Abstract
Heat shock proteins are one type of molecular chaperone. Their primary
responsibility is to help refold proteins that are denatured due to high temperatures
These proteins are essential for organisms living at the edges of their thermal
ranges, for example, many intertidal organisms which may be exposed to potentially
lethal temperatures during low tide periods. An example of such an intertidal
organism is the mussel Mytilus californianus, which is found in both subtidal and
intertidal locations, and can often experience temperatures of 30° C and higher. Such
temperatures were seen in the two artificial tide pools, one subtidal and one intertidal,
which were created for this study. Equal numbers of M. californianus ranging in size
were placed in the two pools and samples were collected over five weeks.
Subsequent analysis showed that Heat shock protein-70 (Hsp 70) is expressed in
response to extreme temperatures. The expressed Hsp 70 was visualized on the
gels as two isoform bands - band 1 and band 2. Comparing the mean band 1 and
band 2 intensities of Hsp 70 for both subtidal and intertidal groups of M. californianus
over five weeks has shown that the intertidal group had elevated levels of Hsp 70 in
comparison to the subtidal group for all weeks compared.
Introduction
In order for proteins to function properly they need to be able to reversibly
change their conformations. As a result, proteins need to be marginally stable. That
is, they need to be able to perform rapid changes in their three-dimensional
configuration while at the same time remain adequately stable at physiological
temperature. However, proteins are often exposed to temperatures above their
normal physiological temperatures and as a result these proteins become denatured.
Proteins can be reversibly or irreversibly denatured. Thus, cells need an elaborate
system to either restore the function of reversibly denatured proteins or to dispose of
irreversibly denatured proteins. One particular class of proteins that are involved in
these "clean up" processes are known as heat shock proteins.
In 1962, Ritossa observed that the chromosomes in the salivary glands of heat
stressed Drosophila melanogaster showed a distinct pattern of "puffing" (Figure 1)
This indicated that some class of genes was being strongly transcribed as a result of
high temperatures (Somero, 1995). This response became known as the heat shock
response and the resulting proteins became known as heat shock proteins. It is now
known that there a number of different classes of heat shock proteins synthesized as
a result of heat stress. The Hsp70 family is highly conserved across species and is
the most widely induced in respond to heat stress.
In nature, the temperature at which the synthesis of heat shock proteins is first
induced, the threshold induction temperature, varies widely among species. This
makes sense since different species are evolutionarily adapted to different
temperatures and have proteins with different thermal sensitivities. Therefore, what
might constitute heat stress for one species might be within the normal range of
temperatures for another. However, induction temperatures are known to change with
seasonal acclimatization and with laboratory acclimation (Somero, 1995). These
changes in the induction temperature can be attributed to induced thermal tolerance
In order to achieve induced thermal tolerance, also known as heat hardening, an
organism must first be exposed to sub-lethal heat stress, which causes the synthesis
of heat shock proteins, and then allowed to recover. If the organism is then exposed
to severe heat stress it may exhibit tolerance to these new higher temperatures.
It should be noted that heat shock proteins are not only synthesized in
response to heat stress. Instead, they may be induced by a number of cellular
stresses. Such stresses include chemical stress and physical stress. Thus it might
be more appropriate to call the "heat shock response" the "stress response." Heat
shock proteins are a subset of a broad class of proteins called molecular
chaperones. In most, if not all, cells molecular chaperones are present during non¬
stressful times and additional proteins are induced in response to stress. In normal
cellular function molecular chaperones are responsible for the proper folding and
compartmentalization of proteins (Figure 2)
The heat shock response does not occur without a great deal of energy being
consumed. The source of ATP use is in the production of the heat shock proteins and
the subsequent re-folding of denatured proteins. Thus, there is strict regulation of
transcription and translation of heat shock proteins. The model of regulation
presently in use suggests that the amount of free heat shock protein in the cell serves
as a signal for the amount of transcription of Hsp genes that is needed (Craig and
Gross, 1991). Furthermore, Craig and Gross suggest that there are three elements
involved in the regulation of heat shock protein production (Figure 3). The first
element is the amount of free heat shock protein. The second element is the
concentration of heat shock factor (HSF). The third element is heat shock element
(HSE), which is the gene regulatory element. Under normal conditions in the cell,
Hsp70 binds to HSF to form a complex. Thus there is very little free HSF or Hsp70 in
a cell without heat stress. As a result, no Hsp70 will be produced. Once heat stress
occurs this complex breaks down and the Hsp70 binds to denatured proteins. HSF is
then free to bind to HSE, the gene regulator, and transcription and translation of
additional Hsp70 occurs. With increased heat stress more and more of the Hsp7O-
HSF complex disassociates. In other words, the level of Hsp70 production is related
to the intensity of the heat stress.
