(Received for publication, November 13, 1994; and in revised form, January 11, 1995)
From the
In striated muscles, contractility is controlled by
Ca binding to the regulatory protein complex
troponin, which is a component of the thin filaments. Troponin is an
allosteric inhibitor acting on tropomyosin to switch the thin filament
between ``on'' and ``off'' states. We have used an in vitro motility assay to examine troponin regulation of
individual actin-tropomyosin filaments moving over immobilized skeletal
muscle heavy meromyosin. The most striking observation is that the
actin-tropomyosin filament appears to be regulated as a single unit. At pCa 9.0, addition of up to 4 nM troponin causes the
proportion of filaments motile to decrease from >85% to 20% with no
dissociation of the filaments from the heavy meromyosin surface or
change in velocity. Increasing Ca
concentration
causes the filaments to be switched back on with half-maximal increase
in the proportion of filaments motile at pCa 5.8-6.0 and
a modest increase in filament velocity. This is an ``all or
none'' process in which an entire filament, up to 15 µm long,
switches rapidly as a single cooperative unit. Thus, the effect of
Ca
upon the thin filament is to recruit motile
filaments.
The Ca regulation of actomyosin in vitro by troponin and tropomyosin has been studied intensively over the
last 25 years, and a generally accepted mechanism for control of
contractility has emerged (reviewed in (1) and (2) ).
Tropomyosin plays the central role in regulation by moving its position
relative to actin, such that when the thin filament is switched
``on,'' it does not impede cross-bridge cycling. To switch
the thin filament ``off,'' tropomyosin moves to prevent
access to the ``strong'' myosin binding sites on actin,
thereby blocking the ATPase cycle. This movement of tropomyosin
relative to actin (3) is controlled by the inhibitory component
of troponin, troponin I, which acts as an allosteric inhibitor, and
this effect is regulated in response to Ca
via the
Ca
binding component of troponin, troponin
C(1) . In vitro, troponin regulates the V
of actin-tropomyosin-activated myosin
Mg
-ATPase while the K
is virtually independent of the degree of
activation(4) . In intact muscle, Ca
primarily controls the force and does not regulate unloaded
velocity(5, 6) . At first glance, the observations in vitro and in live muscle seem to be contradictory; however,
it should be remembered that both types of experiments measure
different properties of bulk material. Analysis of the performance of
individual contractile filaments would provide the opportunity to
elucidate the mechanism in greater molecular detail.
In principle,
the study of the movement of single actin filaments over myosin in
vitro is an ideal technique for investigating the protein switches
that regulate actomyosin. Rather, little work has been done on
Ca-dependent regulation of individual filaments by
skeletal muscle troponin and tropomyosin. Honda and Asakura (7) observed fluorescently labeled actin filaments moving
within an actomyosin gel. They showed that reconstituted rabbit
skeletal muscle thin filaments moved at either maximal or zero velocity
but described little detailed analysis. Wang et al.(8) studied the troponin from Limulus muscle and
observed that high concentrations of troponin halted actin-tropomyosin
filament movement, while Harada et al.(9) noted a
similar effect using rabbit skeletal muscle ``native
tropomyosin.''
We have used the in vitro motility
assay devised by Kron and Spudich (10) to make a detailed
quantitative analysis of skeletal muscle troponin-tropomyosin control
of actin filament movement over immobilized heavy meromyosin (HMM). ()We found that under suitable conditions, where 85% of
actin-tropomyosin filaments were motile, incorporation of troponin at
low Ca
concentrations controlled the proportion of
filaments that were moving without affecting velocity or displacing
actin from myosin. At a fixed inhibitory concentration of troponin,
increasing Ca
concentration released the inhibition
largely by increasing the proportion of filaments, which were motile
back to the level observed with actin-tropomyosin alone.
Actin, troponin, myosin, and tropomyosin were prepared from
rabbit skeletal muscle by standard
procedures(11, 12, 13, 14) . HMM was
prepared from myosin, and skeletal muscle F-actin was labeled with
rhodamine-phalloidin () as described by Kron et
al.(15) . F-actin
-tropomyosin and
F-actin
-tropomyosin-troponin complexes were formed at
10
assay concentration. 100 nM F-actin
, 100
nM skeletal muscle tropomyosin, and 0-80 nM troponin were mixed in 50 mM KCl, 25 mM Imidazole-HCl, 4 mM MgCl
, 1 mM EDTA,
5 mM dithiothreitol, pH 7.4, and incubated for 30-60
min. The complexes were diluted 10-fold immediately prior to infusion
into the motility cell.
