SCRIPTA MEDICA (BRNO) – 73 (6): 381–392, December 2000

A RESPONSE OF YEAST CELLS TO HEAT STRESS:

CELL VIABILITY AND THE STABILITY OF
CYTOSKELETAL STRUCTURES

HOLUBÁ¤OVÁ A., MÜLLER P., SVOBODA A.

Department of Biology, Faculty of Medicine, Masaryk University, Brno

Abstract
The cells of Saccharomyces cerevisiae were exposed to the effect of mild and lethal heat stress and
their viability and changes in their microtubular and actin structures were studied.
An increase in cultivation temperature from 25 °C to 46 °C (lethal heat shock, LHS) resulted,
during 10 min, in a rapid decrease in cell viability. However, a mild increase in incubation temperature
from 25 °C to 37 °C or 41 °C (mild heat shock, MHS) induced cell tolerance to LHS and the cells
were able to survive at 46 °C for up to 30 min. A mild heat shock increased the level of Hsp104 in
cells, as demonstrated by Western blot analysis and indirect immunofluorescence microscopy.
In cell exposed to LHS, immunofluorescence microscopy showed rapid degradation of both
cytoplasmic and nuclear microtubules, but the spindle pole bodies (SPB) remained preserved. On the
other hand, MHS did not produce microtubule degradation. Microtubules remained stable after the
LHS that had been preceded by cell cultivation at 37 °C. It is suggested that stress proteins induced by
MHS were involved in maintaining microtubule stability. The actin cytoskeleton was very sensitive to
heat shock and transfer of cells to both 37 °C and 41 °C resulted in disintegration of actin filaments
and the spreading of actin dots from the bud to the whole cell surface. No stabilisation effect of stress
proteins on actin structures was observed.
Resistance to LHS was also induced by an osmotic shock in the presence of 1M KCl in culture
medium. However, the production of stress proteins in response to osmotic shock was generally slower
than to MHS because the cells became resistant to LHS only after being exposed to 1M KCl for 3 h.

Key words
Saccharomyces cerevisiae, heat shock, Hsp104, actin, microtubules

INTRODUCTION
Physical or chemical stressors induce direct or indirect structural changes in
proteins, which results in protein aggregation and, subsequently, disturbed
functioning of cell compartments. However, the impairment of cell structures
need not always be irreversible. Low-intensity stressors have been found to
increase the synthesis of one protein category, namely, heat-shock proteins (Hsp),
that help the organism to acquire tolerance to stress induced by the initial agents
(1). In the yeast Saccharomyces cerevisiae, great attention has been paid to the
Hsp100/Clp protein family (2), of which Hsp104 has a key role in tolerance to

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heat and other stressors (3). Hsp104, together with Hsp70 and Hsp40, compose
a chaperone complex that facilitates reactivation of heat-damaged proteins (4) and
thus assists in maintaining the essential processes in the cell under stress. An
increased synthesis of heat-shock proteins can also be induced by hypertonic
conditions, ethanol and many other stressors (2,3).
The objective of this study was to investigate responses of the cytoskeletal
structures to stress factors in yeast cells. In Saccharomyces cerevisiae cells, the
response of the cytoskeleton to hypertonic stress reported, in a previous study (5),
was manifested by rapid disintegration of both actin cables and microtubules and
their subsequent repolymerisation within several hours. However, another transfer
of the cells to hypertonic medium was not followed by microtubule disintegration,
which was accounted for by a protective action of Hsp. In this paper, we studied
the effect of heat shock on the stability of microtubules and actin structures with
the aim to find out the extent of Hsp production and the role of these proteins in
the cytoskeleton response to heat stress. It appeared that stress proteins were
involved in maintaining the stability of microtubules but not actin structures.

MATERIALS AND METHODS

Organisms and cultivation
Cells of Saccharomces CCU 21–4–59, were grown on agar MEB medium (2%
malt extract broth, 2 % glucose). Cells incubated in liquid MEB medium at
25 °C were subjected to heat stress when the culture achieved OD(560 nm) equal to
1.0, which corresponded to a density of 107 cells/ml. Protein synthesis was inhibited
by adding cycloheximide (Upjohn Comp.) at a final concentration of 20 µg/ml.

