SPECIAL TOPIC
Stress fiber organization regulated by MLCK and Rho-kinase in
cultured human fibroblasts
Kazuo
Katoh1,
Yumiko
Kano1,
Mutsuki
Amano3,
Kozo
Kaibuchi2,3, and
Keigi
Fujiwara4
1 Department of Structural Analysis, National Cardiovascular
Center Research Institute, Suita, Osaka 565-8565; 2 Division
of Signal Transduction, Nara Institute of Science and Technology,
Ikoma, Nara 630-0101; 3 Department of Cell Pharmacology, Nagoya
University School of Medicine, Showa, Nagoya, Aichi 466-8550, Japan;
and 4 Center for Cardiovascular Research, University of
Rochester, Rochester, New York 14642
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ABSTRACT |
To understand the roles of Rho-kinase and
myosin light chain kinase (MLCK) for the contraction and organization
of stress fibers, we treated cultured human foreskin fibroblasts with
several MLCK, Rho-kinase, or calmodulin inhibitors and analyzed F-actin organization in the cells. Some cells were transfected with green fluorescent protein (GFP)-labeled actin, and the effects of inhibitors were also studied in these living cells. The Rho-kinase
inhibitors Y-27632 and HA1077 caused disassembly of stress fibers and
focal adhesions in the central portion of the cell within 1 h.
However, stress fibers located in the periphery of the cell were not
severely affected by the Rho-kinase inhibitors. When these cells were
washed with fresh medium, the central stress fibers and focal adhesions gradually reformed, and within 3 h the cells were completely
recovered. ML-7 and KT5926 are specific MLCK inhibitors and caused
disruption and/or shortening of peripheral stress fibers, leaving the
central fibers relatively intact even though their number was reduced. The calmodulin inhibitors W-5 and W-7 gave essentially the same results
as the MLCK inhibitors. The MLCK and calmodulin inhibitors, but not the
Rho-kinase inhibitors, caused cells to lose the spread morphology,
indicating that the peripheral fibers play a major role in keeping the
flattened state of the cell. When stress fiber models were reactivated,
the peripheral fibers contracted before the central fibers. Thus our
study shows that there are at least two different stress fiber systems
in the cell. The central stress fiber system is dependent more on the
activity of Rho-kinase than on that of MLCK, while the peripheral
stress fiber system depends on MLCK.
Rho-kinase; stress fiber; contraction; myosin regulatory light
chain; myosin light chain kinase
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INTRODUCTION |
STRESS FIBERS are
seen as bundles of actin filaments and are an actin-myosin-based
contractile system. We have recently developed a method for isolating
stress fibers from cultured cells and reactivating them (11,
12). The method is based on sequentially extracting cells with
low ionic strength and then detergent (Triton X-100) solutions. The
stress fibers isolated by the procedure contained actin, myosin,
-actinin, calmodulin, and myosin light chain kinase (MLCK). A rapid
contraction was achieved by treating isolated stress fibers with Mg-ATP
and Ca2+. We concluded that this contraction was regulated
by the Ca2+-dependent calmodulin/MLCK system.
Rho (Ras homology) proteins are GTPases involved in signal
transduction. Activation of Rho proteins is known to modulate the organization of actin filaments in cells, including formation of stress
fibers and focal adhesions (1, 19). Several proteins are
known to be targets of activated Rho, such as Rho-kinase (ROK
/ROCK II) (8, 17, 18), myosin binding subunit (MBS) of myosin phosphatase (15), p140mDia (24), protein
kinase N (4), and phospholipase D (21). It is
now known that actomyosin contraction can be regulated by Rho-kinase in
two ways. One way is by phosphorylating myosin regulatory light chain
(MRLC) at serine-19 of smooth muscle (3, 16) and
fibroblast (2, 7). Interestingly, serine-19 is the residue
phosphorylated by MLCK. The other way contraction can be regulated by
Rho-kinase is by inhibiting the myosin phosphatase activity via
phosphorylation of MBS of myosin phosphatase (15).
