16 December 2013 Volume 103 Number 25

Applied Physics
Letters

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Cell cytoskeletal conformation under reversible thermal control
Ting-Ya Chang, Chung-Yao Yang, Kai-Wei Liao, J. Andrew Yeh, and Chao-Min Cheng

Citation: Appl. Phys. Lett. 103, 253701 (2013); doi: 10.1063/1.4840955

View online: http://dx.doi.org/10.1063/1.4840955
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 APPLIED PHYSICS LETTERS 103, 253701 (2013)

Cell cytoskeletal conformation under reversible thermal control

Ting-Ya Chang,a) Chung-Yao Yang,a) Kai-Wei Liao, J. Andrew Yeh,b) and Chao-Min Chengc)
Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu 300, Taiwan
(Received 24 September 2013; accepted 24 October 2013; published online 16 December 2013)
In order to assess the role of cytoskeletal structure in modulating cell surface topography during
cell transformation, we investigated cytoskeletal organization of Madin-Darby canine kidney
(MDCK) epithelial cells at different thermal gradients. Specifically, we examined actin
polymerization as a function of temperature in a controlled thermal environment. After applying an
increase in temperature of 5  C, we observed fewer actin filaments in the network, as these
molecular polymers depolymerized. Partial stress fibers of MDCK cells could be rearranged,
but some of them were disrupted irreversibly after a second thermal treatment, and MDCK
cells underwent apoptosis at higher temperatures as well. V C 2013 AIP Publishing LLC.

[http://dx.doi.org/10.1063/1.4840955]

This paper describes temperature-modulated cytos-
keletal conformation changes of Madin-Darby canine kidney
(MDCK) epithelial cells examined using an easy-to-build
(or easy-to-use) reversible temperature control system.
Mechanical stresses generated by internal and external forces
at the cell surface are thought to play a major role in the reg-
ulation of cell growth and behavior.1 These forces are
applied to adhesive contacts formed between the cell and the
extracellular matrix or between neighboring cells, causing
them to migrate, spread, maintain tissue shape, or to resist
the shear forces of blood flow.2–5 Filamentous actin
(F-actin), a major component of cortical cytoplasm and focal
adhesions, is believed to play a key role in the mechanical
response of cells.6–8 The cytoskeletal system plays a role in
the regulation of cell surface functions such as adhesion to a
substrate and receptor mobility.9,10 The lateral movement of
receptors within the plasma membrane and the conveyance
of information to the nucleus are thought to be regulated by
components of the cytoskeletal system, collectively termed
surface-modulating assemblies.11,12 Evidence for such a rela-
tionship has been observed in the capping of lectin by anti-
body receptors on cell surfaces.13,14
Actin filaments can grow or shrink, and consequently
exert forces on surrounding objects by polymerizing actin in
their host cell. In vivo, the dynamic rearrangements of actin are
controlled by a myriad of helper proteins that nucleate, cross-
link, and cap-growing actin filaments.15 This polymerization
process in vitro is strongly influenced by environmental varia-
bles such as pH, temperature, and ionic strength. For example,
individual actin monomers (globular actin or G-actin) poly-
merize completely in the presence of potassium or magnesium
ions to form a double stranded chain of actin (F-actin)
in vitro.16 Two simple approaches for controlling actin poly-
merization include the manipulation of ion concentration or
temperature. Heat shock in living eukaryotic cells is noticeable
when the environmental temperature is 3–8  C above normal.
Hyperthermia (heat shock) induces physiological changes in
the morphology of the nucleoli, cytoplasmic organelles, and
a)
Ting-Ya Chang and Chung-Yao Yang equally contributed to this work.
b)
E-mail: jayeh@mx.nthu.edu.tw
c)
E-mail: chaomin@mx.nthu.edu.tw
the cytoskeleton.17 Responses to hyperthermia are generally
similar in different eukaryotic cell types, however, there is no
information in the literature regarding the similarity of cyto-
skeleton responses to heat shock across cell types.
One physics-based perspective regarding cell structure
considers the cytoskeleton network as a polymer network,18
which can exhibit binding energies on the order of a few
kBT.19 We could hypothesize that the thermal environment
directly affect cellular behaviors even without the signaling
cascades that can be activated by heat shock. In a previous
study,20 we investigated thermally induced changes in the
cytoskeletal conformation of NIH-3T3 fibroblasts. In this
previous work, we found that the cytoskeleton approaches
original polymerized organization following heat shock, but
did not completely return to the original state, (i.e., only a
partial reversal was observed). Unlike the epithelial cells lin-
ing the body structures, fibroblasts are not restricted by a
polarizing attachment to a basal lamina on one side. The
migration over substratum between fibroblasts and epithelial
cells is also different, either as individual cells or as cell
sheets.21 In addition, the physiological environment of epi-
thelial cells (e.g., skin, lung) is different that the environment
for fibroblasts, which are often deep within the body.
Epithelial cells, then, would naturally be exposed to substan-
tially more heat shock by the physical nature of their posi-
tioning. For example, either cold weather or hot drink (or
other external thermal stimulations) exposes epithelial cells
to considerable temperature variance, while fibroblasts are
shielded from major temperature deviations, and typically
stay near 37  C. We have attempted to garner physiological
meaning and purpose of both cell types by comparing cytos-
keletal conformations of both epithelial cells and fibroblasts
as they undergo thermal stimulation. Herein, we expand
upon our previous study by using MDCK epithelial cells
instead of NIH 3T3 fibroblasts. The physiological difference
between these cells is critical, as epithelial cells serve a very
different role in vivo. Our goal here is to gather deeper into
cytoskeletal organization following various heat shock
cycles for different cell types and relate their comparative
differences to possible physiological roles.
MDCK cells were maintained in 90% Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum

0003-6951/2013/103(25)/253701/5/$30.00 103, 253701-1 C 2013 AIP Publishing LLC

V
253701-2 Chang et al.
(FBS) and 100 U/ml penicillin/streptomyocin at 37  C
in a humidified, 5% CO2 incubator. We used 0.25%
trypsin-ethylenediaminetetraacetic acid (EDTA) to enzy-
matically separate the cells for plating on coverslips for
24 h (control group) at a density of 1  104 cells/ml for the
following heat shock protocol. Our process involved
increasing the temperature by 5  C and applying this for 2 h
before reversing the process and lowering the temperature
back to 37  C. Using this specific time-temperature profile,
we were able to induce remodeling of actin filaments. After
24 h of culturing at 37  C, the cells were transferred into a
43  C humidified incubator with 5% CO2 and incubated for
another 2 h at 43  C. Subsequently, we recovered the heat
shock-treated cells at 37  C in a humidified 5% CO2 incuba-
tor for 24 h and repeated the above heat shock cycle (i.e.,
2 h in 43  C incubator). In this experiment, we collected the
cells at various time points and examined the degree of po-
lymerization and depolymerization of F-actin bundles and
the ratio of F-actin and G-actin during alternate temperature
changes. The MDCK cells were cultured in various temper-
ature conditions (e.g., 24 h at 37  C; 24 h at 37  C/2 h at
43  C; 24 h at 37  C/2 h at 43  C/24 h at 37  C; 24 h at
37  C/2 h at 43  C/24 h at 37  C/2 h at 43  C) for cytos-
keletal observation. After temperature treatment, we imme-
diately fixed the cells in a 3.7% formaldehyde solution in
phosphate-buffered saline (PBS) for 13 min at 37  C, and
permeabilized them with 0.3% Triton-X-100 in PBS for
Appl. Phys. Lett. 103, 253701 (2013)
13 min at room temperature. F-actin and G-actin were la-
R
beled with 6 lM Alexa FlourV488 phalloidin (Invitrogen,
Carlsbad, CA, USA) and 0.3 lM Deoxyribonuclease
(DNase; Invitrogen, Carlsbad, CA, USA), respectively. The
nucleus was stained with 40 ,6-diamidino-2-phenylindole
(DAPI; Invitrogen, Carlsbad, CA, USA). Cells were
mounted with fluoromount-G on glass coverslips. Cells
were observed under an epi-fluorescent microscope (Zeiss
Axio Observer, Germany) equipped with a 63 (NA ¼ 1.4)
oil immersion objective and a 10 eyepiece. These studies
suggest that the whole cell as well as subcellular regions
have distinct patterns that retain a reversible equilibrium
directly related to the thermal environment.
Figure 1(a) displays the fluorescence images of MDCK
cells labelled with F-actin (green) and G-actin (red) after cul-
ture in a 37  C incubator for 24 h. The results indicate that
MDCK cells showed F-actin stress fibers within the plasma
membrane. Stress fibers form when a cell makes stable con-
nections to a substrate. To more accurately quantify their
appearance from an imaging perspective, we analyzed the
intensities of individual actin filaments from a spatial distri-
bution perspective.22 We quantified these actin filaments by
examining the fluorescent intensity peaks in the plasma
R
membrane after staining with Alexa FlourV488 phalloidin
(Fig. 1). Examining the intensity profiles through the line
scans from the images allowed us to determine the presence
and absence of F-actin at different temperature. In
FIG. 1. Epi-fluorescence and convo-
lute images of MDCK cells labelled
with F-actin (green), G-actin (red), and
nuclei (blue) at various conditions.
Focusing on a region in which we were
interested in the images (for these
cells); the presence of the F-actin was
quantitatively examined. By rotating
the line regions (once again, we were
interested in) at defined angles, we
compared the plots for a correlation of
the peaks that aligned in a linear form;
if these peaks aligned, then a correla-
tion was made for the presence of the
F-actin. The three horizontal lines
(a, b, c) were analyzed at other angles
as described previously (e.g., by rotat-
ing the line regions). (a) The appear-
ance of these aligned peaks (1, 2, 3)
was markedly showed at 37  C. (b)
The aligned peaks were reduced or dis-
appeared for the cells with increased
temperature to 43  C. (c) The appear-
ance of aligned peaks (1, 2, 3, 4, 5, 6,
7) were distinctly showed for the cells
with reversed temperature controlled
system to 37  C. The intensity analysis
of these images was processed with
deconvolution filtering in ImageJ
(from National Institutes of Health).
253701-3 Chang et al.
Figure 1(a), the continuous intensity peaks at three different