Stress induction
Mild heat stress was induced by heating a 10- ml cell suspension for 30 min at
37 °C and for another 30 min at 41 °C. Strong heat stress was induced by heating
the suspension for 10 to 30 min at 46 °C.
Osmotic stress was induced by adding a KCl solution to a 10-ml cell culture
to achieve a final concentration of 1M; the suspension was divided into two equal
samples which were incubated at 25 °C for 1 and 3 h, respectively.

Cell viability assay

Both the control cells and the cells exposed to heat were, after dilution, seeded
onto MEB agar in Petri dishes and their viability was assessed by counting colony
forming units (CFU).

Western blot assay

A sample of proteins was obtained by either protoplast osmolysis or
mechanical disintegration of cells frozen in liquid nitrogen. Proteins were

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separated on 10 % SDS-polyacrylamide gel with 5 % focusing gel (6),
subsequently blotted to a nitrocellulose membrane (7) and immunoassayed using
the anti-Hsp104 polyclonal antibody SPA-1040 (StressGen) at a dilution of
1:2000, and a secondary antibody (Sigma, anti-rabbit IgG) conjugated with
peroxidase. Hsp104 was detected by the ECL method (Amersham).

Cell fixation and permeabilisation

A culture of exponentially growing cells was fixed, at a 3:1 ratio, with
a fixative containing 15 % formaldehyde, 100 mM KH2PO4 buffer (pH, 6.2), 0.5
mM MgCl2 and 1mM EGTA (8,9,10) for 90 min at room temperature. The culture
was rinsed with phosphate buffer and incubated with NovoZym 234 (1 mg/ml) in
phosphate buffer for 20 min at room temperature. After subsequent rinsing with
buffer, the cells were permeabilised with 1 % Triton X-100 for 3 min.

Immunofluorescence
Fluorescence staining. The permeabilised cells were incubated with 2 %
bovine serum albumin (BSA) for 20 min and, after an addition of the primary
antibody, allowed to stand overnight at 4 °C.
Hsp104 was labeled with the SPA-1040 antibody. Tubulin was stained with
mouse monoclonal anti-tubulin antibody TAT 1 (11); SwAR/FIFC or
SwAM/FITC (Institute of Sera and Vaccines, Prague) were used as secondary
antibodies. The cells were washed free of the antibodies and mixed with
a mounting medium (Vectashield, Vector, USA) with DAPI (4-6-diamino-2-
phenylindole) at a final concentration of 1µg/ml (9).
The actin cytoskeleton was labeled by means of rhodamine-phalloidin
(Molecular Probes) according to Pringle et al (9). The stained cells were viewed
and photographed with a Leitz Laborlux S fluorescence microscope.

RESULTS
1. Induction of thermotolerance after mild heat stress (MHS)
The cells grew fast at 25 °C and also 37 °C was well tolerated. They slowed
down growth at 41 ∞C and ceased growing at 46 °C. Their growth was arrested in
the presence of cycloheximide (Fig. 1).
If the cells grown at 25 °C were transferred to a temperature of 47 °C or higher,
their ability to form colonies decreased rapidly. A certain proportion of the cell
population survived for 10 min but, after 20 min of cultivation at 46 °C, less than
1 % of the cells produced colonies (Fig. 2). Therefore, the temperature of 46 °C was
chosen as a reference lethal heat stress factor (LHS).
If a culture grown at 25 °C was incubated first at 37 °C (30 min) and then at
41 °C (30 min) and subsequently transferred to 46 °C, the proportion of vital cells
markedly increased and the cells survived cultivation at this temperature for

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30 min (Fig. 2). Incubation at 37 and 41 °C was considered to produce mild heat
stress (MHS), which apparently was the reason for an increase in tolerance to
LHS at 46 °C.
Tolerance to LHS was also induced by 60 min of incubation at 37 °C, or 60 min
at 41 °C. An increase in incubation temperature from 25 °C to 37 or 41 °C had,
in itself, no damaging effect on the cells; on the contrary, the cell counts slightly
increased during this incubation (Fig. 1,2). If, during MHS, protein synthesis was
inhibited by cycloheximide, thermotolerance did not develop and the cells
became sensitive to LHS again (Fig. 2).

