Influences of thermal acclimation and acute temperature change on the motility of epithelial wound-healing cells (keratocytes) of tropical, temperate and Antarctic fish
1 Biochemistry Department, Beckman Center, Room 473A, Stanford University
School of Medicine, Stanford, CA 94305-5307, USA
2 Hopkins Marine Station, Pacific Grove, CA 93950, USA
* Author for correspondence (e-mail: somero{at}stanford.edu)
Accepted 4 September 2003
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Summary |
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Key words: Antarctic fish, cell motility, cytoskeleton, notothenioid, keratocytes, temperature
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Introduction |
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Less is known about the effects of temperature on nonspecific immunity,
although available data indicate these effects to be complex and of
potentially great importance to organisms. Low temperatures appear to increase
the efficacy of certain nonspecific immune responses, including cytotoxic cell
lytic activity (Le Morvan et al.,
1995), phagocyte respiratory burst
(Dexiang and Ainsworth, 1991
;
Collazos et al., 1994
) and
macrophage response to activating factor and burst activity
(Le Morvan et al., 1997
).
Thus, there is evidence that nonspecific immunity may respond to cold stress
relatively quickly and protect the organism from further damage by a pathogen
until cold-temperature suppression of specific immunity is overcome.
A potentially important contributor to the nonspecific immune responses is
the wound healing function of cells termed keratocytes, which are terminally
differentiated epithelial cells found in teleosts and amphibians. The motility
of these cells was first described in goldfish (Carassius auratus) by
Goodrich (1924). Subsequent
research, including in vivo studies using Xenopus laevis,
showed that keratocytes migrate across wounded areas to provide a barrier to
infection (Radice,
1980a
,b
;
Euteneuer and Schliwa, 1984
).
Keratocytes may crawl together as a sheet of cells or move individually. The
cells extend large lamellipodia, and motility is driven by actin
polymerization (Theriot and Mitchison,
1991
; Small et al.,
1995
). As is true for other types of actin-based cell motility,
locomotion is thought to occur through the coordination of several distinct
steps. First, the cell extends its leading edge forward, and this protrusion
of the membrane is coupled with adhesion to the substrate. Next, traction
force leads to a forward translocation of the cell body. Lastly, contact with
the substrate at the rear of the cell is dissociated and the rear edge of the
cell is released to follow the forward procession. Many of the cellular
processes involved in keratocyte movement, such as actin polymerization and
membrane fluidity, are strongly affected by the physical and chemical
environment (Cossins et al.,
1987
; Hochachka and Somero,
2002
). Thus, it is to be expected that keratocyte motility and the
wound healing capacity it supports would be highly sensitive to environmental
temperature. The potential scale of these thermal effects is suggested by the
finding that actin-based movement in mammalian fibroblast cells increased with
a Q10 of approximately 4 between 29°C and 39°C
(Hartmann-Petersen et al.,
2000
).
Currently, little is known about thermal effects on keratocyte motility in
ectotherms and how this thermal sensitivity varies among species
evolutionarily adapted to different temperatures or among conspecifics
acclimated to low and high temperatures. To examine these phenomena, we
studied keratocyte motility in four species of fish: two eurythermal species -
a temperate goby, Gillichthys mirabilis (10-35°C), and a desert
pupfish, Cyprinodon salinus (10-40°C) - and two species with
narrow environmental temperature ranges - an Antarctic notothenioid,
Trematomus bernacchii (-1.86°C), and a tropical clownfish,
Amphiprion percula (26-30°C). In order to examine the long-term
effects of acclimation as well as acute effects of experimental temperature,
the first two species were acclimated to multiple temperatures within their
physiological thermal range, and the stenothermal T. bernacchii was
maintained at 5°C, the highest temperature to which this species can be
acclimated (see Somero and DeVries,
1967; Hofmann et al.,
2000
). We describe variation in keratocyte morphology and quantify
keratocyte motility in terms of cell speed and direction in relation to
evolutionary, acclimatory and experimental temperatures. These data provide
insights into thermal effects on nonspecific immunity and into the
physiological determinants of organismal thermal tolerance limits.