Although regulation of MLCK and Rho-kinase activities is well
characterized, each separately by in vitro analyses, it is not easy to
determine the distinct roles of MLCK and Rho-kinase in living cells. In
this study, we have made an attempt at examining different effects of
MLCK and Rho-kinase on the organization of stress fibers. Our study has
revealed that there are two stress fiber systems: 1)
a thick peripheral stress fiber system that is more sensitive to MLCK
and calmodulin inhibitors than to Rho-kinase inhibitors and
2) a thin central stress fiber network that is more
sensitive to Rho-kinase inhibitors than to the other two types. Focal
adhesions associated with the central stress fibers were also sensitive
to Rho-kinase inhibitors. In vitro contraction of peripheral stress
fibers occurred before central stress fibers began to shorten. These
observed differences indicate that not all stress fibers are regulated
in the same way. We suggest that the formation, maintenance, and
contraction of the central stress fibers depend largely on the activity
of Rho-kinase, while the Ca2+-dependent
calmodulin/MLCK system plays a major role in regulating the peripheral
stress fibers.
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METHODS |
Cell culture.
Human foreskin fibroblasts (FS-133), bovine endothelial cells,
and human lung fibroblast (WI-38) cells were cultured with the use of a
1:1 mixture of Dulbecco's modified Eagle medium and a nutrient mixture
of F-12 (GIBCO, Grand Island, NY), pH 7.4, containing 50 U/ml
penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum
(Salmond Smith Biolab, New Zealand) (21). The cells were
maintained at 37°C in a humidified, 5% CO2 atmosphere.
Antibodies.
The following monoclonal antibodies were purchased: anti-
-smooth
muscle actin (Sigma, St. Louis, MO), anti-pan-myosin (Amersham, Amersham, UK), anti-MLCK (Sigma), anti-calmodulin (Upstate
Biotechnology, Lake Placid, NY), and anti-vinculin (Sigma).
Rhodamine-labeled phalloidin was also purchased for F-actin staining
(Molecular Probes, Eugene, OR). A rabbit affinity-purified polyclonal
antibody against the glutathione-S-transferase (GST)-bound
MBS peptide (GST-MBS-N; 1-706 amino acids) was described
previously (14).
Inhibitors.
The MLCK inhibitors ML-7 (Seikagaku, Tokyo, Japan), ML-9 (Seikagaku),
and wortmannin (Wako, Osaka, Japan) were purchased. The calmodulin
inhibitors W-5 (Biomol, Plymouth Meeting, PA), W-7 (Biomol), and
KT5926 (Calbiochem, La Jolla, CA) were also purchased. The Rho-kinase
inhibitors Y-27632 (23) and HA1077 (22, 23)
were kindly provided by Yoshitomi Pharmaceutical Industry (Osaka,
Japan) and Asahi Chemical Industry (Shizuoka, Japan), respectively.
Isolation of stress fibers.
Stress fibers were isolated by a modified method described in our
previous report (11, 12). Briefly, FS-133 cultured for 3-4 days in culture dishes (150 mm in diameter; Greiner,
Frickenhausen, Germany) were quickly washed in ice-chilled
phosphate-buffered saline (PBS) and then extracted for 20-30 min
with gentle agitation in a low-ionic strength solution consisting of
2.5 mM triethanolamine (2,2',2"-nitrilotriethanol; TEA) (Wako), 20 µg/ml Trasylol (Bayer, Leverkusen, Germany), 1 µg/ml leupeptin
(Peptide Institute, Osaka, Japan), and 1 µg/ml pepstatin (Peptide
Institute), pH 8.2 at 4°C. At this stage, most cells lost their
dorsal side and nuclei, but some still had these parts of the cell,
which could be removed by gentle shearing under a phase-contrast
microscope with a stream of extraction buffer. The material attached to
culture dishes was gently washed by Triton X-100-based extraction
buffer [0.05% Triton X-100 (Wako), 20 µg/ml Trasylol, 1 µg/ml
leupeptin, and 1 µg/ml pepstatin in PBS, pH 7.4] for 3-30 min.
Still attached to coverslips were mainly stress fibers, and such
preparations were used for protein localization and contraction studies.