2. Heat shock protein synthesis

The response of cells to heat stress is associated with the synthesis of heat
shock proteins (1). Our experiments were designed to detect one of these proteins,
Hsp104. While, in the lysate of control cells, only traces of this protein were
found, in cells exposed to mild heat stress, the band corresponding to Hsp104 was
much more apparent (Fig. 3). The increase of the Hsp104 in MHS cells was
clearly demonstrated by immunofluorescence microscopy. The control cells
showed only scattered fluorescent dots while the MHS-exposed cells had many
fluorescent dots evenly distributed throughout the cytoplasm (Fig. 4 ) without
apparent co-localisation with any of the organelles or structures. The number of
fluorescent dots increased with the increasing duration of MHS exposure.

3. Changes in the cytoskeleton after heat shock exposure

Cells grown at 25 °C showed a characteristic array of short cytoplasmic
microtubules emanating from the spindle pole body or displayed a strongly
fluorescent spindle (Fig. 5A). Upon the transfer from 25 to 46 °C the cells lost
their cytoplasmic and nuclear microtubules within 10 min. and a point
fluorescence was seen only at regions of the spindle pole bodies (Fig. 5C). When
cells were first exposed to MHS and then transferred to 46 °C, cytoplasmic
microtubules were slightly shortened but nuclear microtubules remained
unaffected (Fig. 5E). This may imply the role of MHS-induced stress proteins in
maintaining the stability of microtubules.
The actin cytoskeleton in the control cells showed a typical asymmetric
arrangement: a cluster of fluorescent patches in the bud and in, the cytoplasm,
actin filaments converging at the site of budding (Fig. 6A). An increase in
temperature up to 37 °C resulted in disruption of actin filaments, while actin
patches remained accumulated in the bud. At 41 °C the patches were seen
distributed along the whole surface of the cell. In cells transferred from 25 °C to
46 °C, actin structures are rapidly degraded and large aggregates of actin without
distinct relation to any cell structure were observed (Fig. 6C). A similar
degradation of actin structures was observed if the cells were first exposed to

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 Fig. 1
Optical density of a S.cerevisiae culture grown at 25 °C, during a gradual increase in temperature
and in the presence of cycloheximide.

Fig. 2
Survival of cells after exposure to a lethal heat shock (LHS) in the control cells (25 °C), in cells
exposed to a mild heat shock (MHS) and in cells incubated in the presence of cycloheximide.
Standard deviation was determined at the 95 % level of significance.

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 Fig. 3
Detection of Hsp104 protein by Western blot analysis in lysates of cells grown at 25 °C (C) and
after exposure to mild heat shock (MHS).

Fig. 4
Hsp104 protein visualised by immunofluorescence microscopy. Cells grown at 25 °C (A) and
cells after exposure to MHS (C) show a great number of fluorescent dots (arrow). B,D, nuclei
stained with DAPI.

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 Fig. 5
Microtubules (MT) in S. cerevisiae cells. A, MTs in control cells (25 °C), note the short, astral
MTs (arrow) and a spindle in a dividing cell (arrow head). C, MTs in cells after exposure to LHS
(25 °C → 46 °C); only fluorescent SPBs are visible (arrow). E, MTs in cells after exposure to
MHS and a subsequent LHS (25 °C → 37 °C, 41 °C → 46 °C). B,C,F, nuclei stained with DAPI.

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 Fig. 6
Actin structures visualised by rhodamine-phalloidin staining. A, actin filaments and patches in
control cells (25 °C). C, actin in cells after exposure to LHS (25 °C → 46 °C); actin patches are
evenly distributed under the whole cell surface. B,D, nuclei visualised by DAPI staining.

