The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response Ga ´ bor Balogh 1 , Ibolya Horva ´ th 1 , Eniko ˜ Nagy 1 , Zso ´ fia Hoyk 2 ,Sa ´ ndor Benko ˜ 3 , Olivier Bensaude 4 and La ´ szlo ´ Vı ´ gh 1 1 Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 2 Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary 3 Outpatient Medical Centre, Municipality of Szeged, Hungary 4De ´ partement de Ge ´ ne ´ tique Mole ´ culaire, Ecole Normale Supe ´ rieure, Paris, France Cellular stress response is a universal mechanism of extraordinary pathophysiological and pharmacological significance [1]. Dysregulation of the stress protein expression is known to play a determining role in the pathology of different human diseases and aging [2]. Identification of the primary sensors that perceive var- ious stress stimuli and of the transducers that carry, amplify and integrate the signals culminating in the expression of a particular heat shock protein (HSP) is therefore of key importance [3,4]. HSP expression in mammalian cells is primarily regulated at the level of transcription and, although not exclusively, is mainly mediated by heat shock fac- tors (HSF), especially HSF1 [5]. The conversion of HSFs to their active, DNA-binding form involves oligomerization to a trimeric state and reversible hyperphosphorylation at multiple sites [6]. The exact mechanism of HSF1 hyperphosphorylation is cur- rently unknown, and the regulation of the mamma- lian heat shock response appears to be more complex Keywords local anesthetics; molecular chaperones; membrane fluidity; membrane microdomains; stress proteins Correspondence L. Vı ´ gh, Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, POB 521, H-6701, Hungary Tel ⁄ Fax: +36 62 432048 E-mail: vigh@brc.hu (Received 18 July 2005, revised 27 September 2005, accepted 3 October 2005) doi:10.1111/j.1742-4658.2005.04999.x The concentrations of two structurally distinct membrane fluidizers, the local anesthetic benzyl alcohol (BA) and heptanol (HE), were used at con- centrations so that their addition to K562 cells caused identical increases in the level of plasma membrane fluidity as tested by 1,6-diphenyl-1,3,5-hexa- triene (DPH) anisotropy. The level of membrane fluidization induced by the chemical agents on isolated membranes at such concentrations corres- ponded to the membrane fluidity increase seen during a thermal shift up to 42 °C. The formation of isofluid membrane states in response to the administration of BA or HE resulted in almost identical downshifts in the temperature thresholds of the heat shock response, accompanied by increa- ses in the expression of genes for stress proteins such as heat shock protein (HSP)-70 at the physiological temperature. Similarly to thermal stress, the exposure of the cells to these membrane fluidizers elicited nearly identical increases of cytosolic Ca 2+ concentration in both Ca 2+ -containing and Ca 2+ -free media and also closely similar extents of increase in mitochond- rial hyperpolarization. We obtained no evidence that the activation of heat shock protein expression by membrane fluidizers is induced by a protein- unfolding signal. We suggest, that the increase of fluidity in specific mem- brane domains, together with subsequent alterations in key cellular events are converted into signal(s) leading to activation of heat shock genes. Abbreviations BA, benzyl alcohol; DPH, 1,6-diphenyl-1,3,5-hexatriene; ERK, extracellular signal-regulated kinase; HE, heptanol; HSF, heat shock factor; HSP, heat shock protein; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; DW m , mitochondrial membrane potential. FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS 6077 than previously thought [7]. The existence of interac- tions between stress-activated signaling pathways and HSPs is well established [8]. The overall interplay of different stress-sensitive signaling pathways ultimately determines the magnitude of the transcriptional activ- ity of HSF1 [2,8,9]. Hitherto, most of the published studies have focused predominantly on the cellular responses to severe heat stress, which causes the unfolding of pre-existing pro- teins and the misfolding of nascent polypeptides [6]. It is suggested therefore that the denaturation of a pro- portion of the cellular proteins during severe heat serves as the primary heat-sensing machinery which triggers the up-regulation of the HSP gene expression. Because mild heat stress is not coupled with the exten- ded unfolding of cellular proteins, it may be assumed that it is sensed by a different mechanism [10]. A num- ber of data support the notion that, indeed, instead of proteotoxicity, a change in the fluidity of membranes may be the first event that signals a change in tempera- ture and may, thus, be regarded as a thermosensor under such conditions [3,4,11–13]. By affecting the membrane microdomain structure and mobility, fever- range hyperthermia may result in the activation of membrane proteins, e.g. multiple growth factor recep- tors [10]. Following such a typical scenario, the activa- tion of growth factor receptors may in turn activate the Ras ⁄ Rac1 pathway, which has been shown to play a critical role in HSF1 activation and HSP up-regula- tion [14]. We have reported that specific alterations in the membrane physical state for prokaryotes and yeasts, can act as an additional stress sensor [11–13]. We assumed that membrane-controlled signaling events might exist temporarily if the adjustment of the mem- brane hyperstructure is completed subsequent to stress [3,4]. Here, we furnish the first evidence that chemic- ally induced membrane perturbations of K562 ery- throleukemic cells, analogously with heat-induced plasma membrane fluidization, are indeed capable of activating HSP formation even at the growth tempera- ture, without causing measurable protein denaturation. We also demonstrate that, just as in response to heat treatment, there are immediate increases in intracellu- lar free Ca 2+ level and mitochondrial membrane potential, DY m , following the administration of mem- brane fluidizers. Hence, it is highly conceivable that changes in the fluidity of the plasma membrane, which is affected considerably by environmental stress, are well suited for cells to sense stress. In a wider sense, even subtle alterations or defects of the lipid phase of membranes (known to be present during aging or under pathophysiological conditions) should influence membrane-initiated signaling processes, leading to a dysregulated stress response. Results Selection of the critical concentrations of membrane perturbers equipotent in fluidization with temperature upshifts We proposed that the lipid phase of membranes plays a central role in the cellular responses that occur during acute heat stress and pathological states [3,4,11–13]. A direct correlation between the membrane fluidization of the lipid region and the HSP response, however, has not been unambiguously established for mammalian cells. By intercalating between membrane lipids the two structurally unrelated membrane fluidizers that we selected benzyl alcohol (BA) and heptanol (HE), we induced a disordering effect by weakening the van der Vaals interactions between the lipid acyl chains [13]. As in the case of heat stress, the initial fluidity increases induced by these membrane perturbants in vivo are fol- lowed by a rapid relaxation period (G. Balogh et al., unpublished results). Thus, for a correct assessment and comparison of the levels of the thermally and chemically induced primary changes in the membrane physical orders, we used isolated membranes. As shown by Fig. 1A, the plasma membrane fraction of K562 cells was labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) and the steady-state fluorescence anisotropy [11–13] was monitored as a function of temperature. Simultaneously, the fluidity changes were recorded at the different concentrations of the two alcohols (Fig. 1B,C). In this way it was possible to determine the critical concentrations of each of the two fluidizers at which their addition to membrane preparations caused increases in the level of membrane fluidity iden- tical to that found after a temperature change to 42 °C. As highlighted by the arrows in Fig. 1A–C, plasma membrane hyperfluidization resulting from heat treat- ment at 42 °C (i.e. a reduction of the steady-state DPH anisotropy value by  0.015 units) can be attained by the administration of 30 mm BA or 4.5 mm HE. The critical concentrations of the membrane perturbers proved to be essentially equipotent in causing mem- brane hyperfluidization in vivo (Fig. 2). The decrease in the lipid order was followed in the membrane interior of the K562 cells by monitoring the DPH anisotropy change. The fluidizing effects of the alcohols in the gly- cerol and upper acyl regions were also determined by means of the charged, not membrane permeable derivative of DPH, 1-(4-trimethylammoniumphenyl)-6- phenyl-1,3,5-hexatriene (TMA-DPH). Membrane fluidity and heat shock response G. Balogh et al. 6078 FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS Membrane fluidizers lower the set-point temperature of HSP-70 synthesis K562 cells were treated at different temperatures in the presence or absence of different concentrations of BA or HE for 60 min. Following a 3-h recovery period at 37 °C, the cells were then labeled with a 14 C amino acid mixture for an additional 60 min to follow the level of the de novo synthesized HSP-70. Co-treatment of the cells with BA or HE during heat stress resulted in a dose and temperature-dependent synthesis of HSP-70 (Fig. 3). Obviously, gradual rising of the tem- perature shifted the peak heat stress response towards the lower alcohol concentration range, indicating a Fig. 2. Membrane fluidity measurements in vivo. K562 cells were labeled with 0.2 lM DPH (¤) or TMA-DPH (h) for 40 or 5 min, respect- ively, and then further incubated with different concentrations of BA or HE. The fluorescence steady-state anisotropy was measured and the differences from the controls were calculated. The arrows indicate the concentrations of the alcohols at which similar levels of HSP-70 syn- thesis were detected at 37 °C. Mean ± SD, n ¼ 6. Fig. 3. HSP-70 induction in K562 cells treated with BA or HE and subjected to heat stress. Cells were treated with various concentrations of BA or HE for 1 h at different temperatures. After a 3 h recovery period, the cells were labeled for 1 h with 14 C protein hydrolysate and, after SDS ⁄ PAGE, prepared for fluorography. The HSP-70 lane of the fluorograph is presented. The arrows indicate the most effective concentra- tions of the alcohols at 37 °C. Fig. 1. Heat stress- or membrane fluidizer-induced changes in isolated plasma membrane fluidity, tested with DPH. Isolated plasma mem- branes were labeled with DPH and (A) the effects of heat or (B) different concentrations of BA or HE on the steady-state fluorescence anisotropy were measured. The arrows indicate the concentrations of the alcohols that exert a fluidizing effect equivalent to that caused by exposure to 42 °C. Mean ± SD, n ¼ 4. G. Balogh et al. Membrane fluidity and heat shock response FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS 6079 cooperative triggering mechanism in the induction of HSP-70 synthesis. The maximum responses at 37 °C were obtained by the administration of 30 mm BA or 4.5 mm HE, these critical concentrations of the fluidiz- ers being exactly those that caused identical levels of in vitro and in vivo plasma membrane fluidization (Figs 1 and 2). In other words, elevation of the plasma membrane fluidity as a consequence either of heat exposure or of chemical membrane perturbations is equally followed by the activation of HSP formation. The higher doses of BA or HE in synergy with heat stress caused a complete inhibition of protein synthe- sis. Thus, at 42 °C the highest tolerable concentrations of BA and HE were 10 and 2 mm, respectively. Effects of heat and membrane fluidizers on the cellular morphology and the cytosolic free Ca 2+ level Heat stress is known to produce distinct morphological changes in mammalian cells [15]. Using electron micro- scopy, a moderate level of membrane blebbing was also detected in the present study when K562 cells were heat shocked at 42 °C or incubated with 30 mm BA or 4.5 mm HE for 1 h. However, no major altera- tions in cell ultrastructure were observed following these treatments (data not shown). The intracellular calcium [Ca 2+ ] i concentration, which is tightly regulated, is known to be a key signa- ling element of the heat shock response in mammalian cells. Whereas the synthesis of HSP-70 has been dem- onstrated to be promoted by an increase in [Ca 2+ ] i , the overexpression of HSP-70 attenuates increases in [Ca 2+ ] i [16,17]. It was earlier documented that mem- brane fluidizer anesthetics may displace Ca 2+ from internal and external binding sites and alter the func- tioning of different Ca 2+ regulatory systems [18,19]. Therefore, we monitored any dose-dependent increa- ses in cytosolic [Ca 2+ ] i following treatment with the membrane fluidizer alcohols and to compare the find- ings with the [Ca 2+ ] i increase resulting from heat shock. By continuous monitoring of Fura-2 fluorescence when the cells were treated with these alcohols at concentra- tions equipotent in membrane fluidization and in the induction of HSP-70, it was found that BA and HE enhanced the level of [Ca 2+ ] i in a closely similar and strictly dose-dependent fashion (Fig. 4A). [Ca 2+ ] i rose to its plateau level within  30 s (from 185 nm to 290 nm and 305 nm). To compare the effects of heat with these alcohols on the free cytosolic Ca 2+ levels, the cells were heated at 42 °C for 5 min. The averaged [Ca 2+ ] i value obtained is displayed by the bar in Fig. 4A. Obvi- ously, the heat stress at 42 °C caused a similar elevation of [Ca 2+ ] i (from 185 nm to 296.5 ± 16.5 nm) to that produced by the corresponding alcohol doses at which equal HSP-70 synthesis was documented. In order to estimate the contribution of intracellular Ca 2+ -mobilizing compound, cells were suspended in a buffer without Ca 2+ , but containing the Ca 2+ chelator EGTA. Whereas the absolute values dropped to about one-third, the pattern of [Ca 2+ ] i obtained by treatment with heat stress and the membrane fluidizer alcohols was not affected by the depletion of external Ca 2+ (Fig. 4B). The effects of heat stress and membrane fluidizers on DW m Together with several other stimuli, via the activation of phospholipase A 2 or by other mechanisms, an intra- cellular free Ca 2+ overload is known to elicit struc- tural and functional changes in the mitochondria. These include swelling, the disruption of electron transport, and the opening of mitochondrial membrane Fig. 4. Intracellular free Ca 2+ concentration increase induced by heat or membrane fluidizers. [Ca 2+ ] i was measured at 37 °Cby using fura-2 ⁄ AM. (A) Time course of [Ca 2+ ] i rise induced in 1.2 mM CaCl 2 -containing buffer by BA or HE or treatment at 42 °C. (B) [Ca 2+ ] i concentrations in Ca 2+ -free buffer containing EGTA, meas- ured in samples treated with alcohol or heat for 5 min. Mean ± SD, *P < 0.05 compared with control, n ¼ 4. Membrane fluidity and heat shock response G. Balogh et al. 6080 FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS permeability transition pores [20]. Recent studies pro- vided evidence that the change in DY m during cellular insults exhibits a biphasic profile and is not associated exclusively with apoptosis. Instead, acting as one of the major checkpoints of cell death pathway selection, mitochondrial hyperpolarization may represent an early and reversible switch in cellular signaling [21,22]. In line with the above reasoning, we addressed the question of whether the strikingly similar changes in [Ca 2+ ] i seen following membrane hyperfluidization induced either by mild heat or by equipotent mem- brane fluidizers are paralleled by similar tendencies in changes in DY m . A two-dimensional display of 5,59,6,69-tetrachloro-1,19,3,39-tetraethyl-benzimidazolyl- carbocyanine iodide (JC-1) red fluorescence vs. green fluorescence illustrates the changes in DY m that occur following membrane manipulations (Fig. 5A). A higher intensity of red fluorescence is supposed to indicate a higher DY m (hyperpolarization). Cells treated with carbonyl cyanide p-chlorophenylhydrazone (CCCP) served as methodological control for mitochondrial depolarization. Figure 5B depicts histograms in which DY m (detected via the J-aggregate fluorescence) is plot- ted against the number of cells. As for heat stress