ABSTRACT
Tigriopus californicus exhibits a heat shock response under
thermal and osmotic stress. At least 3 heat shock proteins (hsps)
are induced under conditions of thermal stress at 30.1°c.
least 2 hsps are induced by T. californicus in 508 and 2x instant
ocean, the latter causing greater hsp induction. The proteins
identified under thermal stress were 2 hsp 60's and 1 hsp 70. The
proteins identified under osmotic stress were 1 hsp 60 and 1 hsp
70. In both conditions hsp synthesis occured rapidly, most likely
under 1 hour. All analysis of protein induction was done by
autoradiography using L-[32S)methionine and was supplemented by
Western analysis. Identification of hsps in T. californicus will
provide the basis for furthur study of hsps in invertebrates and
elucidate the role of hsps in the stress response.
Heat shock proteins (hsps) are highly conserved proteins produced by an
organism under the presence of stress. Stress may come in many different
varieties, including changes in temperature, osmolarity, light exposure, or
oxygen levels. A cellular response within the organism produces a series of
heat shock proteins (hsps), identifiable on the basis of their size and
primary structure, to protect the organism from the deleterious effects of
stress. Functionally, hsps may serve as constitutive proteins that regulate
intracellular traffic of other proteins. Hsps may also function as molecular
chaperones and aid in the assembly of immunoglobulins by protecting an unbound
heavy chain as the light chain is assembled. Finally, hsps may be induced
under shock to transport proteins in and out of intracellular organelles, bind
and protect proteins vulnerable to proteases, remove degraded proteins, or
refold proteins that have lost their functional tertiary structure. More
specifically, many hsps have bound to themselves ATP which when hydrolyzed
provides the energy necessary to refold or repair degraded protein (1).
Since the heat shock response has been shown to be universal, from
prokaryotes to eukaryotes, Tigriopus californicus, a marine crustacean, was
also expected to demonstrate the heat shock response. T. californicus was
chosen as the organism for experimentation because it is easily kept in the
laboratory and available in large quantities. More commonly called copepods,
T. californicus can tolerate tremendous fluctuations in temperature and
salinity. Previous study reports that T. californicus may survive in
seawater temperatures up to 40°C and salt concentrations up to 6008 (2).
During the summer months, increased ambient temperature and a high degree of
evaporation would cause these environmental changes. This study proposed to
examine the heat shock response of T. californicus under various conditions of
temperature and salinity, specifically evaluating what hsps are produced.
MATERIALS AND METHODS
Collection and Storage Conditions. T. californicus were collected
approximately every other week from one high splash pool at Hopkins Marine
Station in Pacific Grove, California. The pool's temperature was found to be
18.9°C. Animals were concentrated using a household strainer and placed in a
5 L transparent container filled with filtered seawater. The container was
kept within the laboratory, most commonly at a temperature of 19°0. The
animals were fed ground mussel 3 times a week and kept near a window that
allowed natural exposure to light and dark. Filtered water was changed
periodically when T. californicus death was apparent.
Determination of the heat shock temperature. Three hundred T.
californicus, without regard to sex, were divided into groups of 20 and placed
in 15 large test tubes (Kimax 5.5 x 1.0cm) containing approximately 5ml of
filtered seawater. The tubes were then placed in a thermal gradient having
temperatures ranging from 9.7°c-42.3°C in order to determine the optimum
temperature for heat shock. Survival under these conditions was then observed
for 4 hours. Observations occurred every 10 min. for the first 2 hours,
followed by observations every 30 min. for the subsequent 2 hours. The
criterion for optimal heat shock temperature was the highest temperature at
which T. californicus sustained survival throughout the 4 hours.
Optimum heat shock temperature was concluded to be 30.1°C. Control
temperature was set at 18.9°C, close to the animal's normal seawater
temperature. All subsequent experiments were carried out using two water
baths, one set at the heat shock temperature and one at the control
temperature.
