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Extracellular Matrix- and Cytoskeleton-Dependent Changes
in Cell Shape and Stiffness
Kiran Bhadriraju*,1 and Linda K. Hansen*,†,2
*Biomedical Engineering Institute, MMC 609 and †Department of Laboratory Medicine and Pathology, MMC 609,
420 Delaware Street S.E., University of Minnesota, Minneapolis, Minnesota 55455
Cell spreading is correlated with changes in impor-
tant cell functions including DNA synthesis, motility,
and differentiation. Spreading is accompanied by a
complex reorganization of the cytoskeleton that can
be related to changes in cell stiffness. While cytoskel-
etal organization and the resulting cell stiffness have
been studied in motile cells such as fibroblasts, less is
known of these events in nonmigratory, epithelial
cells. Hence, we examined the relationship between
cell function, spreading, and stiffness, as measured by
atomic force microscopy. Cell stiffness increased with
spreading on a high density of fibronectin (1000 ng/
cm2) but remained low in cells that stayed rounded on
a low fibronectin density (1 ng/cm2). Disrupting actin
or myosin had the same effect of inhibiting spreading,
but had different effects on stiffness. Disrupting f-ac-
tin assembly lowered both stiffness and spreading,
while inhibiting myosin light chain kinase inhibited
spreading but increased cell stiffness. However, dis-
rupting either actin or myosin inhibited DNA synthe-
sis. These results demonstrate the relationship be-
tween cell stiffness and spreading in hepatocytes.
They specifically show that normal actin and myosin
function is required for hepatocyte spreading and
DNA synthesis and demonstrate opposing effects on
cell stiffness upon disruption of actin and myosin.
© 2002 Elsevier Science (USA)
Key Words: actin; atomic force microscopy; cell
shape; cell spreading; cytoskeleton; extracellular ma-
trix; growth; hepatocyte; myosin; stiffness.
INTRODUCTION
Cell spreading on adhesive proteins such as fibronec-
tin, laminin, or collagen is accompanied by changes in
the structure and composition of the cytoskeleton,
which in turn accommodate and facilitate the change
in shape [1, 2]. Spreading is also accompanied by dis-
tinct changes in cell behavior depending on the cell
type; cells that are allowed to spread undergo growth
and dedifferentiation while cells that are constrained
to remain rounded in culture undergo either apoptosis
or differentiation. Several cell types exhibit this strik-
ing shape–function relationship, including hepatocytes
[3], mouse mammary epithelial cells [4], endothelial
cells [5], fibroblasts [6], and adipocytes [7].
Hepatocytes are the principal cell type of the liver
constituting up to 90% of adult liver by weight, and
they perform several critical metabolic functions in-
cluding the synthesis and secretion of bile and several
serum factors including fibronectin, fibrinogen, trans-
ferrin, and albumin. In vitro, hepatocytes cultured on a
low density of fibronectin, collagen, or laminin [3], or
on a synthetic RGD peptide [8], remain rounded and
exhibit enhanced differentiated function. This rounded
morphology is associated with low DNA synthesis. On
the other hand, when allowed to spread well on a high
density of fibronectin, collagen, or laminin, they dedif-
ferentiate and exhibit enhanced DNA synthesis [3].
Hepatocytes similarly exhibit low DNA synthesis and
enhanced differentiated function when a rounded mor-
phology is induced by disrupting f-actin assembly us-
ing cytochalasin D [9], further demonstrating a connec-
tion between cytoskeletal structure and cell growth.
Disrupting microtubules has a less striking effect on
growth or differentiation [9]. Since the motor protein
myosin associates with actin filaments in nonmuscle
cells [10], it is likely that some of the effects of disrupt-
ing the actin cytoskeleton are in fact due to the disrup-
tion of actomyosin assemblies. Little information, how-
ever, exists on how hepatocyte spreading or function is
influenced upon disrupting myosin activity. When we
measured hepatocyte myosin activity by several meth-
ods, we found a consistent increase with cell spreading
of myosin association with the cytoskeleton, light chain
phosphorylation, and ATPase activity (Bhadriraju and
Hansen, manuscript in preparation). However, hepa-
tocytes do not present prominent stress fibers as seen
in fibroblasts or endothelial cells and are generally not
regarded as force-generating cells, since they possess
neither contractile nor motile behavior. Hence, hepa-
1Current address: Johns Hopkins University School of Medicine,
720 Rutland Avenue, Traylor 715, Baltimore, MD 21205.
2To whom correspondence and reprint requests should be ad-
dressed. Fax: (612) 625-1121. E-mail: hanse066@tc.umn.edu.
