elsevier.com/locate/yexcr
Heat stress-induced localization of small heat shock proteins
in mouse myoblasts: intranuclear lamin A/C speckles
as target for aB-crystallin and Hsp25
Amit S. Adhikari, K. Sridhar Rao, Nandini Rangaraj, Veena K. Parnaik, and Ch. Mohan Rao *
Centre for Cellular and Molecular Biology, Hyderabad AP 500 007, India
Received 3 March 2004, revised version received 14 May 2004, accepted 20 May 2004
Available online 3 July 2004
Abstract
We examined the effect of heat stress on localization of two sHsps, aB-crystallin and Hsp25, and of Hsc70, a member of a different class
of heat shock proteins (Hsps), in both undifferentiated and differentiated mouse C2C12 cells. Under normal conditions, aB-crystallin and
Hsp25 are found in the cytoplasm; only aB-crystallin is also found in the nucleus, distributed in a speckled pattern. Hsc70 is found to be
homogeneously distributed throughout the cell. On heat stress, all these proteins translocate almost entirely into the nucleus and upon
recovery relocate to the cytoplasm. Dual staining experiments using C2C12 myoblasts show that aB-crystallin and Hsp25, but not Hsc70,
colocalize with the intranuclear lamin A/C and the splicing factor SC-35, suggesting interactions of sHsps and intranuclear lamin A/C.
Interestingly, none of these proteins are found in the myotube nuclei. Upon heat stress, only Hsc70 translocates into the myotube nuclei. This
differential entry of aB-crystallin and Hsp25 into the nuclei of myoblasts and myotubes upon heat stress may have functional role in the
development and/or in the maintenance of muscle cells. Our study therefore suggests that these sHsps may be a part of the intranuclear lamin
A/C network or stabilizing this specific network.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Alpha crystallin; hsp25; hsp70; Lamin; Muscle; Colocalization
Introduction
Living cells withstand various types of environmental
stress by selectively over expressing different classes of
proteins collectively known as heat shock proteins (Hsps).
Many of these have also been shown to act as molecular
chaperones in preventing heat- or other stress-induced
aggregation of proteins and assisting in proper folding.
aB-Crystallin is one of the 10 mammalian members of the
sHsp family that have been identified till date [1]. All the
members of this family share a common domain of approximately 80– 100 amino acids called the ‘‘a crystallin domain.’’
aB-Crystallin and human Hsp27 as well as its rodent orthologue Hsp25 have been shown to be stress inducible [2– 4].
aB-Crystallin, which is predominantly present in eye
lens as a hetero-oligomer with aA-crystallin, is also
expressed in significant amounts in nonlenticular tissues
* Corresponding author. Centre for Cellular and Molecular Biology,
Uppal Road Hyderabad AP 500 007, India. Fax: +91-40-2716-0591.
E-mail address: [email protected] (C. Mohan Rao).
0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2004.05.032
such as brain, heart, skeletal muscle, kidney, and skin [5]. It
has a monomeric mass of 20 kDa and forms large homooligomers of 400– 600 kDa. The expression of aB-crystallin
has been shown to be elevated in response to different
stimuli like heat stress, ischemia, oxidative stress, and also
in many pathological conditions like Alzheimer’s, Creutzfeldt – Jakob, and prion diseases [6 –11]. aB-Crystallin and
Hsp27 are found to be associated with Rosenthal fibers in
Alexander’s disease [12,13]. A point mutation (R120G) in
aB-crystallin leads to desmin-related myopathy in humans
[14]. Studies from our laboratory [15] and those of others
[16,17] indicate that the mutation leads to loss of chaperone
activity in vitro, which perhaps is the molecular basis for the
pathology. aB-Crystallin knockout mouse shows extensive
muscle wastage [18], corroborating the importance of sHsps
in muscle maintenance.
The homo- and hetero-oligomers of aA- and aB-crystallin
as well as sHsp27/25 are known to exhibit molecular chaperone-like activity in preventing aggregation of proteins [19 –
24]. Our laboratory has shown that the chaperone-like activity of a-crystallin is temperature dependent. a-Crystallins are
394
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
found to protect enzymes from heat-induced inactivation and
also aid in reactivation of some enzymes [24]. Several studies
have indicated that sHsps, including aB-crystallin and
Hsp27, can interact with intermediate filaments and assist
their assembly in the cytoplasm [25]. This association of aBcrystallin with intermediate filaments has also been shown to
be temperature dependent [26].
Interestingly, aB-crystallin, in addition to its presence in
the cytoplasm, is also present in the nucleus [27]. It translocates from the cytoplasm to the nucleus under heat stress
in NIH3T3 and N1E-115 cell lines [2,28]. Such nuclear
localization suggests a regulatory role for aB-crystallin
during stress.
We have investigated the localization of aB-crystallin and
its possible nuclear targets under normal and heat stress
conditions in C2C12 undifferentiated mouse myoblasts and
differentiated myotubes. We have also studied the closely
related Hsp25 and the unrelated Hsc70 for comparison. Our
study shows interesting differences in localization as well as
heat-induced translocation among the very similar sHsps,
aB-crystallin, and Hsp25 and the unrelated Hsc70 in C2C12
cells. We find that aB-crystallin and Hsp25, but not Hsc70,
colocalize significantly with the intranuclear lamin A/C
speckles in myoblasts. Interestingly, this translocation of
aB-crystallin and Hsp25 in the nucleus appears to be differentiation stage specific, implying its functional significance.
