Plant actin controls membrane permeability

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.     

Nanosecond electric pulses trigger actin responses in plant cells

We have analyzed the cellular effects of nanosecond pulsed electrical fields on plant cells using fluorescently tagged marker lines in the tobacco cell line BY-2 and confocal laser scanning microscopy. We observe a disintegration of the cytoskeleton in the cell cortex, followed by contraction of actin filaments towards the nucleus, and disintegration of the nuclear envelope. These responses are accompanied by irreversible permeabilization of the plasma membrane manifest as uptake of Trypan Blue. By pretreatment with the actin-stabilizing drug phalloidin, the detachment of transvacuolar actin from the cell periphery can be suppressed, and this treatment can also suppress the irreversible perforation of the plasma membrane. We discuss these findings in terms of a model, where nanosecond pulsed electric fields trigger actin responses that are key events in the plant-specific form of programmed cell death.     

The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

Nanopore-facilitated, voltage-driven phosphatidylserine translocation in lipid bilayers--in cells and in silico

Nanosecond, megavolt-per-meter pulses--higher power but lower total energy than the electroporative pulses used to introduce normally excluded material into biological cells--produce large intracellular electric fields without destructively charging the plasma membrane. Nanoelectropulse perturbation of mammalian cells causes translocation of phosphatidylserine (PS) to the outer face of the cell, intracellular calcium release, and in some cell types a subsequent progression to apoptosis. Experimental observations and molecular dynamics (MD) simulations of membranes in pulsed electric fields presented here support the hypothesis that nanoelectropulse-induced PS externalization is driven by the electric potential that appears across the lipid bilayer during a pulse and is facilitated by the poration of the membrane that occurs even during pulses as brief as 3 ns. MD simulations of phospholipid bilayers in supraphysiological electric fields show a tight association between PS externalization and membrane pore formation on a nanosecond time scale that is consistent with experimental evidence for electropermeabilization and anode-directed PS translocation after nanosecond electric pulse exposure, suggesting a molecular mechanism for nanoelectroporation and nanosecond PS externalization: electrophoretic migration of the negatively charged PS head group along the surface of nanometer-diameter electropores initiated by field-driven alignment of water dipoles at the membrane interface.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

Thylakoid membrane responses to moderately high leaf temperature in Pima cotton

Photosynthesis is inhibited by high temperatures that plants are likely to experience under natural conditions. Both increased thylakoid membrane ionic conductance and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) deactivation have been suggested as the primary cause. The moderately heat‐tolerant crop Pima S‐6 cotton (Gossypium barbadense) was used to examine heat stress‐induced inhibition of photosynthesis. Previous field‐work indicated that moderate heat stress (T = 35–45 °C) is associated with very rapid leaf temperature changes. Therefore, a system was devised for rapidly heating intact, attached leaves to mimic natural field heat‐stress conditions and monitored Rubisco activation, carbon‐cycle metabolites, thylakoid ionic conductance, and photosystem I activity. As a proxy for NADPH and stromal redox status the activation state of NADP‐malate dehydrogenase (NADP‐MDH) was measured. In dark‐adapted cotton leaves, heating caused an increase in thylakoid permeability at temperatures as low as 36 °C. The increased permeability did not cause a decline in adenosine 5′‐triphosphate (ATP) levels during steady‐state or transient heating. Rapid heating caused a transient decline in ribulose 1,5‐bisphosphate without a decrease in Rubisco activation. Sustained heating caused a decline in Rubisco activation and also oxidized the stroma as judged by NADP‐MDH activation and this is hypothesized to result from increased cyclic photophosphorylation, explaining the maintenance of ATP content in the face of increased thylakoid membrane ion leakiness.     

Evidence for localized cell heating induced by infrared optical tweezers.

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.     

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.     

Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.     

Formation of large, membrane skeleton-free erythrocyte vesicles as a function of the intracellular pH and temperature

Vesiculation of intact erythrocytes can be induced by decreasing their intracellular pH and then heating the red cell suspension to a critical temperature value. While at intracellular pH 6 vesiculation begins at 45 degrees C, further decrease in the intracellular pH lowers the critical temperature. In addition, the critical temperature value can be modified by varying the length of the interval between titration and heating as well as by changing the temperature during this interval. The vesicles are large (1-3.5 micron in diameter), haemoglobin-containing and completely free of skeletal proteins. Pretreatment of the cells with diamide and 2,4-dinitrophenol had no substantial effect on vesiculation, while N-ethylmaleimide, chlorpromazine and wheat germ agglutinin proved to be inhibitory. Increasing the osmolarity of the incubation medium markedly decreased the critical temperature: red cells suspended in a solution of 600 mosM NaCl vesiculated at 42 degrees C instead of 45 degrees C when the intracellular pH was decreased to 6. We propose that the vesiculation is due to a purely physicochemical molecular mechanism which affects the state and dimension of the membrane skeleton. We also discuss the possible role of an altered haemoglobin-membrane interaction in preventing low pH-induced intramembrane particle aggregation in the membrane skeleton-free vesicles.     