locations (1, 2, and 3) indicate that there were three polymer-
ized F-actins at the plasma membrane (the location we meas-
ured). Compared to cells cultured in a 37  C incubator, cells
receiving heat shock treatment at 43  C produced depolymer-
ization of F-actin fibers within MDCK cells (discontinuous
intensity peaks in Fig. 1(b)). The extracellular thermal sur-
rounding environment affected intracellular cytoskeletal dy-
namics and changed cellular morphology and behavior. The
heat effect was sufficient to destroy the stress fibers made
out of F-actin in the plasma membrane, and depolymerized
F-actin into G-actin. Consequently, compared to the control
(cells cultured at 37  C), heat shocking MDCK cells reduced
the level of F-actin, and the raised the level of G-actin. To
examine reversibility, the cells cultured at 43  C for 2 h were
transferred to a 37  C incubator for another 24 h (for recov-
ery). After recovery for 24 h, the G-actin again polymerized
into F-actin and we observed stress fibers in the plasma
membrane (five continuous peaks in Fig. 1(c)). This implies
that, even after 2 h of heat shock, cells do not die and can
remain viable, and rebuild the actin cytoskeleton at 37  C. It
Appl. Phys. Lett. 103, 253701 (2013)
is important to note that the cytoskeletal response was re-
versible after 24 h of recovery at 37  C due to the high quan-
tity of F-actin.
In order to gather greater insight into the effect of heat
on cells, we transferred already heat-shocked (and recov-
ered) MDCK cells to a 43  C incubator for another 2 h (sec-
ond heat shock treatment; Fig. 2). Intriguingly, some of the
MDCK cells [Figs. 2(a) and 2(b) (a, b, c)] displayed F-actin
stress fibers following this second heat shock treatment
(63.6% 6 9.1%, N ¼ 2, n ¼ 11; data are mean 6 standard
deviation). This indicates that MDCK actin filaments are
irreversibly changed following prolonged or cyclic heat
treatment, and partial stress fibers can be rearranged, but
some of them are disrupted permanently. After the second
heat shock treatment, the MDCK cells were transferred to a
37  C incubator for another 24 h (for recovery). It is well
documented that mammalian cells subjected to cyclic me-
chanical strain transmit this mechanical force into intracellu-
lar signals and induce cellular responses, and may suppress
apoptosis as well.23–25 Li et al. found that cell viability was
not significantly affected by tensile strain at any of the time
FIG. 2. Epi-fluorescence and convo-
lute images of MDCK cells labelled
with F-actin (green), G-actin (red), and
nuclei (blue) after second heat shock
treatment (43  C). The appearance of
these aligned peaks (1, 2, 3, 4) was dis-
tinctly showed at (a) and lines [a, b, c]
in (b). The aligned peaks were reduced
or disappeared at lines [d, e, f] in (b)
and (c). Partial stress fibers of MDCK
cells can be rearranged, but some of
them are disrupted permanently. The
intensity analysis of these images was
processed with deconvolution filtering
in ImageJ (from the National Institutes
of Health).
253701-4 Chang et al.
FIG. 3. Phase contrast images combined with fluorescence images of
MDCK cells cultured on glass coverslips with thermal gradient distribution
at (a) 37  C and (b) 43  C. The apoptotic cells were labelled with Annexin
V-FITC (green) and DAPI (blue). (c) Apoptotic cell ratio on glass slide with
thermal gradient (Fig. S1) at 37  C and 43  C after 2 h culture. Data are
mean 6 standard deviation (N ¼ 3, n ¼ 3), ***p < 0.001, indicating statisti-
cally significant differences between 37  C and 43  C.
points when compared with the unstrained cells.26 These
results indicated that the cell proliferation would be
enhanced by cyclic mechanical stimulation. However, in our
experimental process, the amount of live cells on the cover-
slips and the rate of proliferation decreased. We further
found that cell number was decreased after second heat
shock treatment, which indicated that MDCK cells under-
went progressive apoptosis via thermal effect (versus cyclic
mechanical stimulation—different in vitro cyclic physical-
based stimuli on individual cells). In addition, we created a
thermal gradient to probe the effects of localized thermal
stimulation on such cell behaviors (i.e., apoptosis in this
study) for regional polymerization and depolymerization of
the actin filaments in a single cell. To understand whether
this was a global or local response, we created a thermal
setup (Supplemental Fig. S1)27 to control the spatial distribu-