Material and methods
Cell culture and heat treatment
C2C12, mouse skeletal myoblast cell line, was maintained at subconfluent densities (60 – 70%) in DMEM (Sigma, USA) supplemented with 20% fetal calf serum (Sigma)
at 37jC in a humidified atmosphere containing 5% CO2. For
myogenic differentiation, the growth medium was replaced
with DMEM containing 2% horse serum (Sigma) and the
cells were incubated at 37jC for 72 h. For heat stress,
myoblasts or myotubes plated on cover slips with appropriate culture medium were incubated at 43jC in a water bath
for 70 min. For recovery studies, heat-stressed cells were
incubated at 37jC, 5% CO2 in humidified chamber. Cell
viability, as assessed by trypan blue staining, was greater
than 95%. To investigate whether de novo synthesis of sHsps
occurred during the recovery period of 3 h, cycloheximide
(Sigma), a protein synthesis inhibitor, was added immediately after heat stress at a final concentration of 25 Ag/ml and
cells were kept for recovery at 37jC. Cells were fixed with
3.5% formaldehyde after every hour for 3 h and stained with
antibodies to aB-crystallin and Hsp25 or Hsc70.
Antibodies
Rabbit polyclonal antibody against the carboxy terminal
21 amino acids of aB-crystallin was a kind gift of Dr. Usha
Andley, Department of Ophthalmology and Visual Sciences,
Washington University School of Medicine, USA. Rabbit
polyclonal antibodies against Hsp25, aB-crystallin, and
Hsp70 and rat monoclonal antibody (mAb) against Hsc70
were obtained from Stressgen Biotechnologies, Canada.
Antibodies to recombinant lamins used in this study are
mouse mAb LA2H10 that recognizes intranuclear lamin A/
C speckles and LA2B3 that recognizes peripheral lamin A/C
rim, rabbit polyclonal antibodies to lamin A/C, and lamin
B1, which also stain the peripheral lamina [29]. Monoclonal
antibody to SC-35 was provided by Dr. J. Gall, Carnegie
Institution of Washington, Baltimore, USA. For myogenin,
mouse monoclonal antibody was purchased from Developmental Study Hybridoma Bank, USA.
Immunofluorescence microscopy
C2C12 myoblast or myotubes grown on cover slips in
six well culture dishes were given heat stress as described
above. Cells were washed and then fixed in PBS containing
3.5% formaldehyde immediately after heat stress or after 3
h of recovery or without stress. Formaldehyde-fixed cells
were permeabilized using 0.5% Triton X-100 in PBS at
room temperature for 8 min or with methanol at 20jC for
10 min, blocked in 2% BSA, and incubated in primary
antibody (polyclonal) followed by FITC-tagged or Alexa
488-conjugated secondary antibody. In the case of dual
labeling, the immunostaining of the second protein was
done subsequently with respective primary antibody
(monoclonal), which was followed by the addition of
Cy3-tagged secondary antibody. All the incubations were
for 1 h. Samples were mounted in Vectashield mounting
medium (Vector laboratories, USA) containing DAPI (1 Ag/
ml) as a nuclear counterstain. There was no cross reactivity
of the fluorescent second antibodies in control experiments in which primary antibody was eliminated. Antibody conjugates were from Molecular Probes or Jackson
ImmunoResearch Laboratories, USA. Confocal laser
scanning immunofluorescence microscopy was performed
on a Meridian Ultima scan head attached to an Olympus
IMT-2 inverted microscope fitted with 100, 1.3 NA, or
60, 1.4 NA objective lenses with excitation at 514, 488,
and 351– 364 nm (Argon ion laser). Image analysis, including crossover subtraction, estimation of colocalized
speckles was done using DASY master program V4.19
(Meridian Instruments Inc.). For quantitative analysis of
colocalization, the data from individual sections (0.5 Am
thickness) were queried by test lines through the speckles
and fluorescence intensities of both the dyes were viewed
graphically. The number of overlapping and nonoverlapping peaks was estimated and percent-colocalized peaks
were calculated [29,30]. The images were assembled using
Adobe Photoshop 5.0.
Soluble and insoluble protein fractions from isolated
nuclei and cytoplasm were quantitated by amido black
staining method [31], electrophoresed in a 12% SDS PAGE,
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
immunobloted using specific antibodies, and visualized
using a chemiluminescence kit (Amersham Pharmacia,
USA).
Results
Immunolocalization of aB-crystallin, Hsp25 and Hsc70
We carried out immunolabeling experiments for aBcrystallin using a rabbit polyclonal antibody that was raised
against the 21 amino acid carboxy terminal peptide of aBcrystallin. This antibody has been shown to specifically
recognize aB-crystallin [32]. CHO cells, which do not have
endogenous aB-crystallin, did not show any signal with this
antibody upon immunostaining (data not shown). The
C2C12 cell line, a well-characterized mouse cell line,
exhibited good staining with the aB-crystallin antibody.
The C2C12 mouse myoblasts are known to express aBcrystallin and Hsp25. When myoblasts fuse to form multinucleated myotubes, the levels of aB-crystallin and Hsp25
have been shown to be elevated 10- and 3-fold, respectively
[33,34]. When we added purified aB-crystallin during the
staining of C2C12 cells with aB-crystallin antibody, we
observed a concentration-dependent reduction in the staining, establishing the specificity of the antibody (data not
shown).
Confocal analysis of C2C12 myoblast cells immunostained with this antibody showed that aB-crystallin was
distributed both in the cytoplasm and nucleus. The cytoplasmic distribution of aB-crystallin was uniform, while it
displayed a speckled appearance in the nucleus (Fig. 1A).
395
Interestingly, though Hsp25 shares high homology (43%
identity, 57% similarity) with aB-crystallin, immunostaining with an Hsp25-specific antibody shows that this protein
is mostly found in the cytoplasm with little or no distribution in the nucleus (Fig. 1D).
