©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Motility Analysis of Actin-Tropomyosin Regulation by Troponin and Calcium
THE THIN FILAMENT IS SWITCHED AS A SINGLE COOPERATIVE UNIT (*)

(Received for publication, November 13, 1994; and in revised form, January 11, 1995)

Iain D. C. Fraser Steven B. Marston (§)

From the Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(max) 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). (^1)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.


MATERIALS AND METHODS

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-actinbullet-tropomyosin and F-actinbullet-tropomyosin-troponin complexes were formed at 10times assay concentration. 100 nM F-actinbullet, 100 nM skeletal muscle tropomyosin, and 0-80 nM troponin were mixed in 50 mM KCl, 25 mM Imidazole-HCl, 4 mM MgCl(2), 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 times 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(2), 1 mM EDTA, 5 mM dithiothreitol, pH 7.4) to provide a coating of immobilized HMM on the coverslip. This was followed by 2 times 50 µl of buffer B (A + 0.5 mg/ml bovine serum albumin) and then 2 times 50 µl of 10 nM actinbullet ± 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 actinbullet-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 actinbullet or actinbullet-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^2 of the assay cell.


RESULTS

Motility of Actinbullet-Tropomyosin Filaments

The binding of tropomyosin to the rhodamine-phalloidin-labeled actin (actinbullet) at assay conditions was investigated. At 0.085 ionic strength, tropomyosin remained bound to the actin at the very low concentrations employed in the motility assay, provided that the proteins were premixed at 10times assay concentration (100 nM of each) and then diluted immediately prior to infusion into the flow cell. Binding of actinbullet to tropomyosin is directly demonstrated by cosedimentation and SDS-gel electrophoresis (Fig. 1). Determination of the band intensities by densitometry showed the tropomyosin:actin ratio to be unaffected by dilution prior to infusion into the flow cell. In lanesA and B, the ratio was 0.85 before and 0.86 after dilution, respectively.


Figure 1: SDS-gel electrophoresis demonstrating binding of tropomyosin (Tm) and troponin to actinbullet at motility assay conditions. Samples of the diluted and undiluted actinbulletbullettropomyosin and actinbullet-tropomyosin-troponin complexes were centrifuged for 30 min at 150,000 times g to sediment actinbullet 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 actinbullet 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 actinbullet 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 actinbullet 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.



Incorporation of the Troponin Complex into the Motility Assay

Initially, we introduced troponin at low Ca to investigate its effect on motility. As with tropomyosin, we incorporated the troponin into the assay by premixing with the other thin filament proteins at 10times the final assay concentrations.

Fig. 1(lanesC and D) demonstrates the binding of troponin to actinbullet-tropomyosin at motility assay conditions. Determination of the band intensities by densitometry showed the troponin I:troponin C:actinbullet ratio was 0.29:0.16:1 before and 0.27:0.18:1 after dilution. The tropomyosin + troponin T:actinbullet 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 actinbullet-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 actinbullet-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^2 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 actinbullet-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.



Effect of Increasing Caat a Fixed Inhibitory Concentration of Troponin

Ca controlled the motility of the actinbullet-tropomyosin-troponin filament. Using 10 nM actinbullet, 10 nM tropomyosin, and 5 nM troponin, we varied Ca concentration between pCa 9.0 and 4.5. The results are plotted as frequency histograms in Fig. 4and summarized in Fig. 5.


Figure 4: Distribution of actinbullet-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 actinbullet-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 10M.



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 actinbullet, 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.


DISCUSSION

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.

Effect of Tropomyosin on Motility

Tropomyosin binding is reported to be highly cooperative with 50% bound at 0.2-2 µMin vitro from equilibrium measurements(16, 17) . It might be expected, therefore, that formation of actin-tropomyosin complexes at the 10 nM concentration required for the motility assay would not be possible. In practice, however, we found that tropomyosin and troponin remained bound to the actin at the low concentrations employed in the assay, provided that the proteins were premixed at 10times assay concentration (100 nM of each) and then diluted prior to use (Fig. 1). The apparent disagreement with the equilibrium binding experiments may indicate a kinetic barrier to tropomyosin dissociation during our experiments.

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 Effect of the Troponin Complex on Motility

Low concentrations of troponin, at 10M Ca, stopped actin-tropomyosin filaments moving but did not displace the filaments from the HMM surface. Analysis of the movement clearly showed that troponin had no effect on filament velocity; the major effect being on the proportion of filaments that were motile. This ``switch off'' of filaments was virtually complete at 5 nM troponin and indicated an apparent affinity in the order of 0.5 times 10^9M for troponin binding to actin-tropomyosin. Higher concentrations of troponin produced a displacement of filaments from the HMM, but since this occurred both in the absence and presence of tropomyosin, displacement was clearly not directly related to troponin control of actin-tropomyosin filament motility.

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 HMMbulletADPbulletP(i) 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.


FOOTNOTES

*
This work was supported by the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-171-352-8121 (ext. 3307); Fax: 44-71-823-3392; S.Marston{at}ucl.ac.uk.

(^1)
The abbreviations used are: HMM, heavy meromyosin; , rhodamine-phalloidin.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.