A generalized concept for cell killing by heat

Based on the analysis of many survival curves obtained after hyperthermic treatments of CHO cells at various temperatures, or after consecutive exposure to two different temperatures, a generalized concept has been developed for the action of heat on cell survival. The basic idea of this concept is that cellular inactivation by heat is a two step process. In the first step, heating produces nonlethal lesions. In the second step, the nonlethal lesions are converted into lethal events upon further heating. The conversion of one of the nonlethal lesions in a cell leads to cell death. Based on the assumption that both production and conversion of nonlethal lesions occur at random and depend only on temperature, a mathematical model has been worked out that quantitatively describes cell killing by single heating as well as by step-down or step-up heating. After the cells are heated at a certain temperature for a time t, the surviving fraction is given by the equation S(t) = exp [(p/c) X [1 - c X t - exp(-c X t)]) where p is the rate constant for the production of nonlethal lesions per cell and per unit of time, and c is the rate constant for the conversion of one nonlethal lesion into a lethal event per unit of time. When heating is performed consecutively at two different temperatures; i.e., when a pretreatment at the temperature T1 for the time t1 is followed by a graded exposure to the temperature T for the time t, the surviving fraction is given by the equation S(t1,t) = exp [(p1/c1) X exp(-c X t) X [1 - c1 X t1 X exp (c X t) - exp(-c1 X t1) + (p/c) X [1 - c X t - exp(-c X t)]) where p1 and c1 are the production rate and the conversion rate at the temperature T1 of the pretreatment, and p and c are the corresponding values at the temperature of the second treatment. By fitting the equations given above to the experimental data of many heat survival curves, the values of p and c were determined for the temperature range 39 to 45 degrees C. In this range, the conversion rate c increases exponentially with temperature; the slope corresponds to an activation energy of Ea = 86 +/- 6 kcal/mol. The Arrhenius plot of the production rate p shows an inflection point at 42.5 degrees C. Above that temperature, the activation energy is 185 +/- 14 kcal/mol; below, Ea = 370 +/- 30 kcal/mol was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.     

Mechanisms of laser nanoparticle-based techniques for gene transfection—a calculation study

Cell plasma membranes can be transiently permeabilized to uptake exogenous molecules with high efficiency using a laser nanoparticle-based gene transfection technique. In combination with experimental results, a theoretical model is set up to calculate the temperature distribution and variance around the nanoparticles. This paper also provides a thorough discussion of the underlying mechanisms of cell permeabilization. We find that, rapid heating of the particles and the accompanying extreme temperature rise can lead to microbubble formation around laser-heated particles, which is the origin of photoacoustic effects and other nonlinear optical responses. This transient heat is also capable of causing protein denaturation through thermal inactivation and photochemistry. Furthermore, the dynamic mode that involves the overlapping of bubbles is presented. This mode can significantly increase the plasma membrane permeability of the cells without affecting their viability.     

Nanosecond Electric Pulses Affect a Plant-Specific Kinesin at the Plasma Membrane

Electric pulses with high field strength and durations in the nanosecond range (nsPEFs) are of considerable interest for biotechnological and medical applications. However, their actual cellular site of action is still under debate—due to their extremely short rise times, nsPEFs are thought to act mainly in the cell interior rather than at the plasma membrane. On the other hand, nsPEFs can induce membrane permeability. We have revisited this issue using plant cells as a model. By mapping the cellular responses to nsPEFs of different field strength and duration in the tobacco BY-2 cell line, we could define a treatment that does not impinge on short-term viability, such that the physiological responses to the treatment can be followed. We observe, for these conditions, a mild disintegration of the cytoskeleton, impaired membrane localization of the PIN1 auxin-efflux transporter and a delayed premitotic nuclear positioning followed by a transient mitotic arrest. To address the target site of nsPEFs, we made use of the plant-specific KCH kinesin, which can assume two different states with different localization (either near the nucleus or at the cell membrane) driving different cellular functions. We show that nsPEFs reduce cell expansion in nontransformed cells but promote expansion in a line overexpressing KCH. Since cell elongation and cell widening are linked to the KCH localized at the cell membrane, the inverted response in the KCH overexpressor provides evidence for a direct action of nsPEFs, also at the cell membrane.     

Lethality in mammalian cells due to hyperthermia under oxic and hypoxic conditions.

