Identification and Induction of
Heat Shock Proteins
in Tigriopus californicus
Theresa A. McEvoy
June 3, 1993
Dr. R. P. Levine
Stanford University
Biology 175H
ABSTRACT
Using immunoblotting and enhanced chemiluminescence
techniques, heat shock proteins of molecular weight 60 KD and 70KD
were identified constitutively in Tigriopus californicus under
control conditions of 19 degrees Celsius. Hsp 60 was induced
during separate experiments under conditions of increased
temperature and increased salinity concentrations. Western
analysis could not definitively identify induction of hsp 70 during
heat shock or salinity shock. This study serves as the basis for
further study of heat shock proteins in marine organisms.
Introduction
All organisms possess mechanisms enabling them to endure or
adapt to the common stresses of their environments. In the marine
environment, common stresses may include variable temperatures,
introduction of pollutants, or changes in pH. There are a variety of
ways organisms may cope with stress. At the cellular level, one of
the most important reactions to stress may be the increased
synthesis of additional proteins commonly referred to as heat shock
proteins or hspsé. The increased production of these groups of
proteins in response to stress is commonly referred to as the heat
shock response5. In addition to playing a vital role in normal cell
activities, hsps protect the cells from the damaging effects of
extreme temperature changes or other environmental stress4 . Heat
shock proteins are among the most highly conserved genetic system
known, indicating that they may play an important biological role in
the adaptation and evolution of organisms5. Although heat shock
response has been studied in fruit flies, yeast, chickens, mammals
and bacteria, it has not been rigorously evaluated in marine
organisms, many of which live in highly variable habitats associated
with the periodicity of the tides.
Tigriopus californicus is a marine copepod that thrives in the
high splash pools associated with rocky coastlines. This hearty
crustacean ranges from the coast of Glacier Bay, Alaska, south to
Baja California2. This study identifies two groups of heat shock
proteins in Tigriopus and attempts to observe increased production
of the hsps in response to changes in temperature and salinity
simulating harsher environmental conditions. Tigriopus californicus
was chosen as the test organism for this nine week project because
it is relatively abundant in the tide pools near Hopkins Marine
Station and is easily maintained in the lab at room temperature.
Materials and Methods
Collecting Tigriopus californicus. Copepods were collected
through a small collecting strainer from two high splash pools near
Hopkins Marine Station in Monterey Bay, California. They were
maintained in filtered sea water at room temperature and fed ground
mussel approximately every twenty four hours.
Obtaining protein. Protein samples were obtained by
homogenization of Tigriopus californicus in homogenization buffer
containing 10 ml PBS in 10 mM EDTA, 50uI PMSF, 10ul pepstatin,
1Oul leupeptin, 100ul chymostatin and 50ul ofNP-40 (.5%).
Following centrifugation, supernatant from the sample was boiled
for three minutes and protein content was determined using
Coomassie stain reagent.'
SDS PAGE. Solubilized protein from the sample was separated
according to molecular weight by electrophoresis on a 10% SDS
acrylamide gel. Prestained molecular weight standards were
obtained from Sigma Chemical Corporation (St. Louis, MO.). The hsp
60kD standard was obtained from mouse protein and made available
to the lab by Ildiko Kovacs at Hopkins Marine Station. The hsp 70KD
bovine brain stress protein standard was supplied by StressGen
Biotechnologies Corporation (Victoria, B.C.).
Western Blotting. The western blotting procedure was carried out
according to the protocol described by Harlow and Lanes. The
primary heat shock monoclonal anti-body used for detecting heat
shock proteins of the 60kD family was obtained from StressGen
Biotechnologies Corp. Anti-hsp70 monoclonal antibody 3A3 was
made available to the lab by Shawn Murphy at Northwestern
University. The anti-GOKD antibody was diluted 1:1000. All other
anti-bodies, including the secondary antibodies, were diluted
1:10000. Secondary anti-rabbit lgG peroxidase conjugate and anti¬
mouse lgG peroxidase conjugate were supplied by Sigma Immuno
Chemicals.
