From the Physiology Program, Department of Environmental Health, Harvard
School of Public Health, Boston, Mass.
Correspondence to Dr Ning Wang, Physiology Program, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115. E-mail nwang{at}hsph.harvard.edu
Abstract
Abstract—Mechanical properties of
the cells are important in controlling cell shape, cell migration, and
other functions. To understand how cytoskeletal (CSK) filaments
interact with one another mechanically, mechanical properties of
adherent endothelial cells were analyzed after
treatment with CSK-disrupting drugs. CSK stiffness (the ratio of
applied stress to strain, a measure of cell resistance to shape
deformation), viscosity (an index of intracellular structural damping),
and permanent deformation (a measure of "plasticity") were measured
with magnetic twisting cytometry, by which rotational stress was
applied directly to integrin receptors with ferromagnetic beads coated
with RGD-containing peptide. Treatment with cytochalasin D, which
disrupts actin microfilaments inhibited stiffness by 50% and decreased
permanent deformation from 70% to 50% but had almost no effect on
viscosity. In contrast, nocodazole, a microtubule disrupter, had very
little effect on inhibition of CSK stiffness, decreased viscosity by
25%, and had no effects on permanent deformation.
Acrylamide, an intermediate filament disrupter, had little
effect on inhibition of CSK stiffness, little effect on viscosity, and
no effect on permanent deformation. Taxol, a drug that facilitates
microtubule polymerization, increased stiffness by 10%, increased
viscosity by 10%, and decreased permanent deformation from 70% to
50%. Combinations of cytochalasin D and nocodazole, cytochalasin D and
acrylamide, or all three drugs resulted in a synergistic
effect on inhibition of CSK stiffness and viscosity but not in
permanent deformation. Inhibition of oxidative metabolism
with potassium cyanide had no effects on stress-induced stiffening
response. Inhibition of tyrosine phosphatase with phenylarsine oxide
had no effect on stress-induced stiffening response. We conclude that
higher order mechanical interactions of CSK filaments are important in
determining the mechanical properties of the cell.
One of the
fundamental questions in cellular mechanics is how individual
filamental systems contribute to the overall behavior of cell shape
stability, which has profound implications in regulating vascular tone.
Control of cell and thus nuclear shape is critical for cell growth,
motility, differentiation, and
apoptosis.1 2 3 4 5 6 Loss of shape control is a
hallmark of neoplastic transformation and is associated with
deregulation of both cytoplasmic and nuclear
functions.7 8 While soluble molecules such as
growth factors are important in switching genes on and off, these
molecules alone cannot determine cell shape stability. Because
cytoskeletal (CSK) structural and mechanical alterations appear to be
important in control of cell deformability,9 10
we set out to quantify changes in CSK mechanics using magnetic twisting
cytometry after treatment with different
drugs.11 12 13 We demonstrate here that the
interplay between different CSK filament systems is required for
control of cell mechanics.
Methods
Cell Culture
Magnetic Twisting Cytometry
Results
To determine how individual CSK filament systems interact
with each other mechanically, changes in CSK mechanics were measured
after different filament systems were disrupted.
Endothelial cells were plated on fibronectin for 6
hours before the experiments. RGD-coated ferromagnetic beads were then
added to the well for 15 minutes, and unbound beads were washed away. A
stress of 40 dyne/cm2 was applied, and angular
strain was measured. Cytochalasin D (0.1 µg/mL), which severs
microfilaments and disrupts actin network,14
nocodazole (10 µg/mL), which inhibits microtubule
formation,15 acrylamide (4
mmol/L), which disrupts intermediate filaments, or a combination of
these agents was added to the cells for 15 to 60 minutes. Treatment
with cytochalasin D inhibited stiffness by 50% (P<0.05),
decreased permanent deformation from 70% to 50% (P<0.05),
but had almost no effect on viscosity (P>0.05). In
contrast, nocodazole inhibited stiffness by 20% (P<0.05),
decreased viscosity by 25% (P<0.05), and had no effect on
permanent deformation (P>0.05). Acrylamide
inhibited stiffness by 15% (P<0.05) and viscosity by 10%
(P<0.05) and had no effect on permanent deformation
(P>0.05). A combination of cytochalasin D and nocodazole,
cytochalasin D and acrylamide, or all 3 drugs resulted in
added effects on inhibition of stiffness by 75% to 80%
(P<0.05) and viscosity by 55% to 70% (P<0.05)
but had no added effect on permanent deformation (P>0.05
compared with cytochalasin D effect) (Figure 1
Discussion
Our results indicate that the apparent viscosity is an index of
CSK structural damping (but not of intracellular fluid viscosity),
which can be decreased by disrupting microtubules. This damping process
depends on the mechanical interactions among all 3 filament systems.