Immunofluorescence microscopy.
Cells and stress fiber models were fixed with 1% paraformaldehyde in
PBS for 30-60 min and treated with 10% normal goat serum for
1 h at room temperature. They were then stained with anti-MLCK (1:100), anti-calmodulin (1:100), anti-MBS (1:100), anti-myosin (1:100), or anti-vinculin (1:400) for 60 min. After being washed in
PBS, samples were incubated with fluorescein (Cappel, Durham, NC)-,
rhodamine (Cappel)-, or Texas red (EY-Lab, San Mateo, CA)-labeled goat
anti-rabbit or anti-mouse IgG. Some specimens were double-stained with
one of the antibodies (and the appropriate secondary antibody) and
rhodamine-labeled phalloidin.
Specimens were observed using a Zeiss Axiophot (Carl Zeiss, Germany)
epifluorescence microscope with an apochromat ×63 [numerical aperture
(N.A.) 1.4, oil-immersion] objective lens. Fluorescent images were
photographed by using Kodak T-Max 400 film (Eastman Kodak, Rochester, NY).
Electron microscopy.
For thin-section electron microscopy, isolated stress fiber models on
coverslips were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 30 min
at room temperature. Fixed stress fibers were washed for 30 min in 0.1 M sodium cacodylate buffer and postfixed for 1 h with 1%
OsO4 in the same buffer at 4°C. Samples were dehydrated through a graded series of ethanol (50, 65, 75, 85, 95, 99, 100%) and
embedded in Epon 812. Thin sections were stained with uranyl acetate
and lead citrate. Samples were examined with a 2000FX electron
microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV.
Contraction of stress fibers.
Isolated stress fiber models in a perfusion chamber were first immersed
in a wash solution (10 mM imidazole, 100 mM KCl, and 2 mM EGTA) and
perfused with an Mg-ATP solution (0.1 mM ATP, 3 mM MgCl2, 1 mM EGTA, 75 mM KCl, and 20 mM imidazole, pH 7.2) with 1 mM
CaCl2. Contraction was observed under a phase-contrast
microscope (Zeiss Axiophot with a plan-neofluar ×63, N.A. 1.25, oil-immersion, antiflex objective lens; Carl Zeiss) equipped with a
video-enhanced imaging system for 5-20 min. The video system
consisted of a high-resolution charge-coupled device camera
(C2400-77; Hamamatsu Photonics, Hamamatsu, Japan) and a digital
image processor (Image Sigma-II; Nippon Avionics, Tokyo, Japan), and
images were recorded by a high-resolution laser video disk recorder
(LV-250H; TEAC, Tokyo, Japan).
Green fluorescent protein-labeled actin expression and inhibitor
experiments.
Human foreskin fibroblasts (FS-133) were transfected with the
pEGFP-actin vector obtained from Clontech (Palo Alto, CA) using the
Tfx-50 reagents (Promega, Madison, WI). Transfected cells were
cultured as described in Cell culture. For
time-lapse confocal laser-scanning microscopy, green fluorescent
protein (GFP)-actin-transfected cells were plated on a culture dish (5 cm in diameter) and placed on a temperature-controlled stage at 37°C
(Tokai, Shizuoka, Japan). Culture medium was exchanged every 20 min for
long-term observations.
For inhibitor experiments, FS-133, WI-38, and endothelial cells as well
as GFP-actin-transfected FS-133 cells were treated for 60 min with
Y-27632 (10 µM), HA1077 (30 µM), wortmannin (100 nM), ML-7 (25 µM), ML-9 (100 µM), KT5926 (15 µM), W-5 (100 µM), or W-7 (100 µM). Each of these inhibitors was added to culture medium. Samples
were observed under a confocal laser-scanning fluorescence microscope
(Fluoview with a LUMPlanFl ×60, N.A. 0.90, water-immersion lens;
Olympus, Tokyo, Japan) or a video-enhanced phase-contrast microscope.