920014-4827/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
Experimental Cell Research 278, 92–100 (2002)
doi:10.1006/excr.2002.5557
tocytes presented a unique interest from the perspec-
tive of studying stiffness changes related to shape and
function.
Over the past several years, a high-resolution sur-
face imaging instrument called the atomic force micro-
scope (AFM)3has been increasingly used for measuring
the stiffness of small samples such as living cells [11,
12]. In its essence, the AFM consists of a small flexible
cantilever whose deflection can be measured with sub-
nanometer accuracy as it interacts with the sample
surface. Knowing the cantilever stiffness, these deflec-
tions can be translated into forces on the sample, which
then can be used to measure an apparent sample stiff-
ness assuming a mathematical model for the mechan-
ical behavior of the sample [13]. The AFM has been
used for measuring the stiffness of several different
biological samples including cardiac myocytes [14],
cholinergic synaptic vesicles [15], and gelatin [16].
Techniques that use force mapping have been invalu-
able in generating stiffness maps of biological surfaces
[15, 17, 18]. Using such force mapping techniques, it
has been shown that disrupting the actin cytoskeleton
with cytochalasin D reduces cell stiffness while dis-
rupting the microtubule network does not have a sig-
nificant effect on stiffness [19]. While such studies have
been useful in understanding stiffness distribution
over cells, there are few studies relating global changes
in cell shape to stiffness and function or in response to
changes in myosin activity. The present study was
undertaken with the aim of correlating stiffness mea-
surements with spreading and growth. The correlation
between stiffness and cell spreading was determined.
In addition, the effects of disruption of actin or myosin
activity on both DNA synthesis and cell stiffness were
assessed. These findings are discussed in relation to
possible underlying mechanistic events.
MATERIALS AND METHODS
Cell culture. Hepatocytes were harvested by collagenase perfu-
sion of adult rat livers [20] and used immediately after harvest. Cells
were cultured in a hormonally defined medium containing saturat-
ing amounts of growth factors and supplements: 10 ng/ml epidermal
growth factor (Collaborative Research, Bedford, MA), 5 nM dexa-
methasone (Sigma, St. Louis, MO), 20 mU/ml insulin (Sigma), 100
mg/ml ascorbic acid (Gibco, Gaithersburg, MD), 100 U/ml each of
penicillin and streptomycin (Irving Scientific, Santa Ana, CA), and
50 mg/ml of L-glutamine (Gibco). AFM experiments were performed
at ambient temperature and atmospheric CO2. To maintain pH, a
commercially available culture medium containing a proprietary
phosphate buffer (Gibco) that can maintain physiologic pH under
atmospheric CO2conditions was used along with the above supple-
ments for the AFM experiments and the corresponding spreading
experiments. No effects on cell spreading from ambient temperature
and atmospheric CO2were observed.
Substrates for cell culture were prepared by incubating 12-mm
glass coverslips overnight with fibronectin in a carbonate buffer (15
mM sodium carbonate, 35 mM sodium bicarbonate in water, pH 9.4)
and the nonspecific binding sites were blocked with 1% BSA [21].
Cells were plated at a density of 5000/cm2. For the experiments
studying the effect of actin or myosin, respectively, the drug cytocha-
lasin D (cytoD) at 1
g/ml or the drug ML-9 (1-(5-chloronapthalene-
1-sulfonyl)homopiperazine, HCl), at 7.6
M, was used (both drugs
were from Calbiochem, La Jolla, CA). Stock solutions of the drug
were made in ethanol and contributed a maximum of 0.38% v/v of
ethanol to the final media volume. At this concentration, ethanol by
itself had no effect on cell shape or stiffness (data not presented). All
AFM measurements were performed at room temperature. Cells
were cultured in ambient air at 37°C until the time of AFM. Cells
exhibited a well-spread morphology by 6 h under these conditions,
similar to those cultured in the presence of 5% CO2.
Atomic force microscopy AFM was performed on a Digital Instru-
ments Multimode Microscope (Digital Instruments, Santa Barbara,
CA) in a fluid cell at room temperature by indenting the dorsal
surface of live, adherent cells with a flexible cantilever. All measure-
ments were made with standard silicon nitride cantilevers (Digital
Instruments) with a nominal stiffness of 0.06 N/m. Where required,
fluid exchange was done by means of a syringe using ports provided
in the fluid cell. All force curves were recorded in contact mode. The
AFM was equipped with a Nikon light microscope and video camera,
allowing direct visualization of the cantilever tip on a video monitor.
In this manner the cantilever could be positioned precisely over the
perinuclear region between the spreading edge and the nucleus,
where all measurements were obtained. When performed this way,
all conditions yielded reproducible trends in stiffness.
