Two-dimensional gel analysis of the heat-shock response in marine snails (genus Tegula): interspecific variation in protein expression and acclimation ability
Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950-3094, USA
Accepted 14 June 2006
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Summary |
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Key words: acclimation, heat-shock protein, molecular chaperone, phenotypic plasticity, Tegula, two-dimensional gel electrophoresis
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Introduction |
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As molecular chaperones, Hsps stabilize and refold reversibly denatured
proteins and help degrade irreversibly denatured proteins
(Feige et al., 1996;
Frydman, 2001
;
Hartl and Hayer-Hartl, 2002
;
Young et al., 2004
). Their
role in conferring increased heat tolerance has been established. The onset
temperature (Ton) of Hsp70 synthesis, the major
heat-induced protein in most organisms, correlates in general positively with
a species' temperature range (Feder and
Hofmann, 1999
; Nakano and
Iwama, 2002
; Tomanek and
Somero, 1999
). Ton of Hsp expression, however,
is not fixed and can shift to higher temperatures after laboratory acclimation
(Tomanek and Somero, 1999
) and
seasonal acclimatization to increasing temperatures
(Barua and Heckathorn, 2004
;
Buckley et al., 2001
;
Chapple et al., 1998
;
Dietz and Somero, 1992
;
Hamdoun et al., 2003
;
Hofmann and Somero, 1995
;
Roberts et al., 1997
), an
adjustment I refer to as the phenotypic plasticity of the heat-shock response
(HSR). A shift in Ton in response to acclimation is
equivalent to a decrease in Hsp synthesis, at least at the lower temperatures
that normally elicit a stress response, and is in part due to Hsp70 itself,
such that an increase in steady-state levels will shift the activation of its
own transcription and synthesis towards higher temperatures
(Morimoto, 1998
;
Tomanek and Somero, 2002
;
Voellmy, 2004
). Interestingly,
a heat-sensitive intertidal marine snail species of the genus Tegula
shifts Ton of a Hsp70 band more after acclimation to
higher temperatures than does a heat-tolerant congener
(Tomanek and Somero, 1999
). It
is therefore possible that species that vary in heat tolerance activate the
stress response at a common temperature following acclimation to warmer
temperature. This suggests that species with the highest heat tolerance may
have a relatively lower capacity to further adjust the stress response to
warmer acclimation temperatures. If so, this limit to acclimation capacity of
Hsp expression could explain the limited ability of more heat-tolerant
organisms to increase tolerance to even greater heat stress
(Cavicchi et al., 1995
;
Hoffmann et al., 2003
;
Stillman, 2003
;
Ushakov et al., 1977
;
Zatsepina et al., 2001
).
However, it is not known if these results of a single Hsp band can be
extrapolated to the entire complement of heat-induced proteins. Simply,
previous acclimation studies have only used one-dimensional gel
electrophoresis to analyze heat-induced protein bands that were close to a
molecular mass class that is characteristic for a given Hsp family
(Hofmann and Somero, 1996;
Tomanek and Somero, 1999
),
despite the fact that the Hsp complement has been resolved since the early
days of two-dimensional gel electrophoresis (2D-GE;
Mirault et al., 1978
).
In the present work, I address the effect of acclimation on the synthesis
of the Hsp complement in three cooltemperate Tegula snail
species that differ in their vertical distribution patterns along the subtidal
to mid-intertidal axis in rocky shore habitats and differ in upper lethal
temperatures by approximately 6.5°C
(Tomanek and Somero, 1999).
T. funebralis, which occupies the mid- to low-intertidal zone,
experiences greater and more variable temperatures on a daily scale and is
more heat-tolerant than the two low-intertidal to subtidal zone congeners
T. brunnea and T. montereyi
(Tomanek and Somero, 1999
).
