In vitro Motility Assays with Actin
The interaction between actin and myosin has been studied for years using a variety of techniques, including ultracentrifugation, light scattering, chemical cross-linking, fluorescence, and measurement of the effect of actin on the MgATPase activity of myosin. The sliding actin in vitro motility assay constitutes a relatively recent technique for studying actin-myosin interaction. This assay, developed by Kron and Spudich (1986), takes advantage of the ability to image rhodamine-phalloidin-labeled actin filaments by fluorescence microscopy as they interact with and are translocated by myosin bound to a coverslip surface. The sliding actin in vitro motility assay is among the most elegant biochemical assays, reproducing the most fundamental property of a muscle, the ability of myosin to translocate actin using only the two highly purified proteins. It is a close in vitro correlate of the maximum unloaded shortening velocity of muscle fibers (Homsher et al., 1992). As shown here, it is simple to set up, reproducible, quantitative, and utilizes as little as 1 µg of myosin per assay. The assay is now used routinely in a large number of laboratories studying myosin and actin biochemistry. Although originally developed for studying the conventional class II myosin, it can be adapted to study unconventional myosins also.
This article discusses the design of the assay, describes the equipment required for its setup, and deals with methods for quantification and presentation of the results. Because different myosins exhibit a range of actin translocation speeds from 0.02 to 60µm/s (Sellers, 1999), it will be necessary to discuss modification of the experimental setup for fast and slow myosins. Also, differences between low- and high-duty cycle myosins are discussed. We will describe the instrumentation that we use in our system and elaborate on other options where applicable.
II. MATERIALS AND INSTRUMENTATION
The following reagents are from Sigma Chemical Company (www.sigmaaldrich.com): MOPS (Cat. No. M-5162), EGTA (Cat. No. E-4378), ATP (Cat. No. A- 5394), glucose (Cat. No. G-7528), glucose oxidase (Cat. No. G-6891), catalase (Cat. No. C-3155), and methylcellulose (Cat. No. M-0512). Rhodamine-phalloidin (Cat. No. R415) is from Molecular Probes, Inc. (www.probes.com). Nitrocellulose (superclean grade, Cat. No. 11180) is from Ernest F. Fullam, Inc. (www.fullam.com). Bovine serum albumin (BSA, Cat. No. 160069) and dithiothreitol (DTT, Cat. No. 856126) are from ICN (www.icnbiomed.com). The following items are from Thomas Scientific (www.thomassci. corn): microscope slides (Cat. No. 6684-H30) and microscope 18-mm2 No. 1 thickness coverslips (Cat. No. 6667-F24). Double sticky cellophane tape (Scotch Brand) and Sony sVHS videotapes (ST120) are from local suppliers.
The following instrumentation is used in our laboratory for the following procedures. Zeiss Axioplan microscope and objectives (www.zeiss.com), Air Therm Heater (www.wpiinc.com), intensified CCD camera from Videoscope, International (www.videoscopeintl. com), TR black-and-white videomonitor and sVHS videotape recorder (Model AG7350) from Panasonic (www.panasonic.com), Argus 10 image processor from Hamamatsu Photonics (www. hamamatsu.com), and VP110 digitizer from Motion Analysis (www.motionanalysis.com).
A. Construction of Flow Cells
B. Preparation of Rhodamine-Phalloidin- Labeled Actin
C. Preparation of Sample for Motility Assay
In the protocol just described, myosin is bound to the surface as monomers. If myosin is to be bound as filaments, it is necessary to block with BSA that is in a low ionic strength solution, such as the wash solution. Alternatively, heavy meromyosin or a soluble unconventional myosin can be applied to the flow chamber at either low or high ionic strength. In some cases, myosin or HMM can be attached to the surface via specific antibodies against their carboxyl-terminal sequence (Winkelmann et al., 1995; Cuda et al., 1993; Reck-Peterson et al., 2001), which may also serve the purpose of further purifying the desired isoform of myosin. If actin filaments are binding poorly to the surface, the motility buffer can be augmented with 0.7% methlycellulose (modify motility buffer preparation to add 0.5 ml of 1.4% methylcellulose and add less water to bring the final volume to 1 ml. Note that this solution is very viscous and must be mixed well).