Some samples were fixed with paraformaldehyde and stained
simultaneously with anti-vinculin (Sigma) and rhodamine-labeled phalloidin as described in Immunofluorescence
microscopy. Recovery experiments were done by treating
cells with one of these inhibitors for 1 h and washing them with
fresh medium, and the process of recovery was recorded under time-lapse
confocal microscope or a video-enhanced phase-contrast microscope.
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RESULTS |
Effect of various inhibitors on living fibroblasts.
Stress fibers consist of bundles of actin filaments and can be best
observed in various types of cultured cells. Despite its excellent
detectability, fluorescence microscopy cannot provide accurate size
information for stained structures. Phase-contrast microscopy, on the
other hand, reveals the size of the structure. Because compactness of
the actin filaments in stress fibers causes enough refractive index
difference between the stress fibers and the surrounding cytoplasm, one
can observe the stress fibers by phase-contrast microscopy.
Figure 1a
is a video-enhanced phase-contrast image of a living fibroblast and
shows stress fibers in the basal part of the cell (basal stress fibers;
see Ref. 13). There were many thin stress fibers located
in the central (i.e., away from the lateral cell border) portion of the
cell (Fig. 1a, arrowheads). In addition, there were thicker
phase dense structures associated with the gently arched smooth parts
of the lateral cell border (Fig. 1a, arrows). Because they
may also be stress fibers, we investigated the structure in more detail
by electron microscopy. The stress fiber isolation procedure we had
reported earlier enabled us to stabilize stress fibers while other
cellular structures were effectively removed (12). Figure
1b shows a structure, found at the lateral border of a cell,
that contains parallel microfilaments in a bundle. This ultrastructure
is identical, even at a high magnification, to that of stress fibers
located in the central part of the cell (Fig. 1c). The only
difference between the two was their widths; the peripheral stress
fibers were significantly thicker than the central fibers. Stress
fibers stained with rhodamine-labeled phalloidin also revealed the
peripheral and central stress fibers (Fig. 1d). Note that,
in general, the stress fibers at the cell periphery stain more strongly
than those in the center. The staining intensity of the cell cortex was
much weaker compared with that of peripheral stress fibers. Thus there are two types of basal stress fibers, those located in the central region of the cell (central stress fibers; Fig. 1a,
arrowheads) and those associated with the lateral cell border
(peripheral stress fibers; Fig. 1a, arrows).

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Fig. 1.
Morphology of peripheral and central stress fibers.
a: video-enhanced phase-contrast image of a control living
fibroblast showing the central (arrowheads) and peripheral (arrows)
stress fibers. Electron micrographs show isolated peripheral
(b) and central (c) stress fibers.
Low-magnification micrographs are included so that the peripheral
location of the stress fiber (b) can be clearly shown. Boxed
areas in b and c are enlarged in insets. Note
that the peripheral fiber is thicker than the central stress fibers.
d: control fibroblast fixed and stained with
rhodamine-labeled phalloidin.
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We wondered whether the two types have different properties other than
morphological. Because it is now known that stress fiber formation and
maintenance are regulated by Rho and Rho-kinase, presumably by
controlling the level of MRLC phosphorylation (1, 3), we
thought that the two stress fiber types might respond differently to
various inhibitors that affected MRLC phosphorylation. Living
fibroblasts were treated with Rho-kinase inhibitors, MLCK inhibitors,
or calmodulin inhibitors, and their stress fiber patterns were studied
by fixing and staining cells with rhodamine-phalloidin.
When cells were treated for 1 h with the specific Rho-kinase
inhibitors Y-27632 (Fig. 2,
a-c) or HA1077 (data not shown), the number of central
stress fibers was greatly reduced. Interestingly, the peripheral stress
fibers were hardly affected, and the general cell shape was preserved.
However, peripheral stress fibers were greatly affected when cells were
treated with ML-7, a specific MLCK inhibitor (Fig. 2,
d-f). They appeared to be shortened and disrupted, and the cells became more rounded. Although the number is
reduced, the central stress fibers were still present in these cells.