Furthermore, T. funebralis differs from the two subtidal species in
having (i) a higher Ton and a wider temperature range of
Hsp synthesis, (ii) a faster rate to reach pre-stress levels of Hsp synthesis
after a thermal exposure typical for the mid-intertidal zone, and (iii)
smaller changes in inducible and steady-state levels of Hsp70 and generally
higher levels of heat-shock transcription factor-1 (HSF-1) during acclimation
to increasing temperatures (Tomanek,
2002
; Tomanek and Somero,
1999
,
2000
,
2002
). These interspecific
differences enable T. funebralis, but not T. brunnea, to
better cope with the thermal variation in the mid-intertidal zone
(Tomanek and Sanford, 2003
)
and further support the hypothesis that greater heat tolerance may correlate
with a limited capacity to modify the stress response.
In this study, I tested for the first time how acclimation affects the
incorporation of 35S-labeled methionine and cysteine into over 30
heat-induced proteins or their variants in gill tissue, by separating the Hsp
complement using 2D-GE. My results show that the more heat-tolerant species
displays a far lower capacity to adjust Hsp synthesis following acclimation to
warmer temperatures than the two heat-sensitive species. These results
contradict the general observation that animals from thermally more variable
environments have a greater capacity to acclimate or modify physiological
function in response to changing (here increasing) temperatures than animals
from environments with moderate temperature variation. Furthermore, it has
been proposed that the benefit of acclimation to increasing temperatures is to
prepare the animal for the possibility that the environment will become warmer
(Huey et al., 1999;
Leroi et al., 1994
). I discuss
how the differences in phenotypic plasticity between the Tegula
congeners support this interpretation in the context of the thermal
characteristics of their respective vertical distribution ranges in the
intertidal zone. Finally, the results also suggest that animals from thermally
more-variable relative to less-variable environments may be more sensitive to
future temperature increases.
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Materials and methods |
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Temperature exposure, labeling protocol and sample preparation
After acclimation to 13°C and 22°C I dissected gill tissue under
non-heat-shock-inducing conditions (13°C and 22°C, respectively).
Tissues were placed into microcentrifuge tubes holding 300 µl of filtered
(0.2 µm) seawater containing 10 mmol l1 glucose. Tubes
were pre-incubated to either 13°C or 24°C for 13°C- and
22°C-acclimated animals, respectively. Gill tissue was aerated every 20
min. At the start of the experiment tissues were incubated at 13°C,
24°C, 27°C and 30°C for 2.5 h (for further details, see
Tomanek and Somero, 1999).
Gill tissue was not directly transferred to 27°C and 30°C but instead
incubated first to 24°C (13°C-acclimated animals) and 27°C (for
the 30°C incubation) for 5 min to simulate a more gradual temperature
increase. After incubation at the experimental temperature they were
transferred to a common temperature of 13°C for 15 min before being
incubated with 35S-labeled methionine/cysteine (6.78 MBq
ml1; Perkin Elmer) for 4 h. At the end of the experiment
tissues were washed in filtered seawater, frozen on dry ice and stored at
70°C. Tissues were first homogenized using a sample grinding kit
containing an abrasive grinding resin in 200 µl of lysis buffer containing
8 mol l1 urea, 4% CHAPS and 2% Pharmalyte 3-10 and
subsequently precipitated using the 2-D clean-up kit (both kits were from
Amersham Bioscience, Piscataway, NJ, USA). The precipitate was dissolved in
120 µl of rehydration buffer (8 mol l1 urea, 2% CHAPS,
0.5% IPG buffer pH 47 (Amersham Bioscience), 0.002% Bromophenol Blue).
Levels of 35S-labelled amino acids incorporated into newly
synthesized proteins were determined by precipitation with trichloroacetic
acid (for details, see Tomanek and Somero,
1999
).
Isoelectric focusing, 2D-GE and fluorography
Samples were added (5x105 cts min1) to
the rehydration buffer (see above) during overnight rehydration of Immobiline
DryStrip gels (7 cm long, pH 47; Amersham Bioscience). Rehydrated
strips were run on a Multiphor II electrophoresis system (Amersham Bioscience)
for 1 min at 200 V and for 2 h 50 min at 3500 V before being stored at
70°C.