The recorded force curves were exported to force curve analysis
software (SPMCON) and stiffness was measured as previously de-
scribed [17] using Mathematica software. The method assumes that
the sample deforms upon indentation by a truncated cone, as
F⫽2/
关E/共1⫺
2兲兴i2tan(
),
where Fis the indentation force, Eis the Young modulus (stiffness)
of the sample,
is its Poisson’s ratio, iis the sample indentation
(obtained from the force curve data), and
is the half-opening angle
of the indenter, the AFM cantilever tip in this case. Fitting the AFM
force curves to the above equation yields an apparent cell stiffness in
newtons per square meter [17]. Since a cell is expected to be incom-
pressible (i.e., no change in cell volume when indented), the Poisson
ratio was taken to be 0.5. The manufacturer’s values of 0.06 N/m and
34°were taken for the cantilever stiffness and half-opening angle of
the cone. Due to batch-to-batch variations, the AFM cantilever stiff-
ness can vary from the nominal value. In order to make meaningful
comparisons, all data presented in a figure were acquired with the
same cantilever. It took about 3 min to obtain a force curve on each
cell. Ten force curves, one per cell, on 10 different cells were obtained
representing values at the middle of the time interval for each time
point and the mean stiffness ⫾SD was reported.
In the experiments involving the use of cytoskeleton-disrupting
drugs, the drugs at the final concentration were directly added to
cells in petri dishes 30 min before AFM. After the cells had been
moved to the AFM stage, the drug concentration was maintained
throughout microscopy by perfusing media with the drug through
the AFM fluid-cell port using a syringe.
Microscopy and morphometry. Cells were fixed at the time points
of interest in 0.5% glutaraldehyde and light microscope images were
obtained on a Zeiss Axiophot25 using Varel optics. The fixed cells
were then stained with Coomassie brilliant blue, and spread cell area
3Abbreviations used: ECM, extracellular matrix; AFM, atomic
force microscope, atomic force microscopy; Fn, fibronectin; hiFN,
1000 ng/cm2of Fn; loFN, 1 ng/cm2of Fn; cytoD, cytochalasin D;
ML-9, ML-9 hydrochloride, myosin light chain kinase inhibitor;
MLCK, myosin light chain kinase.
93ATOMIC FORCE MICROSCOPY AND CELL STIFFNESS
was obtained by image analysis of digitized light microscope images
[3] using Optimas (Media Cybernetics, Bothell, WA) software. At
least 50 cells were counted per condition and the mean area ⫾SD is
reported.
For fluorescent actin imaging, cells were cultured on 8-chamber
LabTek slides (Nunc), fixed in 1% paraformaldehyde, rinsed in PBS,
and then incubated for 15 min in 0.2% Triton X-100 and 0.1% BSA in
PBS (“IF buffer”). Rhodamine phalloidin was added at 1
g/ml in IF
buffer for 30 min and then rinsed several times with PBS. Chamber
wells were removed and coverslips were placed onto the glass slides.
Staining was assessed under 100⫻magnification on a Zeiss Axios-
kop fluorescence microscope and images were captured using a
Kodak MDS290 digital camera.
DNA synthesis. DNA synthesis was assayed by measuring
[3H]thymidine incorporation. Cells were cultured in Immulon 2B
96-well plates on hiFN as described above. Cytoskeletal drugs were
added at 24 h after plating and [3H]thymidine (ICN, Costa Mesa, CA)
was added at a final concentration of 10
Ci/ml 48 h after plating.
Parallel plates were used to quantify cell attachment by using the
CyQuant fluorimetric assay (Molecular Probes). DNA synthesis was
quantified from incorporated [3H]thymidine as counts/1000 cells per
24 h and values are presented as a percentage of that without any
drugs ⫾standard deviation.
RESULTS
Cell Spreading and Stiffness
Stiffness measurements generally involve measur-
ing the deformation of a sample under a defined load.
