In Vitro Assays of Processive Myosin Motors

METHODS 22, 373–381 (2000) doi:10.1006/meth.2000.1089, available online at http://www.idealibrary.com on

In Vitro Assays of Processive Myosin Motors Ronald S. Rock, 1 Matthias Rief, Amit D. Mehta, and James A. Spudich Department of Biochemistry, Stanford University School of Medicine, Beckman Center B405, Stanford, California 94305-5307

Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins. © 2000 Academic Press

The molecular motors that are responsible for the transport of cellular cargo face a bewildering array of tasks. Various cargoes must be transported to different locations in the cell, yet the motors are limited to two different classes of tracks. Unlike the situation faced by skeletal muscle myosin II, where a near-crystalline arrangement of actin and myosin is assembled for motility, the cargo transport motors are often required to operate with relatively few motors and in areas containing relatively low densities of tracks. To accomplish these tasks, a large number of classes of motors have evolved, each with 1

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its own set of cargo-binding, biochemical, and mechanical properties (1– 4). Cargo transport motors must face the problem of diffusion. Ideally, only one or a few motor proteins would be attached to each piece of cargo. However, prolonged detachment from the track must not occur, or the cargo will rapidly diffuse away. To overcome this problem, some motor proteins are processive, meaning that they undergo many catalytic cycles with associated mechanical advances for each track-binding event. Hence, one or a few motor molecules can transport cargo for appreciable distances without dissociating from the track. Until recently, the entire myosin family of motor proteins was assumed to be nonprocessive. Here the assays that were used to demonstrate the processivity and to characterize mechanical properties (5) of myosin V (6, 7) are described. Many of the methods outlined below are based on earlier studies of the processive microtubule-based motor kinesin, which has been studied extensively (8 –20).

GLIDING FILAMENT ASSAYS The most straightforward demonstrations of processivity use variations of the gliding filament in vitro motility assay (21, 22). In the gliding filament assay, myosin molecules are randomly adsorbed to a microscope coverslip. Once the coverslip is blocked with bovine serum albumin (BSA) to prevent further, nonspecific protein adsorption, actin filaments, typically labeled with tetramethylrhodamine (TMR)-phalloidin, are bound to the myosin mole373

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cules, and motility buffer containing ATP is introduced. At high motor densities, smooth and continuous movement of the actin filaments is observed for both the processive myosin V and nonprocessive myosin II. However, as the surface density of the motor is dropped, the two classes of myosin exhibit markedly different behavior. For myosin II, below a certain critical density the actin filaments detach from the coverslip surface, since not enough motors are present in a strongly bound state to support the actin. A processive motor, on the other hand, binds and holds an actin filament for many steps and, therefore, is more tolerant of dilution. As myosin V density is dropped to 2.7 molecules ␮m ⫺2, the actin filaments appear tethered to the surface at single points, and exhibit “nodal pivoting,” a diffusive rotation about the contact point, even as translation of the filament through the nodal attachment occurs. Due to the conformational strain required of multiple motors that must remain attached throughout such rotation, this pivoting behavior is a first signature of a processive motor (8). To observe motility under such low surface myosin densities, pretreatment of the coverslip surface is required (8, 9). The myosin V molecule has two catalytic domains at the end of extended, ⬃25-nm-long neck regions. If a given myosin V molecule attaches to the surface by the tail domain, it is possible that at least one of the two heads also attaches to the surface and becomes inactive. Assuming that both heads are required in a hand-over-hand model for processivity (1), movement supported by single processive motors will not be observed unless both heads are active. To reduce the fraction of inactive heads, a large fraction of the coverslip surface is blocked with an inert protein such as BSA. In practice, incubation of the coverslip with 0.01 mg mL ⫺1 BSA is optimal. After adsorbing greater amounts of BSA and then attempting to adsorb myosin V, actin filaments no longer bind to the surface. Moreover, movement and nodal pivoting are not observed with significantly smaller amounts of BSA. Casein has been examined as a preblocking protein as well (8), but does not result in improved motility results with myosin V. The gliding filament assay is ideal for measuring the effect of various parameters, such as ATP concentration, ionic strength, temperature, and myosin head density, on actin velocity (22). At high densities of myosin V, where the observed movement resembles that driven by myosin II, the established method of following either the leading or trailing

