(Received for publication, May 3, 1995; and in revised form, May 31, 1995)
From the
We investigated the role of myosin light chain
(MLC) phosphorylation (MLC-P) in non-muscle contractility
by comparing MLC-P and the contractile properties of wild type 3T3
fibroblasts and 3T3 fibroblasts expressing the catalytic domain of
myosin light chain kinase (tMK). MLC-P is 0.96 mol of
PO
/mol of MLC
in cells expressing tMK compared
to 0.20 mol of PO
/mol of MLC
in control cells.
Expressing tMK also results in a 2-fold increase in cortical stiffness
compared to control cells. Contractile properties were quantified by
growing wild type and transfected fibroblasts in collagen and attaching
the ensuing fibers to an apparatus for performing mechanical
measurements. Serum stimulation resulted in a dose-dependent increase
in force with maximal force generated in the presence of 30% (v/v)
serum. Surprisingly, MLC-P did not increase in wild type cells
following stimulation with 30% serum, and tMK expression did not affect
the contractile properties of fibers made from these cells. Moreover,
the dose responses to serum, maximal force, force-velocity
relationships, and dynamic stiffness were similar in the wild type
cells and fibroblasts expressing tMK. These data demonstrate that
non-muscle cells can generate force without an increase in MLC-P, and
that an increase in MLC-P does not affect the contractile properties of
fibroblast fibers.
Cellular force generation requires energy transduction. ATP
hydrolysis due to the cyclic interaction of actin and filamentous
myosin (i.e. myosin II) is a major pathway for converting
chemical energy into mechanical work(1) . This reaction is
regulated in mammalian smooth muscle (2) and non-muscle (3) cells by the phosphorylation of the 20-kDa light chain of
these myosins (MLC) (
)by the
calcium-calmodulin-dependent enzyme myosin light chain kinase.
MLC
phosphorylation (MLC-P) is essential for force
generation by smooth muscles, and this reaction has also been
implicated in modulating the rate of cross-bridge cycling and the speed
of contraction(2) . Increases in MLC-P have also been
correlated with force generation (4, 5) and filament
dynamics (6) in fibroblasts following serum stimulation.
However, the role of MLC-P in fibroblast contractility is not known
with certainty.
In order to better understand the mechanisms that regulate non-muscle contractility, we compared the contractile properties and MLC-P in wild type 3T3 fibroblasts and fibroblasts expressing the catalytic domain of myosin light chain kinase (tMK). tMK was expressed using a retroviral vector based on the murine Moloney leukemia virus(7) . The contractile properties of the fibroblasts were quantified by growing cells in collagen and performing mechanical studies on the ensuing fibers in myobaths(8) . Although tMK expression substantially increases MLC-P and cortical stiffness, MLC-P did not change with stimulation and force generation. In addition, the contractile properties are similar in the wild type cells and cells expressing tMK following serum stimulation. These data suggest that an increase in MLC-P is not obligatory for force generation by non-muscle cells.
Relative changes
in MLC-P were quantitated in the following manner. Monolayers of wild
type or infected cells grown on 60-mm dishes or cells grown into fibers
in silicone rubber ``molds'' (see below) were washed in
PO-free DMEM and incubated in 1 ml of PO
-free
DMEM containing 50 µCi of
P
for 6 h at 37
°C. Cells grown in dishes or fibers were stimulated by adding fetal
bovine serum (30%, v/v) to the media containing
P
for 5 or 20 min. The cells or fibers were washed twice at the
appropriate times with ice-cold Gey's buffer and extracted, and
the myosin immunoprecipitated as described previously(12) . The
immunoprecipitates were then separated by SDS-PAGE (5%-20% gradient).
Following Coomassie staining, the gels were air-dried and exposed to
x-ray film, and the bands on the autoradiogram representing MLC
were quantified densitometrically. Coomassie Blue staining of the
myosin heavy chain in each of the immunoprecipitates following SDS-PAGE
was also quantified densitometrically. The areas of scans of the light
chains were then divided by the areas of the corresponding scans of the
heavy chain. The MLC-P data were normalized by dividing the ratios at 5
and 20 min following serum stimulation in control and tMK cells by
their respective ratios at time 0.
The length of the fiber was controlled with a Cambridge Technologies
ergometer. Force-velocity relations were measured by imposing constant
shortening velocities on the fiber and measuring the resulting force.
Stiffness was measured by imposing rapid (<1 ms) shortening and
stretching steps (-2.6 to +0.6% L). A
series of 8 step changes at 60-s intervals constituted an experimental
set for the measurement of stiffness; the slope of the peak force versus length curve was taken as stiffness.