From several studies of hyperthermia there have been reports that hypoxic cells are more sensitive to heat than their oxic counterparts. Experimental techniques in this investigation eliminate the effect of pH, trypsinization and cell attachment, when assaying the effects of hyperthermia on cells. Under hypoxic conditions, HeLa S3 and Chinese hamster cell-lines do not have an increased sensitivity to heat compared with oxic cells. HeLa S3 cells are protected against heat by hypoxia. Light-microscopy indicates the rupture of the plasma membrane, occasional nuclear budding, membrane vesicles and granulation of cell contents after heating at 43 degrees C for 3 hours. Scanning electron micrographs show that cells are more rounded after heat treatment and that there is an accompanying decrease in the number of microvilli, suggesting that the mechanism of cell attachment is affected. Heated cells should be delicately handled and subjected to the minimal trauma so that an accurate comparison of survival can be made.     

Scrotal heating stimulates panting and reduces body temperature similarly in febrile and non-febrile rams (Ovis aries)

It is known that heating the ram scrotum stimulates heat loss resulting in a decrease in body temperature and that during fever core temperature increases, but local scrotal thermoeffectors operate to maintain normal scrotal temperature. We have investigated whether scrotal warming influences core body temperature and the panting effector during fever generation. We measured rectal temperature, intrascrotal temperature, scrotal skin temperature and respiratory frequency in four adult Merino rams following intravascular injection of saline or lipopolysaccharide at an ambient temperature of 18-20 degrees C while scrotal skin temperature was maintained at 33 degrees C or elevated to 41 degrees C. Compared to maintaining normal scrotal temperature, heating the scrotum increased respiratory frequency and reduced rectal temperature by a similar amount following LPS as following saline. Fever was associated with decreased respiratory frequency compared to saline at both 33 and 41 degrees C scrotal temperature, suggesting that the fever was generated mainly by decreasing respiratory heat loss. We conclude that scrotal thermal afferent stimulation resulted in an offset for the set-point of body temperature regulation in both normothermic and febrile rams.     

Thermoelasticity of red blood cell membrane.

The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol.     

Influence of heat transmission mode on heating rates and on the selection of patches for heating in a mediterranean lizard

Heliothermy (heat gain by radiation) has been given a prominent role in basking lizards. However, thigmothermy (heat gain by conduction) could be relevant for heating in small lizards. To ascertain the importance of the different heat transmission modes to the thermoregulatory processes, we conducted an experimental study where we analyzed the role of heat transmission modes on heating rates and on the selection of sites for heating in the Mediterranean lizard Acanthodactylus erythrurus (Lacertidae). The study was conducted under laboratory conditions, where two situations of different operative temperatures (38 degrees and 50 degrees C) were simulated in a terrarium. In a first experiment, individuals were allowed to heat up during 2 min at both temperatures and under both heat transmission modes. In a second experiment, individuals were allowed to select between patches differing in the main transmission mode, at both temperatures, to heat up. Experiences were conducted with live, nontethered lizards with a starting body temperature of 27 degrees C. Temperature had a significant effect on the heating rate, with heat gain per unit of time being faster at the higher operative temperature (50 degrees C). The effect of the mode of heat transmission on the heating rate was also significant: at 50 degrees C, heating rate was greater when the main heat transmission mode was conduction from the substrate (thigmothermy) than when heating was mainly due to heat gain by radiation (heliothermy); at 38 degrees C, heating rates did not significantly differ between transmission modes. At 38 degrees C, selection of the site for heating was not significantly different from that expected by chance. However, at 50 degrees C, the heating site offering the slowest heating rate (heliothermic patch) was selected. These results show that heating rates vary not only with environmental temperature but also with different predominant heat transmission modes. Lizards are able to identify and exploit this heterogeneity, selecting the source of heat gain (radiation) that minimizes the risk of overheating when temperature is high.     

Alternatives for natural‐gas‐based heating systems: A quantitative GIS‐based analysis of climate impacts and financial feasibility

The heating of buildings currently produces 6% of global greenhouse gas emissions. Sustainable heating technologies can reduce heating‐related CO₂ emissions by up to 90%. We present a Python‐based GIS model to analyze the environmental and financial impact of strategies to reduce heating‐related CO₂ emissions of residential buildings. The city‐wide implementation of three alternatives to natural gas are evaluated: high‐temperature heating networks, low‐temperature heating networks, and heat pumps. We find that both lowering the demand for heat and providing more sustainable sources of heat will be necessary to achieve significant CO₂‐emission reductions. Of the studied alternatives, only low‐temperature heating networks and heat pumps have the potential to reduce CO₂ emissions by 90%. A CO₂ tax and an increase in tax on the use of natural gas are potent policy tools to accelerate the adoption of low‐carbon heating technologies.     

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat‐stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat‐induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat‐induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.