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Materials and methods |
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Thermal acclimation
Stocks of field-collected G. mirabilis were maintained at Hopkins
Marine Station in a continuous-flow, open-circulation tank containing ambient
water (16°C) from Monterey Bay. Individuals were acclimated to 10°C,
16°C and 25°C (±0.5°C) for a period of 4 weeks in closed
recirculating tanks equipped with a flow-through heater/chiller. C.
salinus were acclimated for 4 weeks at temperatures of
26±0.5°C or 35±0.5°C in closed recirculating tanks in
seawater taken from Monterey Bay that was adjusted to 50%thou salinity. T.
bernacchii were studied at the US McMurdo Station. Fish were maintained
in a continuous-flow, open-circulation tank in ambient water (-1.86°C)
pumped in from McMurdo Sound. T. bernacchii acclimated to 5°C
were held for 3 weeks in a closed recirculating tank heated to
5±0.5°C. A. percula were maintained at 26±0.5°C
for 4 weeks in seawater made from Instant Ocean© (MediaCybernetics,
Silver Spring, MD, USA).
Cell culture
Keratocytes were cultured in L-15 medium made with 50% filtered seawater
(0.2 µm filter) and supplemented with Hepes, 5% antibiotic/antimycotic
(Sigma) and 10% fetal bovine serum (FBS)
(Ream, 2002). Several scales
were removed, using forceps, from 2-3 fish from a single acclimation for each
temperature experiment. In the case of T. bernacchii, scales were
removed from only one fish for each experiment because of limited specimen
availability. The scales were washed in medium for 15 min and then plated
between untreated glass cover slips with fresh medium. Cultures of keratocytes
from G. mirabilis acclimated to 10°C, 16°C and 25°C were
maintained at 12°C, 20°C and 25°C, respectively; keratocytes from
C. salinus and A. percula were maintained at 25°C; and
keratocytes from T. bernacchii acclimated to -1.86°C and 5°C
were maintained at 0°C and 4°C, respectively. Keratocytes cultured
from G. mirabilis matured in 12 h and were viable for 36 h after
maturation; keratocytes from C. salinus and A. percula
matured in 6 h and were viable for 12 h after maturation; and keratocytes from
T. bernacchii matured in 48 h and were viable for 36 h after
maturation.
Supercooling of keratocytes of T. bernacchii
T. bernacchii can be supercooled to -6°C if they are
previously warmed to 0°C to melt any ice crystals present in their body
fluids (DeVries and Cheng,
1992). Keratocytes cultured from T. bernacchii
(-1.86°C) were subjected to supercooling experiments in order to determine
whether or not individual cells in primary culture could withstand
supercooling. Glass cover slips containing keratocytes were placed in seawater
at 0°C and slowly cooled to -6.0°C. Keratocytes were then slowly
returned to 5°C and visualized on an Olympus light microscope to determine
if they were still capable of locomotion.
Video microscopy
Cells were imaged on three different inverted light microscopes (located at
the three different biological laboratories where the research was conducted)
under 10x(2x), 20x(2x) or 40x(2x)
magnification. The Nikon Diaphot microscope (Hopkins Marine Station) was
equipped with a Peltier temperature-controlled stage, whereas the Olympus BH-2
(McMurdo Station) and Nikon Diaphot-300 (Stanford University School of
Medicine) microscopes were equipped with temperature-controlled stages
attached to a circulating water bath. Cell cultures, cell behavior and cell
speed remained consistent across imaging conditions. Temperature was measured
at the center of the surface of the cover slip in the area being imaged using
a thermocouple probe. Temperature was maintained within ±0.5°C of
the designated experimental temperature. The temperature of the cover slip and
surrounding medium was slowly brought from room temperature to experimental
temperature, taking as little as 10 min or as long as 4 h, depending on the
temperature differential. Once the experimental temperature was reached, the
cells were held at that temperature for a minimum of 20 min before recording
data. There was no discernible difference in cell speed or behavior between
cells monitored after 20 min or 4 h atexperimental temperatures. Each cell was
recorded for 15 min and video frames were captured every 15 s, resulting in a
total of 61 frames per cell. Depending on the speed and path of the cell, many
cells crawled out of the field of view. When this occurred, the time lapse was
paused, the stage was moved in order to replace the cell in the field of view,
and the time lapse was then continued. Each pause resulted in a temporal and
spatial discontinuity that was accounted for in the analysis but which often
reduced the total number of useful frames per cell. A total of 20 cells from a
minimum of two cultures were imaged at each experimental temperature. No more
than 12 cells per culture were imaged for a given temperature condition.