All experiments were carried out using coverslips coated with silicone by soaking in 0.2% dichloromethylsilane in chloroform. A flow cell was prepared from a freshly siliconized coverslip and a microscope slide as described by Kron et al.(15) . Assay components and buffers were infused into the flow cell at 30-60-s intervals.
HMM was pretreated to
minimize the presence of ``rigor heads,'' which show ATP
insensitive binding to actin filaments. HMM, F-actin, and ATP were
mixed to final concentrations of 200 µg/ml, 150 µg/ml, and 1
mM, respectively, and incubated on ice for 10 min. The mixture
was centrifuged at 150,000 g to pellet F-actin with
associated ``rigor heads,'' leaving HMM in the supernatant
suitable for use in the motility assay for several hours. Two 50-µl
aliquots of HMM at 100 µg/ml were infused in buffer A (50 mM KCl, 25 mM Imidazole-HCl, 4 mM MgCl
,
1 mM EDTA, 5 mM dithiothreitol, pH 7.4) to provide a
coating of immobilized HMM on the coverslip. This was followed by 2
50 µl of buffer B (A + 0.5 mg/ml bovine serum albumin)
and then 2
50 µl of 10 nM actin
± associated tropomyosin-troponin in buffer A. 50 µl of
buffer C (B + 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 3
mg/ml glucose, 0.5% methylcellulose, ± troponin at assay
concentration) and 50 µl of buffer D (C + 1 mM ATP)
were then infused. Ca
concentration was varied by
incorporating Ca
-EGTA buffers in the final assay
buffers C and D.
The movement of actin-tropomyosin
filaments over the immobilized skeletal muscle HMM was observed under a
Zeiss epifluorescence microscope (63/1.25 objective) with a DAGE-SIT-68
camera and recorded on video tape. We only collected data from cells in
which at least 80% of actin
or
actin
-tropomyosin filaments were motile.
For analysis of filament movement, a sequence of 10 images was collected at 0.65-s intervals via a frame grabber installed in a Macintosh IIci computer. 20 filaments were chosen at random from a printout of the first image and were tracked manually using custom made software (ME Electronics, Reading, United Kingdom), which yielded x and y coordinates, velocity, and time for each step in the sequence.
We determined standard errors in velocity measurements with increasing numbers of actin filaments moving at an average of 3.4 µm/s. Standard error reduced from 0.07 to 0.03 between 5 and 20 filaments tracked but was only reduced by a further 0.013 in tracking another 150 filaments. Velocity, standard error or proportion of motile filaments showed no significant alteration in tracking filaments from separate sequences in the same assay or through longer time intervals.
The density of filaments attached to heavy meromyosin was determined
using a subroutine of the filament analysis program. From each assay,
five images were collected randomly from different areas of the flow
cell, and filament number was counted. The overall average provided a
measure of the filament density in each 100 µm of the
assay cell.
Figure 1:
SDS-gel
electrophoresis demonstrating binding of tropomyosin (Tm) and
troponin to actin at motility assay conditions. Samples of
the diluted and undiluted actin
tropomyosin and
actin
-tropomyosin-troponin complexes were centrifuged for
30 min at 150,000
g to sediment actin
filaments. SDS-gel electrophoresis of the pellets are shown as
digitized images with background subtraction. A and B show that tropomyosin was bound to the actin
in both
the undiluted (100 nM actin + 100 nM tropomyosin) and the 10-fold diluted samples, respectively. C and D show undiluted and 10-fold diluted 100 nM actin + 100 nM tropomyosin + 50 nM troponin.
In the
motility assay at 25 °C, 10 nM tropomyosin reduced the
proportion of actin filaments that were motile from >85%
to 42%. This provided a functional check that actin and tropomyosin
were associated under the conditions of the assay but was clearly
unsatisfactory for subsequent study of troponin regulation. When we
increased the temperature to 28 °C, 10 nM skeletal muscle
tropomyosin could be incorporated with >85% of the filaments
remaining motile. The velocity of motile actin
filaments
was not changed by the inclusion of 10 nM skeletal muscle
tropomyosin at either 25 or 28 °C (Table 1). We chose 28
°C as a suitable temperature for subsequent experiments.
Fig. 1(lanesC and D) demonstrates the binding of troponin to
actin-tropomyosin at motility assay conditions.
Determination of the band intensities by densitometry showed the
troponin I:troponin C:actin
ratio was 0.29:0.16:1 before
and 0.27:0.18:1 after dilution. The tropomyosin + troponin
T:actin
band ratio was 1.09 before and 1.10 after dilution.