Looking at heat shock expression in a particular species can give us insight to
that animal's environment. Many species are often living at the edges of their thermal
ranges. By looking at Hsp 70 expression in animals held in particular environments
we can understand the thermal ranges of species and tease apart why certain
species are found in different parts of the intertidal. In addition, if the pattern of Hsp 70
expression is followed over a period of time we can understand the ability of
organisms to acclimate to thermal stress as well as to changes in their environment.
This study looks at the expression of heat shock proteins in subtidal and intertidal
Mytilus californianus in hopes of understanding some of these patterns.
This study also looks at the expression of heat shock proteins in M
californianus of different sizes. It has been shown that the body temperature of large
mussels rises rapidly and remains high during low tide periods. The body
temperatures of smaller mussels, on the other hand, fluctuate with environmental
conditions. It is possible that mussels of different sizes express Hsp 70 in different
quantities or at different times in order to deal with different body temperature
patterns. Understanding such patterns could provide substantial insight as to how M
californianus and other intertidal organisms survive heat shock and other stress
during development.
Materials and Methods
Aquaria Setup and Mussel Collection
Prior to the start of this experiment, I collected approximately 300 M.
californianus and placed them in an indoor aquarium with a flow-through water
system. These mussels were left in this tank for one week in order for them to
acclimate to a common temperature. During this week two "tide pools" were set up,
one intertidal and the other subtidal. Both pools were made out of kiddy pools. The
bottoms of the pools were covered with rocks and these rocks were then covered with
burlap. There was a drain in the side of each pool below the level of the burlap. This
way the intertidal pool could drain completely and the mussels placed on the burlap
would see complete emersion. Both pools had a flow-through water system. The
subtidal pool was left running all of the time, and the flow-through system for the
intertidal pool was outfitted with a timer which controlled the "tides." Low tide occurred
every day from 10am to 2pm.
ÄAfter the collected mussels had been in the indoor aquarium tank for one week
20 mussels with a wide range of body sizes were collected and kept as a control
group. The remaining mussels were divided into two groups of equivalent size
ranges and numbers (147 per pool) and placed in the two tide pools. Äfter one week
eight mussels ranging in size from small to large were collected from each pool. In
addition to the mussels collected from the two tide pools, eight mussels were also
collected from the intertidal site where all of the mussels were originally collected.
This sample of eight mussels also ranged in body size from small to large. This
collection protocol was repeated weekly for five weeks.
Once these samples were collected, all of the mussels were measured and
dissected. The length, width and height of the shell were measured as well as the full
body weight and the soft body weight. Both gills and the abductor muscle were kept.
One gill from each mussel was then homogenized with a lysis buffer (30ml: 7.5ml
32mM Tris-HCL pH 6.8, 6ml 2% SDS (10% stock solution, 60ul 1mM EDTA (500 mM
stock), 150ul 0.25mg/ml Pefabloc, 60ul 10ug/ml Pepstatin, 60ul 10ug/ml Leupeptin
16.70ml H20) in a 1:5 ratio and the protein concentration was quantified.
Protein Analysis: SDS-PAGE and Western Blot
Next, SDS-PAGE was used to resolve the proteins of each of the samples,
which were run in duplicate. In addition, in each gel 3ul of Hsc 70 were run as a way
to standardize each gel so that they could be compared. When the gels are
transferred and finally exposed to film the resulting "picture" may not have the same
intensity from gel to gel. By having Hsc 70, a band of known quantity, in the gel one
can compare the intensity of that band to the rest of the bands in the gels when
quantifying. Thus the resulting intensities of the bands from gel to gel for the samples
will be standardized. All gels were run for one hour at 200 volts. The proteins on each
gel were then transferred to a nitrocellulose membrane. The transfer was done at 80
volts for one hour and fifteen minutes (Laemmli, 1970).