Image analysis
Movies of keratocytes imaged on the Nikon (Hopkins Marine Station) or
Olympus microscopes were acquired using a Silicon Intensifier Target (SIT)
camera (Nikon; Hopkins Marine Station) or chilled charge-coupled device (CCD)
camera (Olympus) attached to a VCR and recorded in real time to VHS. Images
were pulled from the tapes every 15 s using ImagePro Plus® data analysis
software (MediaCybernetics). Time-lapse video was acquired digitally for
keratocytes imaged on the Nikon (Stanford University Medical School)
microscope using an intensified charge-coupled device (ICCD) camera (GenII
Sys/CCD-c72) with the shutter driver and acquisition capabilities of
MetaMorph® image analysis software (Universal Imaging CorporationTM;
Downingtown, PA, USA). Frames were captured every 15 s and compiled into
time-lapse movies. Keratocytes were tracked by hand using a mouse and the
`point-and-click' method utilized by both software packages, yielding discrete
(x,y) coordinates for the keratocyte at every 15 s interval. The cell
body and leading edge of the lamellipodium were tracked for >60 cells and
no discernible difference was found between the two tracking methods. The cell
body was chosen for simplicity and all cells used in this analysis were
tracked with that method.
Analysis of cell velocity
The speed of a cell was determined as the instantaneous speed over 15 s for
each continuous 15 s interval. All instantaneous speeds measured for all cells
at a given experimental condition were placed into bins and the frequency of
occurrence was determined for each bin. The mean and median instantaneous
velocities over 15 s were also determined for each cell. Autocorrelation
analysis of instantaneous speed over time was used to determine the degree of
memory associated with speed (i.e. are instantaneous speed measurements
independent or related to previous speeds?) where the speed autocorrelation
over k time intervals is given by:
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Keratocyte paths were characterized with respect to directional movement.
Directional movement can be visualized by plotting the trajectory of each cell
as (x,y) coordinates. Each trajectory begins at the origin and is
rotated so that the first step taken by each cell is in the same direction
(x=0). A matrix using non-overlapping intervals was used to determine
all possible path lengths for this analysis. Additionally, the magnitude of
the turns made by keratocytes can be examined by plotting the mean cos
(), or turning angle, against the path length traveled by each cell for
each pair of (x,y) coordinates. This was done using non-overlapping
intervals for all possible path lengths. This method does not reveal
directionality, because it takes into account turning magnitude only.
Mathematical formulas used in these analyses can be found in Ream
(2002
).
Statistical analysis
Calculation of mean speed, median speed, standard deviation and standard
error for each cell was performed using Microsoft Excel®. The statistical
analysis package in Sigma Plot® version 5.0 was used to perform Student's
t-tests to determine whether or not the increase in keratocyte speed
between experimental temperatures was statistically significant. The Microsoft
Excel® statistical software package was employed to run two-way analyses
of variance (ANOVAs) between experimental temperatures within each acclimation
to determine if thermal acclimation had an effect on speed.
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Results |
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Keratocytes from G. mirabilis exhibited the greatest size
variation. Within a single culture, cells of average size (35 µm wide
along the long axis) could be found along with cells as large as
80 µm
and as small as
15 µm in width. For the other species, size variation
within a species was only approximately ±5 µm. Regardless of their
size, G. mirabilis keratocytes displayed the characteristic `canoe
shape' first described by Goodrich
(1924
), which denotes an
elongated, elliptical cell body with a smooth-edged, narrow lamellipodium
running along one side of the cell body and smoothly curving around each end
(Fig. 1A). Some degree of
ruffling was apparent over time and usually occurred at the leading edge of
the cell, although the edges of the lamellipodium were generally smooth.
Shape-changing events in G. mirabilis keratocytes had durations of
1-2 min, occurred in fewer than half of the cells and involved few
lamellipodial rearrangements.
Keratocytes of C. salinus were 35 µm wide and varied little
in size. The keratocytes were less canoe-shaped in appearance due to a
lengthening in the lamellipodia between the cell body and the leading edge,
which gave the cells a fatter appearance. Additionally, the lamellipodia of
these cells ruffled >50% of the time. These ruffles were almost always in
the first third of the lamellipodium at the leading edge of the cell and
appeared as large, rapidly changing, phase-dense regions rather than small
dark creases. Shape changes in C. salinus keratocytes occurred in
slightly fewer than half of the cells, lasted approximately 2-4 min and
involved more lamellipodial rearrangements than in cells of G.
mirabilis and A. percula.