Members of another class of heat shock proteins, Hsp70
and its constitutive isoform, Hsc70, are by far the most
abundant and well-studied proteins for their protective roles
and chaperone activities [35,36]. To study how the sHsps,
aB-crystallin, and Hsp25 differ in their localization with
reference to Hsc70, we have also stained the cells with an
Hsc70-specific antibody. As shown in Fig. 1G, Hsc70 is
localized both in the cytoplasm and the nucleus similar to
aB-crystallin. However, comparison of Figs. 1A and G
shows that the staining patterns of aB-crystallin and
Hsc70 in the nucleus are different. aB-crystallin localizes
in the nucleus with a speckled appearance, excluding
nucleolar regions. On the other hand, the staining of
Hsc70 is more or less uniform.
Effect of heat shock on intracellular localization of sHsps
and Hsc70
To determine the localization of the sHsps and Hsc70
upon heat shock and subsequent recovery, we have incubated C2C12 cells at 43jC for 70 min, followed by recovery
at 37jC for 3 h. Cells were fixed immediately after heat
stress or recovery and immunostained with specific antibodies. It is evident from Figs. 1B, E, and H that after the
heat stress, aB-crystallin, Hsp25, and Hsc70 are exclusively
present in the nucleus with little or no staining in the
cytoplasm, indicating that these heat shock proteins trans-
Fig. 1. Immunolocalization of aB-crystallin, Hsp25, and Hsc70 in C2C12 myoblasts. (A – C) aB-Crystallin, (D – F) Hsp25, and (G – I) Hsc70 were stained with
their respective polyclonal antibodies. Unstressed cells (under normal conditions) or stressed cells (43jC, 70 min) were fixed in 3.5% formaldehyde,
immediately after heat stress or 3 h after recovery at 37jC. Counterstaining of the nucleus with DAPI (AV – IV) for the corresponding cells are as shown. Scale
bar: 10 Am. The arrows indicate speckles in B and E, nucleoli in I.
396
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
locate into the nucleus upon heat shock. However, the
nuclear staining pattern for the sHsps and Hsc70 is different—aB-crystallin and Hsp25 are localized in a speckled
pattern, whereas Hsc70 is stained homogeneously with
intense staining in the nucleolar region. The nuclear localization of Hsc70 upon heat stress has also been reported
earlier [37]. To exclude the possibility of induction of
differentiation upon heat stress, cells were stained for the
differentiation-specific marker, myogenin [38]. We found
that only 2– 3% cells stained positive for myogenin before
and after heat stress (data not shown) showing that the
cells did not undergo differentiation. The intracellular
distribution of aB-crystallin, Hsp25, and Hsc70 was examined after 3 h of recovery from heat shock. As shown in
Figs. 1C and F, both the sHsps were detected in the
cytoplasm on recovery from heat shock. Cytoplasmic
staining of aB-crystallin and Hsp25 after recovery could
be due to de novo synthesis of protein or relocation from
the nucleus to the cytoplasm. To distinguish between these
possibilities, we performed the recovery experiments in the
presence of cycloheximide, a known protein synthesis
inhibitor. We observed that aB-crystallin and Hsp25 were
still detectable in the cytoplasm upon recovery after heat
stress in the presence of cycloheximide. Thus, these results
indicate that the sHsps relocate into the cytoplasm during
recovery. The kinetics of such relocation appears to differ
between the sHsps and Hsc70. After 3 h recovery, both
aB-crystallin and Hsp25 exhibit a staining pattern similar
to that in unstressed cells, whereas most of the Hsc70 is
retained in the nucleus (Fig. 1I), suggesting that the
relocation of Hsc70 after the heat shock is a slower
process compared to that of the sHsps.
aB-crystallin and Hsp25 colocalize with intranuclear lamin
A/C speckles
Both of the small heat shock proteins are detected in the
nuclear insoluble fraction after heat shock. This prompted
us to look for a possibility of their interaction with the
nuclear matrix, the major constituents of which are the
lamins. As described earlier (Figs. 1A – C), aB-crystallin
exhibits a distinct speckled staining pattern in the nucleus,
especially after heat stress. Immunostaining of C2C12
myoblasts with LA2H10, a monoclonal antibody for lamin
A/C, is known to show a speckled distribution of lamin A/
C in the nucleus [29]. We therefore carried out dual
labeling experiments to investigate whether aB-crystallin
colocalizes with lamin A/C speckles. In unstressed cells,
nuclear aB-crystallin colocalized partially with lamin A/C
speckles as shown in Fig. 2A (unstressed cells). Quantitation of the extent of colocalization indicated that about 50%
of lamin speckles were positive for aB-crystallin. Upon
subjecting the cells to heat shock, the extent of colocalization increased to >80% as shown in Fig. 2A (stressed cells).
The percent colocalization of aB-crystallin with lamin A/C
decreased during recovery. Only 30% aB-crystallin colo-
calized with lamin A/C speckles 3 h after recovery (Fig.
2A; after recovery). Colocalization after recovery is less
than that observed in the unstressed cells. As mentioned
earlier, Hsp25 was not detected in the nucleus under normal
conditions and dual staining also did not show any colocalization with lamin speckles in normal cells (Fig. 2B;
unstressed cells). Upon heat stress, Hsp25 translocated into
the nucleus and exhibited colocalization with lamin A/C
speckles (Fig. 2B; stressed cells) to the extent of 65%.
Colocalization of Hsp25 with lamin A/C decreased to less
than 20% upon recovery (Fig. 2B; after recovery). These
results suggest that both the small heat shock proteins, aBcrystallin and Hsp25, might be interacting with intranuclear
lamin A/C. In contrast to the observation with the sHsps,
Hsc70, however, did not show significant colocalization
with lamin A/C speckles in normal (approximately 10%),
heat shock conditions (approximately 20%) as well as upon
recovery (25%) (Fig. 2C; unstressed cells, stressed cells,
and after recovery).
aB-crystallin colocalizes with splicing factor SC-35
speckles
Lamin A/C speckles have earlier been shown to colocalize with mRNA splicing factors such as SC-35 [29] and
mediate spatial organization of SFCs [39]. We have investigated whether aB-crystallin also colocalizes with SC-35 in
normal and heat-stressed myoblasts. Fig. 3 (unstressed cells)
shows that aB-crystallin colocalizes with the splicing factor
SC-35 in both normal conditions and upon heat shock (Fig.