Radiolabeling of T. californicus proteins during heat shock.
Protein synthesis in T. californicus at 18.9°C and during heat shock at 30.1°0
was examined using L-[32S]methionine (Amersham, Arlington, Il1.). Two tubes
each containing approximately 300 T. californicus in 5 ml of filtered seawater
were acclimated at 18.9°C for 24 hours prior to experimentation. At time
zero, 50uCi of radioactive methionine was diluted into 100uL of seawater and
added immediately to the test tube upon transfer to 30.1°C. Control animals
were labeled at 18.9°C, but in an otherwise identical manner. Both tubes were
left undisturbed for 3 hours for the specimens to incorporate the radioactive
methionine. Radioactive labeling of proteins was stopped by washing the
animals 3 times with 2ml of 0.2M PBS-methionine. Homogenization of T
californicus occurred in a 2ml glass homogenizer (Radnoti, Monrovia, CA) using
200uL of homogenization buffer (10mL PBS/10mM EDTA, 50 uL PMSF, 1OuL
pepstatin, 10uL leupeptin, 100uL chymostatin, and 50uL of NP-40). The
homogenized sample was then centrifuged at 18,000G for 10 min. The
supernatant was removed and 10uL samples were used for protein determination
(3) and radioactive count analysis.
The heat shock time course experiment occurred in an identical manner as
above, but with the addition of 2 tubes of T. californicus homogenized at hour
1 and hour 2.
Radiolabeling of T. californicus proteins during salinity shock.
Protein synthesis of T. californicus in 508, lx, and 2x artifical seawater
(Instant Ocean, Aquarium Systems, Mentor, OH) was also examined using L¬
[°98methionine. Three tubes containing T. californicus identical to the ones
described above were acclimated in 508 instant ocean for 24 hours prior to
experimentation. At time zero, 50uCi of radioactive methionine was diluted
into 100uL of 2x seawater and added immediately to the test tube upon transfer
of the animals into 2x instant ocean. Animals in the two remaining tubes were
radiolabeled in the same manner using the appropriate instant ocean
concentration (508 or 1x). The specimens were left undisturbed for 3 hours to
allow for radioactive methionine incorporation. All tubes were kept in a
18.9°C water bath. Washing, homogenization, protein determination, and
radioactive count determination all were done similar to the methods described
in heat shock experimentation.
The salinity time course experiment occurred in an identical manner as
above, with the addition of 4 tubes (2-508 instant ocean and 2-1x instant
ocean) of T. californicus homogenized at hour 2, hour 3, and hour 4.
SDS-PAGE. After the the supernatant was isolated, protein was solubilized
in 2x SDS buffer (4) containing dithiothreitol and boiled for 3 minutes.
Another radioactive count determination was performed following
solubilization. Protein was then either stored in a -20°C freezer or used
immediately for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
electrophoresis). SDS-PAGE (108 polyacrylamide) was carried out by the method
of Laemmli (4). Gels were dried using a vacuum gel drying apparatus (Bio-Rad
Laboratories, Richmond, CA) at 80°C for 2hrs. The gels were then exposed to
Kodak XAR-5 film at room temperature for 24-72hrs. Protein molecular weight
markers were from Sigma Co., St. Louis, Mo.
Immunoblotting. Western blots of T. californicus protein were done
according to standard technique (5). To probe for hsp 60, rabbit anti-hsp 60
from Synechococcus sp. (StressGen, Victoria, B.C., Canada) was used as the
primary antibody followed by an anti-rabbit secondary antibody (Sigma Immuno,
St. Louis, Mo.). To probe for hsp 70, antibody 3A3 (Dr. Sean Murphy,
Northwestern Univ., Illinois) was used as the primary antibody followed by an
anti-mouse IgG from goat as the secondary antibody (Sigma Immuno).
Proteins transferred by Western procedure onto nitrocellulose were
exposed to x-ray film in the same manner as dried gels.