Keratocytes of A. percula were 35 µm wide along the long
axis and had the stereotypical canoe shape. The leading edges of their
lamellipodia were gently rounded, and most ruffling occurred at the ends of
the lamellipodia rather than at the leading edge. Shape changing events in
A. percula keratocytes had durations of 1-2 min, occurred in less
than half of the cells undergoing shape changes and few had lamellipodial
rearrangements.
T. bernacchii keratocytes also displayed the characteristic canoe
shape and had minimal variation in size (35 µm wide along the long
axis). The lamellipodia always appeared smooth and any ruffling was in the
form of small creases in the lamellipodia at the edges of the cell body. The
most notable feature of T. bernacchii keratocytes was their extremely
slow rate of locomotion. Locomotion, lamellipodial ruffling or any manner of
shape change was only detectable using time-lapse microscopy, in contrast to
the cells of other species where dynamic events were often apparent to the
eye. Shape changes occurred in a little over half of the cells, lasted
approximately 4-5 min and involved a large number of lamellipodial
rearrangements.
Keratocyte motility can be characterized in terms of a velocity comprising a magnitude (speed) and a complex directional component (Figs 2, 3). The directional component can be further broken down into persistence, or movement in a single direction, and turning behavior, namely the size of the angles made by a cell as it turns. Both components of velocity as well as the thermal range over which motility was observed, varied among species and, in some cases, with acclimation history.
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The temperature ranges over which keratocyte motility occurred reflected the evolutionary adaptation temperatures of the species (Fig. 4). Keratocytes of T. bernacchii maintained at the normal McMurdo Sound temperature (-1.86°C) or acclimated to 5°C were functional over the full range of measurement temperatures (5-20°C). Keratocytes from T. bernacchii (-1.86°C) that were supercooled to -6.0°C for 10 min had normal motility when they were returned to 5°C. Keratocytes cultured from G. mirabilis acclimated to 10°C, 16°C and 25°C were able to function at 10-35°C. The keratocytes isolated from A. percula maintained at 26°C were also functional at 10-35°C. Keratocytes from C. salinus acclimated to 26°C and 35°C were functional at 10-40°C and 5-40°C, respectively. In all cases, the thermal range of the keratocytes was at least as broad as the thermal range of the fishes' habitats, and, for the two stenothermal species (T. bernacchii and A. percula), the thermal range of keratocyte motility was much greater than the environmental temperature range of the fish.
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Thermal effects on keratocyte speed
Mean speed of keratocyte movement varied among species and as a function of
experimental temperature (Fig.
4). Autocorrelation analysis of instantaneous speeds measured over
15 min varied from cell to cell and showed no strong correlation and no
oscillation in speed for any species examined
(Table 1), indicating that each
instantaneous speed is discrete and is not affected by previous cell
speed.
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G. mirabilis keratocyte motility significantly increased in speed
with increasing temperature from 5°C to 25°C (P<0.001 in
all cases) with a Q10 of 2.5
(Fig. 4A). At 25°C, mean
speed reached a maximum and then decreased at higher temperatures. Variation
in instantaneous speed also increased with increasing measurement temperature
between 5°C and 35°C. Although the mean speed of keratocytes from
individuals held at all three acclimation temperatures exhibited the same
response to experimental temperature (P=0.53), mean speed varied with
acclimation temperature, with cells from the 10°C group most commonly
having the slowest speeds (Fig.
4A). At low experimental temperatures of 5-15°C, keratocytes
from 25°C-acclimated fish moved more rapidly than keratocytes from the two
cold acclimations (16°C and 10°C). Conversely, cells from
cold-acclimated fish (10°C) moved more slowly at warmer temperatures
(20-35°C) than those from the two warmer acclimations (16°C and
25°C). The maximal speed of
45 µm min-1 was reached by
keratocytes from the 16°C acclimation. The mean speed for all the
acclimation groups at 15°C, the experimental temperature that was closest
to the habitat temperatures of the population used, was 11.4 µm
min-1.
