ARTICLE
Received 10 Apr 2012 | Accepted 30 May 2012 | Published 17 Jul 2012

DOI: 10.1038/ncomms1934

Switching of myosin-V motion between thelever-arm swing and Brownian search-and-catch
Keisuke Fujita1,*, Mitsuhiro Iwaki2,3,*, Atsuko H. Iwane1, Lorenzo Marcucci1 & Toshio Yanagida1,3Motor proteins are force-generating nanomachines that are highly adaptable to their everchanging biological environments and have a high energy conversion efficiency. Here weconstructed an imaging system that uses optical tweezers and a DNA handle to visualize
elementary mechanical processes of a nanomachine under load. We apply our system tomyosin-V, a well-known motor protein that takes 72 nm ‘hand-over-hand’ steps composedof a ‘lever-arm swing’ and a ‘Brownian search-and-catch’. We find that the lever-arm swing
generates a large proportion of the force at low load ( < 0.5 pN), resulting in 3 kBT of work. At
high load (1.9 pN), however, the contribution of the Brownian search-and-catch increases todominate, reaching 13 kBT of work. We believe the ability to switch between these two forcegeneration modes facilitates myosin-V function at high efficiency while operating in a dynamic
intracellular environment.

1 Soft Biosystem Group, Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871,

Japan. 2 Graduate School of Medicine, Osaka University, 1-3 Yamadaoka, Suita, 565-0871, Japan. 3 Laboratory for Cell Dynamics Observation, Quantitative
Biology Center, RIKEN, OLABB, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan. *These authors contributed equally to this work. Correspondence andrequests for materials should be addressed to T.Y. (email: yanagida@fbs.osaka-u.ac.jp) or to M.I. (email: iwaki@fbs.osaka-u.ac.jp).
nature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1934Myosin-V is a dimeric motor protein that transports various cargos such as melanosomes, endoplasmic reticulum
and messenger RNAs1,2 by moving processively along
cytoskeletal actin filaments3–5. Because of the cell’s dense and hetero­geneous actin meshwork6, the cargo itself should apply a dynamic
load onto the myosin-V. To overcome this, a single myosin-V converts the chemical free energy of 1 ATP into a directional motion of
36 nm and a maximum force of 2–3 pN (refs 4,7,8). The energy conversion efficiency depends on external load, but can reach ~100%
during a forward step near stall force9.
The minimal unit for myosin-V force generation is a motor orhead domain, which includes the actin and nucleotide-binding site,
and a lever-arm, which has six calmodulin-binding motifs10,11.
A crystal structure and electron micrographs have suggested thelever-arm tilts in conjunction with ATP hydrolysis cycle12,13, leading to the model assumes that the ‘lever-arm swing’ is the dominant
process for force generation14,15. However, recent single-moleculenano-imaging and high-speed atomic force microscopy (AFM)
have revealed that the two head domains alternately step 72 nm(hand-over-hand mechanism)16–18 by combining the lever-arm
swing19 by the lead head with a ‘diffusional Brownian search’ by
the rear head17,20. In general, the lever-arm swing is responsible
for force generation and triggering the Brownian search following
detachment of the rear head. We have argued, however, that thehand-over-hand mechanism in processive myosins also entails aaBrownian search-and-forward-catch component, where attachmentof the detached head to actin to the forward and backward bindingsites is enhanced and suppressed, respectively, in a strain-dependent
manner21,22 and/or by a steric effect between the myosin head andsites23,24, a theory consistent with the A.F. Huxley Brownian ratchetmodel for muscle contraction25. To confirm whether the stroke or
Brownian search is the greater contributor to force generation, wetherefore sought to measure their energetic contribution.
Here we describe a labelling method and measurement systemof high spatio-temporal resolution that uses DNA as a nanometric
tool26, finding that the contribution of the two mechanisms depends
on the load. At low load ( < 0.5 pN), the lever-arm swing is dominantly responsible for force generation, contributing 3 kBT of work.
At high load (1.9 pN), however, the contribution of the Brownian
search-and-catch increases to dominate, reaching 13 kBT of work,whereas that from the lever-arm changes very little. We believe thisswitching mechanism is an important feature that allows myosin-V
to adapt to a cell’s dynamic environment.