Although each myosin has its own characteristic velocity, the velocity of a given myosin can vary with ionic and assay conditions. In general the velocity tends to increase as the ionic strength is raised from 20 to 100mM and increases with temperature. At higher ionic strengths the actin filaments typically begin to become weakly associated with the myosin-coated surface and move erratically. The velocity of actin filament translocation by some myosins, such as vertebrate smooth muscle myosin and Limulus-striated muscle myosin, is increased markedly (two to four times) by the inclusion of 200nM tropomyosin in the motility buffer (Wang et al., 1993; Umemoto and Sellers, 1990). However, tropomyosin inhibits the movement of brush border myosin I (Collins et al., 1990).
Step 3 is optional. It is often included to improve the "quality" of movement by binding unlabeled actin to noncycling myosin heads that would otherwise bind and tether the labeled actin added subsequently. This step can be omitted if the myosin is capable of moving actin filaments smoothly. For myosin V, better quality movement can be obtained by first blocking the surface with 0.1-1.0mg/ml of BSA before adding the myosin.
D. Recording and Quantifying Data Steps and Equipment
A schematic diagram of the equipment setup is shown in Fig. 1. The following describes the equipment used in our laboratory. There is a wide selection of video microscopy equipment represented by many manufacturers.
Given the range of motility rates of different myosins, there is no standard number of frames to average in order to get a good image. If the myosin is moving at 5 µm/s, a 2 or 4 frame average is used along with high illumination levels that can be tolerated because of the short exposure time needed to define a filament path. With slow myosins that may move at rates of less than 0.1 µm/s, it is possible to average 64 frames and to reduce the light intensity so that longer recording periods are possible.
The quantification of the rate of actin filament sliding is perhaps the most difficult part of the motility assay. The method just described requires a fairly expensive apparatus that is accurate, very fast, and can give unbiased results (for an extensive discussion of quantification of data, see Homsher et al., 1992). The user inputs the desired sampling rate and sampling time to collect data from either the live image or a prerecorded image. The computer determines the centroid position of each actin filament in each frame, connects the centroids to form paths, calculates the incremental velocity between each successive data point in a path, and, finally, calculates a mean + SD for each filament path. This process takes only seconds for a field of 25-30 actin filaments. Several investigators use commercial frame grabbers and write their own software for semiautomated tracking of actin filaments (Work and Warshaw, 1992; Marston et al., 1996). There is at least one free downloadable source of semiautomatic tracking software available (http://mCl1.mcri.ac.uk/retrac/).
E. Presentation of Data
Figure 2 shows three frames taken at 1-s intervals of actin filaments moving over a myosin-coated surface as it would appear on the video monitor. Data from such an experiment are presented most commonly as the mean ± SD of the velocity of the population of actin filaments. In general, the SD is typically 10-20% of the mean. There are two cases where merely reporting this number does not always accurately describe what is occurring in the assay. One such case is when something (perhaps a regulatory protein) is affecting the number of filaments that are moving. If, in the absence of the regulatory protein, >95% of the actin filaments are moving at 1 µm/s whereas only 5% of the filaments move at any velocity in the presence of the regulatory protein, reporting only the mean value for the velocity in each case does not reflect the difference that is observed in the assay between the two conditions. A better method for data display for this example is to display all data in the form of a histogram so that one can see that most of the actin filaments are not moving in the presence of the regulatory protein. This display also allows the reader to see whether the regulatory protein affects the speed of movement of the few actin filaments that remain moving. The other case where more complex data display is necessary is if the filaments are moving erratically. Here the mean velocity will underestimate the "instantaneous" velocity and will have a considerably larger standard deviation than that of smoothly moving filaments. One way to display these data graphically is to show a path plot in which the centroid position of the moving actin filaments is plotted in two-dimensional space as a function of time (Fig. 3).
I thank Qian Xu and Takeshi Sakamoto for critical reading of the manuscript.
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