C. salinus keratocyte speed increased with increasing temperature
from 5°C to 35°C, with a Q10 of 2.0, and decreased between
35°C and 40°C. The speeds are statistically different at each
temperature (P<0.001 in all cases) except for cells of individuals
acclimated to 35°C, which moved at the same rate at 25°C and 30°C.
The variation in speed also increased with temperature. C. salinus
keratocytes from both acclimation temperatures exhibited the same response to
experimental temperature (P=0.46), although mean speed varied between
acclimation groups at several measurement temperatures. Keratocytes from both
thermal acclimations did not vary in speed at low (10-15°C) and high
(35-40°C) experimental temperatures but crawled at statistically different
speeds at the middle experimental temperatures (20-30°C;
Fig. 4B). At 20°C, the
cold-acclimated cells moved faster, but at 25-30°C the warm-acclimated
cells were the fastest moving. The mean speed for all the acclimation groups
at 30°C, a common physiological temperature for C. salinus, was
13.8 µm min-1 (Fig.
5). The maximal instantaneous speed was 45 µm
min-1 at 35°C. Both the mean speed at physiological temperature
and the maximal speed were very similar to those of keratocytes from G.
mirabilis.
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Motility of keratocytes of A. percula significantly increased in
speed as temperature increased from 10°C to 35°C
(P<0.001), except between 15°C and 20°C, at which
temperatures the keratocytes exhibited similar speeds (P=0.095;
Fig. 4C). The overall
Q10 was 2.0. The maximal instantaneous speed measured was
45 µm min-1. Variation in instantaneous speed also increased
with increasing temperature between 5°C and 15°C and between 20°C
and 35°C. The mean speed at 30°C, a common physiological temperature,
was 10.8 µm min-1 (Fig.
5).
Keratocytes cultured from T. bernacchii held at -1.86°C also exhibited temperature-dependent rates of motility (Fig. 4D, inset). Keratocyte speeds are statistically different (P<0.05) between the experimental temperatures of 5°C, 10°C and 15°C; however, keratocytes moved at the same speed at 15°C and 20°C (P=0.068). The maximal instantaneous speed measured was 0.046 µm min-1, a small fraction of the maximal velocity of the other species' keratocytes. The mean speed at 5°C, the experimental temperature closest to physiological (-1.86°C) was 0.008 µm min-1. Thus, no temperature compensation of rate of motility was found for keratocytes of T. bernacchii (Fig. 5). Although the mean speed of keratocytes from T. bernacchii held at both acclimation temperatures exhibited the same response to experimental temperature (P=0.63), acclimation groups were statistically different (P<0.01) from each other at each experimental temperature. The strong acclimation effect on the speed of keratocytes from animals acclimated to 5°C is most obvious at the experimental temperatures of 15°C and 20°C, where speed was 10-20-fold greater than in the -1.86°C-acclimated cells. Variability in speeds was enormous, with speeds in the range of 0.065-1.05 µm min-1. Keratocyte speeds were statistically different (P<0.01) at each experimental temperature.
Thermal effects on keratocyte trajectories
Two types of analysis were used to quantitatively describe the directional
component inherent in cell velocity. The first type of analysis plots the
trajectory of each cell, beginning at the origin and oriented such that the
first step taken by each cell is along the x-axis. Trajectories were
compared at all experimental temperatures; however, general trends can be
summarized by comparing trajectories at 10°C and 20°C
(Fig. 6). This analysis made it
possible to visualize path shape and to determine the degree of persistence in
a single direction. There were cells taking very straight and persistent
trajectories at all experimental temperatures and acclimations; however, the
number of individual cells exhibiting this type of directional movement varied
with experimental temperature and acclimation. The second type of analysis,
plotting the cos () of vectors tangential to the cell trajectory
versus path length traveled between each pair of vectors, revealed
turning magnitudes in highly persistent cells that were not obvious in the
trajectory analysis (Fig.
7).
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Keratocytes exhibited a range of turning behaviors that varied with both
experimental and acclimation temperatures. At lower experimental temperatures,
cells from all species traveled in persistently straight trajectories,
regardless of acclimation history. Keratocytes from T. bernacchii had
the greatest persistence at all experimental temperatures of any species
examined, and persistence was not affected by experimental temperature. For
all non-Antarctic species, as experimental temperature increased, persistence
in a single direction decreased and turning associated with random walk
behavior increased. Interestingly, persistent circular trajectories appear
with increasing experimental temperature in the case of keratocytes cultured
from G. mirabilis and C. salinus. G. mirabilis keratocytes
took circular paths only at experimental temperatures of 20°C.
