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Is cytoskeletal tension a major determinant of
cell deformability in adherent endothelial cells?
JACOB POURATI,1 ANDREW MANIOTIS,2 DAVID SPIEGEL,1 JONATHAN L. SCHAFFER,3
JAMES P. BUTLER,1 JEFFREY J. FREDBERG,1 DONALD E. INGBER,2
DIMITRIJIE STAMENOVIC,4 AND NING WANG1
1Physiology Program, Harvard School of Public Health and 2Departments of Pathology
and Surgery, Children’s Hospital and Harvard Medical School, Boston 02115; and
3Departments of Orthopedic Surgery, Brigham and Women’s Hospital, Children’s Hospital,
and Harvard Medical School and 4Department of Biomedical Engineering,
Boston University, Boston, Massachusetts 02215
major determinant of cell deformability: the higher the
initial tension, the stiffer the cell would be. Although it
has long been known that several cell types are under
tension (1, 2, 10, 16), it has not been shown that this
tension plays a role in regulating cell deformability.
The main goal of this study was to show that the CSK
tension influences cellular resistance to shape distortion in a stretch-dependent manner in adherent endothelial cells.
We tested the hypothesis in two parts. First, we
confirmed the presence of initial tension in living
adherent endothelial cells by rapidly cutting them with
a microneedle or by dislodging focal adhesions. The
rationale was that if the CSK is initially tensed, then
the cell would rapidly retract after the cut, as would a
tensed violin string. We found that the cell did retract
rapidly after cutting. Second, we assessed, indirectly,
the effects of changes in CSK initial tension on CSK
stiffness. To do this, we modified the stretchable membrane system of Schaffer et al. (23) so that it could fit
into a magnetic twisting cytometry (MTC) device. Our
rationale was that a rapid uniform distension of the
substrate to which the cell is adherent would increase
the CSK distension and thus increase the tension in the
CSK lattice. The hypothesis predicts that this should
manifest itself by an immediate increase in CSK stiffness. We found that, after a rapid stretch, the cells did
exhibit a stretch-dependent increase in CSK stiffness.
This finding is consistent with the notion that the CSK
is initially tensed and that this tension is a major
determinant of cell deformability.
mechanical tension; shape stability; cell adhesion; shear
deformation; stiffness
Cell cutting. Endothelial cells were plated sparsely in
serum-free medium on coverslips coated with high densities
of fibronectin (500 ng/well) permissive to sustained cell
attachment for 4 h. A coverslip was then placed into a
35-mm-diameter petri dish containing fresh medium. A layer
of mineral oil was layered over the medium to maintain pH.
Then the petri dish was placed on an Omega RTD 0.1°-stable
stage heating ring coupled to a Nikon Diapot inverted microscope. Images were obtained with a Citron videocamera and
recorded on a GYYRE video recorder. Microneedles were
pulled with a Sutter micropipette puller, adjusted to produce
long tips of ,1- to 5-µm diameter, with a length of 40–100 µm.
To determine whether these cells carry an initial tension,
they were cut by a microneedle across the cytoplasm. The
ensuing change of cell shape was quantitated.
Cell-stretching system. A schematic diagram of the cellstretching system is shown in Fig. 1. A 76-µm-thick mem-
CELL DEFORMABILITY and shape control are important in
cell spreading, migration, growth, and apoptosis (3, 7,
15, 17, 26), but the mechanisms by which adherent cells
regulate their deformability and shape are not well
understood. In our previous studies, we have postulated that mechanical tension of the cytoskeleton (CSK)
is a basic determinant of cell shape and function in
adherent cells (12–14, 18, 27, 29). According to this
hypothesis, the level of preexisting mechanical tension
(or initial tension, defined as tension residing in CSK
before mechanical measurements) is predicted to be a
MATERIALS AND METHODS
0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society
C1283
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Pourati, Jacob, Andrew Maniotis, David Spiegel,
Jonathan L. Schaffer, James P. Butler, Jeffrey J. Fredberg, Donald E. Ingber, Dimitrijie Stamenovic, and
Ning Wang. Is cytoskeletal tension a major determinant of
cell deformability in adherent endothelial cells? Am. J. Physiol.