In such a mode for the AFM, the output is in the form
of a force curve, which plots the sample movement
(moved by a piezoelectric motor) on the x-axis vs the
cantilever deflection (or the force exerted by the canti-
lever) on the y-axis. Figure 1 shows two force curves
superimposed, one for a hard surface, glass (curve 1-2-
3), and another for a soft surface, a single hepatocyte
(curve 1-2-4). As the sample is moved toward the can-
tilever (x-axis, moving from right to left with zero de-
fining the point of sample contact with the cantilever),
cantilever deflection (y-axis) will be zero as long as the
cantilever does not touch the sample surface. After the
cantilever comes in contact with the sample surface
(point 2 on the figure), it starts to deflect upward as the
sample further presses into the cantilever (region 2-3
on glass and region 2-4 on the hepatocyte). The abso-
lute value of the slope for this contact region is unity
for a hard surface, as the cantilever does not indent a
hard surface and is thus deflected by as much distance
as the sample moves (region 2-3 in Fig. 1). The absolute
value of the slope is less than unity for a soft surface, as
the cantilever deflection is less than the sample move-
ment due to sample indentation by the cantilever (re-
gion 2-4 in Fig. 1). These differences in slope can be
quantified as stiffness in newtons per square meter if a
mechanical model is assumed for the material proper-
ties of the cell, in this case a Hertzian solid [22], as
described under Materials and Methods. It should be
noted that while in certain modes of the AFM it is
possible to make stiffness maps of the cell surface [18,
19], it was not an objective of this study to map the
distribution of cell surface stiffness. It was rather to
study trends in stiffness under conditions that change
cell function. Stiffness measurements in the perinu-
clear region gave reproducible results for the condi-
tions tested.
Asafirst step in understanding how hepatocyte stiff-
ness is related to shape, stiffness was measured with
increased spreading. Cells were plated on coverslips
coated with 1000 ng/cm2of fibronectin as described
under Materials and Methods. This density of Fn in-
duces significant cell spreading by 6 h. Stiffness was
measured at time points of interest by placing the
coverslips in the fluid cell of the microscope and indent-
ing the dorsal surface of cells with the AFM cantilever
as described above. Cells were imaged in hormonally
defined medium in the absence of any cytoskeleton
disrupting drugs. All measurements were done just
inside the inner boundary of the cell body excluding
both the spreading edge and the nucleus. Area mea-
surements showed that cells increasingly spread for
several hours after plating (Fig. 2A). Stiffness also
increased for several hours after plating (Fig. 2B), mea-
suring 5.34 ⫾3.81 kPa, 10.55 ⫾7.78 kPa, and 30.3 ⫾
12.86 kPa at 45 min, 2 h, and 6 h after plating, respec-
tively. The increases were statistically significant com-
pared to each preceding time point (P⬍0.05, n⫽10
to 14 cells). Previous reports using a different method
have shown a similar relationship between spreading
and indentation stiffness for fibroblasts [23].
Substrate Density and Stiffness
While Fig. 1 demonstrates a correlation between cell
shape and stiffness, it is not possible to distinguish
between possible effects of increased spreading or sim-
ply increased cell culture and adhesion time. To better
distinguish between the possible effects of cell spread-
ing and culture time, spreading was varied indepen-
FIG. 1. Overview of AFM force curves. Two force curves, one for
a hard surface (curve 1-2-3, on glass) and another for a soft surface
(curve 1-2-4, on a single hepatocyte). The various regions of the force
curve are described in the text. The slope of the force curves (i.e., for
region 2-3 on glass and region 2-4 on the hepatocyte) is different for
the two substrates, reflecting differences in their stiffness.
94 BHADRIRAJU AND HANSEN
dent of adhesion time by varying substrate density.
Hepatocytes were plated on a high (1000 ng/cm2)orlow
(1 ng/cm2) density of fibronectin and allowed to spread.
After 10 h, the area of hepatocytes on the high density
of fibronectin (hiFN) was 1192
m2while the area on
the low density of fibronectin (loFN) was 255
m2(Fig.
3A). Stiffness was measured on parallel plates (stiff-
ness of cells on hiFN was measured first followed by
that of cells on loFN). For corresponding times, the
cells on hiFN, which were more spread, had a stiffness
that was 16.21 ⫾5.75 kPa compared to 9.73 ⫾6.02 kPa
for cells on loFN (P⬍0.05, n⫽6 to 8 cells; Fig. 3B).
Thus, increased stiffness was independent of culture
time and instead correlated with increased spreading
on a higher substrate density.
Stiffness and the Actomyosin Cytoskeleton
Previous work has shown that changing actin or
myosin activity changes cortical stiffness and that
changes in cell shape are accompanied by changes in
actin arrangement [9] and myosin activity [24] or lo-
calization [25]. Nonmuscle myosin activity, similar to
that of smooth muscle myosin, is regulated by the state
of its phosphorylation. The principal physiological ki-
nase for nonmuscle myosin is myosin light chain ki-
nase (MLCK), which phosphorylates the 20-kDa regu-
latory light chain of myosin [26]. Several previous
studies have shown that perturbing myosin changes
cell stiffness. Myosin-knockout Dictyostelium exhibit
decreased cortical stiffness [27] and 3T3 fibroblasts
expressing the catalytic domain of myosin light chain
kinase exhibit increased cortical stiffness [28]. Inhibit-
ing myosin light chain kinase activity has also been
shown to change cell shape [29, 30]. Inhibiting myosin
ATPase activity inhibits spreading in fibroblasts [24].