Additionally, keratocytes from individuals acclimated to 10°C were more
likely to have circular trajectories than those acclimated to 16°C, while
fish acclimated to 25°C had the greatest number of cells with circular
trajectories. C. salinus keratocytes were less persistent at lower
experimental temperatures, and at higher experimental temperatures they
exhibited turns of larger radii compared with G. mirabilis
keratocytes, with few C. salinus cells making completely circular
paths at experimental temperatures below 35°C. Furthermore, trajectories
varied with acclimation temperature; keratocytes from cold-acclimated fish
(26°C) were less persistent with more random walks at experimental
temperatures below 20°C and showed a greater incidence of curved and
circular paths at temperatures greater than 20°C than cells from the
warm-acclimated group.
Analysis of the cos () versus path length reveals that the
turning angles between two adjacent video time-lapse intervals (path lengths
20 µm) are less sharp than the turning angles between non-adjacent
intervals spaced further apart, which shows that the turning angle increases
with increased path length [average cos (
)=0.1 to 0.9, depending on
path length]. Thermal acclimation affected the magnitude of directional
changes taken by cells of all species examined. Keratocytes from G.
mirabilis and C. salinus acclimated to 10°C and 26°C,
respectively, displayed greater turning angles than the cells from warmer
acclimations. G. mirabilis cells acclimated to 16°C took the
straightest paths at 20°C, and thermal acclimation had the strongest
effect on turning magnitude at 20°C. The turning magnitudes of keratocytes
cultured from C. salinus exhibited greater turning magnitude at
higher temperatures, and cells acclimated to 35°C took the straightest
paths at 10°C. A. percula keratocytes made larger angle turns
than the other non-Antarctic species, and their turning behavior was
statistically the same (P>0.05) at every experimental temperature.
Thermal acclimation affected turning behavior in T. bernacchii
keratocytes at path lengths greater than 2 µm, with -1.86°C-acclimated
cells making larger turns at low temperatures and 5°C-acclimated cells
making larger turns at high temperatures.
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Discussion |
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For all species except the Antarctic notothenioid T. bernacchii, rates of movement reflected compensation for temperature (Fig. 5). Thus, at common habitat temperatures, the keratocytes of G. mirabilis, C. salinus and A. percula had mean speeds of 11.4 µm min-1 (15°C), 13.8 µm min-1 (30°C) and 10.8 µm min-1 (30°C), respectively. Although we did not directly examine the process of wound healing in this study, we conjecture that conservation of a certain range of motility rates is advantageous for ensuring that the role of keratocytes in the wound-healing process is retained across adaptation temperatures. Therefore, it is likely that keratocytes moving at 11-14 µm min-1 are capable of adequate migration to be effective in wound closure for prevention of infection. Slower rates may not be as effective and more rapid locomotion might be too costly, metabolically, and not yield a sufficient increase in protective value.
Although temperature compensation of keratocyte motility was observed in comparisons among the non-Antarctic species, none of the species studied showed acclimatory temperature compensation of motility. The lack of acclimatory temperature compensation suggests that seasonal variation in wound closure behavior may exist. However, until it is known how pathogen activity varies with temperature, the potential importance, if any, of this seasonal change cannot be established.
The very low speeds recorded for keratocytes of T.
bernacchii are paradoxical in the context of the proposed importance
of conservation of wound-healing behavior. Keratocytes of the Antarctic fish
moved at rates that were less than one percent those of the other species at
their physiological temperatures. We do not interpret these extremely low
rates of movement to be an indication of a `failure' to cold-adapt, because
Antarctic notothenioids have a pronounced level of cold-adapted metabolic and
enzymatic activity in certain tissues
(Somero et al., 1968;
Kawall et al., 2002
) and there
is no a priori basis for assuming that cold adaptation is precluded
in any particular cell type. Thus, the biological significance, if any, of the
extremely slow rates of movement of the keratocytes of T. bernacchii
is not apparent. Likewise, the significance of the increased mobility of
keratocytes from 5°C-acclimated T. bernacchii is unclear. Because
5°C is several degrees above the highest temperatures that this species
experiences (and likely has experienced for 10-15 million years), the effects
of acclimation to 5°C could represent a pathological condition.
