Báo cáo khoa học: The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response pptx

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:
(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