Expression of tMK in active form was demonstrated by
immunoprecipitating myosin from cells grown in tissue culture dishes
and labeled with P
. SDS-PAGE followed by
autoradiography demonstrated a substantial increase in MLC-P in cells
infected with tMK compared to wild type cells or cells infected with
the empty plasmid (Fig.1). Quantitation of the stoichiometry of
MLC-P demonstrated a 4.8-fold increase in MLC-P in tMK cells compared
to control cells (Table1). Phosphopeptide maps of trypsin
digests demonstrated the presence of a phosphopeptide in myosin
immunoprecipitated from tMK cells that had a pI of 4.5, which is the pI
reported for the MLC
phosphopeptide containing the Ser
residue phosphorylated by myosin light chain kinase(15) . This
peptide also comigrated with a peptide present in purified smooth
muscle and macrophage myosins and myosin immunoprecipitated from neo
cells that were phosphorylated in vitro (Fig.2).
Figure 1:
MLC phosphorylation in NIH
3T3 fibroblasts. Wild type cells (WT), neo (NEO), and
tMK cells (tMK CELLS) grown in tissue culture dishes and wild
type cells grown into fibers (WILD TYPE FIBERS) were labeled
with
P
. Wild type cells grown in fibers and
the tMK cells were stimulated with serum for 5 or 20 min and extracted,
and the myosin immunoprecipitated as described under ``Materials
and Methods.'' The immunoprecipitates were separated by SDS-PAGE
and exposed to x-ray film. Note the increased level of MLC-P
(MLC
) in tMK cells compared to the wild type and neo cells
and the absence of an increase in MLC-P following serum stimulation in
wild type fibers or tMK cells. The amount of myosin heavy chain (MHC)
in each of the immunoprecipitates was similar based on Coomassie Blue
staining.
Figure 2:
Phosphopeptide map analysis of the site of
MLC-P in tMK cells. Myosin was immunoprecipitated from tMK cells (tMK) labeled with P. Myosin immunoprecipitated
from unlabeled control cells (NEO) and myosin purified from
tracheal smooth muscle (TSM) and macrophages (MØ) were phosphorylated with purified chicken gizzard
myosin light chain kinase, in vitro, and the MLC
in each sample was digested and analyzed as described under
``Materials and Methods.'' This is a picture of an
autoradiogram of a single isoelectric focussing gel showing the region
around pH 4.5. The lane marked NEO was exposed for a longer
period of time because the immunoprecipitated myosin incorporated
phosphate poorly.
Experiments were then performed to investigate the effect of tMK expression and the resulting increase in MLC-P on the stiffness of the cortical cytoskeleton. Cortical stiffness was quantified using the ``cell poker,'' which measures the force required to indent the free surface of adherent cells(16, 17) . Analysis of histograms of stiffness values obtained by poking individual neo or tMK cells demonstrated a shift to higher stiffness values in tMK cells, and that the mean stiffness in tMK cells was almost twice that in neo cells (Table1).
We then compared the contractile properties of wild type Swiss or NIH 3T3 cells and cells expressing tMK. Fibers composed of these three cell types were mounted under isometric conditions in MOPS-PSS, and the length was adjusted to match the original fiber length in the mold. After stress relaxation, force measurements indicated a steady level of baseline force that was virtually identical in wild type Swiss and NIH 3T3 cells and tMK cells (Table2). Addition of serum increased isometric force (Fig. 3). Force fell below the initial baseline level upon the addition of 1 µM cytochalasin D (Table2), and the extent and time courses for relaxation were very similar in wild type and tMK cells ( Fig.3and Table 2). The latter finding suggests that the intrinsic mechanical activity in all types of fibers was similar. Contracted fibers also relaxed when the serum was replaced with MOPS-PSS, and these contraction/relaxation cycles could be repeated for at least 8 h(8) . Serum induced virtually identical dose-dependent contractions in wild type Swiss or NIH 3T3 cells and tMK cells (Fig.4). Maximal isometric forces, which were produced in response to 30% serum, developed by fibers made from wild type Swiss 3T3 cells, NIH 3T3 cells, and tMK cells were also very similar (Table2).
Figure 3: Contraction-relaxation cycle of wild type (WT) and tMK (tMK) cells. Fibers made from wild type NIH 3T3 cells and tMK cells were mounted in myobaths in MOPS-PSS buffer as described under ``Materials and Methods.'' Addition of calf serum (CS) to a final concentration of 30% resulted in force generation. These contractions could be reversed by washing out the serum or by adding cytochalasin D (Cyto D) to a final concentration of 1 µM. The time courses for contraction and relaxation are virtually identical in the two types of fibers.
Figure 4: Dose-response relationship for serum and force. Fibers made from wild type Swiss (SW) or NIH (NIH) 3T3 cells or NIH 3T3 cells expressing tMK (tMK) were mounted in myobaths in MOPS-PSS buffer (see ``Materials and Methods''). Cumulative dose-response curves were performed by serially replacing the buffer with new buffer containing increasing concentrations of serum. A total number of 6 fibers were analyzed in each group, and there were no statistically significant differences in the data at any serum concentration. The basal and maximal levels of force generated by the three types of fibers are given in Table2, and these values were not significantly different.