The range of temperatures over which keratocyte motility was observed and
the thermal optima of keratocyte speed reflected both evolutionary and
acclimation temperatures (Fig.
4). The ability of keratocytes of T. bernacchii to
survive 10 min of supercooling at -6°C and display apparently normal rates
of movement at 5°C after the bout of supercooling supports the observation
of DeVries and Cheng (1992)
that T. bernacchii can withstand these very low temperatures as long
as ice crystals are absent in the internal fluids and medium. The ability of
keratocytes of T. bernacchii to sustain motility at a temperature of
20°C, which is approximately 15°C above the upper lethal temperature
of this species (Somero and DeVries,
1967
; Hofmann et al.,
2000
), indicates that the mechanisms underlying acute thermal
death may involve complex physiological functions (for example, failure of
synaptic transmission) rather than the survival of individual cells
(Somero, 1996
;
Hochachka and Somero,
2002
).
In only one instance did thermal acclimation affect the functional thermal range of cultured keratocytes: cells cultured from C. salinus acclimated at 26°C did not survive at 5°C, whereas cells from fish acclimated to the warmer 35°C did. This suggests a potential thermal stress for C. salinus acclimated at 26°C, which was responsible for decreasing the range of cellular thermal tolerance.
Effects of experimental temperature were noted on the persistence and
directionality of keratocyte movement, with persistently straight trajectories
giving way to random walks and persistent circular trajectories as
experimental temperature increased. Thermal acclimation also affected turning
behavior, with cells from cold-acclimated specimens generally making turns of
greater magnitude. Although G. mirabilis, C. salinus (35°C
acclimation) and A. percula keratocytes appear very persistent at
5°C and 10°C in the path length versus displacement analysis,
the cos () analysis reveals that they are making large turns and must
be moving in an oscillatory, yet persistent, manner. One of the most
noteworthy results is that thermal acclimation does not affect how the
keratocytes' mean speeds change with experimental temperature but does
influence how the experimental temperature affects directional movement, in
terms of persistence and turning angle, in G. mirabilis and C.
salinus. Speed memory and oscillation are also affected by temperature
acclimation.
Collectively, these results indicate that there is more than one
temperature-sensitive mechanism governing cell motility. The rate-limiting
process(es) responsible for speed is distinct from the mechanism(s) underlying
directionality and persistence, and each mechanism is affected differently by
temperature. Manipulation of temperature may allow a decoupling of these
mechanisms and permit their individual study, allowing a detailed analysis of
the effects of temperature on motility within and between species to be made.
For example, the polymerization and depolymerization of cytoskeletal tubulin
subunits in Antarctic fish occur at a reduced rate compared with those in
mammals (Detrich et al., 2000).
Similar differences in orthologous biochemical systems related to keratocyte
motility may be present in species from different thermal niches. Examining
the thermal responses of such processes as actin polymerization, membrane
fluidity or binding/release of adhesion proteins may lead to insights into the
mechanisms that underlie the thermal sensitivities of keratocyte speed and
directional behavior. However, we have previously shown that there are only
small, conservative changes in the sequences of
-actins from
vertebrates adapted to habitat temperatures ranging over 42°C, and
-actins of Antarctic notothenioids exhibit no apparent adaptation at
the level of primary structure (Ream,
2002
). Therefore, temperature-dependent variation in motility is
probably not attributable to intrinsic variation between actin homologs.
One fundamental question that remains for future analysis is the interplay that may exist between speed and directional behavior in wound healing. It is possible that the variation in trajectory in response to temperature acclimation and experimental temperature has a functional significance, much as we conjectured for the conservation of speed per se, because memory and oscillation are present in a significant number of cells only at physiological temperatures.
In this study, we attempted to isolate external variables and focus solely on thermal effects; however, in situ wound healing occurs in the presence of numerous signaling molecules that are known to influence migration in other cell types. Obviously, temperature will affect not only the rate of diffusion of these molecules but also the transduction of chemical signals. Further examination of keratocyte motility during wound healing in situ may provide information on the interplay of chemical signals and/or an electrical gradient (established across the wound) on the temperature-dependent speed and directionality of these cells. Our initial descriptions of thermal effects on keratocyte function thus open up a number of avenues of study in cell biology and evolutionary physiology that can be addressed using this promising experimental system.
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