at 42 °C and BA at 30 mm, two treatments at which equal extent of membrane hyperfluidization are cou- pled with identical degrees induction of HSP-70 syn- thesis, we observed a noteworthy uniform increase in DY m . The quantification of DY m in arbitrary units in response to gradually increasing heat and increasing concentrations of the membrane fluidizers is displayed on Fig. 6. Both heat treatment and membrane hyper- fluidization with these alcohols led to the closely sim- ilar extent of mitochondrial hyperpolarization. The chemical membrane fluidizers do not exert a measurable effect on protein denaturation Firefly luciferase can be inactivated by heat shock when it is expressed in mammalian cells. The loss of enzymatic activity correlates with the loss of its solu- A B Fig. 5. Flow cytometric analysis of mitochondrial membrane poten- tial of K562 cells after heat treatment, or incubation with BA or CCCP. Cells were left untreated or treated with BA, heat or CCCP for 1 h as indicated. Cells were then stained with JC-1 and assayed by flow cytometry. (A) Dot plots of JC-1 red fluorescence vs. green fluorescence (B) corresponding histograms, in which the J-aggre- gate fluorescence is plotted against the number of cells. Fig. 6. Quantification of the DW m changes caused by gradually increasing heat stress or increasing concentrations of membrane fluidizers. Cell were treated with BA, HE or subjected to heat stress for 1 h as indicated. The samples were analyzed as in Fig. 5. The mean fluorescence intensity of J-aggregates was used to determine the DW m . Mean ± S.D, *P<0.05 compared with con- trol, n ¼ 4. G. Balogh et al. Membrane fluidity and heat shock response FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS 6081 bility and can be taken as direct evidence of protein denaturation. This method served as a sensitive tool with which to test the proteotoxicity of HSP-inducing compounds [23]. In the present study, we used HeLa cells expressing cytoplasmic firefly luciferase. The pres- ence of either 30 mm BA or 4.5 mm HE did not exert a significant effect on luciferase activity when the cells were tested at their growth temperature. In contrast, loss of enzyme activity was detected in cells exposed to 42 °C (Fig. 7). The same tendency was observed in an in vitro protein denaturation assay, using lysates of K562 cells (data not shown). Discussion Whereas the importance of HSPs in the pathogenesis of many diseases is well established together with their potential therapeutic value, our knowledge of the stress sensing and signaling that lead eventually to an altered HSP expression is still very limited [1]. The early finding that most of the stressors and agents with the ability to induce HSPs appeared to be proteotoxic gave rise to the suggestion that protein denaturation may be the sole initiating signal for the activation of HSP genes [24]. In the course of the present study, we treated K562 cells with BA or HE at concentrations that induce a heat shock response at the normal growth temperature, as highlighted by monitoring of the synthesis of the major HSP, HSP-70. The critical concentrations of each of the two fluidizers were selected so that their addition to the cells caused identical increases in the plasma membrane fluidity level, corresponding to the fall in membrane microviscosity induced by heat stress- ing at 42 °C. We have demonstrated that, irrespective of the origin of the membrane perturbations, the formation of isofluid membrane states is accompanied by an essentially identical heat shock response in K562 cells. Heat shock at 42 °C or the administration of 30 mm BA or 4.5 mm HE, structurally distant com- pounds, proved equally effective in the up-regulation of HSP-70 formation. At the cellular level, Ca 2+ is derived from external and internal sources. We assume that the mechanism by which heat stress and these alcohols alter the Ca 2+ homeostasis in the present study basically results from their action on Na + ⁄ Ca 2+ exchangers and subsequent Ca 2+ mobilization from different intracellular Ca 2+ pools [17]. Lipid rearrangement induced changes in membrane permeability, and the activity of mechano- sensitive ion channels during stress may also promote Ca 2+ influx into the cytosol [18]. In parallel with the induction of HSP synthesis, heat stress and the admin- istration of these membrane fluidizers elicited nearly identical elevations of the cytosolic Ca 2+ concentra- tion, in both Ca 2+ -containing and Ca 2+ -free media. It is suggested that the increase in intracellular free Ca 2+ level that occurs during the cellular responses to heat shock, serum or growth factors is due to the release of the Ca 2+ -regulatory compound inositol 1,4,5-triphos- phate and coupled to the activation of phospho- inositide-specific phospholipase C (PLC) [25]. The costimulation of phospholipases such as PLC and PLA 2 by heat shock and the resultant release of lipid mediators could also enhance the subsequent mem- brane association and activation of protein kinase C (PKC), found to drive the phosphorylation of HSFs [18,23]. In separate studies, an intracellular Ca 2+ level elevation was shown to stimulate HSF1 translocation into the nucleus, resulting in HSP-70 expression [26], and proved to be essential for the multistep activation of HSFs [27]. Similar to our findings, an immediate change in intracellular free Ca 2+ level and an in vivo change in membrane lipid order following treatment with the calcium ionophore ionomycin have been repor- ted, in parallel with the activation