Enhanced Chemiluminescence (ECL). Films of the nitrocellulose
membrane were developed in the dark room using Kodak film
according to the protocol for ECL immunodetection supplied by
Amersham International (Buckinghamshire, England).
Heat and Salinity Shock Experiments.
Protein samples used to
detect induced hsps after exposure to heat shock were obtained from
individuals acclimated to 19 degree sea water for twenty-four hours
and then transferred to 32 degree Celsius sea water for a period of
four hours. Similarly, copepods were acclimated at 50% artificial
sea water for twenty-four hours and transferred to 100% or 200%
artificial sea water to test for salinity shock. All salinity shock
experiments were conducted at 19 degrees Celsius.
Results
Heat shock proteins with molecular weights 60 KD and 70 KD
were present in Tigriopus californicus under control conditions at
19 degrees and 100% salinity as shown in figures 1 and 2
respectively. Each figure is a developed film of the nitrocellulose
membrane after enhanced chemiluminescence following Western
blotting. In figure 1, the mouse standard containing hsp 60 is
illuminated in lane 4, and protein obtained from Tigriopus
californicus is in lanes 1 through 3. Lanes1, 2, and 3 contain 12.6
ug, 25.2 ug, and 50.2 ug of protein, respectively. The dark bands
directly across from the dark band of the standard indicate the
presence of hsp 60 in the protein sample. Two bands of protein are
visible in lane 1 in which the most protein was loaded. Figure 2
contains a bovine brain standard of hsp 70 in the far right lane.
Protein extracted from Tigriopus californicus is present in lanes 1
through 3. Again, the dark bands across from the standard indicate
the existence of hsp 70 in the protein sample obtained from
Tigriopus californicus under control conditions. A slightly higher
molecular weight protein also reacted with the anti-hsp70 antibody
as displayed by the three lighter marks directly above the dark
bands.
The results of exposing Tigriopus californicus to 30 degrees
Celsius is shown by the five minute exposure film in figure 3. Here,
the standard was loaded in lane 5 and increasing amounts of protein
extracted from Tigriopus under the control condition of 19 degrees
Celsius were run in lanes 1,2, 3 and 4. Lanes 6 through 10 contain
increasing amounts of protein from copepods acclimated for
twenty-four hours at 19 degrees and then transferred to 30 degrees
Celsius for 3 hours. Equal amounts of protein are loaded in lanes 1
and 6, lanes 2 and 7, lanes 3 and 8, and lanes 4 and 9. The presence
of induced hsp 60 is indicated by the multiple bands visible in the
sample lanes 6 through 10 as compared to the corresponding control
lanes 1 through 4 containing equal protein.
Exposing Tigriopus californicus to salinities of 100% and 200%
for four hours after 24-hour acclimation at 50% instant ocean
induced production of hsp 60 (figure 4) but did not induce production
of hsp 70 (figure 5). In each figure, lane 2 is the control, containing
protein obtained from animals acclimated at 50% instant ocean and
lanes 3 and 4 contain protein of Tigriopus californicus exposed to
the increased salinity concentrations of 100% and 200%,
respectively. Figure 4 displays an overloaded gel which nonetheless
shows multiple bands in the sample lanes 2 (50%), 3 (100%), and 4
(200%) as compared to the standard loaded in lane 1. Although the
copepods were acclimated at 50% artificial sea water, multiple
bands are present in lane 2, perhaps indicating that they had not yet
adapted to the change. Figure 5 shows only single bands in the three
samples loaded in lanes 2, 3, and 4, from which definitive results
cannot be discussed.
Conclusions
Heat shock proteins of the 60kD and 70kD molecular weight
families were identified in samples of protein from Tigriopus
californicus under optimal conditions of 19 degrees Celsius and
100% sea water. These constitutive proteins may enable the
organisms to endure normal ranges of stress introduced by small
changes in their habitat. Identification of these proteins verifies
that cross reaction with the anti-hsp antibodies has occured.
