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ATP-dependent movement of myosin in vitro: characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP- dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP- dependent movement of myosin molecules on actin.

are derived from a dissected Nitella cell. Thus, the actin substratum, aside from its high degree of spatial organization (10,11), is not well defined. It was important, therefore, to define the limits of salt concentrations and other parameters that will support myosin motility along the Nitella substratum. These limits may reflect in part the limits of stability of the Nitella substratum or the actin cables per se, or relate to the presence of auxiliary proteins on the actin cables. In fact, however, the optimal conditions in our system are consistent with the previously described (1,7,26) optimal conditions for the interaction of purified actin and myosin using actinactivated ATPase as an assay, which suggests that the assay is not complicated by the possible presence of auxiliary proteins on the Nitella substratum.
The results presented in this paper establish that the Nitellabased in vitro motility system is a fast, reliable, and quantitative assay for the velocity of myosin movement along actin filaments.

MATERIALS AND METHODS
Materials: Nitella axillaris was cultured (16) from original stocks provided by Dr. L. Taiz (University of California, Santa Cruz) and Dr. P. Richmond (University of the Pacific). In this protocol, 40 ml of chow (a mixture of 16 parts loam, 5 parts leaf mold, 6 parts fine sand, 4 parts steer manure, and 0.02 parts bone meal, by volume), 15 ml of potting soil, and 60 ml H20 were sterilized and then diluted in a 6 x 18 in cylindrical tank filled with distilled H20. After waiting for 2 d to allow particles time to settle, several terminal internodal cells from a stock Nitella culture were added to the tank and it was placed under 100 foot-candies of fluorescent (blue-green) illumination. Cultures normally took 2--4 wk to mature.
Skeletal myosin was prepared from rabbit muscle by the method of Kielly and Harrington (12), and was stored as a stock solution in 0.6 M KCI, 50 mM potassium phosphate, pH 6.5, 0.5 mM dithiothreitol (DTT) a, 0.5 mM EDTA, on ice. N-ethylmaleimide (NEM)-inactivated myosin was prepared by reacting myosin in storage buffer with excess NEM (1 raM) for 24 h on ice. The reaction was stopped by adding 2 mM DTT. In addition, skeletal myosin samples were kindly donated by Dr. S. Lowey (Brandeis University) and Dr. R. Cooke (University of California, San Francisco). Dictyostelium discoideum myosin was prepared as described previously (17) with modifications detailed elsewhere (Griffith, L. M., and J. A. Spudich, manuscript in preparation). Turkey gizzard myosin, with >95% light chain phosphorylation, was a gift of Dr. J. Sellers (National Institutes of Health).

Myosin-Bead Preparation:
In our standard assay, Covaspheres MX particles (1 ,m in diameter; 2.5 x 10 ~° beads/ml; Covalent Technology Corp., Ann Arbor, MI) were diluted 10-fold with 10 mM potassium phosphate, pH 7.0, 0.2 mM DTT, and enough of a concentrated stock solution of myosin (5-10 mg/ml) to give the desired final protein concentration. In all cases, except for the myosin concentration dependence study, 100-200 #g/ml myosin was incubated with the beads. The final buffer conditions were 10 mM potassium phosphate, pH 7.0, 50 mM KCI, 0.2 mM DTT, and 0.03 mM EDTA. Beads were allowed to incubate with myosin on ice for at least 1 h before they were assayed for motility. Most often the beads aggregate into distinct groups ~5-10 t=m in diameter, but the rates of movement are independent of bead diameter between 0.6 and 120 ,m (22,23). While it was possible to disrupt bead aggregates by mild sonication without affecting motility, generally this was not done.