Interestingly, permanent deformation appears to be associated only with
microfilaments and not with microtubules and/or intermediate filaments.
Together with our earlier work indicating that permanent deformation
could depend on chemical remodeling due to actions of various small CSK
severing, cross-linking, and bundling proteins, we suggest that these
remodeling processes that are associated with the microfilament
structure are a source of permanent deformation.
Taxol, a drug that facilitates microtubule polymerization, increased
stiffness by 10% (P<0.05), increased viscosity by 10%
(P<0.05), and decreased permanent deformation from 70% to
50% (P<0.05). Addition of taxol and nocodazole together
abolished the effects of nocodazole, and addition of taxol and
cytochalasin D had the same effects as cytochalasin D alone (not
shown). These results show that taxol specifically modulates
microtubules. The resulting increases in stiffness and viscosity due to
microtubule hyperpolymerization are consistent with our earlier
results in cardiac muscle cells.16 Taxol might
change permanent deformation via interference with the
microfilament-dependent remodeling process.
Previously, we have found that stiffness increases with
increasing applied stresses.11 12 13 We wondered
whether this phenomenon depended on the process of oxidative
metabolism. Treating endothelial cells with
potassium cyanide (2.5 mmol/L for 15 minutes), an oxidative
metabolism inhibitor, had no effect on
inhibition of stress-induced stiffening response (Figure 2
Actin filaments function as both tension generators and
load-bearing elements, depending on their organization and location in
the cell.19 Microtubules function as internal
support struts in structurally compromised cells; however, they appear
to be redundant load-bearing elements (ie, under conditions in which
actin filament integrity is maintained and/or extracellular matrix is
bearing the load). Similar complementary load-bearing roles for
different CSK filaments and cell-substratum contacts have been observed
in past studies.20 Intermediate filaments, which
physically interlink the cell surface with the nucleus and carry
tensile loads, also may be redundant support elements because they can
be induced to retract from the cell surface without producing
large-scale changes of cell shape. Our data support the idea that cells
behave like a tensegrity structure, ie, like a prestressed,
interconnected filament network.19 21 It is
important to emphasize that redundancy of load-bearing elements is
likely a critical property of cell architecture. It provides structural
stability and permits dynamic changes of form and organization of
individual filaments without resulting in collapse of the
cytoskeleton.
In summary, we have demonstrated that the mechanical properties of the
cell depend on the mechanical interactions among all 3 CSK filament
systems.
Acknowledgments
This work was supported by National Institutes of Health grants
HL-33009 and HL-56398 and NASA grant NAG54839. I thank Dr Winfried
Moller for providing the magnetic beads, Drs Jeffrey J. Fredberg and
Donald E. Ingber for advice, and In Lim and Jianxin Chen for
technical assistance.
Received January 24, 1998;
first decision February 11, 1998;
accepted April 2, 1998.
References
1.
Folkman J, Moscona A. Role of cell shape in growth
control. Nature. 1978;273:345–349.[Medline]
[Order article via Infotrieve]
2.
Ingber DE, Folkman J. Mechanochemical switching
between growth and differentiation during fibroblast growth
factor-stimulated angiogenesis in vitro: role of extracellular
matrix. J Cell Biol. 1989;109:317–330.
3.
Ingber DE. Fibronectin controls capillary
endothelial cell growth by modulating cell shape.
Proc Natl Acad Sci U S A. 1990;87:3579–3583.
4.
Emerman JT, Pitelka DR. Maintenance and
induction of morphological differentiation in dissociated mammary
epithelium on floating collagen membranes. In Vitro. 1977;13:316–322.[Medline]
[Order article via Infotrieve]
5.
Singhvi R, Kumar A, Lopez GP, Stephanopoulos GN, Wang
DIC, Whitesides GM, Ingber DE. Engineering cell shape and
function. Science. 1994;264:696–698.
6.
Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE.
Geometric control of cell life and death. Science. 1997;276:1425–1428.
7.
Ingber DE, Madri JA, Jamieson JD. Role of basal lamina
in the neoplastic disorganization of tissue architecture. Proc
Natl Acad Sci U S A. 1981;78:3901–3909.
8.