274 (Cell Physiol. 43): C1283–C1289, 1998.—We tested the
hypothesis that mechanical tension in the cytoskeleton (CSK)
is a major determinant of cell deformability. To confirm that
tension was present in adherent endothelial cells, we either
cut or detached them from their basal surface by a microneedle. After cutting or detachment, the cells rapidly
retracted. This retraction was prevented, however, if the CSK
actin lattice was disrupted by cytochalasin D (Cyto D). These
results confirmed that there was preexisting CSK tension in
these cells and that the actin lattice was a primary stressbearing component of the CSK. Second, to determine the
extent to which that preexisting CSK tension could alter cell
deformability, we developed a stretchable cell culture membrane system to impose a rapid mechanical distension (and
presumably a rapid increase in CSK tension) on adherent
endothelial cells. Altered cell deformability was quantitated
as the shear stiffness measured by magnetic twisting cytometry. When membrane strain increased 2.5 or 5%, the cell
stiffness increased 15 and 30%, respectively. Disruption of
actin lattice with Cyto D abolished this stretch-induced
increase in stiffness, demonstrating that the increased stiffness depended on the integrity of the actin CSK. Permeabilizing the cells with saponin and washing away ATP and Ca21
did not inhibit the stretch-induced stiffening of the cell. These
results suggest that the stretch-induced stiffening was primarily due to the direct mechanical changes in the forces distending the CSK but not to ATP- or Ca21-dependent processes.
Taken together, these results suggest preexisting CSK tension is a major determinant of cell deformability in adherent
endothelial cells.
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CYTOSKELETAL INITIAL TENSION
Fig. 1. Schematic of cell-stretching system. A piece of elastic membrane (76-µm-thick silicone elastomer, Dow Corning) was prestretched
and clamped onto a bottomless well of a 96-well plate with a piece of
3-ml syringe. A platen was placed into a plastic vial, and membrane
well was placed on top of platen. A threaded rod was screwed down
to push membrane well downward through a spacer. Because platen
was stationary, downward movement of membrane well results
in upward stretching of membrane on which cells are attached.
Whole stretching system was placed into magnetic twisting cytometer.
between the rotation of the threaded platen against a platform (i.e., upward movement of the platen) and the strain of
the membrane (Fig. 2). To further determine whether the
stretching of the membrane was uniform, strains in two
orthogonal directions (X and Y) were measured. We found
that the membrane stretching was uniform up to 5% strain of
the membrane with a diameter of 4.4 mm (Fig. 3). The Young’s
modulus of the membrane was found to be 2.7 3 108 dyn/cm2.
There was no breakage, leakage, or buckling of the membrane
after repeated stretches.
Cell cultures for stretching. Bovine capillary endothelial
cells were cultured to confluence, serum deprived, trypsin-
brane of special formulation silicone elastomer (Dow Corning,
Midland, MI) was tightly clamped onto a bottomless 96-well
plate (6 mm ID) by pushing a clamp over the well to
prestretch the membrane. A 4.4-mm-diameter platen was
placed at the bottom of a plastic vial, and the membrane well
was placed above the platen. A threaded rotating shaft was
fixed at the top of the vial by two plastic plugs. A threaded rod
was screwed into the shaft. Turning the rod advanced it
downward against the well. A spacer was placed on the top of
the well to transmit the downward movement of the rod to the
membrane. The top of the platen was shaped so that only the
edge ring came into contact with the membrane. To minimize
friction between the platen and the membrane, a small
amount of glycerin was applied to the platen and to the
membrane. Because the platen was rigid and stationary, this
action stretched the membrane in a controlled fashion.
Calibration of cell-stretching system. Dots were drawn on
the prestretched membrane with a fine-tipped pen. The
positions of dots at different states of stretching were observed with a dissecting microscope and recorded with a
digital charge-coupled device camera connected to a computer
with Photometrics graphics software. Stretch was calculated
as the ratio of the postdisplaced dot relative positions to the
predisplaced relative dot positions (23). Strain was defined as
stretch minus 1. There was a positive, but nonlinear relation
Fig. 3. Calibration of biaxial strain of membrane during stretching.
Fine dots were drawn in black ink in 2 mutually orthogonal directions (X and Y). Positions of dots before and after stretching
membrane were recorded with a digital camera connected to a 386
Gateway computer and Photometrics graphics software. X and Y
strains represent dots close to edge of flat surface of membrane. Dots
close to center of membrane displayed similar results (not shown).
Means 6 SE; n 5 4 wells.