Nite/la Dissection: A Nitella internodal cell (2-4 cm in length and ~ 1 mm in diameter) was trimmed free of branch cells. The cell was rinsed briefly with distilled H20 and placed in 10 ml of dissection buffer (10 mM imidazole, pH 7.0, 25 mM KCI, 4 mM MgCI2, 4 mM EGTA, l0 mM sucrose, and 1 mM ATP). The dissection was performed at 22°C in 50-ram plastic petri dishes on a layer (2-3 mm thick) of Sylgard (Dow Coming Corp., Midland, MI). First, a cell was secured at both ends with pins of tungsten wire (length, 1-3 ram; diameter, 0.003 in) sharpened at one end by electrolysis in 10 M NaOH. Next, the cell was opened with a transverse cut using microscissors (Moria, MCI9B; from Fine Science Tools Ltd., North Vancouver, B.C., Canada). The cell was then cut open along its whole length taking care to disturb as few chloroplasts as possible in the process. Finally, another transverse cut was made at each end of the lengthwise cut, and the central portion was opened and pinned fiat. The cytoplasm was largely washed away in the process of cutting and pinning the cell substratum to the Sylgard. Occasionally, vesicular materials from the Nitella continued to move along the chloroplast rows after the cell was cut open, but this material ran off the ends of the pinned substratum within a few minutes.
Standard Assay,: Myosin-coated beads were mixed 1:3 with dissection buffer containing 0.2 M sucrose and 1 mM DTT. Samples were then drawn into a microcapillary and 0.1-1 t=l was applied to a limited region of the dissected Nitella. The assay was performed at 22"C, except in the temperature dependence study. In the figures, each data point represents the average of 5-15 beads or distinct bead a~dgregates.
Bead movement was recorded with a video camera mounted on a Zeiss photomicroscope and connected to a Panasonic video cassette recorder (Model NV-8050). A 40x water-immersion objective lens was used; the magnification on the monitor screen was 2,000. Our initial experiments used fluoresceinlabeled beads to visualize the movement (22). The beads are easily seen, however, using bright-field optics, and this simpler method of monitoring the beads is now standard. Analysis of movement was performed on replay from the video cassette recorder where it was possible to freeze the motion. The positions of actively moving beads were traced on a piece of plastic placed on the monitor screen; the tape was then advanced and the new positions of the beads were recorded. Because the time is also recorded on the tape, the velocities of the beads were easily determined.

RESULTS
When Covaspheres coated with skeletal myosin were expelled from the microcapillary, they exhibited Brownian motion as ]Abbreviations used in this paper: DTT, dithiothreitol; HMM, heavy meromyosin; NEM, N-ethylmaleimide.

1868
THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984 they settled onto the Nitella substratum. Within 1-2 min, many beads and bead aggregates became attached to the chloroplast rows, ceased their Brownian motion, and began to move unidirectionally along the rows. As reported earlier (22), the direction of movement reversed across each of the two Nitella indifferent zones, consistent with the reverse in polarity of actin filaments on either side of these zones (11). The direction of movement was from the pointed ends of the actin filaments toward their barbed ends, as described previously (22). Myosin-bead movement proceeded over long distances (>100 #m) and at a constant rate independent of the size of the bead aggregate. Over time, often within 20-30 min after dissection, bead movement became less consistent: it sometimes appeared as if the beads were caught by and then broke free from invisible restraints, and some beads stopped moving altogether. The same Nitella could be used for measurements for ~ 1 h after dissection, until few enough beads were still moving smoothly and continuously for the data to be useful.
The rates of movement of beads coated with a fresh myosin preparation were highly reproducible (standard error was ~10% when 10 bead velocities were measured). These rates were consistent from one N. axillaris cell to another. Different skeletal muscle myosin preparations, however, moved at considerably different rates, between 2 to 6 ~m/s. Furthermore, we observed that over a period of weeks, beads coated with myosin from the same preparation moved at progressively slower rates. This effect was reduced by the addition of DTT to the myosin storage buffer, suggesting that oxidation was responsible for the rate decrease. To more directly test the requirement for active myosin, we determined the effect of NEM-inactivated myosin on bead motility when mixed with active myosin. The presence of NEM-myosin dramatically slowed bead movement. When only 20% of the myosin on the beads was NEM-inactivated, movement was slowed by 50%. Therefore, it is likely that the variability of myosin-bead velocity found in beads coated with myosin from different preparations or from a single preparation over time is due to partial inactivation of the myosin.