Fibroblasts treated with other MLCK inhibitors such as ML-9 and KT5926
also exhibited the same response (data not shown). W-5 and W-7 are
calmodulin inhibitors, and cells treated with these drugs have shown
clear and specific reduction in peripheral stress fibers, leaving the
central type unaffected (Fig. 2, g-i). As was the case
for cells treated with the MLCK inhibitors, the calmodulin inhibitors
also induced cell rounding. These observations suggest that the loss of
peripheral stress fibers causes cell rounding. Effects of various
inhibitors on FS-133 cells are summarized in Table
1. These effects were reproducible and
were observed in almost all of the cells treated. To ascertain that
these drug effects were not limited to FS-133 cells, we also treated
bovine endothelial and WI-38 cells in the same way. We observed the
identical drug effects in these cells, suggesting that our findings are not cell type-specific events.

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Fig. 2.
Effects of Rho-kinase (Y-27632), myosin light chain
kinase (MLCK; ML-7) and calmodulin (W-7) inhibitors on peripheral and
central stress fibers of fibroblasts. a-c: cell treated
with Y-27632 and stained with rhodamine-phalloidin showing reduced
number of central stress fibers. Peripheral fibers are present.
d-f: cell treated with ML-7. Rhodamine-phalloidin
staining shows peripheral stress fibers more severely affected than
central fibers. g-i: fibroblast treated with W-7 and
stained with rhodamine-phalloidin. Like the cell treated with the MLCK
inhibitor, peripheral but not central stress fibers were severely
affected. The cells treated with ML-7 (d-f) or W-7
(g-i) lost their spread morphology.
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Association of contractile proteins with drug-resistant stress
fibers.
Stress fibers are not just bundles of actin filaments but contain a
host of actin and myosin binding proteins (5, 6, 12).
Whether or not the drug-resistant stress fibers were still associated
with such proteins, we used the Y-27632-resistant stress fibers and
investigated whether certain of the known stress fiber component
proteins were associated with the remaining fibers. Because contraction
and/or tension production is the major function of stress fibers, we
have selected proteins that are related to stress fiber contraction and
its regulation.
Fibroblasts treated with Y-27632 for 1 h were fixed and stained
with anti-MLCK (Fig. 3a) or
anti-myosin (Fig. 3b) together with rhodamine-phalloidin for
actin visualization. The double-labeled cells (Fig. 3, a and
b) showed that the remaining peripheral stress fibers
contained myosin and MLCK. Calmodulin was also associated with the
stress fibers (data not shown). Thus the remaining peripheral stress
fibers still contain the MLCK/calmodulin-based actin-myosin contractile
system even after Y-27632 treatment. The tight appearance of the
remaining peripheral stress fibers suggests that they may indeed be
under tension. The same observation was made in cells treated with
HA1077, another Rho-kinase inhibitor (data not shown). Thus, from the
standpoint of molecular composition, the remaining stress fibers are
not different from those in control cells.

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Fig. 3.
Distribution of MLCK and myosin
in stress fibers after Y-27632 treatment. Y-27632-treated fibroblasts
were double-stained with rhodamine-phalloidin and anti-MLCK
(a) or rhodamine-phalloidin and anti-myosin
(b).
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Time-lapse observation of living cells treated with inhibitors.
The effects of inhibitors on stress fibers were examined using living
cells. Fibroblasts transfected with GFP-actin were observed using a
confocal time-lapse recording system for up to 1 h while they were
treated with either a Rho-kinase or an MLCK inhibitor. These continuous
observations allowed us to ascertain the origin of remaining stress
fibers. They also allowed us to study closely how each type of stress
fiber was affected by the drugs. Many cells were studied, and the most
prevalent types of stress fiber responses to various inhibitors are
described here. Fibroblasts expressing GFP-actin developed stress
fibers and exhibited the cell shape indistinguishable from those of
nontransfected cells. Figure 4 shows a
series of fluorescence images of the same cell being treated with
Y-27632. Although overexpressed GFP-actin gave a considerable level of
general background fluorescence, both the central and the peripheral
stress fibers were clearly recognized when the Rho-kinase inhibitor was
added (Fig. 4, time 00:00). With time, however, the central stress
fibers gradually disappeared (Fig. 4). On the other hand, the
peripheral stress fibers were not lost (Fig. 4, arrowheads).