3; stressed cells): The percentage colocalization was estimated to be 55% in unstressed and 85% in heat-stressed
cells. Colocalization decreased to less than 40% upon
recovery (Fig. 3; after recovery). It may also be noted that
the staining pattern of the non-snRNP splicing factor, SC35, stained with monoclonal antibody to SC-35, remained
unaltered upon heat stress and on recovery (Fig. 3; stressed
cells and after recovery). Thus, our results show that aBcrystallin also colocalizes with SC-35 in unstressed C2C12
myoblasts. These results are in agreement with the recent
findings of van den Ijssel et al. [27] and van Rijk et al. [40],
who have shown that under normal conditions, aB-crystallin colocalizes with SC-35 as well as the snRNP components Sm and U1A. In addition, our results demonstrate that
the extent of colocalization of aB-crystallin with SC-35
increases upon heat stress compared to that in the unstressed
cells.
Based on the observation that the levels of lamin B
increase upon heat treatment at 45.5jC, but not at 43jC,
Dynlacht et al. [41] suggested that lamin B is a heat shock
protein. Whether such an increase in lamin A/C levels
occurs upon heat stress is not known. Since our study
shows that sHsps, aB-crystallin, and Hsp25 colocalize with
lamin A/C, especially upon heat stress, we have investigated the staining patterns of A- and B-type lamins upon heat
stress.
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
Immunolabeling of C2C12 cells with polyclonal antibodies against lamin B shows its presence in the inner
nuclear rim (Fig. 4D). However, neither aB-crystallin nor
Hsp25 shows a rimlike pattern in normal as well as heat
stress conditions, suggesting that lamin B may not interact
significantly with these sHsps. Under the conditions of
heat stress, used in our experiments (43jC, 70 min), the
staining pattern of lamin B does not alter significantly
(Fig. 4E); further, the staining pattern remains the same
even after 3 h of recovery (Fig. 4F), indicating that lamin
B architecture is not changed upon heat stress. Polyclonal
antibodies to lamin A/C stain the peripheral lamin A/C as
a rimlike pattern as shown in Fig. 4A. Upon heat stress,
this rimlike pattern is perturbed significantly (Fig. 4B),
indicating that the peripheral lamin A/C architecture is
considerably changed upon heat stress. The rimlike staining pattern is not completely recovered even after 3 h of
recovery (Fig. 4C). As our study shows, the internal
speckled lamin A/C architecture interacts with small heat
shock proteins and probably gets stabilized. This interaction might be the reason for the resistance of internal
speckled lamin A/C architecture towards heat stress. It is
possible that the reorganization of this architecture occurs
during early recovery periods, whose significance is not
clear.
aB-crystallin and Hsp25 do not translocate into myotube
nuclei
Individual myoblasts stop dividing and fuse to form
multinucleated myotubes upon serum depletion. Lamin A/
C speckles have been shown to reorganize into a diffuse
network in myotubes [42]. Hence, under normal conditions
of staining, mAb LA2H10 does not stain lamin A/C in
myotube nuclei. As expected, due to reorganization, the
speckled staining pattern observed in myoblasts was absent
in the unstressed myotubes (Fig. 5A). Our experiments also
show that the speckled pattern of lamin A/C does not appear
in myotubes subjected to heat stress conditions (Fig. 5B) or
even after subsequent recovery (Fig. 5C). In contrast to the
intranuclear speckles, the peripheral lamin A/C is seen in
both myoblasts and myotubes. The peripheral staining
pattern remains unaltered during differentiation (Fig. 5D)
as well as upon heat stress to myotubes and subsequent
recovery (Figs. 5E and F).
Since myotubes show altered intranuclear lamin A/C
organization, we have investigated the localization of the
sHsps and Hsc70 in unstressed and heat-stressed myotubes. Interestingly, aB-crystallin as well as Hsc70 are not
detectable in the nuclei of myotubes under normal conditions (Figs. 5G and M), unlike in myoblasts (Figs. 1A
and G). The nuclei of myotubes also do not have detectable levels of Hsp25 (Fig. 5J) as seen previously in
unstressed myoblasts (Fig. 1D). Both aB-crystallin and
Hsp25, which translocate into the nucleus upon heat stress
in myoblasts, do not translocate into the nuclei of myo-
397
tubes when subjected to similar heat stress (Figs. 5H and
K) and upon recovery (Figs. 5I and L). In contrast to the
sHsps, Hsc70 translocates into the nucleus upon heat stress
(Fig. 5N) and continues to remain in the nucleus even after
3 h of recovery (Fig. 5O). Thus, these results demonstrate
that there are marked differences in the localization as well
as the heat shock-induced nuclear translocation of sHsps
and Hsc70 between the undifferentiated and differentiated
C2C12 cells.