tion of thermal environment in liquid, and examined the spe-
cific areas where thermal transitions were most prominent.
Figure 3 displays the fluorescence images of MDCK cells
labeled with DAPI and Annexin V-Fluorescein isothiocya-
nate (FITC) to study cell apoptosis ratio at normal tempera-
tures (37  C) and in our heat shock temperature (43  C)
throughout thermal gradient distribution (Supplemental Fig.
S1).27 Approximately 50% of the cells could survive at the
higher temperature regions (43  C), compared to 85% cell
survival at lower temperature regions (37  C; N ¼ 3, n ¼ 3;
data are mean 6 standard deviation), which indicates cell ap-
optosis is triggered by external signals (i.e., heat shock treat-
ment). It is noted that the cell apoptosis ratio (15%) at the
lower temperature region (37  C region in a 43  C incubator)
was relatively higher than at a normal culture conditions
Appl. Phys. Lett. 103, 253701 (2013)
(<5% at a 37  C incubator). Any disruption in the cytoskele-
ton can lead to adverse cellular effects, and it has been found
that the cytoskeleton is one of the earliest and most sensitive
targets of stress.28 The effects of stress on the cytoskeleton
are important in maintaining proper neural function. At high
temperatures, the lipid bilayer of cell membranes can disso-
ciate, changing membrane potentials, thus inducing many
possible changes in cellular function resulting from the loss
of proteins critical for maintaining bilayer integrity.29
In summary, we developed an easy-to-handle and ro-
bust, reversible thermal system that is capable of controlling
the spatial distribution of thermal environment in liquid.
This allowed us to examine specific areas where thermal
transitions were most prominent for influencing cell cytos-
keletal conformation. In this study, we found that cytos-
keletal conformation of MDCK cells is influenced by
thermal effects. We observed fewer actin filaments in the
plasma membrane after applying an increase in temperature
of 5  C, as these molecular polymers depolymerized. The
actin filaments repolymerized when we reversed the sur-
rounding environment and lowered the temperature back to
37  C, indicating a reversible thermal process for these mo-
lecular polymers. However, following an additional, second
thermal treatment, partial stress fibers of MDCK cells could
be rearranged to the point of permanence. This indicates that
MDCK actin filaments and viability as indicated by greater
apoptosis are altered following repeated heat exposure (com-
pared to cyclic mechanical stimulation).We believe these
current results provide insights applicable to a wide range of
fields including cytoskeletal dynamics in biophysics,
molecular-level polymer responses in polymer physics, and
biomaterial responses in materials and applied physics.
We would like to thank the National Science Council of
Taiwan for financially supporting this research under
Contract No. NSC 101-2623-E-007-005-ET (to J. A. Yeh),
NSC 101-2628-E-007-011-MY3, NSC 102-2221-E-007-031
(to C.-M. Cheng), and the Grant for Interactive
Nano/MicroElectroMechanical Components and Systems
from National Tsing Hua University, Taiwan (to J. A. Yeh
and C.-M. Cheng).
1
L. P. Cramer, T. T. Mitchison, and J. A. Theriot, Curr. Opin. Cell Biol. 6,
82 (1994).
2
K. M. Yamada and S. Miyamoto, Curr. Opin. Cell Biol. 7, 681 (1995).
3
K. Konstantopoulos and L. V. McIntire, J. Clin. Invest. 98, 2661 (1996).
4
R. P. Johnson and S. W. Craig, Curr. Opin. Cell Biol. 8, 74 (1996).
5
S. Huang and D. E. Ingber, Nat. Cell Biol. 1, E131 (1999).
6
J. H.-C. Wang, J. Theor. Biol. 202, 33 (2000).
7
C.-M. Cheng, R. L. Steward, Jr., and P. R. LeDuc, J. Biomech. 42, 187
(2009).
8
R. M. Bellin, J. D. Kubicek, M. J. Frigault, A. J. Kamien, R. L. Steward,
Jr., H. M. vBarnes, M. B. DiGiacomo, L. J. Duncan, C. K. Edgerly, E. M.
Morse, C. Y. Park, J. J. Fredberg, C.-M. Cheng, and P. R. LeDuc, Proc.
9
Natl. Acad. Sci. USA 106, 22102 (2009).
S. Kim, K. Shilagardi, S. Zhang, S. N. Hong, K. L. Sens, J. Bo, G. A.
Gonzalez, and E. H. Chen, Dev. Cell 12, 571 (2007).
10
L. Venticinque, K. V. Jamieson, and D. Meruelo, PLOS One 6, e15895
(2011).
11
G. M. Edelman, Science 192, 218 (1976).
12
D. A. McClain, P. D’Eustachio and G. M. Edelman, Proc. Natl. Acad. Sci.
USA 74, 666 (1977).
13
R. L. Hoover, D. K. Bhalla, S. Yanovich, M. Inbar, and M. J. Karnovsky,
J. Cell Physiol. 103, 399 (1980).
253701-5 Chang et al. Appl. Phys. Lett. 103, 253701 (2013)