In the motility assay at pCa 9.0, we observed that in the
presence of low concentrations of troponin, many of the
actin-tropomyosin filaments stopped moving. Some filaments
were completely stationary, whereas others exhibited
``stop-start'' motion. The velocity of sliding appeared to be
constant. To analyze filament motility quantitatively, we collected a
series of 10 video images at 0.65-s intervals for each assay. Since
there was no clear reason to select any particular population for
analysis, we measured the movement of 20 filaments chosen at random
from each series. Fig. 2shows the tracks of these filaments.
Since some filaments started or stopped between images, we calculated
instantaneous frame-to-frame velocity (9 for each filament) rather than
an average velocity over the whole sequence. At the fastest sampling
rate possible with our instrument (10 s
), filaments
still stopped within 1 frame.
Figure 2:
The effect of troponin on
actin-tropomyosin movement over HMM. The tracks of 20
filaments plotted at 0.65-s intervals from six separate assays with
increasing troponin concentration at pCa 9.0 are shown. The
velocity is proportional to the distance between points in each track.
Tracks with fewer than 10 points are from filaments that stopped or
started during the tracking sequence; examples are indicated by arrows. Immobile filaments appear as singledots; these are highlighted with graycircles.
The results are plotted in Fig. 3. They show that in all assays, the velocity of motile
filaments was unaffected by troponin (Fig. 3B).
Filaments were either motile at an average of 3.4 µm/s or were
stationary. The proportion of filaments that were motile, however,
decreased sharply with increasing troponin concentration (Fig. 3A). We also determined the average number of
filaments per 100 µm attached to the immobilized HMM (Fig. 3A). We found that this remained constant over
the range of troponin concentrations required to reduce the proportion
of motile filaments to 20%. The filament density declined at the higher
troponin concentrations. This decline in density at higher troponin
concentrations was also observed when troponin was added to pure actin
filaments in the absence of tropomyosin, but in these experiments,
troponin did not alter the fraction of filaments motile (Table 1).
Figure 3:
The effect of troponin on
actin-tropomyosin filament motility, velocity, and
attachment to heavy meromyosin. Frame-to-frame velocities were
calculated for each filament (9 values per 10 frame sequence).
Velocities >0.5 µm/s were defined as motile, and the proportion
of filaments moving and mean velocity of motile filaments were
calculated. Plots represent data from 50 filaments taken from four
separate assays. A, proportion of filaments motile and
filament density. B, velocity of motile filaments. Data points
represent means ± standard errors.
Figure 4:
Distribution of
actin-tropomyosin-troponin filament velocities at
increasing Ca
concentrations. The movement of 10
nM actin, 10 nM tropomyosin, 5 nM troponin
filaments was analyzed at a range of Ca
concentrations. Individual frame-to-frame velocities from each
tracked filament are plotted in a frequency histogram. Each histogram
contains data from 60 filaments taken from six separate
assays.
Figure 5:
The
effect of the concentration of Ca on
actin
-tropomyosin-troponin filament motility, velocity, and
attachment to heavy meromyosin. A, proportion of filaments
motile and filament density. B, velocity of motile filaments.
Data points represent means, with standard errors, from six
experiments. The proportion of filaments motile increased with
elevation of Ca
with half-maximal increase requiring
a free Ca
concentration of 10
M.
The
major effect of increasing Ca was to increase the
proportion of filaments that were motile ( Fig. 4(arrows) and 5A), without affecting the
number of filaments attached to the immobilized HMM (Fig. 5A). Half-maximal increase in the proportion of
filaments motile occurred between pCa 6.0 and 5.8, where there
was a sharp rise in % motility.
The frequency histograms in Fig. 4show the mean velocity and standard deviation of the
motile filaments at increasing Ca concentrations, and
these are plotted in Fig. 5B. The velocity is observed
to increase from 3.3 to 5.2 µm/s between pCa 9.0 and 4.5.
Similar experiments were carried out using 10 nM actin, 10 nM tropomyosin, 70 nM troponin I, and 140 nM troponin C at a range of
Ca
concentrations between pCa 9.0 and 4.5.
At pCa 9.0, 40% of the filaments were motile at an average
velocity of 4.2 µm/s. The proportion of filaments motile increased
to almost 80% at pCa 4.5 with half-maximal increase again
observed between pCa 6.0 and 5.8. The velocity of motile
filaments, however, showed no appreciable change (Table 1).
To
investigate further the apparent cooperativity in the
``switching'' of filaments, we re-analyzed our data from the
Ca titration experiments to study the behavior of
filaments that were longer or shorter than average. The proportion of
filaments <2 and >10 µm that were motile at each
Ca
concentration was recorded. There was no
significant difference between short, long, and total filaments. Less
than 20% of the filaments, short or long, were motile at pCa
9.0, and half-maximal increase was observed between pCa 6.0
and 5.8, with >80% of filaments moving at pCa 4.5.
