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
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
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
No. 1 thickness coverslips (Cat.
No. 6667-F24). Double sticky cellophane tape (Scotch
Brand) and Sony sVHS videotapes (ST120) are from
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
A. Construction of Flow Cells
B. Preparation of Rhodamine-Phalloidin-
- Prepare nitrocellulose-coated coverslips by first
placing 3µl of a 1% solution of nitrocellulose in
isoamylacetate directly on a No. 1 thickness 18-mm2 coverslip and spreading with the broad side of the
micropipette tip. Dry the coverslip to create the
film and use within 1 day. Some investigators use
silicon coating of the coverslips (Fraser and Marston,
- Place two 5 × 25-mm strips of double sticky
Scotch cellophane tape about 10mm apart on a 25 × 75-mm glass microscope slide. Place a nitrocellulosecoated
coverslip with the coated side down onto the
tracks. Press gently to create a tight seal.
C. Preparation of Sample for Motility Assay
- Place 60µl of 3.3µM rhodamine-phalloidin (in
methanol) into an Eppendorf tube and dry using a
- Redissolve the rhodamine-phalloidin powder in
3-5 µl of methanol.
- Add 85µl of 20mM KCl, 20mM MOPS (pH 7.4),
5mM MgCl2, 0.1mM EGTA, and 10mM DTT
(buffer A). To make 100ml of buffer A, add 1 ml of
2M KCl, 1 ml of 2M MOPS (pH 7.4), 0.5ml of 1M MgCl2, 0.02 ml of 0.5M EGTA, and 154mg DTT.
Bring to 100ml with H2O and adjust pH to 7.4.
- Add 10µl of a freshly diluted 20µM F-actin solution
(in buffer A) and incubate for an hour on ice. The
rhodamine-phalloidin actin can routinely be used
as is at this stage.
- If a lower background fluoresence is desired, centrifuge
for 15min at 435,000g in a TL-100 ultracentrifuge
(Beckman Instruments), remove the
supernatant, and gently resuspend the pink rhodamine-
phalloidin-labeled actin pellet in buffer A
using a pipettor tip that has been cut to widen the
- The rhodamine-phalloidin-labeled actin solution is
stable for several weeks.
- Wash solution: 50 mM KCl, 20 mM MOPS (pH 7.4),
5 mM MgCl2, 0.1 mM EGTA, and 5 mM DTT; to make
100ml, add 2.5 ml of 2M KCl, 1 ml of 2M MOPS, (pH
7.4), 0.5 ml of 1M MgCl2, 0.02ml of 0.5M EGTA, and
77mg of DTT. Bring volume to 100ml with H2O and
pH to 7.4.
- Blocking solution: 1 mg/ml BSA in 0.5M NaCl,
20mM MOPS (pH 7.0), 0.1mM EGTA, and 1 mM DTT solution. To make 100ml of blocking solution,
add 10ml of 5M NaCl, 1 ml of 2M MOPS (pH 7.4),
0.02ml of 0.5M EGTA, 15.4mg of DTT, and 100mg of
BSA. Bring to 100ml with H2O and pH to 7.0.
- Rhodamine-phalloidin-labeled actin solution: 20nM rhodamine-phalloidin-labeled actin. To make 1 ml,
take 10µl of 2µM rhodamine-phalloidin-labeled
actin, 10µl of 500mM DTT, and 980µl of wash
- ATP-actin wash: 1 mM ATP and 5µM F-actin
(unlabeled) in wash solution. To make 1 ml, add 10µl of 0.1M ATP and 50µl of 100µM F-actin to 940µl of
- 4X stock solution: 80mM MOPS (pH 7.4), 20mM MgCl2, and 0.4mM EGTA. To make 100ml, add 4ml of
2M MOPS (pH 7.4), 2ml of 1M MgCl2, and 0.08ml of
0.5M EGTA. Bring volume to 100ml with H2O and pH
- 1.4% methycellulose solution: Dissolve 1.4g of
methylcellulose in a final volume of 100ml of H2O by
stirring overnight. Occasionally it is necessary to
homogenize the solution with a glass-Teflon homogenizer
to aid in solubilization. Dialyze the dissoved
methylcellulose against 4 liters of H2O overnight.
Divide into 10-ml aliquots and freeze at -20°C.
- Motility buffer: 50mM KCl, 20mM MOPS (pH
7.4), 5mM MgCl2, 0.1mM EGTA, 1 mM ATP, 50mM DTT, 2.5mg/ml glucose, 0.1mg/ml glucose oxidase,
and 0.02mg/ml catalase. To make 1 ml, add 250µl of
4X stock solution, 10µl of 0.1M ATP, 25µl of 2M KCl,
100µl of 0.5M DTT (prepare fresh each day by adding
77mg DTT to 1 ml of H2O), 20µl of 125mg/ml glucose,
20µl of 5mg/mol glucose oxidase, 1 µl of 20mg/ml
catalase, and 573 µl of H2O. If methylcellulose is to be
used in the motility buffer (see later), then add 500µl
of 1.4% methylcellulose and 73 µl of H2O.
- Apply 0.2mg/ml myosin in 0.5M NaCl, 20mM MOPS (pH 7.0), 0.1 mM EGTA, and 1 mM DTT to fill
the flow chamber. Wait 1 min.
- Wash with 75 µl of blocking buffer. Wait 1 min.