The contractile properties of the two types
of fibers were characterized further by imposing constant shortening
velocities and measuring the consequent force responses as described
previously(8) . The velocity-force relations were fitted with
the Hill equation, (P + a) (V + b) = b(P
+ a), using a nonlinear least squares routine
(Origin) and taking P
as the initial force. For
contractions induced by 30% serum (n = 24), the
velocities at zero force (V
) were virtually
identical, as was the dimensionless curvature parameter a/P
, for wild type and tMK fibers (Table2). Fiber stiffness was measured by imposing rapid (<1
ms) step changes in length. A plot of the peak force responses against
the imposed step length change showed that the peak force responses of
fibroblast fibers were linear in the region where stretches were
imposed, and this linear range extended to short step
decreases(8) . Stiffness values were not significantly
different in fibers composed of wild type and tMK cells (Table2).
Finally, MLC-P following serum stimulation was
quantified by immunoprecipitating myosin from cells grown in monolayer
or from cells grown into collagen fibers. Most studies were performed
on cells grown on dishes because it was much more difficult to handle
the P
-labeled fibers. Similar phosphorylation
results were obtained with either cells grown on dishes or into fibers.
As shown in Fig.1, there was no detectable increase in MLC-P
following serum stimulation of wild type cells grown into fibers.
Statistical analysis of the normalized phosphorylation data (Table1) at times 0, 5, and 20 min (force generation plateaued
at 20 min, Fig.3) did not demonstrate statistically significant
differences in these data (p > 0.05, n =
4). Moreover, MLC-P was initially high and did not increase further
following serum stimulation of tMK cells grown in culture ( Fig.1and Table 1).
We investigated the role of MLC-P in regulating the
contractile properties of non-muscle cells by comparing the contractile
properties and time courses of MLC-P in fibers composed of wild type
and fibroblasts expressing the catalytic domain of myosin light chain
kinase. To do so, we used a retroviral system to express the catalytic
domain of myosin light chain kinase in 3T3 fibroblasts. Proteolytic
digestion of purified myosin light chain kinase (18) or
insertion of a stop codon following lysine 793 (9) results in a
constitutively active, Ca-calmodulin independent form
of the enzyme that retains its substrate specificity for
MLC
. Fig.1demonstrates a high level of MLC
phosphorylation in unstimulated cells expressing tMK, thereby
verifying that tMK is expressed in active form in the infected cells.
Based on the well-characterized relationship between MLC-P and
smooth muscle contraction (2) and previous studies on
non-muscle cells(4, 5) , we had anticipated (a) an increase in MLC-P during force generation by
fibroblasts and (b) that a constitutive increase in MLC-P
would enhance the mechanical properties (i.e. maximal force,
time to peak force, V, stiffness). Our data do
not support either of these predictions. Both types of fibers contract
and relax at the same rate (Fig.2), they generate similar
levels of isometric force in response to increasing levels of serum
stimulation (Fig.3), and the contractile parameters (Table 2) are virtually identical despite substantial differences
in the levels of MLC-P. Moreover, there is no increase in MLC-P
following serum stimulation (Fig.1) in fibers composed of
either cell type. These data demonstrate that MLC-P is neither required
for force generation nor does increasing MLC-P affect the contractile
properties of fibroblast fibers.
In contrast, increasing MLC-P increases cortical stiffness. The cell poker quantifies the force required to indent the free surface of adherent cells, which is determined mainly by the cortical cytoskeleton (16, 17) . Myosin II is an important contributor to cortical stiffness in Dictyostelium discoideum(17) , and MLC-P has been shown to stabilize myosin filaments in mammalian cells(19) . Thus, the observation that myosin hyperphosphorylation increases cortical stiffness establishes two important points. First, it demonstrates for the first time that cortical stiffness is determined, at least in part, by the level of MLC-P. Second, it establishes that an increase in MLC-P has a local cellular effect. Surprisingly, this local effect does not appear to be transmitted to the fiber as a whole.
Perhaps the most striking
aspect of this study is that the apparent dissociation between
contractile parameters, which reflect an actomyosin-dependent
system(8) , and MLC-P. Most of the contractile parameters
measured in these fibers are very similar to those measured in smooth
muscle, a tissue whose contractile properties have previously been
shown to be regulated by MLC-P(2) . With the exception of
maximal isometric force, which is considerably lower than that reported
for smooth muscles (0.2-1.0 mN/mmversus 10-200 mN/mm
, (8) ), V
, the curvature parameter a/F
and stiffness values are comparable
in the fibroblast fibers and smooth muscle fibers(8) .
Nevertheless, there is no increase in MLC-P under conditions that
result in force generation in these fibers, nor does increasing the
level of MLC-P by expressing tMK affect the contractile properties.
These data suggest that the molecular mechanisms involved in regulating
energy transduction and determining the contractile responses of
fibroblast fibers and smooth muscles are fundamentally different.
Additional studies are required to determine the basis for these
differences.