end of an actin filament may be used to find the velocity (21, 22). However at low motor densities, where filaments rotate as they move and their trailing ends no longer follow the filament contour, this measurement becomes difficult. The pivot point must be determined and the length of the actin filament on either side of the attachment followed over a given time interval. In some cases the free ends of long actin filaments will drift out of the focal plane. These filaments are not scored until the trailing end of the filament is pulled into focus near the attachment point, otherwise errors may be introduced if a spurious filament “end” is identified while the actual end remains out of focus. The location of the nodal attachment point may be found by generating an average image of all frames in a movie of a moving filament. The attachment point will have fluorescent intensity from all frames in the movie and will therefore correspond to the point of maximum intensity in the average image. Image analysis packages, such as NIH Image, may be used to automate parts of the velocity measurement procedure. Myosin V velocities are found not to vary over several decades of myosin density (Fig. 1). This density independence is in contrast to the behavior of myosin II, which exhibits a drop in velocity as the density is decreased (23). Such data are consistent with myosin II having a low duty ratio, or ratio of the strongly bound state time to the total actinactivated ATPase cycle time, while myosin V has a high duty ratio. A high duty ratio for myosin V is consistent with the property of processivity, in which case the duty ratio for a given myosin head need be at least 0.5.

FIG. 1. Measured actin filament velocity as a function of myosin V surface density, calculated as described under gliding filament assay, at 2 mM ATP. The velocity shows no sign of increase as density is raised, as is the case for kinesin, but not myosin II.

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The assay described below is similar in many respects to previously described methods (21, 22).

QUANTITATIVE MEASURES OF PROCESSIVITY

Gliding Filament Assay Buffers AB: 25 mM imidazole hydrochloride, pH 7.4, 25 mM KCl, 4 mM MgCl 2, 1 mM EGTA, 10 mM dithiothreitol (DTT). ABSA: AB containing 1 mg mL ⫺1 BSA. Motility: ABSA containing variable concentrations of ATP, 1 mM phosphocreatine, 0.1 mg mL ⫺1 creatine phosphokinase (an ATP regeneration system), 25 ␮g mL ⫺1 glucose oxidase, 45 ␮g mL ⫺1 catalase, and 1% (w/v) glucose (an oxygen scavenger system).

Although the nodal pivoting behavior provides qualitative evidence that a motor is processive, demonstrating this requires quantitative, statistical evidence to exclude coincident colocalization or aggregation of nonprocessive motors as the agent of motility. Such evidence should include a statistically robust description of actin binding and motility under a broad range of myosin V densities. The landing assay and the continuous movement assay (8, 18) are two such independent indicators of processivity in motor proteins. The landing assay examines the rate at which actin filaments in solution land and move on a myosin-coated surface. As the myosin density is reduced, the landing rate decreases. The functional form of this decrease provides a high-end estimate for the number of molecules required for the observed motility. For example, if only one motor molecule is required for continuous motility, and assuming that an insignificant amount of actin is depleted from solution over the course of the assay, the landing rate will be pseudo-first-order in myosin density. A higher-order dependence excludes a single processive motor as the agent of observed motility. A plot of log(landing rate) versus log(myosin density) gives a straight line of slope N, where N is the order of the landing process (Fig. 2). The fraction of protein that is active does not affect the functional dependence at issue. The continuous movement assay examines the probability of an actin filament moving greater than

Procedure 1. Construct a flow cell from a microscope slide, two strips of 3M double-stick tape, and a nitrocellulose-coated coverslip. Flow cell volumes are typically 10 –15 ␮L, and a two- to threefold excess of solution is introduced in each step. 2. Infuse a solution of 0.01 mg mL ⫺1 BSA. Incubate for 2 min. 3. Rinse with AB, followed by myosin V diluted into AB. Incubate for 2 min. 4. Rinse with ABSA, followed by a solution of 5 ␮g mL ⫺1 actin labeled with TMR-phalloidin. Incubate for 2 min. 5. Rinse with ABSA. 6. Infuse motility buffer. In many cases, it is more meaningful to report an estimate of the number of motor molecules adsorbed per unit area in the flow cell, rather than the protein concentration. Assuming that half of the molecules loaded land on each face of the flow cell, and that all molecules adsorb, the protein concentration times the flow cell volume divided by the area of both flow cell faces provides a high-end estimate of the molecules per unit area. As discussed above, nodal swiveling behavior provides circumstantial evidence that single molecules drive filament translation. When attempting to measure the actin sliding velocity supported by a single motor molecule, actin filaments that do not demonstrate nodal pivoting should be discarded, as they are likely attached by at least two motors. Often this dual-point attachment is clear when the actin filament slides past one of the two attachments, because nodal pivoting suddenly commences.