of stress-activated protein kinase, an enhanced HSF (heat shock element) interaction and the increased synthesis of HSP-70 [28]. Ca 2+ can be released from internal Ca 2+ stores, through channels in the endoplasmic reticulum. Spatio- temporal studies are in progress in our laboratory to elu- cidate the role and contribution of intracellular Ca 2+ reservoirs (i.e. endoplasmic reticulum and mitochon- dria) to the cytosolic rise of this ion observed upon heat shock and administration of different membrane fluidizers. Fig. 7. In vivo protein denaturation assay. The effects of heat or BA or HE treatment on protein denaturation were monitored by meas- urement of the activity of cytosolic luciferase expressed in HeLa cells. Cells were treated with 30 m M BA (¤), 4.5 mM HE (n)or submitted to heat-shock at 42 °C(n). At different time points cells were lysed and analyzed for luciferase activity. Enzyme activity of control cells was taken as 100%. Mean ± SD, n ¼ 3. Membrane fluidity and heat shock response G. Balogh et al. 6082 FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS Both heat treatment and membrane hyperfluidiza- tion with the simultaneous induction of the synthesis of HSPs were parallel by closely similar extent of mitochondrial hyperpolarization. While representing early and reversible steps in apoptosis [21,22], the documented change in DW m which peaked at the dose (or concentration) of the stressors that elicited the maximum HSP response may be assumed with high probability to serve as a key event in the stress signa- ling of K562 cells. Mitochondrial hyperpolarization can develop in several ways, including the Ca 2+ -over- load activated dephosphorylation of cytochrome c oxidase, and is a likely cause of subsequent reactive oxygen species production [21]. The composition of reactive oxygen intermediates and their compartmen- talization during activation of the stress response by heat or membrane perturbants await further studies. As an indication of their delicate and hitherto unex- plored interrelationship, disruption of HSF1, while resulting in a reduced HSP expression also increased DW m in renal cells [29]. On the other hand, the over- production of HSP-70 by heat shock prevented the H 2 O 2 -induced decline in mitochondrial permeability transition and the swelling of the mitochondria [29]. Previous studies on the regulation of the heat shock response in different prokaryotic model organisms revealed that the threshold temperature of activation of the major heat shock genes is significantly lowered by BA treatment [12,13]. Whereas BA stress activated the entire set of heat shock genes when the solubility of the most aggregation-prone protein homoserine trans-succinylase was tested, it failed to cause in vivo protein denaturation in Escherichia coli cells [13]. The overexpression of a desaturase gene in Saccharomyces cerevisiae, or the addition of exogenous fatty acids, can change the unsaturated ⁄ saturated fatty acid ratio and exert a significant effect on the expression of heat shock genes [11]. The HSP co-inducer bimoclomol and its derivatives, just like other chaperone inducers and coinducers, appear to be nonproteotoxic [20,30–32]. It has been suggested that bimoclomol and related com- pounds selectively interact with acidic membrane lipids, modifying those membrane domains where the thermally or chemically induced perturbation of the lipid phase is sensed and transduced into a cellular sig- nal, leading to the enhanced activation of heat shock genes [20]. In the present study, we tested the possible effects of BA and HE on protein stability at non-heat- shock temperatures via the heat-induced inactivation of heterologously expressed cytoplasmic firefly lucif- erase in HeLa cells. Neither of the fluidizers exerted measurable effect on protein denaturation. Taken together, the above findings lend further support to the view that, besides the formation of denatured pro- teins, alterations in the lipid phase of cell membranes, alone or together with consequent elevation of the intracellular cytosolic Ca 2+ level and DY m , may parti- cipate in the sensing and transduction of environmen- tal stress into a cellular signal. It has been demonstrated that shear stress-induced fluidity changes in endothelial cells are sufficient to initi- ate signal transduction [33], i.e. changes in lipid dynam- ics in the plasma membrane can serve as a link between mechanical force and chemical signaling. In fact, BA has been shown to mimic the effect of step-shear stress by increasing ERK and JNK activities. In contrast, the experimental reduction of the membrane fluidity by cho- lesterol administration resulted in the opposite effect. Cell activation by shear stress is hypothesized to occur via the lipid modification of integral and peripheral membrane proteins, or signaling complexes organized in cholesterol-rich microdomains (rafts, focal adhesions, caveoli, etc., see [34]). The phospholipid bilayer is able to mediate the shear stress-induced activation of mem- brane-bound G proteins, even in the absence of G-pro- tein receptors, similarly by changing the composition and physical properties of the lipid phase [35]. The mechanisms highlighted above conceivably also operate in the present case. The heat-induced activation of kinases such as Akt has been shown to increase HSF1 activity. Enhanced Ras maturation by heat stress was associated with a heightened activation of extra- cellular signal-regulated kinase (ERK), a key mediator of both mitogenic and stress signaling