For the second dimension, strips were equilibrated twice for 15 min in 10 ml of equilibration buffer (50 mmol l1 Tris-HCl, pH 8.8, 6 mol l1 urea, 30% glycerol, 2% SDS, 0.002% Bromophenol Blue); first with 100 mg dithiothreitol and second with 250 mg iodoacetamide. Strips were placed on ExcelGel 1214% gradient gels (Amersham Bioscience) and run according to instructions. Afterwards gels were incubated in Amplify fluorographic reagent (Amersham Bioscience) for 30 min and subsequently dried overnight at 60°C (for the first 2 h) using a vacuum.
Image and statistical analyses
Gels were exposed to Hyperfilm (Amersham Bioscience) for 72, 48, 24, 12 and
6 h to obtain a range of exposure intensities, scanned on a densitometer
(Sharp JX-330) and analyzed using Image Master 2D software (Amersham
Bioscience). Specific spot volumes were normalized against total spot volume
within a single gel and square root-transformed to normalize values for
statistical analysis. Similar intensities were found for film that was exposed
for 48 h after incubation at 13°C and 24°C, 24 h after 27°C
incubation and 12 h after 30°C incubation. Expression values of a single
Hsp of all species from all acclimation and incubation temperatures were
fitted into a general linear model to estimate the average effect for each
species, each acclimation and incubation temperature within a species, and an
interaction effect between each combination of acclimation and incubation
temperature (Software R, version 1.9.1). All treatments had an
N-value of three (only exception: T. funebralis incubated to
24°C after acclimation to 22°C, N=1). The P-values
(0.05 and
0.10) that are reported in
Table 1 are for the average
effect of acclimation on Hsp expression within an incubation temperature for a
particular species.
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Results |
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The three Tegula species showed several clusters of Hsps between 68 and 90 kDa (Fig. 2A): one cluster with nine proteins of 71 kDa contained the most highly expressed proteins. There was a single 74 kDa protein. Two clusters contained four proteins of 76 kDa (T. funebralis and T. brunnea only) and up to six of 77 kDa. There were two (T. brunnea and T. montereyi) or three (T. funebralis) 88 kDa proteins. T. montereyi synthesized a cluster of four 72 kDa proteins, but lacked the 76 kDa cluster (Fig. 2A). T. brunnea did not express one of three heat-induced 40 kDa proteins (Fig. 2B). All species shared five proteins of about 2425 kDa (Fig. 2C).
|
Hsp71.8 and 74.1 (Fig. 4) were heat-induced as well as constitutively expressed at 13°C (preliminarily confirmed with western analysis, data not shown). Acclimation to 22°C led to an increase in acute synthesis under heat-shock in both T. brunnea and T. montereyi (Hsp74.1 only; Table 1). The synthesis of both proteins changed with acclimation in T. funebralis (Table 1). Synthesis levels of Hsp71.8 decreased at an incubation of 24°C; Hsp74.1 levels increased overall (significant main effect for acclimation).
|
Acclimation had the greatest effect on synthesis of Hsp77s in T. brunnea (Hsp77.2; 77.4 and 77.5; Fig. 5), and a lesser effect in T. montereyi (Hsp77.1) and T. funebralis (Hsp77.4; Table 1). T. montereyi and T. funebralis expressed two additional proteins (Hsp77.1 and 77.6) that I did not detect in T. brunnea (Fig. 2A).
|
Gill tissue of T. montereyi expressed a unique cluster of four 72 kDa proteins. Acclimation led to the attenuation of Hsp72.4 synthesis only (Fig. 5 and Table 1).