FIG. 2. Actin filament landing rates as a function of myosin V surface density. The fits for movement driven by one molecule and two molecules are shown.

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its length before detaching. Once attached, an actin will either detach once the single attachment point has reached the end of the filament, or will encounter a second attachment point that will continue to hold it to the surface. The probability that a filament moves greater than its length is therefore a function of both the motor surface density and the number of motors required for motility. In all cases, the probability of such movement is one at high motor density, and drops to zero as the motor density is decreased. Qualitatively, if only one motor is required, the decrease will be gradual, with sharper decreases as more motors are required. Quantitatively, the probability of continuous movement is the Poisson probability that, if at least N motor units (N being the requisite number) are present in a given area, at least 2N can be found in that area. The actual area, which can be viewed as the average surface area sampled by a moving filament, is not relevant as long as this area does not change between the different protein densities used; it is incorporated as a fit parameter ␳ 0, and it does not affect curve shape. For a processive motor, N ⫽ 1, and the relevant expression is 1 ⫺ (␳/␳ 0)exp(⫺␳/␳ 0)/[1 ⫺ exp(⫺␳/␳ 0)], where ␳ is the surface density of the motor (Fig. 3). One advantage of this pair of assays is that one set of experiments can provide the data necessary for both forms of analysis, even though the behaviors assayed are independent. Each filament that lands can be scored as a landing event and followed to gauge its movement distance. The assays provide statistical evidence that one functional myosin V molecule, and not a chance colocalization of more

FIG. 3. Continuous movement assay. The fraction of moving actin filaments that travel greater than their length is plotted against myosin V surface density. The curve fit is obtained from a model where a single motor protein is sufficient to drive movement (see text).

than one nonprocessive motors, is sufficient to support actin filament motility over lengths on the order of 1 ␮m. An article by Hancock and Howard further explains the theoretical basis of these assays (18). Landing and Movement Assay The landing assay is identical to the gliding filament assay, except that the actin is added (at a final concentration of 5 ␮g mL ⫺1) along with the motility buffer and not beforehand. Filaments are scored as “landed” when they touch down and move for ⬎0.5 ␮m. To facilitate the scoring of landing events, a movie segment of 30 s is looped repeatedly, with a grid overlay that defines a fixed observation area. Landing events are measured from multiple movie segments until a sufficient number of events are tabulated at each density to provide statistically robust determinations.

THE ISSUE OF AGGREGATES While the above movement assay excludes coincident colocalization of nonprocessive motors, it remains consistent with the hypothesis that solution aggregates underlie the motility observed. If motility is driven by a small population of aggregates, then the landing and movement observations will be similar to those with processive motors. While surface-induced aggregation of protein can be excluded on kinetic grounds (8), the possibility of solution aggregates must be addressed. In the case of kinesin, aggregates were separated by a sucrose density gradient ultracentrifugation step (8). The myosin V purification protocol includes a gel-filtration step that would potentially remove aggregates (6). To exclude the possibility of aggregation occurring after purification, a stock solution of myosin V may be depleted of aggregates in a sedimentation velocity protocol immediately prior to performing the landing assay. Myosin V is a 12 S protein (5), and any dimers can be expected to have an S value nearly twice as large. If the stock solution is sedimented by centrifugation such that a significant fraction of the 12 S species pellets, then an even higher fraction of any dimers will have pelleted, as indicated in Fig. 4. A landing assay performed after this spin will show lower landing rates if the aggregates are responsible for motility, but the same landing rate if a single motor is involved. The data for

IN VITRO ASSAYS OF PROCESSIVE MYOSIN MOTORS

such a set of landing assays are shown in Fig. 5. In this case, the landing rates do not fall below those measured for the zero spin control, as expected for a processive motor. This excludes either a slow equilibrium aggregation or a slow nonequilibrium aggregation. Fast aggregation, or aggregation that takes place on the time scale of loading myosin V in the landing assay, can be excluded since myosin V exhibits a single, sharp boundary in sedimentation velocity experiments (5). Moreover, high resolution images of myosin V molecules did not indicate the protein oligomerizes (7). A slow or fast nonequilibrium aggregation would result in a significant fraction of protein, if not all the protein, appearing as aggregates.

