Is cytoskeletal tension a major determinant of cell
deformability in adherent endothelial cells?
Jacob
Pourati1,
Andrew
Maniotis2,
David
Spiegel1,
Jonathan L.
Schaffer3,
James P.
Butler1,
Jeffrey J.
Fredberg1,
Donald E.
Ingber2,
Dimitrijie
Stamenovic4, and
Ning
Wang1
1 Physiology Program, Harvard
School of Public Health and
2 Departments of Pathology and
Surgery, Children's Hospital and Harvard Medical School, Boston 02115;
and 3 Departments of
Orthopedic Surgery, Brigham and Women's Hospital, Children's
Hospital, and Harvard Medical School and
4 Department of Biomedical
Engineering, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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 stress-bearing 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 Ca2+ 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 Ca2+-dependent processes.
Taken together, these results suggest preexisting CSK tension is a
major determinant of cell deformability in adherent endothelial cells.
mechanical tension; shape stability; cell adhesion; shear
deformation; stiffness
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INTRODUCTION |
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 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.
 |
MATERIALS AND METHODS |
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 cell-stretching system is shown in Fig.
1. A 76-µm-thick membrane 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.

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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.
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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 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 × 108
dyn/cm2. There was no breakage,
leakage, or buckling of the membrane after repeated stretches.

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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. , Strain measured
in X direction; , strain measured
in Y direction. Means ± SE;
n = 4 wells.
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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 ± SE;
n = 4 wells.
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Cell cultures for stretching.
Bovine capillary endothelial cells were cultured to confluence, serum
deprived, trypsinized, 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.
 |
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).

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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 ± SE; n = 20 cells.
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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 extensions retracted rapidly
(<10 s) toward the cell center
(n = 20 cells) when the
focal adhesion was dislodged. This retraction was inhibited by
pretreatment with Cyto D (1 µg/ml for 30 min;
n = 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).

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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-Asp-coated 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 ± SE; n = 6 wells
for 2.5% strain; n = 8 wells for 5%
strain. A dozen other experiments showed similar results.
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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.

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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 ± SE; n = 4 wells. Two other
independent experiments showed similar results.
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To determine whether the stiffening response depended on membrane
integrity, ATP, or Ca2+, the
stiffness was measured when the cells were permeabilized with saponin
(25 µg/ml) for 8 min; intracellular ATP and
Ca2+ 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.

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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
Ca2+ were washed away. Same cells
were twisted again before and after permeabilization. Note that removal
of ATP and Ca2+ did not have any
significant effects on stretch-induced stiffening response. Means ± SE; n = 6 wells. An independent
experiment showed similar results.
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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 ± SE;
n = 8 wells. An independent experiment
showed similar results.
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 |
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 Ca2+, 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 prevented with
Cyto D pretreatment, which might not inhibit
Ca2+ 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
K+ and
Ca2+ 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
Ca2+-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 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, 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.
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ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health Grants
HL-33009, CA-45548, HL-56398, and AR-41352.
 |
FOOTNOTES |
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.
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