In response to heat, all species synthesized at least two proteins of about 88 kDa (Fig. 2A, not seen in all gels). Acclimation attenuated the synthesis of Hsp88.1 in T. brunnea and T. montereyi, but not in T. funebralis (Fig. 6 and Table 1). Synthesis of the two additional proteins, Hsp88.2 and Hsp88.3, was downregulated in T. montereyi and T. brunnea, respectively (Table 1).
|
Three proteins of about 40 kDa were synthesized in response to heat in T. funebralis and T. montereyi (Fig. 2B). T. brunnea did not synthesize one of the proteins (Hsp40.3). Acclimation attenuated the acute synthesis of Hsp40.2 in T. brunnea and of Hsp40.1 in T. montereyi (Fig. 6 and Table 1).
|
Species differed not only in how acclimation affected acute Hsp synthesis
but also in the Ton of Hsp synthesis within the fairly
narrow range of incubation temperatures used in the present study, although
the study was not designed to detect possible differences in
Ton at lower temperatures (<24°C;
Tomanek and Somero, 1999). For
three out of the four shared Hsp77s T. brunnea and T.
montereyi induced synthesis at 24°C, whereas T. funebralis
did not initiate synthesis until 27°C after acclimation to 13°C and
22°C (Fig. 5). Following
acclimation to 13°C, the synthesis of all four Hsp76s was induced at
24°C in T. brunnea, but at 27°C in T. funebralis
(Fig. 5). Hsp40.1 showed a
significant effect with an increase in incubation temperature to 24°C in
T. brunnea and T. montereyi, and with an increase to
27°C in T. funebralis (Fig.
6).
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Discussion |
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The choice of acclimation temperatures (13°C and 22°C) was based on
a previous study in which we used one-dimensional gel electrophoresis
(Tomanek and Somero, 1999). In
this study we found that an increase in acclimation temperature by 5°C can
change Ton and the temperature of maximal Hsp synthesis
(Tpeak) for some Hsps. The upper acclimation temperature
(22°C) was chosen because it is the highest temperature that the two
subtidal species can tolerate over the 21-day acclimation period and is only a
few degrees below temperatures at which T. funebralis shows the first
signs of mortality when kept constantly submersed (personal observation).
Although the results of the previous and the present study differ in detail,
both show a greater shift in Ton (attenuation of acute Hsp
synthesis) for the highly expressed Hsp70s in T. brunnea and T.
montereyi than in T. funebralis. In the present study, however,
I was able to resolve the bands of acutely expressed proteins into a
complement of over 30 protein variants that are represented by spots that
differ in molecular mass and isoelectric point (pI).
Variation within the complement
When compared to the Hsp complements of tropical fish of the genus
Poeciliopsis, the eurythermal teleost Gillichthys mirabilis,
Drosophila and intertidal limpets, Tegula snails show a
surprisingly high number of Hsp variants (Garbuz et al.,
2003,
2002
;
Kültz, 1996
; Norris et
al., 1997
,
1995
;
Sanders et al., 1991
;
White et al., 1994
). At this
point I do not know if these variants are true isoforms (paralogous homologs)
or represent post-translationally modified proteins. But a common molecular
mass, proximate isoelectric points (pI), similar expression patterns and known
post-translational modifications (PTM) of Hsps suggest that several of the
proteins are more closely related to each other and are members of a common
Hsp family. For example, Hsp71.1-4 are the most highly expressed Hsp70s, and
the synthesis of three out of the four was significantly attenuated in T.
brunnea and T. montereyi after acclimation to 22°C
(Table 1). These proteins are
likely paralogs of Hsp70.
Several highly induced Hsps are clustered around a similar molecular mass
and pI (Hsp77s, 76s and 72s; Figs
1,
2). Since there is no report on
the clustering of so many isoforms within an Hsp family, it is possible that
some of these variants represent PTMs. For example, the mammalian BiP (or
Grp78) binds to unfolded proteins in the endoplasmic reticulum and its
function is in part regulated via phosphorylation
(Gething, 1997); and a
mammalian mitochondrial Hsp70 is characterized by its
Ca2+-dependent autophosphorylation activity
(Leusteck et al., 1989
).