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Fig. 2. Calibration of rotation of platen (upward movement of platen)
and actual strains of membrane. Strain is defined as stretch minus 1.
Stretch is defined as ratio of distance between 2 dots after distension
to distance between same 2 dots before distension. p, Strain measured in X direction; j, strain measured in Y direction. Means 6 SE;
n 5 4 wells.
CYTOSKELETAL INITIAL TENSION
RESULTS
Initial tension is present in living adherent cells. To
confirm whether living adherent endothelial cells carry
initial tension, we observed shape changes after a cut
by a microneedle attached to a micromanipulator. We
reasoned that initial tension in the CSK, if any, must be
in static mechanical equilibrium (9), but when the cell
is cut, the static equilibrium is upset and a rapid
deformation must ensue. The results showed that the
initial separation between two parts of the cell increased rapidly after the cut, like a recoil of an elastic
material. The rapid retraction period generally lasted
,10 s, followed by a slow retraction period that occurred over the course of minutes. However, both the
fast and the slow phase of the retractions were completely prevented when the cell was pretreated with
cytochalasin D (Cyto D, 1 µg/ml) for 30 min (Fig. 4A).
The rapid retraction might be attributable to the
sudden release of the initial tension and the ensuing
passive mechanical creep of the associated mechanical
structures; however, an alternative explanation is that
displacements observed after the cut were an active
response to cell injury. To minimize cell injury, we used
a method that was developed by Albrecht-Buehler (1).
We placed a microneedle underneath the basal surface
of the cell and rapidly dislodged focal adhesions under
long processes extending from the cell body. As in the
cutting experiment, we observed that the long exten-
sions retracted rapidly (,10 s) toward the cell center
(n 5 20 cells) when the focal adhesion was dislodged.
This retraction was inhibited by pretreatment with
Cyto D (1 µg/ml for 30 min; n 5 20 cells; Fig. 4B).
Mechanical distension alters cell stiffness. Increasing
the distension of the membrane substrate increased
cell stiffness: 2.5% membrane strain resulted in ,15%
increase in the stiffness (P , 0.05), and 5% membrane
strain resulted in ,30% increase in the stiffness (P ,
0.05 compared with 2.5% strain; P , 0.01 compared
with control; Fig. 5). Stretching the cells and holding
the stretch for 3 min at 5% strain increased the
stiffness by another 10% (data not shown).
To confirm that the CSK actin lattice contributed to
the observed stretch-induced stiffening, adherent cells
were stretched before and after addition of Cyto D,
which disrupts the actin lattice. Addition of Cyto D (1
µg/ml for 30 min) resulted in a 40% reduction in
stiffness from the control (Fig. 6). Cyto D also completely prevented the effects of the stretch on cells.
These data demonstrate that the stretch-induced stiffening response required the presence of the microfilament lattice.
To determine whether the stiffening response depended on membrane integrity, ATP, or Ca21, the
stiffness was measured when the cells were permeabilized with saponin (25 µg/ml) for 8 min; intracellular
ATP and Ca21 were then clamped at zero. In intact
cells, a 5% stretch increased the stiffness by ,30% (P ,
0.005). Addition of saponin in the absence of stretch
increased the stiffness by 5% (P , 0.01), consistent with
our earlier results (30). A 5% stretch in the presence of
saponin resulted in a 25% increase in stiffness (P ,
0.05). There was no significant difference in stiffness in
the absence or presence of saponin for stretched cells
(Fig. 7). Furthermore, a 5% strain in cells pretreated
with the inhibitor of oxidative metabolism 2,4-dinitrophenol (DNP, 1 mM for 15 min) still induced .20%
increase in stiffness (Fig. 8), demonstrating that DNP
had no effect in inhibiting the stiffening response.
Therefore stretch-induced increases in cell stiffness
appear to be not dependent on chemical changes but
dependent on mechanical changes.
DISCUSSION
The most significant finding of this study is that a
rapid stretch of adherent endothelial cells resulted in a
prompt increase in CSK stiffness. In addition, the cell
cutting and dislodging results confirmed earlier findings that adherent cells are initially tensed. Both responses were inhibited by disruption of the actin lattice,
suggesting that the presence of an intact actin lattice
is required for stress transmission throughout the cell.