Fig. 7
Effect of prior conditioning by osmotic shock or MHS on survival of cells exposed to LHS.

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MHS and then to LHS. Therefore, no effect of stress proteins on the stability of
actin cytoskeleton was demonstrated.

4. Effect of osmotic stress on the induction of thermotolerance

Experiments were designed to find out whether stress proteins induced by
other stressors could provide increased resistance of yeast cells to heat stress.
To a culture of cells grown at 25 °C the potassium chloride was added to
a 1M concentration. Samples were taken at 1 h and 3 h of cultivation, and both
were transferred to 46 °C. It appeared that the cells exposed to an osmotic shock
for one hour were sensitive to LHS, as were the control cells. However, the cells
exposed to an osmotic shock for 3 h were thermotolerant similarly to the MHS-
exposed cells (Fig. 7).
The immunoblotting assay showed that, at 1 h of incubation with 1M KCl, the
amount of Hsp104 present was low but increased markedly at 3 h of incubation
(Fig. 8).

DISCUSSION
The results of our experiments with thermosensitivity and thermotolerance in
S. cerevisiae were generally in agreement with the published data (12,13) with
only a slight difference in the values of lethal temperature. This may have been
a specific feature of the strain used. The rapid synthesis of Hsp104 was also in
accordance with the findings published previously (3). The protein was
distributed throughout the whole cytoplasm and its preferential location in the
nucleus (12), was not confirmed. Findings in animal cells have shown Hsp co-
localised with microtubules (14), but no such co-localisation was observed in our
study. This can be explained by a small size of microtubular structures or by
a small size of the yeast cell. The fact that osmotic shock resulted in an increased
tolerance of yeast cells to temperature is a new finding that indicates that heat-
shock proteins which are synthesised in response to osmotic stress are of the same
category as the proteins produced in response to heat stress, and this in agreement
with the literature data (2,3).
The biochemical consequences of heat and osmotic stress, including the
identification of the STRE-genes in yeasts, have been well documented (15), but
cytological correlates of the response of yeast cells to stress are, so far, poorly
defined.
The effects of temperature on the changes in cytoskeletal components have
been repeatedly reported, but with inconsistent results. The general idea is that
eukaryotic cells respond to heat stress by disruption of their cytoskeletal elements
(16). In some mammalian cells, an elevated growth temperature has led to actin
disintegration but not to microtubule degradation (17,18). In other experiments,
hyperthermia has induced a depolymerisation of actin, microtubules and

389
 Fig. 8
Detection of Hsp104 by western blot analysis in cells subjected to osmotic shock. C, control cells
(25 ∞C); OS(1), cells incubated in the presence of 1M KCl for 1 h; OS(3), cells grown in the
presence of 1M KCl for 3 h.

intermediate, filaments, the latter was due to the disappearance of integrin (19).
Heat stress has been reported to cause disruption of microtubules in plant cells,
and the protective role of Hsps has not been seen (20). Our experiments showed
that yeast cells behaved in a manner similar to other eukaryotic cells in that heat
stress induced disintegration of both microtubules and actin structures, and the
same response was induced by an osmotic shock (5).
A previous MHS, which had induced Hsp synthesis, maintained the stability
of microtubules exposed to LHS. A similar prior conditioning, which facilitated
microtubule stability, was also induced by osmotic stress.
Hsps seem to play an important role in the regulation of microtubule formation
(21). In the absence of Hsps, abnormal, bent microtubules have been produced
(22) or different types of microtubular dysfunction have occurred (23,24). It is
probable that, under normal conditions, Hsp concentration in cells is low and
exposure to any stress results in easy degradation of microtubules. However, an
increase in Hsp concentration due to prior conditioning leads to a much higher
stability of microtubules.
The resistance of microtubules to stress differs in relation to their type.
Cytoplasmic microtubules seem to be most sensitive, nuclear microtubules are
less sensitive and the most resistant microtubules are those related to the SPB.
This difference in sensitivity may be due to the involvement of different
microtubule-associated proteins or heat-shock proteins (21).