In addition, myosin cellular localization changes dur-
ing spreading. It is redistributed to the spreading edge
of mouse embryos in vitro [31] and rapidly associates
with polymerized actin in activated platelets as they
change shape [25].
Similar to myosin, disrupting normal actin organi-
zation disrupts cell shape and stiffness in many cell
types. Cytochalasin D, a fungal toxin that inhibits ac-
tin polymerization by binding to the plus end of actin
and preventing it from polymerizing, affects cortical
stiffness and cell shape. Disrupting f-actin integrity
with cytochalasin has previously been shown to reduce
the stiffness of fibroblasts [19]. In addition to the effect
on stiffness, it has been shown that disrupting the
actin cytoskeleton in chondrocytes disrupts spreading
by both inhibiting the spreading process and causing
spread cells to round up [9, 32, 33]. While the majority
of stiffness studies have been done on either Dictyoste-
lium or stromal cell types such as fibroblasts and
smooth muscle cells, there is little information avail-
able on the dynamics of cell stiffness in an epithelial
cell type such as hepatocytes. In light of the known
information about the dependence of cortical stiffness
on actomyosin integrity, and the relation between
shape and function, we hypothesized that disrupting
actin and myosin activity would change hepatocyte
shape and stiffness.
Cells were cultured as described under Materials
and Methods. Parallel plates were either fixed with
glutaraldehyde for morphometry or assessed by AFM
for stiffness. Cells were photographed using Varel op-
tics to enhance the contrast of the spreading edge, and
arrowheads indicate the location of nuclei. It should be
noted that many hepatocytes are binucleated, as indi-
cated by double arrowheads. CytoD almost completely
inhibited cell spreading when added at the time of
plating (Fig. 4, “cytoD-t0”). When added to spread cells
5.5 h after plating, there was retraction of the cell
edges and a distinct blebbing of the cell surface within
30 min (Fig. 4, “cytoD-t6”). To look at the effect of the
drug on spread cells in closer detail, spreading was
quantified upon adding the drug to spread cells. CytoD
FIG. 3. Effect of substrate density on hepatocyte spreading and
stiffness. (A) Hepatocyte spreading on a high (1000 ng/cm2) and low
(1 ng/cm2) density of fibronectin. Each value represents the mean cell
area ⫾standard deviation of at least 50 cells. (B) Hepatocyte stiff-
ness on a high (1000 ng/cm2) and low (1 ng/cm2) density of fibronec-
tin. Each value represents the mean stiffness ⫾standard deviation
of 6 to 8 cells.
FIG. 2. Change in hepatocyte spreading and stiffness with time
on a high density (1000 ng/cm2)offibronectin. (A) Cells were cultured
as described under Materials and Methods and fixed with 0.5%
glutaraldehyde. They were then stained with Coomassie blue and
the spread area was measured using digital image analysis. Each
value represents the mean cell area ⫾standard deviation of at least
40 cells. (B) Increase in hepatocyte stiffness with increased spread-
ing over time. Each value represents the mean stiffness ⫾standard
deviation of 10 to 14 cells.
95ATOMIC FORCE MICROSCOPY AND CELL STIFFNESS
significantly diminished spread cell area when added
at the time of plating and also when added to cells that
were allowed to spread for 5.5 h and assessed 30 min
later (n⫽61,36 for drug-treated and control cells,
respectively; P⬍0.05, Fig. 5A). To study the corre-
sponding effect on stiffness, AFM was performed on
non-drug-treated cells 6 h after plating and on drug-
treated cells 30 min after drug addition, which was
5.5 h after plating. The addition of cytochalasin D
decreased stiffness by more than half to 38.5% of that
of untreated cells (n⫽9, 7 for drug-treated and control
cells, respectively; P⬍0.05, Fig. 5B).
Since myosin forms an integral part of the actomyo-
sin cytoskeleton, a similar study was done using an
MLCK inhibitor, ML-9, to see the effect of inhibiting
normal myosin activity on hepatocyte shape and stiff-
ness. ML-9 is a specific inhibitor of MLCK with a Ki
(concentration for half-maximal inhibition) of 3.8
M
[34]. While ML-9 can also inhibit protein kinases A and
C, its Kifor MLCK is 8.4 times lower than that for
protein kinase A and 7.1 times lower than that for
protein kinase C [35]. ML-9 (7.6
M) partially inhib-
ited hepatocyte spreading when added at the time of
plating (Fig. 6, “ML9-t0”). When added to spread cells
5.5 h after plating, there was a distinct cell retraction
within 30 min (Fig. 6, “ML9-t6”). When spreading was
quantified, ML9 was found to significantly inhibit
spreading when present throughout the 6-h culture
and to cause significant cell retraction within 30 min
when added at 5.5 h (n⫽55, 36; P⬍0.05, Fig. 7A).