Phosphorylation is commonly observed to regulate the activity and cause the
variation in synthesis patterns of small Hsps
(Arrigo and Landry, 1994
;
Norris et al., 1997
). The
extent and role of PTMs must be addressed further to assess their importance
for creating the patterns of Hsp variants in Tegula and their
contribution to the differences in phenotypic plasticity within a cluster of
Hsps.
Interspecific differences in phenotypic plasticity of Hsp synthesis were not only common between T. funebralis on the one hand and the two subtidal species on the other, but the plasticity of the Hsp77 cluster varied even between T. brunnea and T. montereyi (Fig. 2A and Table 1). After acclimation, the synthesis of three out of four variants changed in T. brunnea, but only one did in T. montereyi. Furthermore, these two species either expressed the Hsp76 (T. brunnea) or Hsp72 (T. montereyi) cluster (Fig. 2A), but not both.
Two constitutively expressed Hsps, 71.8 and 74.1, were also expressed
during heat-shock (Fig. 4). I
confirmed their identity with an anti-Hsp70 antibody that I had used
previously (Tomanek and Somero,
2002). The synthesis of both proteins changed with acclimation in
all three species; the direction of change differed among the species in case
of Hsp71.8, but all species showed higher levels of Hsp74.1 synthesis after
acclimation to 22°C (Fig.
4). Although acclimation affected the synthesis of several
heat-induced proteins in T. funebralis, their expression levels were
comparatively low. Thus, changes in the synthesis of Hsp71.8 and 74.1 suggest
that the effect of acclimation on Hsps depends on differences in their
expression pattern (constitutive and inducible vs inducible
only).
Phenotypic plasticity and heat tolerance
Although T. funebralis tolerates higher temperatures
(Tomanek and Somero, 1999),
its ability to modify the stress response during thermal acclimation is
limited in comparison to T. brunnea and T. montereyi (Figs
3,
4,
5,
6,
7 and
Table 1). In addition to the
evidence presented here, previous work showed that T. funebralis does
not adjust steady-state levels of two Hsp70 bands, Hsp90 and the heat-shock
transcription factor 1 (HSF1) after acclimation
(Tomanek and Somero, 2002
). On
the other hand, T. funebralis recovers from a heat-shock at a faster
rate (Tomanek and Somero,
2000
), has higher steady-state levels of HSF1
(Tomanek and Somero, 2002
),
survives thermally stressful low-tide periods better than T. brunnea
(Tomanek and Sanford, 2003
),
and synthesizes Hsps over a much wider temperature range than the two subtidal
species (Tomanek and Somero,
1999
). All of these interspecific differences suggest that T.
funebralis is adapted to cope with the greater thermal variation that it
experiences in the mid-intertidal zone, but has a limited ability to modify
the synthesis of the major stress proteins. It has been suggested that the
function of acclimation is not `to maximize performance at warm
temperatures, but rather to protect against the possibility that the
environment will become even hotter'
(Huey et al., 1999
;
Leroi et al., 1994
). T.
funebralis may have lost the capacity to modify the acute expression of
many of its Hsps, in part because it is adapted to the frequent and extreme
temperature changes that are typical for the mid-intertidal zone. By occupying
a thermal zone that becomes warmer on a daily basis, for example during midday
low-tide periods, T. funebralis may have opted for maximal possible
biochemical protection from thermal insults, and thereby almost eliminated the
need for any further modifications with increasing acclimation temperature.
The interpretation of acclimation as a means by which to prepare for a warmer
environment thus provides a conceptual framework to explain the limited
acclimatory capacity in more eurythermal animals.