pathways, in response to subsequent growth factor stimulation [36]. Given the importance of the plasma membrane in link- ing growth factor receptor activation to the signaling cascade, it is likely that any alteration in surface mem- brane fluidity could greatly influence ERK activation. In fact, ERK activation in aged hepatocytes is reduced in response to either proliferative stimuli or stressful treatments [37]. The level of membrane-associated PKC is also reduced in elderly, hypertensive subjects [38]. It is proposed that this effect is strictly controlled by age- related alterations in fluidity and the polymorphic phase state of the membranes [38]. Thus, strategies aimed at altering the physical state of the membranes can be used to enhance stress responsiveness in aged cells or in disease conditions such as diabetes, where reduced HSP levels are causally linked to stiffer, less fluid membranes as a result of glycation, oxidative stress or an insulin deficiency [39]. Finally, heat and other types of stress are associated not only with changes in the tension, fluidity, permeab- ility or surface charges of membranes, and in lipid and protein rearrangements, but are also coupled with the G. Balogh et al. Membrane fluidity and heat shock response FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS 6083 formation of lipid peroxides and lipid adducts [40]. It may be noted that 4-hydroxynonenal a highly reactive end-product of lipid peroxidation, is an inducer of HSPs and has been suggested to play an important role in the initial phase of stress-mediated signaling in K562 cells [41]. In conclusion, our results strongly indicate that the membranes of mammalian cells play a critical role in thermal sensing as well as signaling. The exact mech- anism of the perception of membrane stress imposed on K562 cells by BA and HE, coupled with the activa- tion of HSP expression, awaits further studies. We propose that, rather than the overall changes in the physical state of membranes, the appearance of specific microdomains [34] with an abnormal hyperfluid state, locally formed nonbilayer structures [38] or changes in the compositions of particular lipid molecular species involved directly in lipid–protein interactions [3,4], are potentially equally able to furnish a stimuli for the activation of heat shock genes [42]. Identification, by single molecule microscopy [43], of the critical local membrane microdomains that may act as primary thermosensors during heat stress is in progress in our laboratory. Experimental procedures Cell culture K562 cells were cultured in RPMI-1640 medium supple- mented with 10% fetal calf serum and 2 mm glutamine in a humidified 5% CO 2 , 95% air atmosphere at 37 °C and routinely subcultured three times a week. Membrane fluidity measurements The plasma membrane fraction of K562 cells was isolated according to Maeda et al. [44]. Isolated plasma membranes were labeled in 10 mm Tris, 10 mm NaCl (pH 7.5) with 0.2 lm DPH at a molar ratio of  1 : 200 probe–phospho- lipid for 10 min, and steady-state fluorescence anisotropy was measured as in [45]. When the temperature dependence of fluidity was followed, the temperature was gradually (0.4 °CÆmin )1 ) increased and the anisotropy data were col- lected every 30 s. DPH-labeled membranes were incubated with different concentrations of BA or HE for 5 min at 37 °C, and DPH anisotropy was measured at 37 °C. For in vivo fluidity measurements, K562 cells were labe- led with 0.2 lm DPH or TMA-DPH, for 40 min or 5 min, respectively, and incubated further with BA (0–50 mm)or HE (0–6 mm) for an additional 5 min. Steady-state fluores- cence anisotropy was determined as in [45]. In vivo protein labeling Cells (1 mL of 10 6 ÆmL )1 ) were treated with different con- centrations of BA or HE for 1 h at various temperatures, as indicated in Fig. 3. The cells were then washed and fur- ther incubated in complete medium for 3 h at 37 ° C. The medium was next replaced with 1 mL buffer A (1.2 mm CaCl 2 , 2.7 mm KCl, 1.5 mm KH 2 PO 4 , 0.5 mm MgCl 2 , 136 mm NaCl, 6.5 mm Na 2 HPO 4 ,5mmd-glucose) contain- ing 10 lL 14 C protein hydrolysate (Amersham CFB25, radioactive concentration 50 lCiÆmL )1 ) and the cells were incubated for 1 h at 37 °C. Following this, the cells were harvested and resuspended in sodium dodecyl sulfate sam- ple buffer. Proteins were separated on 8% SDS ⁄ PAGE and prepared for fluorography. Measurement of intracellular free Ca 2+ level K562 cells were washed in buffer A and loaded with 5 mm Fura-2 ⁄ AM at 37 °C for 45 min. They were then washed with buffer A and placed in the measuring cell at D 510 ¼ 0.25 at 37 °C and treated with BA or HE or subjected to 42 °C. The fluorescence signal was measured with a PTI spectrofluo- rometer (Photon Technology International, Inc., South Brunswick, NJ, USA) with emission at 510 nm and dual exci- tation at 340 and 380 nm (slit width 5 nm). The autofluores- cence from the cells not loaded with the dye was subtracted from the Fura-2 signal. The rate of leakage this fluorescent dye at 37 °C and the method of determining [Ca 2+ ] i are des- cribed in [46]. When the contribution of the intracellular Ca 2+ mobilization was tested, the cells were resuspended in buffer A without Ca 2+ , but containing 10 mm EGTA. Measurement of DW m DW m was analyzed as in [47], by using the fluorescent lipo- philic cation, JC-1. K562 cells (0.5 · 10 6 ) were incubated with JC-1 (5 lgÆmL )1 ) during the last 15 min of any treat- ment in the dark and were immediately analyzed with a FACScan flow