The mechanism of Ca control of striated
muscle by troponin and tropomyosin has been determined from studies of
bulk material in solution or in intact muscle fibers. The motility
assay provides an ideal system to extend these studies at the level of
the individual thin filament. Studies to date (7, 8, 9) have shown that troponin,
tropomyosin, and low Ca
concentrations stop filaments
moving; however, those experiments have usually involved rather high
concentrations of tropomyosin and troponin, and little analysis of
movement quality has been attempted.
In this study, we have investigated troponin-tropomyosin control of thin filament motility based on the following criteria. 1) We have demonstrated that tropomyosin and troponin are actually bound to the actin filaments at motility assay conditions. 2) We have set experimental conditions such that at least 80% of filaments are motile in control assays. 3) Results obtained from different motility cells under the same conditions are consistent (see Table 1). 4) Filaments are randomly chosen for analysis of movement; thus, there are no a priori selection rules that might bias results. 5) A complete analysis of filament movement is made on a second-to-second time scale, from which we have determined velocity, proportion of filaments motile, and density of filaments attached to the HMM substrate.
The skeletal muscle actin-tropomyosin
complex is largely in the ``off'' state under typical test
tube experimental conditions (18, 19) and consequently
gives a lower activation of myosin Mg-ATPase than
actin itself. We observed a corresponding effect in the motility assay
at 25 °C where the incorporation of tropomyosin ``switched
off'' a considerable proportion of the actin filaments. The
problem could be avoided by using tropomyosin from smooth muscle, which
is predominantly in the ``on'' state at normal ionic
strengths(20, 21) . Alternatively we found that by
increasing the assay temperature to 28 °C, which favors the
``on'' state with skeletal muscle tropomyosin(22) ,
we could obtain a satisfactory and consistent level of motility with
>85% of the filaments motile.
The motility of actin-tropomyosin-troponin filaments was
regulated by Ca, and the major effect of increasing
Ca
was to increase the proportion of filaments that
were motile. Ca
also had a significant effect upon
the velocity of filament movement. Since this velocity change did not
occur when troponin was titrated against actin-tropomyosin, it seems
likely that the velocity effect was due to a separate
Ca
-dependent process. It is known that there is a
Ca
-dependent interaction between troponin T and
troponin C that influences ATPase activity(23, 24) .
When we examined Ca
control of the motility of
actin-tropomyosin-troponin I-troponin C filaments (Table 1), we
found the same effect on % motility as with whole troponin but no
effect upon velocity, thus supporting the hypothesis of dual regulatory
effects mediated by troponin I and troponin T.
The troponin
regulation results from the motility assay may readily be interpreted
in terms of the mechanism established from solution experiments. Since
troponin switches off actin filaments without causing them to
dissociate from the HMMADP
P
surface, the
troponin-tropomyosin switch does not appear to control weak
cross-bridge binding states; thus, the immobile filaments remain
attached to myosin via these weak binding sites. Troponin and
tropomyosin must therefore control strong cross-bridge binding states
in the in vitro motility assay as predicted by experiments in
solution and in intact
muscles(18, 25, 26, 27) .
The results show that troponin acts upon actin-tropomyosin in an ``all or none'' fashion to switch the filament between the ``on'' and ``off'' states, and there is no evidence for intermediate states(1, 2) . Solution experiments have indicated a regulatory unit of 7-14 actins controlled by one troponin-tropomyosin complex(1, 2, 28) , while indirect experimentation in muscle fibers has suggested that cooperativity may extend over the entire thin filament(29) . In the motility assay, we can see the behavior of individual actin filaments directly, and it is clear that the troponin-tropomyosin switch is extremely cooperative such that the entire filament is controlled as a single unit. This phenomenon is independent of the length of the filament (from <2 to 15 µm). We do not know whether this is due to positive cooperativity of troponin binding, such that filaments are either saturated with troponin or troponin-free, or due to cooperative interactions along the thin filament(17, 29) . The apparently high cooperativity may also relate to effects induced by myosin cross-bridge binding(1, 28) .
Our results are in accord with the
observations that Ca primarily controls maximum
isometric force in permeabilized muscle fibers rather than unloaded
contraction velocity (5, 6) and moreover indicate that
it does so by recruiting thin filaments in an ``all or none''
fashion. It is, however, not certain whether the latter prediction is
compatible with current models of the dynamics of muscle regulation.