Cell cutting might cause cell injury that could lead to
release of molecules, such as Ca21, which in turn might
induce cell retraction. However, we also observed cell
retraction when long processes of the cell were dislodged from the substrate. This detachment technique
probably caused much less cell injury but yielded
essentially equivalent findings. Furthermore, cell retraction after the cut or detachment was completely
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ized, and plated in defined medium overnight on membrane
dwells that were precoated with human serum fibronectin
(Cappel) at 2 µg/well (30). To ensure that cells were plated
only onto the part of the membrane which was uniformly
stretched (4.4-mm diam), a rubber tube of 4.4 mm ID was
inserted onto the well just before the cells were plated at
20,000/well. This rubber dam was removed before twisting
experiments. The cells were plated 4–10 h and were subconfluent during the whole experiments.
In studies analyzing the role of membrane integrity and
ATP-dependent biochemical processes, cells were permeabilized with saponin as previously described (25, 30). Briefly,
cells were cultured overnight onto the membrane well. They
were washed once in a CSK stabilization buffer (50 mM KCl,
10 mM imidazole, 1 mM EGTA, 1 mM MgSO4, 0.5 mM
dithioreitol, 5 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl
fluoride, and 20 mM PIPES, pH 6.5). Cells were then incubated in the same buffer containing saponin (25 µg/ml; Sigma,
St. Louis, MO) for 8 min at 37°C, and mechanical properties
were measured before and after stretching the membrane.
MTC. The mechanical properties were quantitated using
MTC as described previously (29–31). Ferromagnetic beads
(4.5-µm diam, provided by Dr. W. Moller, Germany) were
coated with Arg-Gly-Asp peptides, which bind specifically to
integrin receptors. These beads were added to each membrane well at 20 µg/well (avg 2 beads/cell) for 15 min. The well
was then washed once with 1% BSA-DMEM to remove
unbound beads. An initial magnetic stress (torque/bead volume) of 60 dyn/cm2 was applied to the cells through the beads
and held for 60 s. Corresponding changes in the angular
strain (a form of shear strain) of the beads were measured.
Stiffness was defined as the ratio of shear stress to shear
strain. The well membrane was then rapidly stretched for 10
s, the same torque was applied, and the mechanical measurements were repeated.
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CYTOSKELETAL INITIAL TENSION
prevented with Cyto D pretreatment, which might not
inhibit Ca21 release. Although we cannot entirely rule
out other interpretations, the results presented here
are consistent with the interpretation that preexisting
tension was present in the living adherent endothelial
cells that we studied.
Despite the fact that the membrane was stretched in
a short time interval (,10 s) and stiffness was measured within 70 s, there remain the possibilities that
the CSK might have remodeled in response to stretch
and affected CSK stiffness. For instance, intracellular
K1 and Ca21 have been shown to be activated within
seconds after mechanical deformation (5). Other intracellular responses, such as transient elevation of inositol lipids, could also happen on the order of 30 s.
Although we cannot exclude these possibilities, we
performed tests that showed that stretch-induced stiffening occurred in ATP- and Ca21-free permeabilized
cells (Fig. 7). Furthermore, stretch-induced stiffening
also persisted in intact cells in which oxidative metabolism was inhibited. In addition, this stretch-induced
stiffening was prevented after cells were treated with
Cyto D, demonstrating that this response was dependent on the presence of intact actin lattice. Therefore,
although other mechanisms cannot be ruled out, we
favor the interpretation that stretch-induced stiffening
response was primarily due to increase in the distending forces within the CSK.
These findings extend previous studies showing that
initial tension may play an important role in regulating
cell deformability (i.e., cell shear stiffness). For example, it has been shown that highly spread endothelial cells are stiffer than less spread cells (30, 31), but
there may be many processes besides CSK tension,
such as actin polymerization and CSK remodeling,
which could have influenced CSK stiffness. In contrast,
the study presented here minimized the effects of these
processes. In another study, CSK tension in airway
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Fig. 4. Cell retraction after cutting or detachment. A: cell cutting. Column 1, intact living adherent endothelial cells
undergoing complete cutting; column 2, intact living cells undergoing partial cutting; column 3, living cells
pretreated with cytochalasin D (Cyto D, 1 µg/ml for 30 min) undergoing complete cutting (A, before cut; B, right
after cut, time 0; C, 18 s after cut for column 1, 4 s after cut for column 2, 81 s after cut for column 3; D, 30 s after cut
for column 1, 24 s after cut for column 2, 105 s after cut for column 3). Partial cut in column 2 was a small slit
initially (B, arrow) but enlarged with time (C and D). Note that for Cyto D-treated cells (column 3), there was no
apparent retraction after cut. Several dozen other cells showed similar results after cutting. B: cell retraction after
cells were detached from basal surface with or without Cyto D pretreatment. Initial, initial cell length measured
from a fixed point on cell body to tip of long process to be detached; After, cell length measured between same 2
points on cell, ,10 s after detachment; Cyto D Initial, initial cell length pretreated with Cyto D for 30 min (1 µg/ml);
Cyto D After, ,10 s after detachment (in presence of Cyto D). Means 6 SE; n 5 20 cells.
