Two developmental stages of Neurospora crassa utilize similar mechanisms for responding to heat shock but contrasting mechanisms for recovery.

At the heat shock temperature of 45 degrees C, there is a transient induction of the synthesis of heat shock proteins and repression of normal protein synthesis in cells of Neurospora crassa. Both conidiospores and mycelial cells resume normal protein synthesis after 60 min at high temperature. At the RNA level, however, these two developmental stages responded with different kinetics to elevated temperature. Heat shock RNAs (for hsp30 and hsp83) accumulated and declined more rapidly in spores than in mycelia, and during recovery spores accumulated mRNA that encoded a normal protein (the proteolipid subunit of the mitochondrial ATPase), whereas mycelia showed no increase in this normal RNA (for at least 120 min). Therefore, the resumption of normal protein synthesis in spores may depend upon accumulation of new mRNAs. In contrast, mycelial cells appeared to change their translational preference during continued incubation at elevated temperature, from a discrimination against normal mRNAs to a resumption of their translation into normal cellular proteins, exemplified by the ATPase proteolipid subunit whose synthesis was measured in the heat-shocked cells.     

Concurrent collapse of keratin filaments, aggregation of organelles, and inhibition of protein synthesis during the heat shock response in mammary epithelial cells

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

Heat shock response of the rat lens

The sequence relationship between the small heat shock proteins and the eye lens protein alpha-crystallin (Ingolia, T. D., and E. E. Craig, 1982, Proc. Natl. Acad. Sci. USA, 79: 2360-2364) prompted us to subject rat lenses in organ culture to heat shock and other forms of stress. The effects on protein synthesis were followed by labeling with [35S]methionine and analysis by one- and two-dimensional gel electrophoresis and fluorography. Heat shock gave a pronounced induction of a protein that could be characterized as the stress protein SP71. This protein probably corresponds to the major mammalian heat shock protein hsp70. Also two minor proteins of 16 and 85 kD were induced, while the synthesis of a constitutive heat shock-related protein, P73, was considerably increased. The synthesis of SP71 started between 30 and 60 min after heat shock, reached its highest level after 3 h, and had stopped again after 8 h. In rat lenses that were preconditioned by an initial mild heat shock, a subsequent shock did not cause renewed synthesis of SP71. This effect resembles the thermotolerance phenomenon observed in cultured cells. The proline analogue azetidine-2-carboxylic acid, zinc chloride, ethanol, and calcium chloride did not, under the conditions used, induce stress proteins in the rat lens. Sodium arsenite, however, had very much the same effects as heat shock. Calcium ionophore A23187 specifically and effectively induced the synthesis of the glucose-regulated protein GRP78. No special response to stress on crystallin synthesis was noticed.     

玉米胚发育过程中热激蛋白的合成

35S-Met标记玉米胚蛋白合成结果表明,热激处理(42℃)与对照(25℃)的蛋白合成趋势相近,热激抑制16 DAP的蛋白合成,增加22和34 DAP蛋白合成.SDS-PAGE自显影图谱表明,热激诱导16DAP的胚合成86.4、80.0、73.2 kD等3种分子量较高的热激蛋白,22DAP后热激诱导合成86.4、80.0、73.2、24.4、18.2、16.8和13.6 kD等7种分子量的热激蛋白.2D-PAGE自显影图谱进一步显示,热激诱导22和28 DAP的胚合成近20种热激蛋白,其中超过10种为小分子热激蛋白.特异热激蛋白BiP(HsP70)、PDI(HsP60)Western blot表明,这2种热激蛋白在玉米胚发育过程均有高水平的表达,热激对其合成影响不明显.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

Physiological responses of Escherichia coli exposed to different heat-stress kinetics

The effects of heat-stress kinetics on the viability of Escherichia coli were investigated. Cells were exposed to heat-stress treatments extending from 30 to 50°C, with either a slope (40 min) or a shock (10 s), both followed by a 1-h plateau at 50°C in nutritive medium. A higher survival rate was observed after the slope than after the shock, when both were followed by a plateau, so the heat slope induced a certain degree of thermotolerance. This tolerance was partly (i) linked to de novo protein synthesis during the subsequent plateau phase, and (ii) abolished after rapid cooling from 50 to 30°C, which means that cellular components with rapidly reversible thermal properties are involved in this type of thermotolerance. The heat-slope-induced thermotolerance was chiefly linked to the maintenance of the plasma membrane integrity (preservation of structure, fluidity, and permeability), and not to GroEL or DnaK overexpression. Moreover, the high level of cell mortality induced by the heat shock could be related to changes in the membrane integrity.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Destabilization and recovery of a yeast prion after mild heat shock

Yeast prion [PSI⁺] is a self-perpetuating amyloid of the translational termination factor Sup35. Although [PSI⁺] propagation is modulated by heat shock proteins (Hsps), high temperature was previously reported to have little or no effect on [PSI⁺]. Our results show that short-term exposure of exponentially growing yeast culture to mild heat shock, followed by immediate resumption of growth, leads to [PSI⁺] destabilization, sometimes persisting for several cell divisions after heat shock. Prion loss occurring in the first division after heat shock is preferentially detected in a daughter cell, indicating the impairment of prion segregation that results in asymmetric prion distribution between a mother cell and a bud. Longer heat shock or prolonged incubation in the absence of nutrients after heat shock led to [PSI⁺] recovery. Both prion destabilization and recovery during heat shock depend on protein synthesis. Maximal prion destabilization coincides with maximal imbalance between Hsp104 and other Hsps such as Hsp70-Ssa. Deletions of individual SSA genes increase prion destabilization and/or counteract recovery. The dynamics of prion aggregation during destabilization and recovery are consistent with the notion that efficient prion fragmentation and segregation require a proper balance between Hsp104 and other (e.g., Hsp70-Ssa) chaperones. In contrast to heat shock, [PSI⁺] destabilization by osmotic stressors does not always depend on cell proliferation and/or protein synthesis, indicating that different stresses may impact the prion via different mechanisms. Our data demonstrate that heat stress causes asymmetric prion distribution in a cell division and confirm that the effects of Hsps on prions are physiologically relevant.     