Interestingly, the cell continued to exhibit membrane-associated
activities such as formation of new lamellipodia (Fig. 4, arrow) and
extension of processes at the periphery.

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Fig. 4.
Effects of Y-27632 on living fibroblasts expressing green
fluorescent protein (GFP)-labeled actin. The drug was added at
time 0, and the cell was imaged by confocal time-lapse
microscopy using GFP-actin fluorescence. Time of drug treatment (in
hours:minutes) is indicated for each plate. Note that central stress
fibers are gradually lost, while peripheral fibers (arrowheads) remain.
Note also that new cell extensions (arrow) are being formed in the
presence of the Rho-kinase inhibitor.
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Our data show that central stress fibers disassemble in cells treated
with Y-27632 and that 1 h of treatment is sufficient to remove
practically all of them. Concomitant with the loss of central
stress fibers, focal adhesions were also lost (compare Fig.
5, a and b). These
drug effects were reversible. When cells incubated with the Rho-kinase
inhibitor for 1 h were washed with fresh culture medium and
incubated, central stress fibers as well as focal adhesions were
gradually reorganized (Fig. 5c). Complete recovery was
achieved in ~3 h (Fig. 5d).

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Fig. 5.
Recovery of stress fibers and focal adhesions in cells being washed
after 1 h of Y-27632 treatment. Untreated control cells
(a), cells treated with Y-27632 for 1 h (b),
and cells washed with fresh medium for 1 (c) or 3 h
(d) were fixed and then double-stained with
rhodamine-phalloidin (red) and anti-vinculin (green). Yellow staining
indicates overlap of the two colors where F-actin and vinculin
colocalize. Before drug treatment, cells had well-developed stress
fibers and large focal adhesions in both peripheral and central cell
regions (a). After 1 h of Y-27632 treatment, cells had
peripheral stress fibers but only a few central stress fibers and small
focal adhesions at the cell periphery (b). After cells were
washed with fresh medium, stress fibers and focal adhesions were
gradually reorganized at the central portion. Cells recovered for 1 (c) or 3 h (d) are
shown.
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We similarly analyzed the effect of ML-7, a specific inhibitor for
MLCK, using fibroblasts transfected with GFP-actin (Fig. 6). Our earlier experiments (Fig. 2,
d-f) indicated that ML-7 affected the peripheral stress
fiber more severely than the central type. As shown in Fig. 6, the
length of peripheral stress fibers gradually shortened, many of them
within 40 min. Fibroblasts also rounded up during the treatment with
ML-7, presumably due to the loss of peripheral fibers. However, in
contrast to the Y-27632 treatment that caused disassembly of the
central stress fiber (Fig. 5), cells treated with ML-7 for 40 min to
1 h exhibited many central stress fibers, albeit they appeared
thinner and less in number. Cells being treated with ML-7 did not show
peripheral membrane activities, such as membrane ruffles and
lamellipodia/filopodia extension. This is quite contrary to the effect
of the Rho-kinase inhibitor shown in Figs. 3-5.

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Fig. 6.
Effects of ML-7 on living fibroblasts expressing
GFP-actin. The drug was added at time 0, and the cell was
imaged by confocal time-lapse microscopy using GFP-actin fluorescence.
Time of drug treatment (in hours:minutes) is indicated for each plate.
Peripheral stress fibers were gradually shortened and/or disrupted.
Although the number of central stress fibers decreased slightly, they
persisted throughout the observation period. Note that the cells are
more rounded and that motile activities at the cell border are
inhibited by the drug.
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Recovery of cells from ML-7 treatment was also studied. Fibroblasts
treated with ML-7 for 1 h were washed with fresh medium at
time 0 and observed with a video-enhanced phase-contrast
microscope for 1 h (Fig.
7a). The most obvious response
was rapid extension of lamellipodia, followed by cell spreading. To
observe stress fibers and focal adhesions, we fixed recovering cells
and stained them simultaneously with rhodamine-phalloidin and
anti-vinculin. After being treated with ML-7 for 1 h, cells were
rounded and had no obvious arched stress fibers at the cell periphery
(Fig. 7b, time 0). The lateral cell border
receded so that it presumably rested on straight central stress fibers.