Next, Western Blots were run on each of the membranes. The membranes
were placed in a blocking buffer (250ml: 25ml 1XTBS, 12.5g 5% non-fat dried milk,
0.05g 0.02% Thimesosol, 0.250ml 0.1% Tween, add H2O) for one hour. The primary
alpha antibody MA3-OO1 was then added to the membranes for one hour. The
membranes were then washed with 1XTBS with and without Tween. The second
antibody, A8-4000, was then added for 30 minutes, and the membranes were then
washed again. Finally a Protein A-peroxidase mixture was added to the membranes
for one hour followed by the washes (Laemmli, 1970). The membranes were then
exposed to equal volumes of Enhanced Chemiluminescence (ECL) reagents 1 and 2
for one minute and then wrapped in Saran wrap (Pierce Chemical Company, Super
Signal: Western Blotting Substrates Handbook). Chemiluminescence is the reaction
that occurs between an enzyme like horseradish peroxidase (HRP) and a
chemiluminescent molecule. This reaction results in an intensified light emission
which allows the band images to be captured on pre flashed, X-ray film (Kodak) when
exposed. During the course of this study exposed time was set at 30 seconds and
the film was then immediately developed.
then scanned the developed film into a computer using Lab Scan, a digital
computer scanning program. Each band on each gel was then quantified using
Image Master 1-D Prime software. The results for each gel were standardized using
the Hsc7O quantity for that particular gel. Finally, the statistical program Systat was
used to compare the subtidal and intertidal treatments overall and per week.
Temperature Data
Temperature data were collected using StowAway XTI data logger chips stored
in waterproof containers. Four data loggers were set out at the start of the experiment.
One was placed in the subtidal pool to record water temperatures. The remaining
three were placed in the intertidal pool, one to record water temperatures, one to
record the body temperatures of a small mussel, and one to record the body
temperatures of a large mussel. All of the data loggers were pre tested for accuracy
and were then set to record the temperature every ten minutes throughout the course
of the study. At the end of the experiment the data were retrieved. Only data from the
small intertidal mussel data logger could be retrieved.
Results
Figures 4 and 5 show the mean band intensities for the two Hsp 70 isoforms -
bands 1 and 2 - for the subtidal and intertidal treatments for weeks 1-3. The control
point corresponds to the original twenty mussels that were dissected after being held
in an indoor tank for one week.
Figure 4 shows that for all three weeks the intensity of Hsp 70 - band 1 for the
intertidal treatment was higher then the intensity of Hsp 70 - band 1 expression for the
subtidal group. Statistical analysis using Systat shows that overall this difference is
statistically significant (p-value= 0.008). Statistical analysis was also done to make
pairwise comparisons of both treatments for each week. For band 1 intensity
differences between treatments were significant only for week 2 (p-value = 0.021).
Since there are presently no field data for week 1, the statistical tests did not
incorporate this "treatment.
Figure 5 shows the mean band 2 intensity data. Again, the intertidal treatment
group had higher intensities of Hsp 70 expression for all three weeks. Overall, the
treatment effects were statistically significant (p-value =0.000). Statistical
comparisons were then done by week. For the band 2 intensities both weeks 1 and 3
were statistically different (week 1 p-value = 0.019, week 2 p-value = 0.005). The p¬
value for week 2 (p-value = 0.061) was close to being statistically significant. Once
again, there are presently no field data for week one and, therefore, this group was not
included in the statistical tests.
Figure 6 shows the body temperatures of a small mussel in the simulated
intertidal pool over the course of the study. The peaks corresponds to highest
temperatures seen during low tide periods. The base line is roughly 15°C, the
temperature of the water coming through the flow-through system.
Discussion
The results collected thus far suggest that the intertidal mussels are indeed
experiencing heat stress. Both bands 1 and 2 are showing elevated levels of Hsp 70
expression in the intertidal treatment over the subtidal treatment. These results are in
agreement with the data that G. E. Hofmann and G. N. Somero gathered during their
1995 and 1996 studies. In comparison to the control mussels the Hsp 70 level after
one week has increased. However, what is not clear is why the subtidal group seems
to follow the same pattern of Hsp 70 expression but at a lower level. It is possible
that the stress of being moved from the intertidal, being placed indoors for a week and
then being returned to an environment, which may have no had a well established
food supply, was sufficient to induce Hsp 70 expression. Add to that stress the heat
stress seen by the intertidal group daily and you could explain the patterns of
expression, as well as the elevated levels of expression in the intertidal group.