cytometer (Becton-Dickinson) equipped with a 488 nm argon laser. Dead cells were excluded by forward and side scatter gating. JC-1 aggregates were detectable in the FL2 (585 ± 21 nm), and JC-1 monomers were detect- able in the FL1 (530 ± 15 nm) channel. Data on 10 4 cells per sample were acquired and analyzed with Cell Quest software. The mean fluorescence intensity of J-aggregates was used to determine the DW m . Estimation of the level of in vivo protein denaturation in response to heat stress and membrane fluidizing alcohols The effects of heat or BA or HE treatment on protein denaturation were monitored via measurement of the Membrane fluidity and heat shock response G. Balogh et al. 6084 FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS activity of luciferase expressed in HeLa cells as in [20]. The cells were incubated at 37 °C with 30 mm BA or 4.5 mm HE or at 42 °C for 30 min. Immediately after treatment, the cells were cooled to 4 °C and lysed. Luciferase activity was measured as described in [48]. Statistical analysis All data are expressed as mean ± SD. Student’s paired t-test (a ¼ 0.05) with the Bonferroni adjustment was used to compare groups. Acknowledgements This work was supported by grants from the Hungar- ian National Scientific Research Foundation (OTKA: TS 044836, T 038334) and Agency for Research Fund Management and Research Exploitation (RET OMFB00067 ⁄ 2005 and Bio-00120⁄ 2003 KPI). References 1 Pockley GA (2001) Heat shock proteins in health and disease: therapeutic targets or therapeutic agents? Expert Rev Mol Med 2001, 1–21. 2 Calderwood KS (2005) Regulatory interfaces between the stress protein response and other gene expression programs in the cell. Methods 35, 139–148. 3 Vigh L, Maresca B & Harwood J (1998) Does the mem- brane physical state control the expression of heat shock and other genes? Trends Biochem Sci 23, 369–373. 4 Vigh L & Maresca B (2002) Dual role of membranes in heat stress: as thermosensors they modulate the expres- sion of stress genes and, by interacting with stress proteins, re-organize their own lipid order and functional- ity. In Cell and Molecular Responses to Stress (Storey, KB & Storey, JM, eds), pp. 173–188. Elsevier, Amsterdam. 5 Holmberg IC, Tran EFS, Eriksson JE & Sistonen L (2002) Multisite phosphorylation provides sophysticated regulation of transcription factors. Trends Biochem Sci 27, 619–627. 6 Sarge PK, Murphy SP & Morimoto RI (1993) Activa- tion of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA- binding activity, and nuclear localization and can occur in the absence of stress. Mol Cell Biol 13, 1392–1407. 7 Trinklein ND, Murray JI, Hartman SJ, Botstein D & Myers RM (2004) The role of heat shock transcription factor1 in the genome-wide regulation of the mamma- lian heat shock response. Mol Biol Cell 15, 1254–1261. 8 Dorion S & Landry J (2002) Activation of the mitogen- activated protein kinase pathways by heat shock. Cell Stress Chaperones 7, 200–208. 9 Gabai VL & Sherman MY (2002) Interplay between molecular chaperones and signaling pathways in survi- val of heat shock. J Appl Physiol 92, 1743–1748. 10 Park HG, Han SI, Oh SY & Kang HS (2005) Cellular responses to mild heat stress. Cell Mol Life Sci 62, 10–23. 11 Carratu L, Franceschelli S, Pardini C, Kobayashi GS, Horvath I, Vigh L & Maresca B (1997) Membrane lipid perturbation sets the temperature of heat shock response in yeast. Proc Natl Acad Sci USA 93, 3870–3875. 12 Horvath I, Glatz A, Varvasovszki V, Torok Z, Pali T, Balogh G, Kovacs E, Nadasdi L, Benko S, Joo F et al. (1998) Membrane physical state controls the signaling mechanism of the heat shock response in Synechocystis PCC 6803: identification of hsp17 as a ‘fluidity gene’. Proc Natl Acad Sci USA 95, 3513–3518. 13 Shigapova N, Torok Z, Balogh G, Goloubinoff P, Vigh L & Horvath I (2005) Membrane fludization triggers membrane remodeling which affects the thermotolerance in Escherichia coli. Biochem Biophys Res Comm 328, 1216–1223. 14 Han SI, Oh SY, Woo SH, Kim KH, Kim JH, Kim HD & Kang HS (2001) Implication of small GTPase Rac1 in the activation of c-junN-terminal kinase and heat shock factor in response of heat shock. J Biol Chem 276, 1889–1895. 15 Welch JW & Suhan PJ (1985) Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filaments in rat fibroblasts after heat-shock treatment. J Cell Biol 101, 1198–1211. 16 Kiang JG, Carr FE, Burns MR & McLain DE (1994) HSP-72 synthesis is promoted by increase in [Ca 2+ ] i or activation of G proteins but not pH i or cAMP. Am J Physiol Cell Physiol 267, C104–C114. 17 Kiang GK, Ding XZ & McClain DE (1998) Overexpres- sion of HSP-70 attenuates increases in [Ca 2+ ] i and pro- tects human epidermoid A-431 cells after chemical hypoxia. Toxicol Appl Pharmacol 149, 185–194. 18 Kultz D (2005) Molecular and evolutionary basis of the cellular stress response. Annu Rev Physiol 67, 225–257. 19 Grant RL & Acosta D Jr (1994) A digitized fluores- cence imaging study on the effects of local anasthetics on cytosolic calcium and mitochondrial membrane potential in cultured rabbit corneal epithelial cells. Toxicol Appl Pharmacol 129, 23–35. 20 Qian L, Song X, Ren H, Gong J & Cheng S (2004) Mito- chondrial mechanism of heat stress-induced injury in rat cardiomyocyte. Cell Stress Chaperones 9, 281–293. 21 Perl A, Gergely P , N agy Gy Koncz A & Banki K (2004) Mito- chondrial hyperpolarization: a checkpoint of T-cell life, death and autoimmunity. Trends Immunol 25, 360–367. 