The limited plasticity of acute Hsp synthesis in T. funebralis
also suggests a possible explanation for the observation that more
heat-tolerant organisms are less capable of increasing tolerance to even
greater heat stress. Clones and siblings of five organismal groups, including
Coelenterata, Arthropoda, Echinodermata and Amphibia, which
showed greater heat tolerance initially, acquired less of an increase in
tolerance after acclimation to higher temperatures
(Ushakov et al., 1977);
strains and populations of Drosophila that were found or raised in
warmer thermal habitats or temperatures during laboratory selection showed not
only higher heat tolerance but also a limited short-term capacity to increase
tolerance to greater heat stress, e.g. heat-hardening
(Cavicchi et al., 1995
;
Hoffmann et al., 2003
;
Zatsepina et al., 2001
); and
more heat-tolerant porcelain crabs of the genus Petrolisthes show a
lower capacity to acclimate the upper thermal limits of cardiac function
(Stillman, 2003
). To date, no
other cellular mechanism has been proposed to explain this relationship.
Interspecific variation in Hsp expression and implications for vertical zonation
An important objective of this study and our previous studies has been to
elucidate how the interspecific variation in Hsp expression patterns
contributes to setting the vertical distribution limits of Tegula
congeners. Our previous comparisons of the Ton of Hsp
synthesis and the higher body temperatures that snails experience in the
mid-intertidal zone suggested that T. funebralis activates the HSR
frequently under natural conditions
(Tomanek and Somero, 1999).
One of the hallmarks of the HSR is the strong and preferential synthesis of
Hsps at elevated temperatures, while the synthesis of non-stress proteins is
suppressed (Lindquist, 1993
;
Storti et al., 1980
).
Fig. 1A illustrates the
attenuation of synthesis of most non-stress proteins at a temperature that
T. funebralis frequently experiences in the mid-intertidal zone
(30°C). Thus, T. funebralis not only has to bear the costs of
increased chaperoning activity but also of the disruption of protein
homeostasis during midday low-tide periods that are most likely to activate
Hsp synthesis. We know from studies on Drosophila and yeast that
there are substantial metabolic costs associated with the HSR
(Krebs and Feder, 1997
;
Sanchez et al., 1992
). This
may explain the slower growth rates shown by T. funebralis in
comparison to T. brunnea and T. montereyi
(Frank, 1965
;
Paine, 1969
;
Watanabe, 1982
).
Two-dimensional gel analysis improves the identification and detection of Hsp
synthesis and will allow us to clarify when and to what extent the de
novo synthesis of Hsps occurs under natural conditions, and thus to
assess the costs of being able to cope with a thermal niche as extreme as the
mid-intertidal zone.
The present study raises several new questions that have to be answered to
better understand the complex role of the interspecific variation in Hsp
synthesis and acclimation ability in setting limits to the thermal environment
an organism can occupy. What do the many heat-induced protein variants
represent: Hsp-families, Hsp-isoforms or their PTMs? What limits phenotypic
plasticity on the cellular level: higher levels of HSF1, the interaction of a
multi-chaperone complex with HSF1, or regulatory steps downstream of HSF1
binding to the Hsp promoter (Buckley et
al., 2001; Morimoto,
1998
; Tomanek and Somero,
2002
)? Why does acclimation affect Hsps differently (e.g. Hsp77s),
even among species that are similar in heat tolerance: are the likely
explanations transcriptional effects or organelle-specific functions? What are
the relevant PTMs of heat-induced proteins in Tegula and how do they
contribute to the interspecific differences in heat tolerance? More
specifically, are the Hsp76 and Hsp72 clusters homologous, and if not, how do
these proteins differ and what is their function (thus explaining the
interspecific differences in Ton of Hsp76 shown in
Fig. 5)?
Another urgent issue is the question of how global climate change will
affect organisms that differ in heat tolerance and phenotypic plasticity.
Although more heat-tolerant organisms seem poised to cope better with an
increase in average temperature and the occurrence of thermal extremes, their
physiological limits are close to their maximal body temperatures
(Somero, 2002) and they show a
limited capacity to further modify these limits, so they may turn out to be
particularly vulnerable to even a small increase in temperature.
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Acknowledgments |
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Footnotes |
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References |
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