CYTOSKELETAL INITIAL TENSION
Fig. 6. Effects of microfilament lattice disruption on stretch-induced
response. Stiffness was measured in adherent endothelial cells; same
cells were then rapidly stretched at 5% for 10 s, and stiffness was
measured again. Cyto D (1 µg/ml for 30 min) was added to same cells
to disrupt microfilament lattice and stiffness was measured again
before and after another 5% strain (S). It appears that disruption of
microfilament lattice abolished stretch-induced stiffening response.
Means 6 SE; n 5 4 wells. Two other independent experiments
showed similar results.
Fig. 7. Effects of membrane permeabilization on stretch-induced
response. Adherent endothelial cells were stretched at 5% strain and
stiffness was measured before and after stretch. Then saponin (25
µg/ml for 8 min) was added to same cells to permeabilize cells, and
ATP and Ca21 were washed away. Same cells were twisted again
before and after permeabilization. Note that removal of ATP and Ca21
did not have any significant effects on stretch-induced stiffening
response. Means 6 SE; n 5 6 wells. An independent experiment
showed similar results.
Fig. 8. Effects of oxidative metabolism inhibition on stretch-induced
response. Adherent endothelial cells were treated with 2,4-dinitrophenol (1 mM) for 15 min before experiments. A rapid 5% stretch was
applied to cells and stiffness was measured. Means 6 SE; n 5 8 wells.
An independent experiment showed similar results.
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Fig. 5. Stretch-induced stiffening depends on degree of stretching.
Endothelial cells were plated on membrane wells overnight in
defined medium in absence of growth factors or serum. Arg-Gly-Aspcoated beads were bound to adherent, spread, and subconfluent cells
for 15 min and unbound beads were washed away. An initial stress of
60 dyn/cm2 was applied. Membrane was rapidly stretched for 10 s;
same stress was applied and stiffness was measured again. Different
wells were used for 2.5% strain and 5% strain. Means 6 SE; n 5 6
wells for 2.5% strain; n 5 8 wells for 5% strain. A dozen other
experiments showed similar results.
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CYTOSKELETAL INITIAL TENSION
discrete but nonpretensed models of percolation that
analyze phase transitions and connectivity within networks (8) do not appear to be consistent with our
results.
In summary, we have presented evidence that stretching adherent endothelial cells on an elastic membrane
results in an increase in CSK stiffness. This is likely to
be the result of an increase in passive CSK tension due
to increased cell distension. Therefore distending stress
of the CSK appears to be a key determinant of cellular
deformability.
This work was supported by National Institutes of Health Grants
HL-33009, CA-45548, HL-56398, and AR-41352.
Address for reprint requests: N. Wang, Physiology Program,
Harvard School of Public Health, 665 Huntington Ave., Boston, MA
02115.
Received 1 October 1997; accepted in final form 11 February 1998.
REFERENCES
1. Albrecht-Buehler, G. Role of cortical tension in fibroblast
shape, and movement. Cell Motil. Cytoskeleton 7: 54–67, 1987.
2. Bray, D., and J. G. White. Cortical flow in animal cells. Science
239: 883–888, 1988.
3. Chen, C. S., M. Mrksich, S. Huang, G. M. Whitesides, and
D. E. Ingber. Geometric control of cell life and death. Science
276: 1425–1428, 1997.
4. Coughlin, M. F., and D. Stamenovic. A tensegrity structure
with buckling compression elements: application to cell mechanics. ASME J. Appl. Mech. 64: 480–486, 1997.
5. Davies, P. F., and S. Tripathi. Stress mechanisms in cultured
cells: an endothelial paradigm. Circ. Res. 72: 239–245, 1993.