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 Yeast actin is known to be very sensitive to even mild stress (9). Actin cables
disappear first; this is followed by the disappearance of the asymmetric
arrangement of actin patches. We observed a similar response of actin structures
regardless of whether the heat shock was mild or lethal. We failed to demonstrate
any stabilizing effects of the heat-shock proteins synthesized during the prior heat
conditioning on actin structures. Slaninová et al. (5) made similar findings in
yeast cells exposed to osmotic shock. However, this actin sensitivity is apparently
not a characteristic feature of cells in general because, in animal cells repeatedly
exposed to heat shock, actin structures have not undergone depolymerisation (18).
Sanchez et al. (3) showed that, in S. cerevisiae, Hsp104 plays a role not only
in thermotolerance but also in other kinds of stress. We confirmed this finding by
demonstrating that an increase in thermotolerance could also be induced by
osmotic stress. This implies either that stress signaling (15,25,26) may induce the
production of a wide range of stress proteins or that these proteins may exert their
protective roles in different kinds of stress.

ACKNOWLEDGEMENT
We thank Mrs M. Zacharníková for her technical assistance and Z. DoleÏalová for her valuable
comments. This work was supported by grant no. 204/00/0394 from the Grant Agency of the Czech
Republic.

Holubáfiová A., Müller P., Svoboda A.

ODPOVùë KVASINKOV¯CH BUNùK NA TEPLOTNÍ ·OK: ÎIVOTASCHOPNOST BUNùK

A STABILITA CYTOSKELETU

Souhrn
Kvasinky Saccharomyces cerevisiae byly vystaveny úãinku mírného a letálního teplotního
stresu a byla sledována Ïivotaschopnost bunûk a zmûny mikrotubulÛ a aktinov˘ch struktur.
Bylo zji‰tûno, Ïe zv˘‰ení inkubaãní teploty z 25 °C na 46 °C vede jiÏ bûhem 10 min k rychlé
ztrátû Ïivotaschopnosti (letální teplotní ‰ok). Mírné zv˘‰ení inkubaãní teploty z 25 °C na 37 °C nebo
41 °C (mírn˘ teplotní ‰ok) navodí toleranci bunûk k letálnímu teplotnímu ‰oku a buÀky v této
teplotû pfieÏívají aÏ 30 min. Jak jsme prokázali Western blotting assay, mírn˘ teplotní ‰ok zv˘‰í
v buÀkách hladinu stresového proteinu Hsp104. Tento protein byl zfietelnû detekován také metodou
nepfiímé imunofluorescence v cytoplasmÛ bunûk vystaven˘ch mírnému teplotnímu ‰oku.
Letální teplotní ‰ok 46°C zpÛsobuje v kontrolních buÀkách rychlou degradaci cytoplasmatick˘ch
a jadern˘ch mikrotubulÛ. V cytoplasmû fluoreskují pouze organizaãní centra mikrotubulÛ (SPB).
Mírn˘ teplotní ‰ok (37–41°C) mikrotubuly nedegraduje. Mikrotubuly zÛstávají stabilní i po letálním
teplotním ‰oku, kterému pfiedcházela pfiedkultivace bunûk pfii 37°C. Stresové proteiny, které se
vytváfií pfii zv˘‰ení inkubaãní teplotû, se tedy evidentnû podílejí na udrÏování stability mikrotubulÛ.
Aktinov˘ cytoskelet je na teplotní ‰ok velmi citliv˘; pouh˘ pfienos bunûk do 37–41°C vede
k rozpadu aktinov˘ch vláken a k dislokaci aktinov˘ch teãek z rostoucích pupenÛ. Nepozorovali
jsme Ïádné stabilizující úãinek stresov˘ch proteinÛ na aktin.
Zjistili jsme, Ïe resistenci bunûk na teplotní ‰ok 46°C navodí také osmotick˘ ‰ok (1M KCl).
Stresové proteiny se zde tvofií zfiejmû pomaleji, neboÈ termoresistence se objeví v buÀkách aÏ po
3 hodinách po aplikaci ‰oku.

391
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