With the bead concentration in our standard assay (where beads were diluted 10-fold to a concentration of 2.5 x 109 beads/ml; see Materials and Methods), bead velocity was constant as long as the myosin concentration in the bead incubation was at least 20/~g/ml. This result was true for smooth muscle myosin and Dictyosteliurn myosin (data not shown) as well as for skeletal myosin (Fig. 1). Below 20 #g/ml there was a sharp decrease in bead velocity, and no movement at all was observed when beads were incubated with <10 pg/ml of myosin in the standard assay. The minimum concentration of myosin required for movement was found to depend on bead concentration (Fig. l). When onefourth the normal amount of beads was used, incubation with myosin at 5 pg/ml caused the beads to move. Conversely, when four times as many beads were used, the myosin concentration required for movement was 30 #g/ml. We tested the relative rates of movement of myosin from skeletal muscle, smooth muscle and a nonmuscle cell (Fig. 2). The greatest velocity was observed with skeletal muscle myosin. The data shown in Fig. 2 derive from skeletal muscle myosin preparations that moved from 3 to 6/~m/s. As mentioned above, different preparations moved at different rates, but all preparations moved between 2 and 6 pm/s. Phosphorylated gizzard smooth muscle myosin, in contrast, moved at only 0.1-0.6 pm/s. Dictyostelium myosin moved at 0.5-1.5 pm/s. These rates of movement are thus characteristic of the type of myosin used. In addition, the rates of movement of the different myosins correlate with their relative actin-activated ATPase activities (2,17).
The high reproducibility of the velocity measurements show that this assay is clearly a useful tool for investigating factors affecting myosin motility. To find the optimal assay conditions (defined as giving the maximum velocities) and to define boundary conditions outside of which the assay does not work, we varied several experimental parameters. As shown in Fig. 3, there is a sharp decrease in velocity below pH 6.8; above pH 7.5, there is a more gradual decline. From pH 6.8 to pH 7.5, velocity is relatively constant. Imidazole buffer and Tris-HC1 buffer gave the same results. We also investigated the effect of the MgCl2 concentration in the dissection buffer ( Fig. 4). At l mM MgCI2, no movement occurred. This may be because ATP, an effective chelator of Mg 2+, was present at 1 mM as well, and free Mg 2. may be required for movement. Bead velocity was maximal at 4 mM MgC12 and then gradually declined at concentrations of up to 20 mM, the highest tested. A third dissection buffer parameter we explored was KCI concentration. Dissection is usually performed in 25 mM KC1, but normal movement occurs in KCl.concentrations as low as l0 mM or as high as 70 mM. At K~'l concentrations of 70-100 raM, myosin-beads sometimes moved. The variability at these KCI concentrations could reflect effects on the Nitella substratum that differ somewhat from cell to cell. At 150 mM KC1 and above no movement was observed.
Myosin-bead movement on actin cables was also found to depend on ATP concentration (Fig. 5). The velocity was halfmaximal at an ATP concentration of 50 pM. The final parameter tested was the temperature at which the assay is performed. As Fig. 6 shows, velocity was found to be strongly temperature-dependent. When graphed as an Arrhenius plot the slope indicates activation energies of 90 kJ/mol from l0 to 22°C and 40 kJ/mol from 23 to 39°C.  The logarithm of the velocity is plotted versus the inverse of the temperature for skeletal muscle myosin. Temperature was controlled by circulating temperature-regulated water through coils surrounding the microscope objective and within the dissection chamber. The temperature was measured at the time of the experiment by placing a thermocouple adjacent to the Nite//a.

DISCUSSION
Myosin-bead movement on Nitella actin provides a quantitative assay for the fundamental function of the myosin molecule. In most cells, the rates of the relevant movements are not easily determined by direct microscopic examination. This in vitro assay allows the direct visual measurement of movement under defined buffer conditions with purified myosins. The assay is simple and fast and gives reproducible values for myosin velocity. In addition, microgram levels of myosin are sufficient to assay its movement. We normally deposit ~10 ng of myosin onto the Nitella substratum, but even smaller quantities can be reliably assayed. Skeletal muscle myosin beads move at a velocity that is notably similar to the maximal relative rate of movement of myosin and actin in the muscle sarcomere (3,6,19). The maximal rate of rabbit skeletal muscle contraction at 25°C is about five lengths per second which corresponds to a relative movement of myosin along actin filaments of ~6 zm/s (3). Rabbit skeletal myosin-coated beads in our in vitro assay move at 2-6 zm/s at 22°C. Furthermore, smooth muscle myosin-beads move at one-tenth that velocity, in keeping with the I 0-fold slower rate of contraction of smooth muscle (5,21). These results suggest that the assay is a reliable measure of the maximal rate of cellular movements that are actin-and myosin-based.