RESULTS
Temperature for heat shock and hsp induction. At 30.1°C T.
californicus sustained swimming activity without instant or gradual death as
in higher temperatures. Therefore, all heat shock experiments used 18.9°0
seawater as the control condition and 30.1°C as the heat shock condition.
Quantitative differences in protein induction between thermally stressed and
nonstresssed T. californicus are apparent when autoradiographs of SDS¬
polyacrylamide gels are compared. Figure 1 is an autoradiograph of an SDS¬
PAGE gel of T. californicus. Each lane contains 87,161 counts per minute
(cpm) of radiolabeled protein as determined by a Packard 2200CA scintillation
counter. Lane A shows protein incorporation of radioactive methionine at the
control temperature (18.9°C). Lane B shows protein incorporation of
radioactive methionine at the heat shock temperature (30.1°C). Proteins
indicated 1 and 2 show increased induction over basal levels in the heat shock
condition. One or more smaller hsp 70's were induced under heat shock
(protein 1) and two hsp 60's were induced (protein 2). One hsp 60 at 58kD,
produced constituently in the control, was enhanced in the heat shock
condition while another hsp 60 at 60kD, faintly produced in the control, was
greatly enhanced under heat shock. Figure 2 is an autoradiograph of a Western
nitrocellulose membrane containing equal T. californicus protein. Proteins
indicated 1 and 2 all show increased induction over basal levels under heat
shock similar to Fig. 1. Lane pairs (A and A', B and B', C and C', D and D')
of control and heat shock protein (respectively) show decreased overall
protein synthesis at 18.9°C in the face of increased hsp production at 30.1°c.
Figure 3 is an autoradiograph of a heat shock time course. All four lanes
were loaded with equal cpm (236,468cpm) of radioactive protein. Proteins 1,
2, 3, and 4 show increased synthesis after 1, 2, and 3 hours under heat shock
as shown in lanes B, C, and D. Again as in Fig. 1, induction of one or more
smaller hsp 70's (protein 1) occurs along with induction of 2 hsp 60's: one at
58kD and one at 60kD (prptein 2). Protein 3 indicates an induced protein of
49kD not apparent in the control and not seen in other autoradiographs. All
proteins 1-3 are consistently synthesized over the 3 hour time course with the
exception of hsp 70 (protein 1) at hour 2 (lane C) where protein induction
returns to levels similar to that of the control.
Hsp induction under various salinity conditions.
Quantitative
differences in protein induction between osmotically stressed and nonstresssed
T. californicus are apparent when autoradiographs of SDS-polyacrylamide gels
are compared. Figure 4 is an autoradiograph of a salinity time course. Lane
A shows the protein production of T. californicus in the control condition of
508 instant ocean. Lanes B, C, and D show protein production in lx instant
ocean at 2, 3, and 4 hours respectively. Lanes E, F, and G show protein
production in 2x instant ocean at 2, 3, and 4 hours respectively. Proteins 1
and 2 show the levels of induced hsp 60 and hsp 70. Lanes B, C, and D show
the lowest levels of hsp 60 and hsp 70 throughout the 4 hour time course.
Lane A shows a moderate increase of one hsp 60 and one hsp 70 in 508 instant
ocean. Lanes E, F, and G show maximum production of hsp 60 and hsp 70 at hour
2 that being to decline at hour 3 and 4. Lanes E, F, and G also show
fluctuations of overall protein synthesis, highest at hour 2 in 2x and close
to control levels at hour 4 in 2x.
DISCUSSION
Optimum temperature for heat shock as noted by Lindquist (1) usually
occurs 10-15°C above optimum growth temperature. The temperature for heat
shock in T. californicus was set at 30.1°C, 11.2°C higher than its normal
splash pool temperature of 18.9°C. Shown by the autoradiographs in Figures 1,
2, and 3 heat shock at 30.1°C induced production of three hsps. A
constitutive hsp 60 of 58kD was enhanced under heat shock while a larger hsp
60 of 60KD was dramatically enhanced. This doublet of hsp 60's was confirmed
by Western analysis which showed one fluorescent band in the control and two
in the heat shock condition. At the same time a series of smaller hsp 70's
are produced under heat shock, yet how many hsps was left unresolved due to
the inability of accurate Western analysis. Resolution by 2-D analysis may
elucidate how many hsp 70's are present.