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OPTICAL TRAPPING

1. Sediment an aliquot of myosin V stock solution (100 ␮L) for 0 –1 h in a Beckman TLA 100.1 rotor at 184,000g. Under these conditions, myosin V completely sediments in ⬃1.5 h. 2. Use the supernatant in the landing and continuous movement assays as described above. 3. Save a sample of the supernatant so that the protein concentration can be measured by a Bradford assay, SDS–PAGE densitometry, or any other convenient protein assay. This measured protein concentration is used when calculating the surface density in either the landing assay or the continuous movement assay. 4. Repeat the protocol with varying centrifugation times.

For high-resolution records of myosin V stepping, optical trapping techniques may be used for studies on the single-molecule level. Two classes of experimental geometries have been developed for optical trapping studies of molecular motors, the dual-bead and the single-bead assays (Fig. 6) (9, 10, 24 –28). In the dual-bead assay, developed initially to study nonprocessive motors, bead handles are attached to either end of an actin filament that is then stretched to tension. This dumbbell is then lowered onto a raised surface platform that is sparsely coated with myosin. Binding events are apparent from the bead displacement (24), the decrease in the Brownian motion of the beads (25), or a loss in correlation of Brownian motion of the two beads (27). Since the filament is pretensioned before a motor binds, it is possible to track the small displacement induced when a single nonprocessive motor binds and moves its track (29). This geometry is inverted in singlebead assays, first developed to examine the processive kinesin motors. Here, the single optically trapped bead is sparsely coated with a motor and then lowered onto a surface-bound track. Even though the system is not pretensioned before initial binding, continued movement by a processive motor will add tension until bead movement reflects motor movements (30). For myosin V, either the single- or double-bead assay may be used. In practice, each assay has strengths and weaknesses. The single-bead assay is

FIG. 4. Calculated fraction of protein remaining in the supernatant after sedimentation in an ultracentrifuge, for a 12 S protein (solid line) and a 20 S protein (dashed line). The calculation was based on the geometry of a Beckman TLA100.1 rotor, 100 ␮L sample volumes, and centrifugation at 184,000g.

FIG. 5. Observed actin filament landing rates for 0-min (open circles), 40-min (closed circles), and 60-min (closed squares) centrifugation runs. Note that the landing rates remain independent of spin time, not expected if slow-forming aggregates were driving motility.

Presedimentation Landing Assay

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procedurally simpler; one need trap a single bead and then probe the surface, as opposed to trapping two beads, connecting an actin filament between them, stretching the filament to tension, and then probing the surface. Moreover, the force applied in the single-bead assay is delivered by a single optical trap, while that applied in a dual-bead assay comes from both, complicating the measurement of this force. Finally, the bead has a well-defined surface area, allowing an estimate of the average number of adsorbed motor proteins. Even if this number depends on other factors, such as the fraction of protein that remains active and is successfully adsorbed, the functional dependence of behavior on motor-to-bead ratios is the measurement of interest (see below) and remains independent of these factors. Drawbacks of the single-bead assay include the following: the motor protein may be subject to radiation damage from trapping light and its initial binding and early travel will be absorbed by bead rotation and other compliant elements until they are pulled to tension. The dual-bead assay allows the system to be pretensioned so that the first steps are apparent, and allows sensitive detection of contact with the surface platform, as both beads move when the actin filament is deflected. On the other hand, the total force applied to the motor protein is obtained from the sum of the distances moved by the two beads times their respective trap stiffnesses. Constant-force feedback systems (see below) are thus difficult to implement with this geometry. Moreover, the sur-

FIG. 6. Schematics of the single-bead and dual-bead assays. In the upper figure, the bead would be held by a single laser trap.