6. De Lanerolle, P., N. Wang, M. O’Donnell, S. Cai, E. Elson,
and D. E. Ingber. Control of cytoskeletal mechanics by myosin
light chain kinase phosphorylation (Abstract). Mol. Biol. Cell 6:
370A, 1995.
7. Folkman, J., and A. Moscona. Role of cell shape in growth
control. Nature 273: 345–349, 1978.
8. Forgacs, G. On the possible role of cytoskeletal filamentous
networks in intracellular signaling: an approach based on percolation. J. Cell Sci. 108: 2131–2143, 1995.
9. Fung, Y. C., and S. Q. Liu. Elementary mechanics of the
endothelium of blood vessels. ASME J. Biomech. Eng. 115: 1–12,
1993.
10. Harris, A. K., P. Wild, and D. Stopak. Silicone rubber substrata: a new wrinkle in the study of cell locomotion. Science 208:
177–179, 1980.
11. Hubmayr, R., S. A. Shore, J. J. Fredberg, E. Planus, R. A.
Panettieri, Jr., W. Moller, J. Heyder, and N. Wang. Pharmacological activation changes stiffness of cultured human airway
smooth muscle cells. Am. J. Physiol. 271 (Cell Physiol. 40):
C1660–C1668, 1996.
12. Ingber, D. E. Cellular tensegrity: defining new rules of biological
design that govern the cytoskeleton. J. Cell Sci. 104: 613–627,
1993.
13. Ingber, D. E., and J. Folkman. Mechanical switching between
growth and differentiation during fibroblast growth factorstimulated angiogenesis in vitro: role of extracellular matrix. J.
Cell Biol. 109: 317–330, 1989.
14. Ingber, D. E., and J. D. Jamieson. Cells as tensegrity structures: architectural regulation of histodifferentiation by physical
forces transduced over basement membrane. In: Gene Expression
During Normal and Malignant Differentiation, edited by L. C.
Anderson, C. G. Gamberg, and P. Ekblom. Orlando, FL: Academic, 1985, p. 13–22.
15. Ingber, D. E., D. Prusty, Z. Sun, H. Betensky, and N. Wang.
Cell shape, cytoskeletal mechanics, and cell cycle control in
angiogenesis. J. Biomech. 28: 1471–1484, 1995.
16. Kolodney, M. S., and R. B. Wysolmerski. Isometric contraction by fibroblasts and endothelial cells in tissue culture: a
quantitative study. J. Cell Biol. 117: 73–82, 1992.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
smooth muscle cells has been altered at a fixed state of
spreading by adding bronchoconstrictors or bronchodilators (11); it was found that CSK stiffness increases in
cells treated with bronchoconstrictors and decreases in
cells treated with bronchodilators over time scales of
,1 min. These changes in CSK stiffness are thought to
be mediated through activation or deactivation of actomyosin apparatus, thus changing the active tension in
the CSK. However, addition of contractile agonists to
the smooth muscle cells may also trigger processes,
such as phosphorylation of talin and paxillin (19),
which in turn may affect CSK stiffness by altering focal
adhesion complexes. CSK stiffness has also been increased by overexpression of myosin light-chain kinase
in fibroblasts (6). However, overexpression of myosin
light-chain kinase might activate processes other than
actomyosin cycling, which in turn could affect the
architecture and mechanics of the CSK. Moreover, in
all these previous studies, the passive components of
the CSK tension had not been manipulated. In contrast, by applying rapid mechanical stretches to the
cells, we were able to minimize the time available for
active cellular responses.
It appears that the results presented here are not
easily explained by linear continuum models of cellular
mechanics. Models suggested in the literature include
linear elastic or viscoelastic half-space models (22, 28),
models in which continuum mechanical properties of
the CSK are deduced from the mechanical properties of
individual actin filaments (20), and models depicting
the adherent cell as a viscous, viscoelastic, or elastic
cytoplasm enclosed by an elastic membrane (9, 21, 24).
Given that stretching was rapid, cell volume would not
change very much. Accordingly, a 5% strain (i.e., an
,10% increase in cell basal surface area) would result
in an ,10% reduction in cell height. Any linear continuum model would predict, at most, a 10% increase in
stiffness. This is so because force transmission between
the bead and the substratum would take place mainly
through the portion of the cell underneath the bead. In
the case of the model of viscous cytoplasm enclosed by
linearly elastic membrane, a 10% decrease in cell
height would produce an even smaller fractional increase in stiffness. However, we found that a 5% stretch
produced a disproportionate (20–30%) increase in CSK
stiffness (Figs. 5–7).