Pienta KJ, Partin AW, Coffey DS. Cancer as a disease
of DNA organization and dynamic cell structure. Cancer Res. 1989;49:2525–2535.
9.
Barbee KA, Davies PF, Ratneshwar L. Shear
stress-induced reorganization of the surface topography of living
endothelial cells imaged by atomic force microscopy.
Circ Res. 1994;74:163–171.
10.
Pourati J, Maniotis A, Spiegel D, Schaffer JL, Butler
JP, Fredberg JJ, Ingber DE, Stamenovic D, Wang N. Is cytoskeletal
tension a major determinant of cell deformability in adherent
endothelial cells? Am J Physiol. 1998;274:C1283–C1289.
11.
Wang N, Butler JP, Ingber DE. Mechanotransduction
across the cell surface and through the cytoskeleton.
Science. 1993;260:1124–1127.
12.
Wang N, Ingber DE. Control of cytoskeletal stiffness by
extracellular matrix, cell shape and mechanical tension. Biophys
J. 1994;66:2181–2189.[Medline]
[Order article via Infotrieve]
13.
Wang N, Ingber DE. Probing transmembrane mechanical
coupling and cytomechanics using magnetic twisting cytometry.
Biochem Cell Biol. 1995;73:327–335.[Medline]
[Order article via Infotrieve]
14.
Cooper JA. Effects of cytochalasin and phalloidin on
actin. J Cell Biol. 1987;105:1473–1478.
15.
Danowski BA. Fibroblast contractility
and actin organization are stimulated by microtubule
inhibitors. J Cell Sci. 1989;93:255–266.
16.
Hirofumi T, Wang N, Narishige T, Ingber DE, Zile MR,
Cooper G IV. Cytoskeletal mechanics in pressure-overload cardiac
hypertrophy. Circ Res. 1997;80:281–289.
17.
Choquet D, Felsenfeld DP, Sheetz MP. Extracellular
matrix rigidity causes strengthening of integrin-cytoskeleton linkages.
Cell. 1997;88:39–48.[Medline]
[Order article via Infotrieve]
18.
Plopper G, Ingber DE. Rapid induction and isolation of
focal adhesion complexes. Biochem Biophys Res Commun. 1993;193:571–578.[Medline]
[Order article via Infotrieve]
19.
Ingber DE. Cellular tensegrity: defining new rules of
biological design that govern the cytoskeleton. J Cell
Sci. 1993;104:613–627.[Medline]
[Order article via Infotrieve]
20.
Joshi HC, Chu D, Buxbaum RE, Heidemann SR. Tension and
compression in the cytoskeleton of PC12 neurites. J Cell
Biol. 1985;101:697–705.
21.
Stamenovic D, Fredberg JJ, Wang N, Butler JP, Ingber
DE. A microstructural approach to cytoskeletal mechanics based on
tensegrity. J Theor Biol. 1996;181:125–136.[Medline]
[Order article via Infotrieve]
© 1998 American Heart Association, Inc.
Third Workshop on Structure and Function of Large
Arteries: Part I
Mechanical Interactions Among Cytoskeletal Filaments
Key Words: microfilaments • microtubules • cytoskeleton • cytometry
Bovine capillary endothelial cells
isolated from adrenal cortex were cultured as
described.3 Quiescent confluent monolayers were
exposed to trypsin/EDTA and washed in DMEM containing 1% BSA. Cell
aliquots were pelleted and resuspended in defined medium consisting of
DMEM, transferrin (5 µg/mL, Collaborative Research), high-density
lipoprotein (10 µg/mL, Bionetics Research Institute), 1% BSA, and
basic fibroblast growth factor (2 ng/mL, Takeda Chemical
Industries).