face area sampled by the trapped actin filament can only be approximated. The expected behavior of processive motors in an optical trapping assay is that a motor will take multiple steps per actin binding event and that bead movement records will resemble staircases. This behavior is observed for myosin V, as shown in Fig. 7. The large step size for this motor protein allows the facile tabulation of dwell times preceding both forward and backward steps. To estimate the motor step size from the stepwise advances of the trapped bead, the effect of compliant linkages that absorb some of the motor advance must be removed. One way to overcome this problem is to characterize the compliant linkages and correct for them quantitatively, but this can be difficult to perform with precision, especially if the elements are non-Hookean (29). To circumvent this problem, a large-amplitude oscillation may be applied to one of the beads in the dual-trap assay. At one end of the oscillation, all compliant elements will be pulled taut, and the bead position will be clipped and fail to follow the full oscillation of the trap. Of course, the motor will be unable to step against the applied load in this situation. However, stepping will occur in the other half of the oscillation cycle, where slack is introduced into the system. Hence, the motor can step when the load is low, and its step distance is measured when the load is high and system elasticity has been removed. The motor step size is then given by stepwise advances in the clipped bead position level (Fig. 8). A more straightforward technique to eliminate the compliance problem in step size measurements is to employ a constant-force feedback loop. In this manner, the load on the myosin V molecule is constant as stepping occurs, so that all compliant elements are strained to the same degree throughout

FIG. 7. Sample data trace of myosin V stepping, taken at 10 ␮M ATP with the dual-bead geometry. The unbound baseline level is set at 0 nm. The myosin steps to a stall level at about 90 nm, or approximately 3 pN, and then detaches.

IN VITRO ASSAYS OF PROCESSIVE MYOSIN MOTORS

the data record, preventing them from absorbing part of the motor step (31, 32).

OPTICAL TRAP ANALYSIS OF PROCESSIVITY Optical trapping methods open a whole new realm of statistical demonstrations of processivity. Instead of relying on diffusive encounters between actin and myosin, where the surface area that encounters an actin filament is an unknown from experiment to experiment, the optical trap allows a defined surface area to be probed. For processive motors, binding and movement events will be observed only when the surface platform (in the dual-bead assay) or the bead (in the single-bead assay) contains one or more motors. Nonprocessive motors require at least two, and perhaps many more, motors within the sampled

FIG. 8. Oscillation experiment. (a) The actin filament experiences alternating taut (top) and slack (bottom) cycles as the left bead is oscillated. (b) The tether length is measured during the taut phase, while stepping occurs during the slack phase. This tether length advances in discrete steps. Clipping of the bead position is indicated by the dark dots, which are averaged across a single bound level. (c) Histogram of steps sizes between the levels so measured.

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surface area. The Poisson probability that one or more motors may be found on the bead is given by 1 ⫺ exp(⫺ ␭ f ), where f is the number of motors per bead, and ␭ is a fit parameter. A ␭ greater than one implies that some of the motors may be damaged or not absorbed. However, it is not relevant to the central question of functional dependence; the shape of the curve is expected to change with N, a measurement unaffected by the value of ␭. The probability that a bead has two or more motors is given by 1 ⫺ exp(⫺ ␭ f ) ⫺ ( ␭ f )exp(⫺ ␭ f ) (9). The results for myosin V in a single-bead assay are shown in Fig. 9. A one-molecule model provides a best fit ␭ ⫽ 5, with an X 2 value of 0.2; a two-molecule model provides a best fit ␭ ⫽ 1.7 with an X 2 of 4.9; and a threemolecule model provides a best fit ␭⫽1 with X 2 ⫽ 10.56. As the hypothesis test requires X 2 ⬍ 4, only the one molecule model is consistent with the data. The value of ␭ ⫽ 5 implies that one of five molecules is active, successfully adsorbed on a bead, and observed. This value of ␭ is higher than the measured values for kinesin (which can approach unity (16)), but is significantly lower than that obtained for a processive dynein, where ␭ ⬃300 (33). This singlebead assay, unlike the surface assays mentioned above, allows a direct estimate of the area in which motors would need to be detected. In the dual-bead assay, the fraction of the platform surface that is sampled in a given attempt may only be approximated. The optical trapping assays also provide independent evidence that aggregates are not responsible

FIG. 9. Fraction of beads moving in single bead experiments. The fits to the N ⫽ 1 model (solid line) and N ⫽ 2 model (dashed line) are shown.