As the weeks went on the subtidal group began to see an established
community of algae in their pool. The intertidal group also saw the establishment of
an algal community, but to a much lesser extent then the subtidal group. It is possible
that this well-established algal community means that the subtidal pool is seeing
high levels of nutrients and food, and subsequently the mussels in this pool might
also be seeing high levels of food. These high food levels could give the mussels a
larger energy budget from which to produce proteins like Hsp-70. Thus, the
establishment of these algal communities is one way to explain the lower levels of
Hsp 70 expression during week three. However, this is not the only hypothesis which
might explain the lower levels.
It is possible that around week three the mussels had begun to acclimate to
their new environment and thus were less stressed. It is also possible that the lower
levels of Hsp 70 expression in week 3 can be attributed to environmental conditions.
The temperature data show that during the first two weeks the small mussel was
seeing temperatures around 35°C almost every day. During the 3rd week, however,
there were very few days where the temperatures got higher then 20°C. These lower
temperatures could be sufficient to lower the Hsp 70 expression. This hypothesis
could also explain the mirror pattern of the subtidal treatment. Even though the pools
are designed as a flow-through system the rate of drainage is less the rate of water
flow. This is done so that water will stay in the pool. As a result, it is possible that
during hot sunny days the subtidal water was actually heating up causing heat stress.
However, without the data logger information for this pool it is impossible to know
exactly what the water temperatures were like during the course of this study. This
hypothesis can be supported using Hofmann and Somero's (1995) study of Mytilus
trossulus. This study showed seasonal variation of Hsp 70 expression in M. trossulus.
In order to determine if environmental conditions are responsible for the
patterns of Hsp 70 expression 1 will need to analyze the data for weeks 4 and 5. The
temperature data for these weeks look very similar to the data from weeks 1 and 2.
Therefore, if the expression is linked to the environmental conditions, I would expect to
see an increase in the levels of Hsp 70 expression. However, if acclimation has
occurred it would be more likely that the levels of Hsp 70 expression will remain low
during weeks 4 and 5.
It is interesting to note the relation of the field data to the subtidal data for both
the band 1 and band 2 intensities. At week 2 the field Hsp 70 expression is below the
level of subtidal Hsp 70 expression. However, at week 3 the field level of Hsp 70
expression is above the level of Hsp 70 expression for the subtidal group. I am not
sure how to explain this pattern. One possibility is that by week 3 the subtidal group
has settled in its new environment and is seeing more food then the field mussels.
This will be determined by malate dehydrogenase (MDH) quantification. MDH is an
enzyme used in the breakdown of food. By quantifying the amount of MDH present in
a organism we can understand how much food it was collecting. This amount of
food, as well as the MDH quantity, will define the energy budget of the mussels. If the
subtidal mussels see higher food levels and thus have higher energy budgets this
might also allow these mussels to expend more energy on the production of Hsp 70,
This connection might be one way of explaining the pattern of Hsp 70 expression
seen in week 3.
Future Work
Further studies to be done this summer will include analysis of the samples for
weeks 4 and 5. I will also complete the analysis of the field data set so that this group
can be compared to the two pool treatments. The MDH analysis will also be done so
that we can understand the energy budget of the three groups. This summer Iwill
also analyze the band intensities based on the size of the mussels in order to
determine if there is any difference in Hsp 70 expression between small and large
mussels.
Conclusion
Thus far, results have shown that subtidal vs. intertidal mussels do differ in
their expression of Hsp 70 for at least the first three weeks. I am not sure how the
field data will correspond to the above two treatments. However it seems that the
mean band intensities are usually below the mean band intensities for the subtidal
group. Week three is the only exception to this pattern so far and analysis of the
samples for weeks 4 and 5, as well as MDH quantification should help to clarify this
paradox. Finally, comparison of Hsp 70 expression by body size will give clearer
understanding of how mussels deal with heat stress during development.