In addition to hsp production, general protein synthesis was reduced
under thermal stress as seen in Fig. 2. Most likely this reduction of overall
protein synthesis occurs so that amino acids are exclusively used to
synthesize only proteins that are needed - hsps - and to prevent the
unnecessary production of proteins that would inevitably undergo thermal
degradation. All these events occur within 1 hour as shown in the time course
(Fig. 3) where all three hsps are greatly enhanced at hour 1. The
inconsistent results of the time course in regards to 1) the thermally induced
49kD protein (Fig. 3, protein 3) not seen in other autoradiographs and 2) hsp
70 reduction (Fig. 3, lane C) after 2 hours of heat shock are anomalies that
need to be reconfirmed.
Under varying salinity conditions T. californicus one would expect the
condition of lx instant ocean to be the least shocking to T. californicus due
to its similar salt concentration to actual seawater. This assumption indeed
proved to be true. Induction of hsps occurred only in 508 and 2x instant
ocean. Even though the animals were acclimated into 508 seawater for 24hrs.,
they still maintained hsp production. Shown by autoradiography (Fig. 4) and
supported by Western analysis of hsp 60 and hsp 70, exposure to 2x instant
ocean caused the most dramatic induction of hsp 60 and hsp 70 followed by 508
instant ocean. As seen in the time course, T. californicus in 2x instant
ocean appeared to adapt somewhat after 4 hours of exposure (Fig. 4, Lane G)
which closely resembled exposure to 508 instant ocean (Fig. 4, Lane A). This
adaptation in 2x instant ocean is interesting when looking at the induction of
smaller proteins over the 4 hour time course. In contrast to heat shock where
smaller proteins were reduced, under osmotic stress smaller non-hsps are
synthesized in increased amounts.
Possibly these smaller proteins are
10
utilized by the organism to overcome its hypo-osmotic pressure by increasing
its internal solute concentration (6).
At least two different hsps, hsp 60 and hsp 70, have been shown to
undergo induction when T. californicus has been exposed to thermal and osmotic
stress. It may be assumed that other types of stressors including pH
variance, anoxia, heavy metals, and pollutants will also cause the induction
of these hsps (7). As discussed in the introduction, these hsps are vital to
an organism's survival under stress. Hsp 60, for instance, may 1) keep
polypeptides in their unfolded state to facilitate their transport across
membranes or 2) control proper folding and unfolding of denatured proteins
(8). Hsp 70, minimally examined in invertebrates, comes in two main varieties
in eukaryotes: hsp 73 that is constitutively produced and hsp 72 that is
produced under stress (8). In the T. californicus, induction of a smaller hsp
70 was apparent, especially under heat shock conditions. These two hsps
expedite intracellular trafficking of proteins and stabilize protein structure
under normal conditions, but also facilitate synthesis and assembly of new
proteins that have been destroyed by shock and remove irreversibly denatured
proteins (8). Since hsps are so highly conserved and are present in all
organisms, understanding the mechanism by which they operate will provide
significant insight into not only how T. californicus deals with stress, but
how many organisms deal with stress.
11
ACKNOWLEDGEMENTS
I would to thank Julie Golden and Jan Von Kampen for being so helpful
and accomodating. I would like to thank Theresa MCEvoy and Christine Shin for
putting up with my wise cracks and Dan Lee for helping me make them. I would
like to thank all the Hopkins students for making this quarter the best
quarter I've ever had at Stanford. Thanks to all the Hopkins professors and
special thanks to Dr. R. Paul Levine for being a great advisor and a good
friend.