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for the apparent processive behavior. The value of ␭ ⫽ 5 means that an aggregate would need to be present in 20% of the protein concentration, as well as 100% active and adsorbed to the beads, meaning that dimers would need to constitute 40% of the total protein, or trimers 60%. These situations are inconsistent with the results from the presedimentation assays, as well as the analytical ultracentrifugation results, which excluded large fractions of aggregates. Dual-Bead Assay The dual-bead assay has been described in detail previously (29). As in the gliding assay, a 2-min incubation of the flow cell with 10 ␮g mL ⫺1 BSA is used to preblock the surface. A biotin–avidin system is used to attach actin filaments to the beads (in place of NEM-myosin-coated beads), a system first described by Ishijima et al. (34). The preparation of these reagents is described here.

Biotinylated Actin Stocks F-buffer: 5 mM Tris/HCl, pH 7.4, 50 mM KCl, 0.2 mM CaCl 2, 1 mM MgCl 2, ⫾0.5 mM DTT. G-buffer: 5 mM Tris/HCl, pH 7.4, 0.2 mM CaCl 2, 0.5 mM DTT, 0.1 mM ATP. F-actin: 5 mg/ml (116 ␮M). Biocytin maleimide: 6.6 mM (3.5 mg/mL) in dimethyl sulfoxide (DMSO). Procedure 1. Sediment 10 ␮L F-actin by spinning at 184,000g for 10 min. Resuspend the pellet in 100 ␮L F-buffer without DTT. Repeat three times. 2. Add 1 ␮L biocytin stock, and incubate for 1 h at room temperature. 3. Sediment the actin, and resuspend in 100 ␮L F-buffer with DTT. Repeat three times. For storage, the actin may be depolymerized by dialysis into G-buffer, and frozen at a final concentration of ⬃10 mg mL ⫺1.

Neutravidin Beads Stocks Neutravidin: 5 mg mL ⫺1 in 50 mM phosphatebuffered saline (PBS), pH 8.0 (Molecular Probes, Eugene, OR).

Tetramethylrhodamine–BSA: 6 mg mL ⫺1 in 50 mM PBS, pH 8.0 (Sigma, St. Louis, MO). Procedure 1. Sediment 10 ␮L biotinylated polystyrene beads (1 ␮m in diameter, 1% solids, Molecular Probes) at 14,000g for 1 min. 2. Resuspend beads in 10 ␮L PBS. Repeat spins three times. 3. Add 5 ␮L neutravidin. Incubate 1 h at room temperature. 4. Add 0.3 ␮L tetramethylrhodamine–BSA. Incubate 10 min. 5. Sediment beads at 14,000g, 1 min, and resuspend in 10 ␮L ABSA. Repeat 10 times. Thorough washing at this stage is critical to prevent the excess neutravidin from binding to the flow cell. These beads and biotinylated actin filaments may be used as a direct replacement of the NEM–myosin linkage system. Biotinylation does not affect measured gliding filament speeds. Single-Bead Assays Stocks Beads: 0.35 ␮m diameter (Polysciences), diluted to 0.01% solids in AB. Preblock: 98 ␮L ABSA plus 2 ␮L tetramethylrhodamine–BSA. Myosin V: 2 ␮g mL ⫺1 in AB. Motility: ATP, 1% glucose, 45 ␮g mL ⫺1 catalase, 25 ␮g mL ⫺1 glucose oxidase in AB. Procedure 1. Add 1 ␮L preblock to 100 ␮L beads. Mix well, and incubate 5 min. 2. Add 1 ␮L myosin V, and incubate 5–30 min. This yields a stock of beads with two molecules of myosin V per bead. This density can be varied as desired. 3. Prepare a flow cell with a slide, an uncoated coverslip, and two strips of 3M double-stick tape. 4. Infuse a solution of 1 ␮L neutravidin diluted to 20 ␮L in AB. 5. Add a solution of 5 ␮g mL ⫺1 biotinylated actin in AB, and rinse with 20 ␮L ABSA immediately. 6. Dilute 1 ␮L myosin V-coated beads to 100 ␮L in motility buffer, and add to the flow cell.

IN VITRO ASSAYS OF PROCESSIVE MYOSIN MOTORS

CONCLUSION Many of the properties that were first observed in the kinesin family of motors are now appearing in the myosin motors. For example, both families contain members that can move along their tracks in either direction, and both have examples of processive and nonprocessive motility. Given the requirements of cargo-transporting motors, it is likely that more examples of processive myosins will begin to emerge. Therefore, the assays described here will likely become a part of the routine characterization of other nonmuscle myosins.

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