To look at the corresponding effect on stiffness, AFM
measurements were performed. Unlike in the case of
cytoD, the disruption of myosin light chain phosphor-
ylation using ML-9 increased stiffness more than 3
times to 374% of that of controls (n⫽8 to 10 cells; P⬍
0.05, Fig. 7B).
Actin structure in the presence or absence of cytoD
and ML9 was assessed to ascertain the drugs’effects
on the cytoskeleton. Hepatocytes were cultured on
loFN for6horhiFN in the presence or absence of
cytoD and ML9 for either the entire 6-h culture period
or the last 30 min of culture. Cells were fixed and
stained with rhodamine–phalloidin to visualize the ac-
tin cytoskeleton. Hepatocytes on hiFN for 6 h possess
actin stress fibers along the spreading edge which is
notably absent in the rounded cells on loFN (Fig. 8).
ML9 had little effect on actin structure for the 30-min
exposure, but inhibited stress fiber formation when
added for the full culture period. CytoD resulted in
disruption of stress fibers into a punctate pattern
throughout the cytoplasm when added at either time
(Fig. 8).
Actomyosin Activity and DNA Synthesis
The results presented here demonstrate that dis-
rupting either actin or myosin inhibits cell spreading
in hepatocytes and perturbs cell stiffness. Previous
work had shown a strong correlation between hepato-
cyte spreading and DNA synthesis [3, 8]. Specifically, it
has been shown that disrupting f-actin assembly using
cytoD inhibits hepatocyte spreading and DNA synthe-
FIG. 4. Effect of cytochalasin D on hepatocyte morphology. Cells plated on hiFN were treated with 1
g/ml cytochalasin either at the time
of plating (cytoD-t0) or at 5.5 h (cytoD-t6) and fixed at 6 h. These are compared to control cells without any drug treatment (no drug).
Arrowheads point to the nucleus. Photos were obtained using Varel optics.
FIG. 5. (A) Effect of cytochalasin D on hepatocyte area. Cells
were plated onto hiFN, treated with 1
M cytochalasin D at the time
of plating (cytoD-t0) or at 5.5 h after plating (cytoD-t6), and fixed at
6 h. The spreading is compared to control cells without any drug
treatment (no drug). Cells treated as in Fig. 4 were fixed with
glutaraldehyde and stained with Coomassie blue, and cell areas were
quantified by digital image analysis. (B) Effect of cytochalasin D on
hepatocyte stiffness. Cells were treated with 1
g/ml of cytochalasin
D at 5.5 h (cytoD-t6) and stiffness was measured at 6 h. Stiffness is
compared to that of control cells without any drug treatment (no
drug). Each bar is the mean ⫾standard deviation of 8–10 cells for
stiffness measurements.
96 BHADRIRAJU AND HANSEN
sis [3]. Disrupting myosin, which needs f-actin assem-
bly for force generation, has a similar effect on growth
in fibroblasts [36]. It was hence of interest to study
whether disrupting myosin perturbs hepatocyte
growth. In order to study this, [3H]thymidine incorpo-
ration was compared between 48 and 72 h after plating
in hepatocytes in the presence of 7.6
M ML-9 or 1
g/ml cytochalasin D. Both drugs had the same effect
of inhibiting DNA synthesis. When added at 24 h after
plating, cytoD inhibited hepatocyte DNA synthesis to
12.6% of cells without drug, and ML-9 inhibited it to
22% of cells without drug (Fig. 9). Similar inhibition
was seen when drugs were added at earlier times after
plating (data not shown).
DISCUSSION
This study addresses the relationship between cell
shape changes, stiffness, and the actomyosin cytoskel-
eton. While many previous efforts have focused on in-
vestigating the contribution of actin to cell shape and
stiffness, there have been fewer studies exploring the
same with regard to myosin. The results presented
here show that stiffness increases with cell spreading
in the absence of any drugs. The addition of actin or
myosin disrupting drugs inhibited cell spreading (Figs.