22 Zaragoza A, Diez-Fernandez C, Alvarez AM, Andres D & Cascales M (2001) Mitochondrial involvement in G. Balogh et al. Membrane fluidity and heat shock response FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS 6085 cocaine-treated rat hepatocytes: effect of N-acetylcysteine and deferoxamine. Br J Pharmacol 132, 1063–1070. 23 Torok Z, Tsvetkova NM, Balogh G, Horvath I, Nagy E, Penzes Z, Hargiati J, Bensaude O, Csermely P, Crow JH et al. (2003) Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase. Proc Natl Acad Sci USA 100, 3131–3136. 24 Hightower LE & White FP (1981) Cellular responses to stress: comparison of a family of 71–73-kilodalton pro- teins rapidly synthesized in rat tissue slices and canava- nine-treated cells in culture. J Cell Physiol 108, 261–275. 25 Calderwood SK & Stevenson MA (1993) Inducers of the heat shock response stimulate phospholipase C and phospholipase A 2 activity in mammalian cells. J Cell Physiol 155, 248–256. 26 Ding XZ, Smallridge RC, Galloway RY & Kiang JG (1996) Increases in HSF1 translocation and synthesis in human epidermoid A-431 cells: Role of protein kinase C and [Ca 2+ ]i. J Invest Med 44, 144–153. 27 Price BD & Calderwood SK (1991) Ca is essential for multistep activation of the heat shock factor in permea- bilized cells. Mol Cell Biol 11, 3365–3368. 28 Sreedhar AS & Arinivas UK (2002) Activation of stress response by ionomycin in rat hepatoma cells. J Cell Biochem 86, 154–161. 29 Yan L-Y, Rajasekaran NS, Sathyanarayanan S & Benjamin IJ (2005) Mouse HSF1 disruption perturbs redox state and increases mitochondrial oxidative stress in kidney. Antioxidants Redox Signaling 7, 465–471. 30 Vigh L, Literati NP, Horvath I, Torok Zs Balogh G, Glatz A, Kovacs E, Boros I, Ferdinandy P, Farkas B, Jaszlits L et al. (1997) Bimoclomol: a novel non-toxic, hyroxylamine derivative with stress protein inducing activity and wide cytoprotective effects. Nature Med 3, 1150–1154. 31 Yan D, Saito K, Ohmi Y, Fujie N & Ohtsuka K (2004) Paeoniflorin, a novel heat shock protein-inducing com- pound. Cell Stress Chaperones 9, 378–389. 32 Sachidhanandam SB, Lu J, Low KSY & Moochala SM (2003) Herbimycin A attenuates apoptosis during heat stress in rats. Eur J Pharmacol 474, 121–128. 33 Butler PJ, Tsou TC, Li JYS, Usami S & Chien S (2001) Rate sensitivity of shear-induced changes in the lateral diffusion of endothelial cell membrane lipids: a role for membrane perturbation in shear-induce MAPK activa- tion. Am J Physiol 280, C962–C969. 34 Vereb G, Szollosi J, Matko J, Farkas T, Vigh L, Matyus L, Waldmann TA & Damjanovich S (2003) Dynamic, yet structured: the cell membrane three dec- ades after the Singer–Nicolson model. Proc Natl Acad Sci USA 100, 8053–8058. 35 Gudi S, Nolan JP & Frangos JA (1998) Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition. Proc Natl Acad Sci USA 95, 2515–2519. 36 Shack S, Gorospe M, Fawcett TW, Hudgins WR & Holbrook NJ (1999) Activation of the cholesterol path- way and Ras maturation in response to stress. Oncogene 18, 6021–6028. 37 Guyton GZ, Gorospe M, Wang X, Kokkonen YD, Liu GC, Roth GS & Holbrook NJ (1998) Age-related changes in activation of MAPK cascades by oxidative stress. J Invest Dermatol 3, 23–27. 38 Escriba PV, Sanchez-Dominguez JM, Alemany R, Perona JS & Ruiz-Gutierrez V (2003) Alterations of lipids, G proteins, and PKC in cell membranes of elderly hypertensives. Hypertension 41, 176–182. 39 Hooper PL & Hooper JJ (2005) Loss of defense against stress: diabetes and heat shock proteins. Diabetes Tech- nol Ther 7, 204–208. 40 Garbe TR & Yukawa H (2001) Common solvent toxicity: autooxidation of respiratory redox-cyclers enforced by membrane derangement. Z Naturforsch 56c, 483–491. 41 Cheng JZ, Sharma R, Yang Y, Singhal SS, Sharma A, Saini MK, Sing SV, Zimniak P, Awasthi S & Awasthi YC (2001) Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress. J Biol Chem 276, 41213–41223. 42 Vigh L, Escriba P, Sonnleitner A, Sonnleitner M, Piotto S, Maresca B, Horva ´ th I & Harwood LJ (2005) The sig- nificance of lipid composition for membrane activity: new concepts and ways of assessing function. Prog Lipid Res 44, 303–344. 43 Schutz GJ, Kada G, Pastushenko VP & Schindler H (2000) Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J 19, 892–901. 44 Maeda T, Balakrishnan K & Mehdi SQ (1983) A simple and rapid method for the preparation of plasma mem- branes. Biochim Biophys Acta 731, 115–120. 45 Torok ZS, Horvath I, Goloubinoff P, Kovacs E, Glatz A, Balogh G & Vigh L (1997) Evidence for a lipochaperonin: Association active protein-folding GroESL oligomers with lipids can stabilzie membranes under heat shock conditions. Proc Natl Acad Sci USA 94, 2192–2197. 46 Kiang JG (1991) Effect of intracellular pH on cytosolic free [Ca 2+ ] in human epidermoid A-431 cells. Eur J Pharmacol 207, 287–296. 47 Khaled AR, Reynolds DA, Young HA, Thompson CB, Muegge K & Durum SK (2001) Interleukin-3 withdra- wal induces an early increase in mitochondrial mem- brane potential unrelated to the Bcl-2 family: roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase. J Biol Chem 276, 6453–6462. 48 Nguyen VT & Bensaude O (1994) Increased thermal aggregation of proteins in ATP-depleted mammalian cells. Eur J Biochem 220, 239–246. Membrane fluidity and heat shock response G. Balogh et al. 6086 FEBS Journal 272 (2005) 6077–6086 ª 2005 The Authors Journal compilation ª 2005 FEBS . The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response Ga ´ bor Balogh 1 , Ibolya Horva ´ th 1 ,. indicate that the membranes of mammalian cells play a critical role in thermal sensing as well as signaling. The exact mech- anism of the perception of membrane