12
LITERATURE CITED
1. Linquist, s. 1986. The Heat Shock-Response. Ann. Rev. Biochem. 55:
1151-1167.
2. Haderlie, E. C., D. P. Abbott, R. L. Caldwell. 1980. Three Other
Crustaceans: A Copepod, a Leptostracan, and a Stomatopod. In Morris,
E. C. Haderlie, D. P. Abbott. 1980. Intertidal Invertebrates of
California. 26: 631-632.
Lowry, O. H., N. J. Rosenbrough, A.L. Farr, and R. J. Randall.
1951. Protein determination with the Folin phenol reagents. J. Biol.
Chem. 193: 265-275.
4. Laemmli, U. K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature. 227: 680-685.
5. Harlow, E., L. David. 1988. Antibodies: A Laboratory Manual. 12:
471-504.
6. Burton, R. S. 1991. The Journal of Experimental Zoology. 259: 166-
173.
7. Lindquist, s., E. A. Craig. 1988. The Heat-Shock Proteins. 22:
631-633.
8. Welch, W. J. 1990. The Mammalian Stress Response: Cell Physiology
and Biochemistry of Stress Proteins. In Morimoto, Tissieres,
Georgopoulos. 1990. Stress Proteins in Biology and Medicine. 10:
242-268.
13
FIGURE LEGENDS
Fig. 1. Autoradiograph of equally counted (87,161cpm) radiolabeled protein
from T. californicus in lanes A and B. Lane A: total protein labeled with
radiolabeled methionine at 18.9°c.
Lane B: total protein labeled with
radioactive methionine at 30.1°c.
Dashed lines indicate proteins induced over basal levels. Protein molecular
weight markers (Sigma Co., St. Louis, I11.) are at 84kD and 58kD. Gel exposed
for 72hrs.
Fig. 2. Autoradiograph of a nitrocellulose membrane containing equal amounts
of T. californicus protein: 10ug in lanes A and A', 15ug in lanes B and B',
25ug in lanes C and C', and 58ug in lanes D and D'. Lanes A-D: protein
labeled with radioactive methionine at 18.9°C. Lanes A'-D': protein labeled
with radioactive methionine at 30.1°C. Dashed lines indicate proteins induced
over basal levels. Molecular weight markers explained in legend to Fig. 1.
Membrane exposed for 96hrs.
Fig. 3. Heat shock time course. Autoradiograph of equally counted
(236, 468cpm) radiolabeled protein from T. californicus. Lane A: control
protein labeled with radioactive methionine at 18.9°C. Lanes B, C, D: heat
shock protein labeled with radioactive methionine at 30.1°C and homogenized at
1, 2, and 3 hours respectively.
Dashed lines indicate proteins induced over basal levels. Molecular weight
markers explained in legend to Fig. 1. Gel exposed for 24hrs.
14
Fig. 4. Salinity shock time course. Autoradiograph of equally counted
(248,240cpm) radiolabeled protein from T. californicus. Lane A: control
protein labeled with radioactive methionine in 508 instant ocean. Lanes B, C,
and D: shocked protein labeled with radioactive methionine in lx instant ocean
and homogenized at 2, 3, and 4 hours respectively. Lanes E, F, and G: shocked
protein labeled with radioactive methionine in 2x instant ocean and
homogenized at 2, 3, and 4 hours respectively. Dashed lines indicate proteins
induced over basal levels. Molecular weight markers explained in legend to
Fig. 1. Gel exposed for 48hrs.
protein
protein 2
-84kD
-58kD
gue
protei
prote
84kD
58kD
18.9°
30.1°C
Figure
protein
protein
protein
Figure 3
ABC D
84KD
5880
48.580

30.180
protein 1-
protein 2-
50%
Figure 4
9
BCDEF
Zhr. 3hr. 4hr 2hr 3hr 4hr.
84kD
58kD