5A and 7A). While both drugs also significantly
changed stiffness, they had opposite effects. Disrupting
actin assembly decreased cell stiffness to almost a
third of controls (Fig. 5B), whereas inhibiting myosin
light chain kinase activity with the drug ML-9 in-
creased stiffness by more than 3 times (Fig. 7B). Both
drugs also completely inhibited DNA synthesis (Fig. 9).
Hence, these data show for the first time shape and
stiffness relationships in hepatocytes, a noncontractile,
nonmotile cell type, and present the effect of inhibiting
myosin on hepatocyte growth.
The stiffness of cells in the absence of any drugs was
found to increase with spreading, not only when the
extent of spreading was changed by Fn density (Fig. 3),
but also by allowing cells to spread over time (Fig. 2).
Previous studies with endothelial cells have shown
that integrin bonds formed by RGD-coated magnetic
beads experience a greater resistance to twist in a
magnetic field at the surface of spread cells on a high
density of fibronectin than of round cells on a low
density of fibronectin [37]. The experiments presented
here are a different mode of assaying cell stiffness, as
they measure the resistance of cells to indentation
(transverse stiffness). They too show a similar increas-
ing trend in stiffness with spreading. Several lines of
evidence presented below suggest that at least part of
this increase in transverse stiffness with spreading is
due to increased actomyosin contractile forces. In-
creased actomyosin contractile events in muscle fibers
can be seen as increased transverse stiffness [38],
showing that changes in cytoskeletal tension can be
detected by indentation measurements. Directly per-
turbing myosin changes cell stiffness, as knocking out
myosin in Dictyostelium results in decreased cortical
stiffness [27]. Further, there is evidence from both the
FIG. 6. Effect of ML-9 on hepatocyte morphology. Cells plated on hiFN were treated with 7.6
M ML-9 either at the time of plating
(ML9-t0) or at 5.5 h (ML9-t6) and fixed at 6 h. These are compared to control cells without any drug treatment (no drug). Arrowheads point
to the nucleus. Photos were obtained using Varel optics.
FIG. 7. (A) Effect of ML-9 on hepatocyte area. Cells were treated
with 7.6
M ML-9 at 5.5 h (ML9) and fixed at 6 h. They were
subsequently stained with Coomassie blue and morphometry was
done by computerized image analysis. Spreading is compared to
control cells without any drug treatment (no drug). (B) Effect of ML-9
on hepatocyte stiffness. Cells were treated with ML-9 as above and
stiffness was compared to that of control cells without any drug
treatment (no drug). Each bar is the mean ⫾standard deviation of
8–12 cells.
97ATOMIC FORCE MICROSCOPY AND CELL STIFFNESS
literature [24] and our own group (Bhadriraju and
Hansen, manuscript in preparation) to suggest that
myosin ATPase activity increases with cell spreading,
which could be reflected in an increased cell stiffness.
The disruption of another major component of the cy-
toskeleton, the microtubule network, has been shown
to not significantly affect cell stiffness [19]. Put to-
gether, these data suggest that the increase in stiffness
with spreading seen in Figs. 2 and 3 is at least in part
due to actomyosin contractile forces.
To directly test the relative effects of the actin and
myosin cytoskeleton on shape and stiffness, drugs were
used to disrupt their function. Cytochalasin D is an f-
actin disrupter that inhibits spreading in many cell types
including hepatocytes. During this study we found that
ML-9, an inhibitor of myosin light chain kinase, also
inhibits hepatocyte spreading (Fig. 6). Interestingly both
drugs also caused spread cells to round up (Figs. 5A and
7A). Several previous studies have noted rounding of
spread cells upon cytochalasin addition [32, 33, 39]. Since
actin and myosin are together required for myosin gen-
erated force, these results suggest that such forces are
necessary to not only cause cell spreading, as some of the
theories of cell spreading assume, but also to preserve
preformed adhesions. Adhesion bonds that strengthen
upon being stressed have been previously incorporated in
mathematical models [40]. Indeed, there has been direct
experimental evidence to show that integrin bonds be-
come strengthened upon being stressed [41], as might be
expected to happen during spreading, thereby stabilizing
adhesions.
In addition to cell shape, the actomyosin disrupting
drugs also had a significant effect on stiffness. The
addition of cytoD decreased cell stiffness (Fig. 5B).
CytoD disrupts actin filaments by capping the growing
FIG. 8. The effect of actomyosin disrupting drugs on the hepatocyte actin cytoskeleton. Hepatocytes were cultured for 6 h onto 8-chamber
glass slides (LabTek, Nunc) coated with hiFN or loFN. Cytochalasin D (CD) or ML-9 was added to cultures on hiFN for either the last 30 min
of culture (5.5–6h)orthefull6hofculture (0–6 h). At 6 h, cells were fixed and stained with rhodamine–phalloidin. Staining was assessed
under 100⫻magnification.
