Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Tissue engineering wikipedia , lookup
Extracellular matrix wikipedia , lookup
Signal transduction wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cell culture wikipedia , lookup
Endomembrane system wikipedia , lookup
Cell encapsulation wikipedia , lookup
Cellular differentiation wikipedia , lookup
List of types of proteins wikipedia , lookup
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.