If linear continuum models of cellular mechanics are
inappropriate to explain our observations in adherent
cells, then either a nonlinear continuum model or an
approach that emphasizes the discrete, as opposed to
the continuous, nature of the CSK microstructure
needs to be used. If the former, then the elastic properties of the continuum would have to be assigned on an
ad hoc basis to account for the dependence of cell
stiffness on cell distension reported here. If the latter,
in contrast, nonlinear behavior of the CSK may be an
intrinsic property conferred by the microstructural
architecture (4, 27). In that case, nonlinearity of the
individual discrete elements is not precluded, but it is
not necessary to postulate such nonlinearity to account
for the essential features of the data. Interestingly,
CYTOSKELETAL INITIAL TENSION
24. Schmid-Schonbein, G. W., T. Kosawada, R. Skalak, and S.
Chien. Membrane model of endothelial cells and leukocytes. A
proposal for the origin of cortical stress. ASME J. Biomech. Eng.
117: 171–178, 1995.
25. Sims, J., S. Karp, and D. E. Ingber. Altering the cellular
mechanical force balance results in integrated changes in cell,
cytoskeletal, and nuclear shape. J. Cell Sci. 103: 1215–1222,
1992.
26. Singhvi, R., A. Kumar, G. Lopez, G. N. Stephanopoulos,
D. I. C. Wang, G. M. Whitesides, and D. E. Ingber. Engineering cell shape and function. Science 264: 696–698, 1994.
27. Stamenovic, D., J. J. Fredberg, N. Wang, J. P. Butler, and
D. E. Ingber. A microstructural approach to cytoskeletal mechanics based on tensegrity. J. Theor. Biol. 181: 125–136, 1996.
28. Theret, D. P., M. J. Levesque, M. Sato, R. M. Nerem, and
L. T. Wheeler. The application of a homogeneous half-space
model in the analysis of endothelial cell micropipette measurements. ASME J. Biomech. Eng. 110: 190–199, 1988.
29. Wang, N., J. P. Butler, and D. E. Ingber. Mechanotransduction
across the cell surface and the cytoskeleton. Science 260: 1124–
1127, 1993.
30. Wang, N., and D. E. Ingber. Control of cytoskeletal stiffness by
extracellular matrix, cell shape, and mechanical tension. Biophys. J. 66: 1281–1289, 1994.
31. Wang, N., and D. E. Ingber. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry. Biochem. Cell Biol. 73: 327–335, 1995.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.1 on June 18, 2017
17. Lauffenburger, D. A., and A. F. Horwitz. Cell migration:
a physically integrated molecular process. Cell 84: 359–369,
1996.
18. Maniotis, A. J., C. S. Chen, and D. E. Ingber. Demonstration
of mechanical connection between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc.
Natl. Acad. Sci. USA 94: 849–854, 1997.
19. Pavalko, F. M., L. P. Adam, M.-F. Wu, T. L. Walker, and S. J.
Gunst. Phosphorylation of dense-plaque proteins talin and
paxillin during tracheal smooth muscle contraction. Am. J.
Physiol. 268 (Cell Physiol. 37): C563–C571, 1995.
20. Satcher, R. L., and C. F. Dewey. Theoretical estimates of
mechanical properties of the endothelial cell cytoskeleton. Biophys. J. 71: 109–118, 1996.
21. Sato, M., M. J. Levesque, and R. M. Nerem. An application of
the micropipette technique to the measurements of the mechanical properties of cultured bovine aortic endothelial cells. ASME
J. Biomech. Eng. 109: 27–34, 1987.
22. Sato, M., D. P. Theret, L. T. Wheeler, N. Ohshima, and R. M.
Nerem. Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic
properties. ASME J. Biomech. Eng. 112: 263–268, 1990.
23. Schaffer, J. L., M. Rizen, G. L. L’Italien, A. Benbrahim, J.
Megerman, L. C. Gerstenfeld, and M. L. Gray. Device for the
application of a dynamic biaxially uniform and isotropic strain to
a flexible cell culture membrane. J. Orthop. Res. 12: 709–719,
1994.
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