FIG. 9. The effect of actomyosin disrupting drugs on DNA syn-
thesis in hepatocytes. Cytochalasin (cytoD) or ML-9 was added at
24 h after plating and DNA synthesis was measured by [3H]thymi-
dine incorporation between 48 and 72 h. Values are mean ⫾stan-
dard deviation of triplicates from a representative experiment ex-
pressed as the percentage of controls without drug.
98 BHADRIRAJU AND HANSEN
ends. Stiffness studies in other cell types show a de-
crease in stiffness upon cytoD treatment [19, 42, 43]. It
not only disrupts actin filament organization but pre-
sumably also reduces cortical tension as a result of
disrupting myosin association with actin. Unlike the
case of cytoD, ML-9 dramatically increased stiffness
(Fig. 7B) in the absence of any effect on actin morphol-
ogy (Fig. 8), although both drugs inhibited spreading.
These data show that stiffness measurements reveal
changes in the cytoskeleton that are decoupled from
gross cell shape. Further evidence for this is that when
cells were induced to round up by partial trypsiniza-
tion, there was not a statistically significant change in
stiffness compared to spread cells (data not presented),
unlike in the case of drug-treated cells.
The reason for the increased stiffness upon ML-9 treat-
mentisnotclearalthoughsomepossiblemechanismscan
be speculated upon. Nonmuscle and smooth muscle my-
osin share many common features in their mode of reg-
ulation. Myosin association with actin in nonmuscle and
smooth muscle cells requires light chain phosphorylation
[44]. Actin-associated myosin hydrolyzes ATP to generate
force through cycles of attachment and detachment with
actin filaments. At high myosin light chain phosphoryla-
tion levels, cross-bridge detachment is rate-limited by
ADP release and ATP attachment (for the next ATPase
cycle). There are, in general, two conditions under which
a low myosin motor activity is associated with a high
level of stiffness. When ATP is depleted from muscle
fibers, myosin cannot detach from actin filaments and
goes into a tightly bound state characterized by a large
increase in stiffness. This state, called rigor, is also seen
in nonmuscle cells upon ATP depletion [27]. There is also
considerable evidence to show that the rate of ATPase
cycling is dependent on the extent of myosin light chain
phosphorylation. Myosin association with actin requires
light chain phosphorylation. If actin-associated myosin is
dephosphorylated,this can produce a state of slow cycling
while maintaining force. The stiffness contributed by ac-
tomyosin assemblies is a reflection of cross-bridge stiff-
ness. Slow cycling can possibly increase the lifetime of
actomyosin cross-bridges, thereby increasing the mea-
sured stiffness as seen here. It is also possible that ML-9
causes a structural rearrangement of the cytoskeleton
(e.g., bunching up) during cell rounding that shows up as
increased stiffness.
Finally, the fact that inhibiting myosin has as strong
an inhibitory role on DNA synthesis as inhibiting f-actin
does (Fig. 9) points to a key role for normal myosin activ-
ity in hepatocyte growth activation. Previous work had
shown a strong correlation between shape and cell cycle
regulation in several cell types including hepatocytes
[45], capillary endothelial cells [46], and smooth muscle
cells [47]. Previous work also suggests that pathways
involving myosin activation impinge on downstream sig-
nals of the growth pathway including MAP kinase,
p27kip1, and cyclin D1 [36, 46]. In light of the connection
between cell shape and entry into the cell cycle shown
before, and myosin activity and growth presented above,
it is likely that inhibiting myosin might impinge on cell
cycle regulating proteins in hepatocytes. We are cur-
rently investigating the possible role of myosin on cyclin
D1 and MAP kinase regulation.
In conclusion, the results presented here show that
both during spreading and in the presence of actin and
myosin disrupting drugs, hepatocytes undergo changes
in cell stiffness that can be detected by atomic force mi-
croscopy. The data also show that both actin and myosin
are required for hepatocyte spreading and for maintain-
ing the spread shape. Also, the inhibition of hepatocyte
DNA synthesis upon myosin inhibition shows that the
motor protein plays a key role in the growth of a noncon-
tractile, nonmotile cell type. Future studies will focus on
the role of myosin in growth pathways.
The authors thank Kristine Groehler, Lisa Jungers, and Diane
Tobolt for expert technical assistance. This project was supported by
a grant from the University of Minnesota Graduate School and from
the National Science Foundation (MCB9808205).
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Received August 20, 2001
Revised version received April 3, 2002
Published online June 20, 2002
100 BHADRIRAJU AND HANSEN