Interestingly, ML-7 treatment did not appear to affect focal adhesions,
because many anti-vinculin staining plaques were present and their size
and morphology were comparable to those in control cells (Fig.
5a). Cells allowed to recover for 45 min had well-developed
arched peripheral stress fibers and began to spread (Fig.
7b, 45 min). Note the presence of filopodia in this cell. By
2 h of recovery, cells looked normal with the fully developed
stress fiber system (Fig. 7b, 120 min).

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Fig. 7.
Recovery of peripheral stress fibers and lamellipodia in
cells washed after 1 h of ML-7 treatment. a:
time-lapsed images of a recovering cell viewed by video-enhanced
phase-contrast microscopy. Time of recovery (in hours:minutes) is
indicated for each plate. With time, the cell spread as lamellipodia
were formed at the cell periphery. b: recovering cells were
fixed (0, 45, and 120 min of recovery) and double-stained with
anti-vinculin (green) and rhodamine-phalloidin (red). Yellow staining
indicates where the two colors overlap. Peripheral stress fibers and
focal adhesions were gradually reformed as the cell respread. Full
recovery was observed in 2 h.
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Our data show that the peripheral and central stress fiber systems have
different drug sensitivity. The peripheral type was more sensitive to
MLCK inhibitors than to Rho-kinase inhibitors, while the central stress
fibers were more sensitive to Rho-kinase inhibitors than to MLCK
inhibitors. These results indicate that there may be some physiological
differences between the two types of stress fibers. Using a contractile
stress fiber system, we investigated whether the mode of contraction
was different between these stress fibers.
Contraction of isolated peripheral and central stress fibers.
Stress fiber models were made by extracting cells with Triton X-100
(12). These models contracted when Mg-ATP was added in the
presence of Ca2+. To observe the mode of contraction of
stress fibers, we used a video-enhanced phase-contrast microscope and
time-lapse recording. Contraction of peripheral stress fibers (Fig.
8, arrowheads) occurred before the
central fibers (Fig. 8, arrows) began to contract. In fact, the central
fibers began to contract when the peripheral fibers had almost finished
contacting. The speed of contraction of each type of stress fibers was
almost identical (for the speed of isolated stress fiber contraction,
see Ref. 12). These observations indicate that the onset
of contraction is differently regulated between the two types of stress
fibers.

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Fig. 8.
Contraction of isolated stress fibers. Contraction of
isolated peripheral (arrowheads) and central (arrows) stress fibers are
shown. These video-enhanced phase-contrast images are from time-lapsed
recordings. Arrowheads indicate the two ends of a peripheral stress
fiber. Arrows indicate the ends of a central stress fiber. Contraction
of the peripheral stress fiber started before the central fiber began
to shorten. Time intervals are indicated in seconds after Mg-ATP
addition.
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DISCUSSION |
Stress fibers are commonly thought of as cytoskeletal structures
found along the basal plasma membrane. However, they are also found in
other parts of the cell. In addition to the basal stress fibers located
in the basal portion of the cell, we have shown the presence of apical
stress fibers that run along the apical plasma membrane
(13). The two types of stress fibers are also found in in
situ cells (10). The ends of stress fibers are anchored to
the plasma membrane. Both ends of basal stress fibers are anchored to
focal adhesions. Our previous study revealed that one end of the apical
stress fiber is anchored to the apical plasma membrane via a structure
we termed the apical plaque (13) and that the other end is
anchored to the basal membrane, presumably to the focal adhesion. In
cells forming a monolayer, such as endothelial cells in situ, we have
found that apical stress fibers run between the apical plaque and the
lateral cell-cell adhesion site (9). Furthermore, we have
some preliminary data indicating the presence of stress fibers spanning
between the lateral cell adhesion and the focal adhesion (Kano and
Katoh, unpublished observation). The categorization discussed here is
solely based on morphological features of stress fibers. Whether or not
there are differences in their molecular composition, contractility,
stability, and other features is not known.