The major weakness of the assay is that the Nitella substratum is not biochemically defined. There could be trace amounts of Nitella enzymes or proteins that modify the myosin and affect its motility. However, we have no results to date to suggest that this complication is a serious one. The cytoplasmic contents of the Nitella cell are diluted 2,000-4,000-fold in the dissection buffer, which significantly reduces the probability of enzymatic modification during the course of the assay. Another weakness of the assay is that Nitella actin could differ in important ways from other actins. For example, Nothnagel et al. (20) suggest that Nitella actin does not bind DNase I. However, others (Vale, R., A. G. Szent-Gy6rgyi, and M. P. Sheetz, manuscript submitted for publication) have been able to confer calcium dependence on 1870 THE JOURNAL OF CELL BIOLOGY • VOLUME 99, 1984 skeletal myosin movement by adding the tropomyosin-troponin complex from rabbit skeletal muscle to the Nitella actin cables. This result indicates that the Nitella actin does not differ significantly from other actins, in keeping with the known highly conserved nature of this protein throughout evolution (13,27). We have recently shown that highly purified reconstituted, oriented actin filaments support movement of myosin-coated beads (24). The development of this totally defined assay will eliminate these two major concerns about the Nitella-based assay system.
A third problem with the assay is that the nature of the myosin on the bead surface is not well defined, and further studies arc underway to clarify what configuration is required to support movement. Since the assay is carded out in 25 mM KCI, some thick filament formation, even at low myosin concentrations, probably occurs. We have not ruled out the possibility that some form of aggregation such as that found in thick filaments may be necessary. Movement of the myosin-coated beads is quantitatively similar to the bead movement reported earlier (22) using heavy meromyosin (HMM) preparations. However, in experiments using HMM preparations, only a small number of beads were moving at any one time; they moved shorter distances; and it took much longer to observe a given number of moving beads than in experiments using myosin. We wish to emphasize that although our initial observations of bead movement used HMM preparations, those preparations were not purified free from all traces of myosin. As reported (22), we estimated from SDS acrylamide gels that myosin represented ~ 1% of the protein in the preparations used. Numerous experiments have suggested that it is unlikely that this level of contamination could account for the movement that we have seen repeatedly with HMM preparations made as described in the Sheetz and Spudich report (22). However, all experiments to obtain movement with highly purified HMM have failed, for reasons that are not yet apparent. Thus, the critical question as to whether HMM free of any trace of myosin can move must await further experimentation.
There are good correlations between the velocity of muscle shortening and of actin-activated myosin ATPase activity (2). With regard to ATP concentration dependence, however, the half-maximal velocity of muscle shortening under zero load occurs at ~200 zM ATP (4), whereas the half-maximal value for actin-activated HMM ATPasc in vitro occurs at 6 zM (18). In the case of the ATPase measurement, each myosin is expected to behave independently, so that a half-maximal rate of hydrolysis should be a reflection of 50% of the myosin ATP-binding sites being filled. On the other hand, in the case of the measurement of the velocity of shortening, the myosin molecules are not behaving independently. A small percentage of the molecules lacking ATP in their active sites would be expected to result in very strong actin-myosin interactions (rigor bonds), which should exert a substantial drag on the relative movement of thick and thin filaments. Thus, considerably more than 50% saturation of the ATP-binding sites may be required for half-maximal velocity of shortening. Our myosin-bead assay would be expected to mimic muscle shortening in this regard. The value we obtained for half-maximal velocity of movement of skeletal myosin-coated beads was 50 #M ATP, which, like the value for muscle shortening, is considerably higher than the ATP concentration required for half-maximal ATPase activity.
With regard to temperature dependence, the activation