Discussion
Small heat shock proteins are necessary for several
cellular functions and particularly in stress tolerance. Although a vast number of stress-response or heat shock
proteins have been discovered, their individual or coordinated functions in vivo are not completely understood. aBCrystallin, one of the major eye lens proteins, is also present
in many nonlenticular tissues including muscles. In our
laboratory, we have been investigating the in vitro chaperone activity of aA- and aB-crystallin particularly in the
context of disease (for a review, see Refs.[23,24]). In an
attempt to understand the in vivo functionality of aBcrystallin, we have investigated its localization in both
undifferentiated and differentiated C2C12 cells, under normal and heat-stressed conditions. We have also investigated
the localization of another closely related sHsp, Hsp25, and
Hsc70, a member of a different class of heat shock proteins,
to understand how they differ in their behavior in normal
and heat stress conditions. The myoblast – myotube system
of C2C12 cells is particularly relevant—aB-crystallin and
Hsp25 are abundantly present in the skeletal muscle; point
mutation in aB-crystallin (R120G) causes myopathic conditions, which involve muscle wastage [14]. aB-crystallin
knockout experiments have shown that this protein is
essential in maintaining muscle integrity [18]. aB-crystallin
has also been reported to protect the C2C12 cells from
differentiation-induced apoptosis by inhibiting caspase-3
maturation, whereas the mutant form R120G is defective
in such a protective function [43].
Our present study shows that though aB-crystallin and
Hsp25 are closely related sHsps, Hsp25 is exclusively
present in the cytoplasm of the myoblasts, whereas aBcrystallin is present in significant levels in the nucleus in
addition to the cytoplasm. The unrelated Hsc70 is also
found both in the cytoplasm and the nucleus. However,
the difference in the immunostaining patterns of aB-crystallin (speckled pattern) and Hsc70 (more or less homogeneous pattern) suggests that their localization in specific
subnuclear compartments and therefore their targets and
functions might be different. van den Ijssel et al. [27] have
reported speckled localization of aB-crystallin in the nucleus of some human cell lines; transfection of the mutant
R120G aB-crystallin disrupts such speckled localization of
aB-crystallin. These studies indicate an important functional
398
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
role for the speckled localization of aB-crystallin in the
nucleus of unstressed or normal cells.
Our study shows that upon heat stress, the closely related
sHsps, aB-crystallin, and Hsp25, as well as the unrelated
Hsc70, translocate into the nucleus. The immunostaining
pattern of both the sHsps shows speckled distribution,
indicating that aB-crystallin as well as Hsp25 is enriched
in specific subnuclear compartments of C2C12 myoblasts
Fig. 2. Colocalization of sHsps and Hsc70 with lamin A/C. C2C12 myoblasts were stained with monoclonal antibody (LA2H10) to lamin A/C and polyclonal
antibody to (A) aB-crystallin, (B) Hsp25, and (C) Hsc70. Unstressed cells (under normal conditions) or stressed cells (43jC, 70 min) were fixed in 3.5%
formaldehyde immediately after heat stress or 3 h after recovery at 37jC. The heat shock proteins were labeled green and lamin A/C, red. Confocal overlays of
the doubly stained cells are shown in the merged panel, where the yellow color highlights structures stained by both the antibodies in a single optical section of
0.5 Am. The arrows indicated colocalized speckles. Counterstaining of the nucleus with DAPI (blue) is also shown. Scale bar: 10 Am.
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
399
Fig. 2 (continued ).
Fig. 3. Colocalization of aB-crystallin with splicing factor SC-35. Unstressed cells (under normal conditions) or stressed cells (43jC, 70 min) were fixed in
3.5% formaldehyde immediately after heat stress or 3 h after recovery at 37jC. aB-crystallin (green) and SC-35 (red) were stained with rabbit polyclonal and
mouse monoclonal antibodies, respectively. The merged areas in the overlays are seen yellow in a single section of 0.5 Am. Counterstaining of the nucleus with
DAPI (blue) is also shown. Scale bar: 10 Am.
400
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
Fig. 4. Localization of lamin A/C and lamin B—Effect of heat shock. (A – C) Lamin A/C stained with rabbit polyclonal antibody, (D – F) lamin B stained with
rabbit polyclonal antibody. Counterstaining of the nucleus with DAPI (AV – FV) is also shown. Scale bar: 10 Am.
upon heat stress. Earlier studies have shown that aBcrystallin translocates from the cytoplasm to the nucleus
in NIH3T3 fibroblasts [2] and N1E-115 neuroblastoma cells
subjected to stress; however, immunostaining showed a
homogeneous distribution [28] rather than a speckled appearance. In neonatal cardiomyocytes of rat, Hsp25 but not
Fig. 5. Localization of lamin A/C, aB-crystallin, Hsp25, and Hsc70 in differentiated C2C12 cells (myotubes)—Effect of heat shock. Upper panel: myotubes
stained for lamin A/C. (A and D) Unstressed myotubes and (B and E) stressed myotubes (43jC, 70 min), (C and F) after 3 h recovery at 37jC. Lamin A/C were
stained with LA2H10 and LA2B3 monoclonal antibodies. Lower panel: unstressed myotubes (under normal conditions) or stressed myotubes (43jC, 70 min)
were fixed in 3.5% formaldehyde immediately after heat stress or 3 h after recovery at 37jC. (G – I) aB-crystallin, (J – L) Hsp25, and (M – O) Hsc70 were
stained with their respective polyclonal antibodies. Counterstaining of the nucleus with DAPI (AV – OV) is also shown. Scale bar: 10 Am.
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
aB-crystallin translocates into the nucleus upon heat stress
[44]. All these studies show that the localization of sHsps
under normal as well as stress conditions varies considerably in different cells types. Whether these differences are
due to the species- and/or tissue-specific recruitment of
various heat shock proteins for some specific, unknown
function is not clear.
Currently, there is considerable interest in understanding
the role of nuclear architecture in the regulation of nuclear
functions [46]. The organization of the nucleus into specific
domains or compartments involved in DNA replication,
transcription, and splicing has been well documented
[47,48]. The major structural proteins in the nucleus are
the lamins, which are type V intermediate filament proteins
that form a network or lamina underlying the inner nuclear
membrane and are also present in the interior of the nucleus
[46,49]. There is definitive evidence for the involvement of
the lamins in DNA replication [50,51] and organization of
transcription [39,52]. There are two kinds of lamins, the Btype lamins and the A-type lamins (A and C). Novel
internal lamin A/C structures that colocalize with RNA
splicing factor compartments (SFCs) are called lamin
speckles [29]. These structures are proposed to be involved
in the spatial organization of transcription sites and SFCs
[39].
