the hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock...

The hyperfluidization of mammalian cell membranes actsas a signal to initiate the heat shock protein responseGabor Balogh1, Ibolya Horvath1, Eniko Nagy1, Zsofia Hoyk2, Sandor Benko3, Olivier Bensaude4

and Laszlo Vıgh1

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

4 Departement de Genetique Moleculaire, Ecole Normale Superieure, 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. Vıgh, Institute of Biochemistry, Biological

Research Centre, Hungarian Academy of

Sciences, Szeged, POB 521, H-6701,

Hungary

Tel ⁄ Fax: +36 62 432048

E-mail: [email protected]

(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 Ca2+ concentration in both Ca2+-containing and

Ca2+-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; DWm, 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 Ca2+ level and mitochondrial membrane

potential, DYm, 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 14C 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 14C 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


cooperative triggering mechanism in the induction of

HSP-70 synthesis. The maximum responses at 37 �Cwere 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 Ca2+

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 [Ca2+]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 [Ca2+]i,

the overexpression of HSP-70 attenuates increases in

[Ca2+]i [16,17]. It was earlier documented that mem-

brane fluidizer anesthetics may displace Ca2+ from

internal and external binding sites and alter the func-

tioning of different Ca2+ regulatory systems [18,19].

Therefore, we monitored any dose-dependent increa-

ses in cytosolic [Ca2+]i following treatment with the

membrane fluidizer alcohols and to compare the find-

ings with the [Ca2+]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 [Ca2+]i in a closely similar and

strictly dose-dependent fashion (Fig. 4A). [Ca2+]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 Ca2+ levels, the cells

were heated at 42 �C for 5 min. The averaged [Ca2+]ivalue obtained is displayed by the bar in Fig. 4A. Obvi-

ously, the heat stress at 42 �C caused a similar elevation

of [Ca2+]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

Ca2+-mobilizing compound, cells were suspended in a

buffer without Ca2+, but containing the Ca2+ chelator

EGTA. Whereas the absolute values dropped to about

one-third, the pattern of [Ca2+]i obtained by treatment

with heat stress and the membrane fluidizer alcohols

was not affected by the depletion of external Ca2+

(Fig. 4B).

The effects of heat stress and membrane

fluidizers on DWm

Together with several other stimuli, via the activation

of phospholipase A2 or by other mechanisms, an intra-

cellular free Ca2+ 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 Ca2+ concentration increase induced by

heat or membrane fluidizers. [Ca2+]i was measured at 37 �C by

using fura-2 ⁄AM. (A) Time course of [Ca2+]i rise induced in 1.2 mM

CaCl2-containing buffer by BA or HE or treatment at 42 �C. (B)

[Ca2+]i concentrations in Ca2+-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.



permeability transition pores [20]. Recent studies pro-

vided evidence that the change in DYm 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

[Ca2+]i seen following membrane hyperfluidization

induced either by mild heat or by equipotent mem-

brane fluidizers are paralleled by similar tendencies

in changes in DYm. 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 DYm that occur

following membrane manipulations (Fig. 5A). A higher

intensity of red fluorescence is supposed to indicate

a higher DYm (hyperpolarization). Cells treated with

carbonyl cyanide p-chlorophenylhydrazone (CCCP)

served as methodological control for mitochondrial

depolarization. Figure 5B depicts histograms in which

DYm (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

DYm. The quantification of DYm 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 DWm 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 DWm. Mean ± S.D, *P < 0.05 compared with con-

trol, n ¼ 4.



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, Ca2+ is derived from external

and internal sources. We assume that the mechanism

by which heat stress and these alcohols alter the Ca2+

homeostasis in the present study basically results from

their action on Na+⁄Ca2+ exchangers and subsequent

Ca2+ mobilization from different intracellular Ca2+

pools [17]. Lipid rearrangement induced changes in

membrane permeability, and the activity of mechano-

sensitive ion channels during stress may also promote

Ca2+ 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 Ca2+ concentra-

tion, in both Ca2+-containing and Ca2+-free media. It

is suggested that the increase in intracellular free Ca2+

level that occurs during the cellular responses to heat

shock, serum or growth factors is due to the release of

the Ca2+-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

PLA2 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 Ca2+ 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 Ca2+ 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].

Ca2+ can be released from internal Ca2+ 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 Ca2+

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 mM 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.



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 DWm 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 Ca2+-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

DWm in renal cells [29]. On the other hand, the over-

production of HSP-70 by heat shock prevented the

H2O2-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 Ca2+ level and DYm, 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



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% CO2, 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 106ÆmL)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. Themedium was next replaced with 1 mL buffer A (1.2 mm

CaCl2, 2.7 mm KCl, 1.5 mm KH2PO4, 0.5 mm MgCl2,

136 mm NaCl, 6.5 mm Na2HPO4, 5 mm d-glucose) contain-

ing 10 lL 14C 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 Ca2+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 D510 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 [Ca2+]i are des-

cribed in [46]. When the contribution of the intracellular

Ca2+ mobilization was tested, the cells were resuspended in

buffer A without Ca2+, but containing 10 mm EGTA.

Measurement of DWm

DWm was analyzed as in [47], by using the fluorescent lipo-

philic cation, JC-1. K562 cells (0.5 · 106) 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 104 cells

per sample were acquired and analyzed with Cell Quest

software. The mean fluorescence intensity of J-aggregates

was used to determine the DWm.

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



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).

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the hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock...

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