14 22
J. Glick, Z. Malik, and N. Garber, Microbios 32, 181 (1981). Q. Yi and M. G. Coppolino, BioTechniques 40, 745 (2006).
15 23
P. Sheterline, J. Clayton, and J. C. Sparrow, Actin (Oxford Universirty M. Haga, A. Chen, D. Gortler, A. Dardik, and B. E. Sumpio, Endothelium
Press, Oxford, U.K., 1999). 10, 149 (2003).
16 24
M. Hazar, R. L. Steward, Jr., C.-J. Chang, C. J. Orndoff, Y. Zeng, M.-S. T. Lee and B. E. Sumpio, Biotechnol. Appl. Biochem. 39, 129 (2004).
25
Ho, P. R. LeDuc, and C.-M. Cheng, Appl. Phys. Lett. 99, 233701 (2011). K. Nishimura, W. Li, Y. Hoshino, T. Kadohama, H. Asada, S. Ohgi, and
17
W. J. Welch and J. P. Suhan, J. Cell Biol. 101, 1198 (1985). B. E. Sumpio, Am. J. Physiol. Cell Physiol. 290, C812 (2005).
18 26
B. Wagner, R. Tharmann, I. Haase, M. Fischer, and A. R. Bausch, Proc. S. Li, X. Jia, V. C. Duance, and E. J. Blain, Eur. Cell Mater. 21, 508 (2011).
27
Natl. Acad. Sci. USA 103, 13974 (2006). See supplementary material at http://dx.doi.org/10.1063/1.4840955 for
19
A. R. Bausch and K. Kroy, Nat. Phys. 2, 231 (2006). thermal gradient simulation.
20 28
C.-M. Cheng and P. R. LeDuc, Adv. Mater. 20, 953 (2008). E. J. Luna and A. L. Hitt, Science 258, 955 (1992).
21 29
D. W. Hamilton and D. M. Brunette, Exp. Cell Res. 309, 429 (2005). J. E. Smith, Vet. Pathol. 24, 471 (1987).