Nuclear protein redistribution in heat-shocked cells (original) (raw)
Related papers
European journal of biochemistry / FEBS, 1980
Distribution and 32P labeling of nuclear and nucleolar phosphoproteins were studied in Chinese hamster ovary cells incubated at supranormal temperature (1 h at 43 degrees C). The heat shock induced the phosphorylation of a nucleolar protein with a molecular weight of 95,000. Similarly, in the non-nucleolar fraction of the nucleus, phosphorylation of a 54,000-Mr protein was induced while a protein with a molecular weight of 35,000 was rapidly dephosphorylated. Except for these definite changes, the labeling pattern of proteins that were 32P labeled prior to the heat shock was not affected. During recovery at 37 degrees C, the efficiency of labeling was reduced (10-50% of control values accoring to the subcellular fraction) and certain changes in labeling of phosphoproteins were detected. The nucleolar protein of 95,000 Mr was no longer phosphorylated. Phosphorylation of the 54,000-Mr protein, induced by heat shock, continued during the first 4 h of recovery at 36 degrees C and then s...
Initial characterization of heat-induced excess nuclear proteins in HeLa cells
Journal of Cellular Physiology, 1992
Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus. © 1992 Wiley-Liss, Inc.
The nuclear matrix is a thermolabile cellular structure
Cell Stress & Chaperones, 2001
Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at T m ϭ 48ЊC and 55ЊC with an onset temperature of approximately 40ЊC. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with T m ϭ 48ЊC, indicating increased exposure of hydrophobic residues at this transition. The T m ϭ 48ЊC transition has a similar T m to those predicted for the critical targets for heat-induced clonogenic killing (T m ϭ 46ЊC) and thermal radiosensitization (T m ϭ 47ЊC), suggesting that denaturation of nuclear matrix proteins with T m ϭ 48ЊC contribute to these forms of nuclear damage. Following heating at 43ЊC for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei isolated from heat-shocked cells and is a form of indirect thermal damage.
Content of nonhistone protein in nuclei after hyperthermic treatment
Journal of Cellular Physiology, 1993
When nuclei were isolated from Chinese hamster ovary cells after being heated, there was a large increase in the amount of 3H-tryptophan labeled nonhistone protein in the nucleus relative to the whole cell. After 15 min or 30 min of heating at 45.5°C, the nuclear nonhistone protein content increased by 1.6 or 1.8, respectively. In contrast, when the nuclear nonhistone protein content was determined in the intact cell by using autoradiography to quantify 3H-tryptophan labeled protein in the nucleus and cytoplasm in sections of fixed cells, the nuclear nonhistone protein content increased by only 1.14 or 1.28 for 15 or 30 min at 45.5°C, respectively. Therefore, heat does not induce a massive movement of cytoplasmic protein into the nucleus. © 1993 Wiley-Liss, Inc.
Biology of The Cell, 1995
Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at 0°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated. nuclear matrix /protein composition /stabilization / heating /sodium tetrathionate
Effect of thermotolerance on heat-induced excess nuclear-associated proteins
Journal of Cellular Physiology, 1993
Earlier studies reported that thermotolerance had two effects on the heat-induced increase in nuclear-associated proteins (NAPs); reduction in NAP levels immediately following hyperthermia and facilitation of NAP recovery to control levels. It has also been demonstrated that there are two phases of thermotolerance; one that requires newly synthesized proteins (protein synthesis dependent thermotolerance; PSDT), and another that does not (protein synthesis independent thermotolerance; PSIT). This study was designed to determine if these two phases of thermotolerance affected NAP binding in a similar or different manner. The results demonstrated that protein synthesis during thermotolerance development was not required to reduce NAP levels measured immediately following hyperthermia, but was required to facilitate NAP recovery to control levels following hyperthermia. Reducing NAP levels was the predominant mechanism by which thermotolerance protected cells from this lesion at 43.0°C while facilitated NAP recovery predominated in protecting against exposure to 45.5°C. The facilitated recovery of NAPs required only proteins synthesized following thermotolerance induction and prior to the second heat challenge. Proteins synthesized following the second heat challenge were not requisite. Finally, the processes that facilitate NAP recovery were inhibited at 3°C, suggesting that they are enzymatically mediated. © 1993 Wiley-Liss, Inc.
Nucleic Acids Research, 1979
Incubation of Chinese Hamster Ovary (CHO) cells for one hour at 430C results in several obvious changes in protein distribution and protein synthesis. One major protein of the cytoplasm (molecular weight 45,000 daltons), also present as a minor component in the nucleus, rapidly disappeared while several proteins, especially high molecular weight peptides, were induced by heat shock. Localization of the proteins in the cytoplasm, extranucleolar chromatin and nucleolar bodies has been carried out. Different sets of induced proteins appear in each subcellular compartment. Four hours after restoration of the normal temperature, the normal pattern of protein synthesis was observed. The 45,000 dalton protein reappeared first. Relations between structural and functional alterations and changes in protein distribution are suggested.
Cell biology international reports, 1992
The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were see...
Changes in nuclear proteins induced by heat shock in Drosophila culture cells
FEBS Letters, 1985
Nuclear proteins of normal and heat-shocked Drosophila cells were analysed by two-dimensional electrophoresis. The computerized processing of the gels allowed us to detect 6 proteins strongly induced by the heat treatment, but which were different from the usually described heat-shock proteins. The possible role of these proteins in genetic regulation is discussed, as is the value of this type of approach for the study of other genetic regulation phenomena.