Our biochemical studies using Western blot analysis
(data not shown) on cellular fractions showed that while
in normal cells aB-crystallin is predominantly found in the
soluble fraction, upon heat stress it is found mainly in the
nuclear insoluble fraction. Thus, these results indicate interaction of aB-crystallin with lamins, the major constituents
of the nuclear insoluble fraction, upon heat stress. Using
nuclear fractionation studies and microscopic studies on the
isolated nuclei and nuclear matrix preparations from
encysted embryos of Artemia franciscana, Willsie and
Clegg [45] have shown that Hsp70 and an sHsp, p26,
associate with lamins. The levels of lamin B, present in
cells in the inner periphery of the nucleus, are increased
twofold in U-1 melanoma and HeLa cell lines at elevated
temperatures of 45.5jC but not at 43jC [41]. Our studies do
not indicate any significant changes either in the levels or in
the rimlike staining pattern of lamin B in C2C12 cells
subjected to heat stress at 43jC. However, the peripheral
lamin A/C generally visualized as a rim by polyclonal
antibodies is disrupted upon heat shock. Thus, our results
show that the architecture of lamin A/C is more dynamic in
nature compared to that of the peripheral lamin B during
heat shock.
As mentioned earlier, immunostaining of aB-crystallin
exhibits a speckled pattern inside the nucleus of C2C12
myoblasts both in normal and heat shock conditions. Hsp25,
translocated into the nucleus upon heat stress, also shows a
speckled pattern. The aB-crystallin and Hsp25 speckles
colocalize with intranuclear lamin A/C speckles upon heat
stress, suggesting that these sHsps play a role in formation
and/or stabilization of the dynamic lamin A/C architecture.
401
Earlier studies have shown the association of internal lamin
A/C speckles with SFCs and their role in mediating spatial
organization of SFCs and RNA polymerase II mediated
transcription [29,39]. Interestingly, our study shows that
aB-crystallin colocalizes with the splicing factor SC-35 as
well. In the light of the above, the interaction of sHsps with
speckles in unstressed cells and their enhancement upon
heat stress appear to be critical for the functional integrity of
the speckle domains.
We have also studied the intracellular localization of aBcrystallin, Hsp25, and Hsc70 in the serum deprivationinduced differentiated myotubes of C2C12 cells. Striking
differences in the localization of sHsps and Hsc70 are seen
upon differentiation. Unlike in the case of myoblasts, aBcrystallin and Hsp25 do not translocate into the nuclei of
myotubes, whereas Hsc70 continues to do so upon subjecting the myotubes to heat stress. This assumes greater
significance considering earlier findings of a reorganization
of lamin A/C speckles to a diffuse network after differentiation of C2C12 myoblasts to myotubes [42]. In addition,
our observation that there is no alteration in the staining
pattern of both lamin A/C intranuclear speckles (LA2H10)
and peripheral rim (LA2B3) upon heat stress in myotubes
suggests a more stable lamin A/C architecture after differentiation. Thus, lack of nuclear translocation of sHps, aBcrystallin, and Hsp25 in myotubes signifies an important
differentiation stage-specific role for these sHsps. aB-crystallin and Hsp25 not only colocalize with the intranuclear
lamin A/C speckles but also with the splicing factor, SC-35,
in myoblasts. However, the speckled staining pattern of the
splicing factor, SC-35, does not change upon differentiation
of C2C12 myoblast to myotubes, despite the distinct
changes in the intranuclear lamin A/C organization. Thus,
it appears that aB-crystallin and Hsp25 stabilize the specific
internal speckled architecture of lamin A/C that harbors the
splicing factor (SC-35) compartments rather than directly
interacting with the splicing factor.
Acknowledgments
We thank Dr. Usha Andley for kindly providing us the
aB-crystallin antibodies and Bh. Murlikrishna and S.
Thanumalayan for help with lamin antibodies. We thank
Dr. B. Raman and Dr. T. Ramakrishna for useful discussions
and for critical reading of the manuscript. Amit S. Adhikari
acknowledges the University Grants Commission, New
Delhi, India, for senior research fellowship.
References
[1] J.M. Fontaine, J.S. Rest, M.J. Welsh, R. Benndorf, The sperm outer
dense fiber protein is the 10th member of the superfamily of mammalian small stress proteins, Cell Stress Chaperones 8 (2003) 62 – 69.
[2] R. Klemenz, E. Frohli, R.H. Steiger, R. Schafer, A. Aoyama, Alpha
402
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
B-crystallin is a small heat shock protein, Proc. Natl. Acad. Sci.
U.S.A. 88 (1991) 3652 – 3656.
S. Dasgupta, T.C. Hohman, D. Carper, Hypertonic stress induces
alpha B-crystallin expression, Exp. Eye Res. 54 (1992) 461 – 470.
M.W. Head, E. Corbin, J.E. Goldman, Coordinate and independent
regulation of alpha B-crystallin and hsp27 expression in response to
physiological stress, J. Cell Physiol. 159 (1994) 41 – 50.
S.P. Bhat, C.N. Nagineni, aB subunit of lens-specific protein a-crystallin is present in other ocular and non-ocular tissues, Biochem.
Biophys. Res. Commun. 158 (1989) 319 – 325.
J.R. Duguid, R.G. Rohwer, B. Seed, Isolation of cDNAs of scrapiemodulated RNAs by subtractive hybridization of a cDNA library,
Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 5738 – 5742.