Ferromagnetic microbeads (4.5 µm in diameter) were
precoated with a synthetic RGD (Arg-Gly-Asp)-containing peptide
(Peptite 2000, Telios) in a carbonated buffer (50 µg/mL protein per
milligram beads) to facilitate protein absorption onto the
beads.3 Endothelial cells were
plated in defined medium at 3x104 cell per well
on fibronectin-coated (500 ng/cm2; this coating
density promotes maximum cell spreading)3 plastic
dishes (96-well Removawells, Immunolon II, Dynatech) for 4 to 6 hours
before twisting experiments. Beads were added to each well at 20 µg
per well (
60 000 beads per well, average 2 beads per cell) for 15
minutes, and then unbound beads were washed away with 1% BSA/DMEM. The
well was placed into the magnetic twisting cytometer and maintained at
37°C. A brief (10 µs) but strong (1000 G) magnetic pulse was
applied to magnetize all surface-bound beads in the horizontal
direction.11 12 13 A twisting torque then was
applied with a weak vertical homogeneous magnetic field (0
to 30 G). The extent of bead rotation (angular strain) was measured by
an in-line magnetometer that measured the magnitude of the bead
magnetic vector in the horizontal direction. Stress was calibrated in a
viscous standard, and angular strain was measured as the beads rotated
in place in response to applied stress.12 13
Stiffness is defined as the ratio of stress to angular strain. Apparent
viscosity was calculated as the product of time constant after
stress release and stiffness. Permanent deformation was calculated as
the percent angular strain sustained after stress
release.12 
). Because the stiffness probed through
nonadhesion molecules such as scavenger receptors was about 12
dyne/cm2 (not shown), and assuming that
stiffnesses are additive and the rest of the stiffness is CSK
stiffness, these results show that about 95% of the CSK stiffness was
inhibited when all 3 drugs were added simultaneously.
Therefore, all 3 filament systems interact mechanically to contribute
to the stiffness of the entire CSK and thus of the whole cell.
View larger version (15K):
[in a new window]
Figure 1. Mechanical role of microfilaments, microtubules,
and intermediate filaments on living adherent
endothelial cells. Apparent stiffness (A), apparent
viscosity (B), and permanent deformation (C) were measured in adherent
endothelial cells (plated for 6 hours) after they were
treated with cytoskeletal disrupters. Applied stress was 40
dyne/cm2. CytD indicates 0.1 µg/mL cytochalasin D; Noc,
10 µg/mL nocodazole; Acry, 4 mmol/L acrylamide;
Taxol, 15 µmol/L taxol; CytD+Noc, 0.1 µg/mL cytochalasin D and
10 µg/mL nocodazole; CytD+Acry, 0.1 µg/mL cytochalasin D and 4
mmol/L acrylamide; and CytD+Acry+Noc, 0.1 µg/mL
cytochalasin D and 10 µg/mL nocodazole and 4 mmol/L
acrylamide. Mean±SE, n=3. The doses for the drugs chosen
in this study such as cytochalasin D, nocodazole, or taxol had specific
saturating effects in disrupting microfilament lattice or modulating
microtubule structures. However, acrylamide had an effect
on more than just intermediate filaments. ). A recent article suggests that
stress-induced stiffening response might be dependent on tyrosine
dephosphorylation.17 To test this
possibility, we added RGD-coated beads for 15 minutes, washed away
unbound beads, and then treated cells with a low dose of phenylarsine
oxide (PAO, 5 nmol/L for 10 minutes). Surprisingly, inhibition of
tyrosine phosphatase with PAO had no effect on stress-induced
stiffening response (Figure 3). Treating
the cells with a much higher dose of PAO (10 µmol/L for 10
minutes) did not have an effect on the stiffening response either (not
shown). The reason for this discrepancy might lie in earlier
experiments: the bead was added for seconds only before the force was
applied.17 The focal adhesion complex was still
in the process of being formed, ie, different linking proteins were
still in the process of being recruited to the bead; thus, the
force-dependent stiffening response observed in their experiments might
reflect the strengthening process in the focal adhesion complex that
depends on tyrosine dephosphorylation. In contrast, in
our experiments, beads were allowed to bind to the cells for 15 minutes
before twisting, and by this time the recruitment of the linking
proteins to the focal adhesion is already almost
complete.18 Thus, our observed stress-induced
stiffening response might reflect the process of mechanical
rearrangement of CSK filaments.
View larger version (17K):
[in a new window]
Figure 2. Effects of oxidative metabolism
inhibition on stiffening response. Endothelial cells
were plated for 4 hours on high density of fibronectin. Cells were
treated either with potassium cyanide ( 
, 2.5 mmol/L for 15
minutes), an oxidative metabolism inhibitor, or
dissolving buffer (
). Potassium cyanide had no effect on inhibition
of stress-induced stiffening response. Mean±SE, n=4.
View larger version (16K):
[in a new window]
Figure 3. Effects of tyrosine phosphatase inhibition on
stiffening response. Endothelial cells were plated for
4 hours on high density of fibronectin. A low dose of phenylarsine
oxide ( , 5 nmol/L for 10 minutes) or control buffer () was added.
Inhibition of tyrosine phosphatase with phenylarsine oxide had no
effect on stress-induced stiffening response. Mean±SE, n=4.
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