Inducible forms of hsp 60 found under both heat shock and
salinity shock may enhance the survival rate of Tigriopus
californicus for several hours or longer during an increase in
temperature of 13 degrees Celsius and increased salinity
This
concentrations of up to four times the original concentration.
production of proteins enabling survival under harsh conditions
is an
important component of this species' ability to live in the high
splash pools. This study is only an initial investigation of heat
shock proteins in marine organisms and provides the basis for
correlating successful survival of organisms with heat shock
protein synthesis and activity.
Literature Cited
1. Bradford, M., 1976. Anal. Biochem. 72:248-254.
2. Haderlie, C., D. Abbott, and R. Caldwell. 1980. Intertidal
Invertebrates of California. Stanford University Press.
Stanford, CA. pp. 632-635.
3. Harlow, E. and D. Lane. 1988. Antibodies, A Laboratory Manual.
Cold Spring Harbor Laboratory. pp. 474-506.
4. Langer, T., and L. Neupert. 1991. Heat Shock Proteins hsp60 and
hsp70: Their Roles in Folding, Assembly and Membrane
Translocation of Proteins. Current Topics in Microbiology and
Immunology. 167: 3-24.
5. Lindquist, S., and E.A. Craig. 1988. The Heat Shock Proteins:
Annual Review of Genetics. 2: 631-666.
6. Morimoto, Tissieres, Georgopoulos (ed.). 1990. Stress Proteins in
Biology and Medicine. Cold Spring Harbor Laboratory Press pp. 1-
20.
Figure Legends
Figure 1. Film developed for one minute after enhanced
chemiluminescence following standard western blot procedure
showing identification of hsp 60 family in T. californicus
acclimated at 19 degrees and 100% sea water. A 10ul sample of
mouse standard is loaded in the far right lane (lane 4) and increasing
amounts of sample protein are loaded in lanes 1 through 3,
respectively. A prestained molecular weight sample was loaded in
lane 5, not shown in the figure.
Figure 2. Film developed for five minutes after western blotting
and enhanced chemiluminescence using anti-hsp 70 antibody 3A3.
Sample protein from organisms acclimated at 19 degrees was loaded
in lanes 1 through 3 and 10ul of hsp 70 standard extracted from
bovine brain (StressGen Technologies Corp.) was loaded in lane 4
Figure 3. Film developed after enhanced chemiluminescence of
protein obtained from T. californicus under control conditions and
under heat shock conditions of 30 degrees Celsius. Control protein
is loaded in increasing amounts in lanes 1 through 4. Heat shocked
protein is loaded in lanes 6 through 10, and the hsp60 standard is
located in lane 5. Equal amounts of protein are loaded in lanes 1 and
6, lanes 2 and 7, lanes 3 and 8, and lanes 4 and 9.
Figure 4. Film developed after western blotting and enhanced
chemiluminescence of protein extracted from T. californicus
acclimated at 50% artificial sea water (lane 2), and transferred to
100% and 200% artificial sea water (lanes 3 and 4 respectively).
Hsp 60 standard is located in lane 1.
Figure 5. Results of the salinity shock experiment probing for
hsp70. Lane 1 contains the hsp70 standard. Lanes 2 contains the
control condition of acclimation at 50% artificial sea water. Lanes
3 and 4 contain protein extracted from copepods transferred to 100%
and 200% artificial sea water.
Figure 1.
84kI
58kl
Figure 2.
Figure 3.
Figure 4.
3
Figure 5.
Acknowledgements
wish to extend thanks to Dr. R. P. Levine for his immense
enthusiasm and dedicated interest in this project. I would also like
to thank Julie Golden for her incredible patience in teaching me the
necessary technical skills of gel electrophoresis and western
blotting. Additional thanks are extended to everyone who worked in
Dr. Levine's lab for sharing precious lab space with me and providing
an enjoyable work environment! And finally, greatful thanks are
offered to the entire staff at Hopkins Marine Station and the other
students participating in the spring program, for their help and
encouragement.