M. Chiesi, S. Longoni, U. Limbruno, Cardiac alpha-crystallin: III.
Involvement during heart ischemia, Mol. Cell. Biochem. 97 (1990)
129 – 136.
K. Renkawek, W.W. de Jong, K.B. Merck, C.W. Frenken, F.P. van
Workum, G.J. Bosman, Alpha B-crystallin is present in reactive glia
in Creutzfeldt – Jakob disease, Acta Neuropathol. (Berl.) 83 (1992)
324 – 327.
K. Kato, S. Goto, K. Hasegawa, Y. Inaguma, Coinduction of two lowmolecular-weight stress proteins, alpha B crystallin and HSP28, by
heat or arsenite stress in human glioma cells, J. Biochem. (Tokyo) 114
(1993) 640 – 647.
P.J. Groenen, K.B. Merck, W.W. de Jong, H. Bloemendal, Structure
and modifications of the junior chaperone alpha-crystallin. From
lens transparency to molecular pathology, Eur. J. Biochem. 225
(1994) 1 – 19.
K. Renkawek, C.E. Voorter, G.J. Bosman, F.P. van Workum, W.W. de
Jong, Expression of alpha B-crystallin in Alzheimer’s disease, Acta
Neuropathol. (Berl.) 87 (1994) 155 – 160.
T. Iwaki, A. Kume-Iwaki, R.K.H. Iiem, J.E. Goldman, aB-crystallin
is expressed in non-lenticular tissues and accumulates in Alexander’s
diseases brain, Cell 57 (1989) 71 – 78.
T. Iwaki, A. Iwaki, J. Tateshi, Y. Sakaki, J.E. Goldman, aB-crystallin
and 27-kd heat shock protein are regulated by stress conditions in the
central-nervous system and accumulate in the Rosenthal fibers, Am. J.
Pathol. 143 (1993) 487 – 495.
P. Vicart, A. Caron, P. Guicheney, Z. Li, M.C. Prevost, A. Faure, D.
Chateau, F. Chapon, F. Tome, J.M. Dupret, et al., A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related
myopathy, Nat. Genet. 20 (1998) 92 – 95.
L.V. Kumar, T. Ramakrishna, C.M. Rao, Structural and functional
consequences of the mutation of a conserved arginine residue in alphaA
and alphaB crystallins, J. Biol. Chem. 274 (1999) 24137 – 24141.
M.P. Bova, O. Yaron, Q. Huang, L. Ding, D.A. Haley, P.L. Stewart, J.
Horwitz, Mutation R120G in alphaB-crystallin, which is linked to a
desmin-related myopathy, results in an irregular structure and defective chaperone-like function, Proc. Natl. Acad. Sci. U.S.A. 96 (1999)
6137 – 6142.
M.D. Perng, P.J. Muchowski, P. van Den Ijssel, G.J. Wu, A.M.
Hutcheson, J.I. Clark, R.A. Quinlan, The cardiomyopathy and lens
cataract mutation in alphaB-crystallin alters its protein structure,
chaperone activity, and interaction with intermediate filaments in
vitro, J. Biol. Chem. 274 (1999) 33235 – 33243.
J.P. Brady, D.L. Garland, D.E. Green, E.R. Tamm, F.J. Giblin, E.F.
Wawrousek, aB-Crystallin in lens development and muscle integrity:
a gene knock out approach, Invest. Ophthalmol. Vis. Sci. 42 (2001)
2924 – 2934.
J. Horwitz, Alpha-crystallin can function as a molecular chaperone,
Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 10449 – 10453.
T.X. Sun, B.K. Das, J.J. Liang, Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin, J. Biol. Chem. 272 (1997) 6220 – 6225.
M. Ehrnsperger, S. Graber, M. Gaestel, J. Buchner, Binding of nonnative protein to Hsp25 during heat shock creates a reservoir of
folding intermediates for reactivation, EMBO J. 16 (1997) 221 – 229.
[22] S.A. Datta, C.M. Rao, Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates,
J. Biol. Chem. 274 (1999) 34773 – 34778.
[23] C.M. Rao, B. Raman, T. Ramakrishna, K. Rajaraman, D. Ghosh, S.
Datta, V.D. Trivedi, M.B. Sukhaswami, Structural perturbation of
alpha-crystallin and its chaperone-like activity, Int. J. Biol. Macromol. 22 (1998) 271 – 281.
[24] C.M. Rao, T. Ramakrishna, B. Raman, Alpha-crystallin: a small heat
shock protein with chaperone activity, PINSA 68 (2002) 349 – 365.
[25] M.W. Head, L. Hurwitz, K. Kegel, J.E. Goldman, AlphaB-crystallin
regulates intermediate filament organization in situ, NeuroReport 11
(2000) 361 – 365.
[26] P.J. Muchowski, M.M. Valdez, J.I. Clark, AlphaB-crystallin selectively targets intermediate filament proteins during thermal stress, Invest.
Ophthalmol. Vis. Sci. 40 (1999) 951 – 958.
[27] P. van den Ijssel, R. Wheelock, A. Prescott, P. Russell, R.A. Quinlan,
Nuclear speckle localisation of the small heat shock protein alphaBcrystallin and its inhibition by the R120G cardiomyopathy-linked
mutation, Exp. Cell Res. 287 (2003) 249 – 261.
[28] K.E. Wiesmann, A. Coop, D. Goode, H.W. Hepburne-Scott, M.J.
Crabbe, Effect of mutations of murine lens alphaB crystallin on
transfected neural cell viability and cellular translocation in response to stress, FEBS Lett. 438 (1998) 25 – 31.
[29] G. Jagatheesan, S. Thanumalayan, B. Muralikrishna, N. Rangaraj,
A.A. Karande, V.K. Parnaik, Colocalization of intranuclear lamin foci
with RNA splicing factors, J. Cell. Sci. 112 (1999) 4651 – 4661.