- Wash with 75 µl of wash solution, followed by 75 µl
of ATP-actin wash solution. Wait 1 min. This step is
- Wash with 75 µl of wash solution, followed by 75 µl
of rhodamine-phalloidin-labeled actin solution.
Wait 1 min.
- Initiate reaction by the addition of 75 µl of motility
- Place slide on microscope stage and image.
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.
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
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
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.
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
D. Recording and Quantifying Data Steps
|FIGURE 1 Schematic diagram of equipment setup.
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
- Place the microscope slide under a 100X, 1.4 NA
Plan-Neofluor objective in an Axioplan microscope
(both from Carl Zeiss, Germany) equipped for epifluorescence.
Other microscopes, including ones with an
inverted format, are also suitable and a variety of
objectives can be used, but note that high numerical
apertures are required for maximal brightness. Illumination
is via a 100-W mercury lamp. An IR filter should
be placed between the source and the sample to attenuate
heat; neutral density filters are useful to attenuate
light intensity if needed. A filter set designed for rhodamine
fluorescence measurements should be utilized
in the filter cube. For quantitative work it is necessary
to control the temperature of the assay. This can be
accomplished in several ways. The most inexpensive
way is to create an air curtain using a hair dryer. Other
methods include fabricating a water jacket for the
objective and to use a circulating water bath to regulate
temperature or creating an environmental box
heated with an Airtherm heater (World Precision
Instruments). In our experience, commercial stage
heaters are not sufficient as oil immersion objectives
act as large heat sinks.
- Image actin filaments using an ICCD 350F
intensified CDD camera (Videoscope International).
Other low-light systems are possible, such as an
SIT camera or intensified SIT camera. Several manufacturers sell this equipment. These camera
systems produce an analog output that can be viewed
on a standard black-and-white video monitor (TR
Panasonic) and recorded on an AG7350 sVHS recorder
(Panasonic). It is also possible to collect data digitally
using a cooled CCD camera linked to a computer.
- It is useful to process the raw image using an
image processor such as the Argus 10 or Argus 20
(Hamamatsu Photonics) to perform frame averaging
and/or background subtraction. There are several
commercial software packages that can do this also.
Display the processed image on another video
- Determine the movement of individual actin
filaments using an automated tracking system equipped
with a VP110 digitizer from Motion Analysis. Many
investigators have created there own software routines
to track actin filaments in a semiautomated manner.
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
E. Presentation of Data
|FIGURE 2 One-second intervals of actin filaments moving over a myosin-coated surface.
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).
|FIGURE 3 Actin filaments plotted in two-dimensional space as a function of time. A) smoothly moving
actin filaments; B) actin filaments moving more erratically; C) a mixture of moving and nonmoving actin
filaments; D) nonmoving actin filaments.
- Actin filaments are moving erratically or only a
fraction of the actin filaments are moving. The cause
of this phenomena is usually noncycling heads in the
preparation. Using the actin-ATP wash solution
described in Section III,C,3 usually helps or eliminates
the problem, but if erratic motility persists, do the following.
Bring the myosin solution to 0.5 M in NaCl and
add actin to a final concentration of 10µM, ATP to
2mM, and MgCl2 to 5mM. Immediately sediment
at 435,000g for 15mn in a Beckman TL-100 ultracentrifuge.
Remove the supernatant and use it for the
- Actin filaments shear quickly into small dots.
Several things can contribute to this phenomenon.
Poor-quality myosin containing a significant number
of noncycling heads might be a problem. See Pitfall 1
for advice on how to remove these. Decreasing the
density of myosin heads on the surface and/or increasing
the ionic strength of the assay solution also sometimes
helps, as does decreasing the light intensity. In
general, myosin bound to the surface as monomers
tend to shear less than myosin bound as filaments.
- Actin filaments appear wobbly when they move
or are moving in a back and forward type manner.
If the assay does not contain methylcellulose, any
portion of the actin filament that is not bound along its
length by myosin will experience Brownian motion
and appear very wobbly. Even though these filaments
may be moving, their movement will be erratic and
difficult to quantify. Increasing the density of myosin
on the surface, decreasing the ionic strength of the
assay, or using methylcellulose in the assay buffer
usually helps. The back and forward motion of the
actin filaments seen in the presence of methylcellulose
is merely Brownian motion in the presence of the
viscous solution where the actin filament is restricted
to move mostly along its long axis. If the actin filament
is not bound it will move back and forward. Increasing
the density of myosin or decreasing the ionic
strength of the solution should help.
- Actin filaments photobleach rapidly. Decrease
the light intensity if possible and use image processing to do frame averaging to improve the signal-to-noise
ratio. Degas the solutions. Make sure the glucose,
glucose oxidase, and catalase components of the motility
buffer are good. The presence of 50mM DTT also
aids in preventing photobleaching.
- Actin filaments leave comet tail-like images as
they move. If you are frame averaging, merely
decrease the number of frames averaged. If not, the
problem is likely to be encountered when the actin
filaments are moving fast and a non-CCD type camera
is used. The streaking or persistence in this case is
related to the fact that the tube cameras effectively
average about four frames in producing their image.
The persistence can be attenuated by increasing the
light level or by switching to a lower magnification
I thank Qian Xu and Takeshi Sakamoto for critical
reading of the manuscript.
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