Acknowledgment
would like to thank George Somero, Lars Tomanek, Mark Denny and Brian
Helmuth for all of their insight and guidance. Without their help none of these results
would have been possible.
References
Craig, E. A. and Gross, C. A. Is Hsp70 the cellular thermometer? Trends in
Biochemical Science. 1991; 16:135-140
Dahlhoff, E. P. and Menge, B. A. Influence of phytoplankton concentration and wave
exposure on the ecophysiology of Mytilus californianus. Marine
Ecology
Progress Series. 1996; 144:97-107.
Gosling, E. The mussel Mytilus: ecology, physiology, genetics and culture.
Developments in Aquaculture and Fisheries Science. 1992. Elsevier
Science Publishers, Amsterdam; vol. 25.
Hofmann, G. E. and Somero, G. N. Evidence for protein damage at
environmental temperatures: seasonal changes in levels of ubiquitin
conjugates and hsp70 in the intertidal mussel Mytilus trossulus. The
Journal of Experimental Biology. 1995; 198:1509-1518.
Hofmann, G. E. and Somero, G. N. Protein ubiquitination and stress protein
synthesis in Mytilus trossulus occurs during recovery from tidal emersion.
Molecular Marine Biology and Biotechnology. 1996; 5(3):175-184.
Laemmli, E. K. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature. 1970; 227:680-685.
Menge, B. A. et al. Benthic-pelagic links and rocky intertidal communities:
Bottom up effects on top-down control? Ecology. 1997; 94:14530-
14535
Roberts, D. A., G. E. Hofmann and G. N. Somero. Heat-shock protein expression in
Mytilus californianus: acclimatization (seasonal and tidal-height
comparisons) and acclimation effects. Biol. Bull. 1997; 192:309-320.
Somero G. The Heat Shock Response. Lecture Notes for Ecological and
Evolutionary Physiology. 1998.
Welch, W. How cells respond to stress. Scientific American, May 1993, 56-64.
Figure Legends
Fig. 1 Puffed chromosomes in the salivary glands of a heat stressed Drosophila
melanogaster. From Welch et al. Scientific American, May 1993.
Fig. 2 The role of molecular chaperones.
Fig. 3 Hsp 70 regulation. Adapted from Somero 1998.
Fig. 4 This figure shows the mean band 1 intensity of Hsp 70 expression for the
intertidal, subtidal, and field groups over the first three weeks. The
asterisk denotes statistically significant differences between the
intertidal and subtidal treatments.
Fig. 5 This figure shows the mean band 2 intensity of Hsp 70 expression for the
intertidal, subtidal, and field groups over the first three weeks. The
asterisk denotes statistically significant differences between the
intertidal and subtidal treatments.
Fig. 6 Body temperature data for a small intertidal M. californianus. Note the
less extreme temperatures during week three.
HEAT-SHOCKED CHROMOSOME

Figure 1
COMPLEXING WITH STRESS PROTEINS
UNFOLDED
PROTEIN
CHAIN
898
00
STRESS
PROTEINS
SELF-ASSEMBLY
ASSISTEL
SELF-ASSEMBLY



FUNCTIONAL
NONFUNCTIONAL
FOLDED PROTEINS
FOLDED PROTEINS
PROTEIN FOLDING occurs spontaneously because of thermodynamic constraints
imposed by the protein's sequence of hydrophilic and hydrophobic amino acids.
Although proteins can fold themselves into biologically functional configurations
(self-assembly), errors in folding can occasionally occur. Stress proteins seem to
help ensure that cellular proteins fold themselves rapidly and with high fidelity.
Weichctal¬
SCIENTIFIC AMERICAN May 1993
Figure 2
Dative
Protei
Hse70 gene
6


Heat
Stas
Figure 3
Ven
Potein

Hp mR
5
gene
HSE
5
1.2
1.1
O.9
0.8
0.7
0.6
0.5
0.4
0.3
0
Hsp 70 Band Intensity
*

2
Week
Figure 4
+ Subtidal
+ Intertidal
+ Field
4 Control
Hsp 70 Band 2 Intensity
*
R
*
Week
Figure 5
- Subtidal
+ Intertidal
+ Field
Control
O
O
C
O
—


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