[30] W. Schul, R. van Driel, L. de Jong, A subset of poly(A) polymerase is
concentrated at sites of RNA synthesis and is associated with domains
enriched in splicing factors and poly(A) RNA, Exp. Cell Res. 238
(1998) 1 – 12.
[31] A.W. Henkel, S.C. Bieger, Quantification of proteins dissolved in
electrophoresis sample buffer, Anal. Biochem. 223 (1994) 329 – 331.
[32] U.P. Andley, Z. Song, E.F. Wawrousek, T.P. Fleming, S. Bassnett,
Differential protective activity of alphaA- and alphaB-crystallin in
lens epithelial cells, J. Biol. Chem. 275 (2000) 36823 – 36831.
[33] Y. Sugiyama, A. Suzuki, M. Kishikawa, R. Akutsu, T. Hirose, M.M.
Waye, S.K. Tsui, S. Yoshida, S. Ohno, Muscle develops a specific
form of small heat shock protein complex composed of MKBP/
HSPB2 and HSPB3 during myogenic differentiation, J. Biol. Chem.
275 (2000) 1095 – 1104.
[34] H. Ito, K. Kamei, I. Iwamoto, Y. Inaguma, K. Kato, Regulation of the
levels of small heat-shock proteins during differentiation of C2C12
cells, Exp. Cell Res. 266 (2001) 213 – 221.
[35] N. Benaroudj, B. Fang, F. Triniolles, C. Ghelis, M.M. Ladjimi,
Overexpression in Escherichia coli, purification and characterization
of the molecular chaperone HSC70, Eur. J. Biochem. 221 (1994)
121 – 128.
[36] S.M. Leung, G. Senisterra, K.P. Ritchie, S.E. Sadis, J.R. Lepock, L.E.
Hightower, Thermal activation of the bovine Hsc70 molecular chaperone at physiological temperatures: physical evidence of a molecular
thermometer, Cell Stress Chaperones 1 (1996) 78 – 89.
[37] K. Ohtsuka, H. Nakamura, C. Sato, Intracellular distribution of
73,000 and 72,000 Dalton heat shock proteins in HeLa cells, Int. J.
Hypertherm. 2 (1986) 267 – 275.
[38] A.B. Lassar, S.X. Skapek, B. Novitch, Regulatory mechanisms that
coordinate skeletal muscle differentiation and cell cycle withdrawal,
Curr. Opin. Cell Biol. 6 (1994) 788 – 794.
[39] R.I. Kumaran, Bh. Muralikrishna, V.K. Parnaik, Lamin A/C speckles
mediate spatial organization of splicing factor compartments and
RNA polymerase II transcription, J. Cell Biol. 159 (2002) 783 – 793.
[40] A.E. van Rijk, G.J. Stege, E.J. Bennink, A. May, H. Bloemendal,
Nuclear staining for the small heat shock protein alphaB-crystallin
colocalizes with splicing factor SC35, Eur. J. Cell Biol. 82 (2003)
361 – 368.
[41] J.R. Dynlacht, M.D. Story, W.-G. Zhu, J. Danner, Lamin B is a
prompt heat shock protein, J. Cell. Physiol. 178 (1999) 28 – 34.
[42] Bh. Muralikrishna, J. Dhawan, N. Rangaraj, V.K. Parnaik, Distinct
A.S. Adhikari et al. / Experimental Cell Research 299 (2004) 393–403
[43]
[44]
[45]
[46]
changes in intranuclear lamin A/C organization during myoblast differentiation, J. Cell. Sci. 114 (2001) 4001 – 4011.
M.C. Kamradt, F. Chen, S. Sam, V.L. Cryns, The small heat shock
protein alpha B-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation, J. Biol. Chem.
277 (2002) 38731 – 38736.
F.A.J.M. van de Klundert, M.L.J. Gijsen, P.R.L.A. van den Ijssel,
L.H.E.H. Snoeckx, W.W. de Jong, aB-crystallin and Hsp25 in neonatal cardiac cells—Differences in cellular localization under stress
conditions, Eur. J. Cell Biol. 75 (1998) 38 – 45.
J.K. Willsie, J.S. Clegg, Small heat shock protein p26 associates with
nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from
stressed cells, J. Cell. Biochem. 84 (2002) 601 – 614.
D.K. Shumaker, E.R. Kuczmarski, R.D. Goldman, The nucleoskeleton: lamins and actin are major players in essential nuclear functions,
Curr. Opin. Cell Biol. 15 (2003) 358 – 366.
403
[47] A.I. Lamond, W.C. Earnshaw, Structure and function in the nucleus,
Science 280 (1998) 547 – 553.
[48] T. Misteli, D.L. Spector, The cellular organization of gene expression,
Curr. Opin. Cell Biol. 10 (1998) 323 – 331.
[49] N. Stuurman, S. Heins, U. Aebi, Nuclear lamins: their structure, assembly, and interactions, J. Struct. Biol. 122 (1998) 42 – 66.
[50] J. Meier, K.H. Campbell, C.C. Ford, R. Stick, C.J. Hutchison,
The role of lamin LIII in nuclear assembly and DNA replication,
in cell-free extracts of Xenopus eggs, J. Cell. Sci. 98 (1991)
271 – 279.
[51] R.D. Moir, M. Montag-Lowy, R.D. Goldman, Dynamic properties of
nuclear lamins: lamin B is associated with sites of DNA replication,
J. Cell Biol. 125 (1994) 1201 – 1212.
[52] T.P. Spann, A.E. Goldman, C. Wang, S. Huang, R.D. Goldman, Alteration of nuclear lamin organization inhibits RNA polymerase IIdependent transcription, J. Cell Biol. 156 (2002) 603 – 608.