In the present study, we show different responses among basal stress
fibers to various inhibitors. As summarized in Table 1, the central
stress fiber is more sensitive to Rho-kinase inhibitors than to MLCK
and calmodulin inhibitors. On the other hand, the peripheral stress
fiber is more susceptible to the same set of MLCK and calmodulin
inhibitors than to the Rho-kinase inhibitors. These inhibitor studies
indicate that activities of kinases that regulate myosin function are
necessary to maintain the stress fiber system in the cell and that two
kinase systems control two different sets of stress fibers. Thus it
appears that cells have a sophisticated system for maintaining this
cytoskeletal structure.
The reason for cells to use different kinase systems for the peripheral
and the central stress fibers is not clear. It is likely that the
difference reflects the structural and/or functional differences of
these stress fibers. Besides the fact that the peripheral stress fibers
are generally thicker than the central ones, there is no other obvious
structural difference between the two types. To our knowledge, there
has been no study focused on the molecular compositions of the two
stress fiber types. This study provides a rationale for carefully
analyzing the composition of various types of stress fibers.
Our present study has revealed that the two types of stress fibers have
different contractile properties. When stress fiber models were
reactivated, peripheral stress fiber contraction was almost complete
when the central ones began to shorten. Because the components, such as
ATP and Ca2+, in the reactivation solution should be
equally accessible to all the stress fibers in the model, the
difference in their reactivation time might be an indication that the
contraction of the two stress fiber systems is regulated differently.
In a separate study, we found that isolated stress fibers could be
reactivated by activating either MLCK or Rho-kinase and that MLCK
initiated contraction faster and more extensively than Rho-kinase
(10a). It is possible that the contraction of peripheral
stress fibers that are sensitive to MLCK inhibitors is regulated
largely by MLCK, while the contraction of central stress fibers that
are sensitive to Rho-kinase inhibitors depend mainly on Rho-kinase.
This hypothesis predicts delayed reactivation time for central stress
fibers, and our data are in agreement with this. The hypothesis also
predicts that the contraction of the peripheral and the central stress
fibers may be inhibited by MLCK and Rho-kinase inhibitors,
respectively, and that the two kinases may have different localization
patterns. However, we observed that the contraction of both stress
fiber types was inhibited by either type of inhibitor alone
(10a), and our localization studies so far showed that both
kinases were present in both stress fiber types without any clear-cut
differences. These results were obtained from both fixed cells and
isolated stress fibers as well as different cell types. Thus, at
present, there is no easy explanation for the observed difference in
the stress fiber reactivation times.
Our data show that Rho-kinase activity is necessary for maintaining the
organization of the central stress fibers, including the focal
adhesions associated with them. However, because there are multiple
targets of Rho-kinase in the cell, it is not easy to determine the
mechanism for the observed effects of Rho-kinase inhibitors. It is
possible that the target of Rho-kinase is not a component of the stress
fiber but is a component of the focal adhesion. This could cause stress
fibers to lose tension needed to maintain their structure, making them
disassemble. Our study also showed that the peripheral stress fibers
and the formation of lamellipodia and filopodia were severely affected
by MLCK but not Rho-kinase inhibitors. Activities of MLCK were also
needed to maintain the spread morphology of the cell. Because MLCK is a
specific kinase for the regulatory light chain of myosin, our results
indicate that actomyosin contraction is necessary to maintain cell
spreading and to form lamellipodia and filopodia. How contraction is
required for developing cell extensions is an interesting puzzle that
needs to be resolved.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by grants-in-aid for Scientific
Research from the Ministry of Education, Science, and Culture of Japan,
grants from the Ministry of Health and Welfare of Japan, special
coordination funds of the Ministry of Education, Culture, Sports,
Science, and Technology of Japan, and a grant from the Organization for
Pharmaceutical safety and Research.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. Katoh, Dept. of Structural Analysis, National Cardiovascular Center Research Institute, 5 Fujishirodai, Suita, Osaka 565-8565, Japan (E-mail: katoichi{at}ri.ncvc.go.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 November 2000; accepted in final form 12 February 2001.
 |
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