Effect of mitogenic stimulation and heat shock on protein syntheses in human T cells

Effects of moderate (42 degrees C, 1 hour) and strong (44 degrees C, 1 hour) heat shocks on resting (TR) and phytohemagglutinin stimulated human T-cells (TP) were studied. Both treatments were shown to cause in the latter considerable fall of the level of protein synthesis, as compared to resting cells. Mitogen-stimulated cells stopped their proliferation irreversibly and part of them (approx: 40%) died after even mild shock (at 42 degrees C). Following heat treatment in both the cell types the synthesis of heat shock 70 and 90 kDa proteins was induced which was much more pronounced in TR. These and earlier results of the authors allow a conclusion that involvement of cells in active proliferation may decrease their resistance to stress, and that this phenomenon coincides with the diminishing in synthesis and accumulation of stress proteins.     

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.     

Heat shock and ceramide have different apoptotic pathways in radiation induced fibrosarcoma (RIF) cells

Heat shock induces various cellular responses including inhibition of protein synthesis, production of heat shock proteins (HSPs) and induction of thermotolerance. The molecular mechanisms of the processes have not been well understood. It has been proposed that ceramide formation during heat shock mediates heat shock induced apoptosis. We examined whether C2-ceramide mimicked the cellular response to heat shock in RIF-1 cells and their thermotolerant derivative TR-RIF-1 cells. Discernible effects between heat shock and C2-ceramide treatments were observed in cellular changes such as total protein synthesis, HSP synthesis, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activity and PARP cleavage. Heat shock immediately inhibited cellular protein synthesis, which was recovered by synthesizing HSPs first and then whole proteins later. Heat shock also activated SAPK/JNK and increased PARP cleavage in dose-dependent manner. Thermotolerant TR-RIF-1 cells responded to heat shock more insensitively than RIF-1 cells. On the other hand, C2-ceramide treatment did not accompany any changes induced by heat shock. No discernible differences between RIF-1 and TR-RIF-1 cells were observed by C2-ceramide treatment. We tried to figure out how C2-ceramide interacts with cellular membrane and found that exogenous C2-ceramide was incorporated into the outer monolayer and flipped into the inner monolayer of human erythrocytes in ATP-dependent manner. However, the rate of C2-ceramide incorporation was similar in control and thermotolerant cells. In summary, thermotolerant cells are resistant to heat shock induced apoptotic signaling but not resistant, rather sensitive to membrane disturbing C2-ceramide mediated apoptosis. These results suggest that heat shock and ceramide have different signal transduction pathways.     

Stage dependent synthesis of heat shock induced proteins in early embryos of Drosophila melanogaster

Summary The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35°C, 37°C and 40°C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.     

Differences in preferential synthesis and redistribution of HSP70 and HSP28 families by heat or sodium arsenite in Chinese hamster ovary cells

Since both heat and sodium arsenite induce thermotolerance, we investigated the differences in synthesis and redistribution of stress proteins induced by these agents in Chinese hamster ovary cells. Five major heat shock proteins (HSPs; Mr 110, 87, 70, 28, and 8.5 kDa) were preferentially synthesized after heat for 10 min at 45.5 degrees C, whereas four major HSPs (Mr 110, 87, 70, and 28 kDa) and one stress protein (33.3 kDa) were preferentially synthesized after treatment with 100 microM sodium arsenite (ARS) for 1 hr. Two HSP families (HSP70a,b,c, and HSP28a,b,c) preferentially relocalized in the nucleus after heat shock. In contrast, only HSP70b redistributed into the nucleus after ARS treatment. Furthermore, the kinetics of synthesis of each member of HSP70 and HSP28 families and their redistribution were different after these treatments. The maximum rates of synthesis of HSP70 and HSP28 families, except HSP28c, were 6-9 hr after heat shock, whereas those of HSP70b and HSP28b,c were 0-2 hr after ARS treatment. In addition, the maximum rates of redistribution of HSP70 and HSP28 families occurred 3-6 hr after heat shock, whereas that of HSP70b occurred immediately after ARS treatment. The degree of redistribution of HSP70b after ARS treatment was significantly less than that after heat treatment. These results suggest that heat treatment but not sodium arsenite treatment stimulates the entry of HSP70 and HSP28 families into the nucleus.     

Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the splicing of mRNA precursors is disrupted by a severe heat shock. Mild heat treatments prior to severe heat shock protect splicing from disruption, as was previously reported for Drosophila melanogaster. In contrast to D. melanogaster, protein synthesis during the pretreatment is not required to protect splicing in yeast cells. However, protein synthesis is required for the rapid recovery of splicing once it has been disrupted by a sudden severe heat shock. Mutations in two classes of yeast hsp genes affect the pattern of RNA splicing during the heat shock response. First, certain hsp70 mutants, which overproduce other heat shock proteins at normal temperatures, show constitutive protection of splicing at high temperatures and do not require pretreatment. Second, in hsp104 mutants, the recovery of RNA splicing after a severe heat shock is delayed compared with wild-type cells. These results indicate a greater degree of specialization in the protective functions of hsps than has previously been suspected. Some of the proteins (e.g., members of the hsp70 and hsp82 gene families) help to maintain normal cellular processes at higher temperatures. The particular function of hsp104, at least in splicing, is to facilitate recovery of the process once it has been disrupted.