Results
Lever-arm swing and Brownian search-and-catch under load.
Figure 1a shows our measurement system. One end of a doublestranded DNA (DNA handle, ~60 nm in length) was biotinylated
and attached to the amino terminus of a myosin-V head via
neutravidin. The opposite end was labelled with digoxigenin (DIG)

Nanometry

Antibody bead
Optical tweezer

HaloTag
BiotinDigoxigenin
DNA (60 nm)

Load

NeutrAvidin(Not to scale)
DNA handle

Motor domain

MyosinV
Directional motion

Actin filament

b
250

150
100

20 nm per division

Displacement (nm)

200

50
0

100 ms per division
1,000 ms per division

Figure 1 | Detection of the lever-arm swing and Brownian search-and-catch by a single myosin-V dimer. (a) Optical trap assay. Inset: illustration of the
DNA-handle linking the myosin head and bead. The structure of the myosin head is obtained by modifying the figure from ref. 27. (b) Processive steps of
single myosin-V molecules at low ATP concentrations (3 µM). Consecutive 20 + 57 nm sub-steps movements are seen. Grey trace, data low-pass filtered
at 400 Hz. Individual steps (red line) were detected by a running t-test (Methods). Black trace, data median-filtered. Inset: sub-steps low-pass filtered at
200 Hz and enlarged. The trace was corrected by an attenuation factor (Methods).
nature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1934

and attached to a 200-nm fluorescent polystyrene bead coated with
DIG antibodies. The bead was optically trapped and the position
was detected with nanometric and millisecond spatio-temporal
resolution. To prevent steric hindrance from the bead, the lengthof the DNA-handle was designed to be longer than the size of the
myosin-V dimer (30–40 nm).
Using this system at low ATP concentrations (3 µM) and low
sampling rate (12.8 Hz), myosin-V was seen to take 77 nm processive
steps (Supplementary Fig. S1). The distribution of the dwell times
between adjacent steps obeyed successive two rate-limiting reactions(k = 2.4 s − 1), consistent with an ATP-binding rate of k = 2.1–2.7 s − 1(refs 28, 29) and second-order rate constants for ATP binding to actomyosin-V of 0.7–0.9 µM − 1 s − 1 (refs 28, 29).These results strongly
suggest myosin-V linked to the optically trapped bead via the DNA
handle and walked in a hand-over-hand fashion16.
When the time resolution was improved to ~2 ms (Methods),
we were able to observe 20 nm sub-steps (Figs 1b and 2a). Steps
were resolved by computer-aided analysis based on a running t-test(Methods). Because the duration of the sub-steps was 123 ms, whichis comparable to the first-passage time for a Brownian head to reacha forward actin-binding site 77 nm away under load (Supplementary
Fig. S2a–c), and the expected bead displacement from the lever-arm
swing is 20 nm (refs 30–33), we concluded the 77 nm steps to becomposed of a 20-nm lever-arm swing and 57 nm Brownian search
sub-step. Using 60-nm gold nanoparticles and no load, Dunn et al.
reported the Brownian search to be 17 ms (ref. 17), a magnitudeless than our result. Differences in the experimental designs likely
explain this discrepancy, as the drag coefficient for our beads was
3.3-fold (200 nm/60 nm in diameter) larger than theirs, while theload applied slowed the diffusion time of the Brownian head exponentially34. We rule out the possibility of artificial steps caused bydetachment of the rear head because the displacement expected bydetachment of the rear head is negligible (Supplementary Fig. S3).
A second peak at 65 nm in the step size histogram (Fig. 2a) can be
explained by convolving the 57 nm Brownian-search and 77 nm stepwhen the 20 nm lever-arm swing could not be resolved (see Supplementary Discussion). The third peak at − 20 nm is probably due tolever-arm reversals (back steps), as these were seen to mostly couple
with subsequent 20 nm lever-arm swings (Fig. 2b)33. Furthermore,correlating − 20 nm steps with subsequent 20 nm steps suggests that
inchworm-like steps, which have been observed in myosin-VI (ref. 35),
were negligible (Fig. 2b). Schematically, inchworm-like steps
would occur from the state 2→5 transition seen in Fig. 2c. Because
the inchworm-like mechanism requires two ATP for every 72 nm
step of a head (36 nm displacement of a tail), whereas the handover-hand mechanism only requires one (Supplementary Fig. S4),
we suggest myosin-V is designed to be an energy-efficient motor.
ATP dependency of the stability in the lead head. In the case ofphysiologically saturating ATP concentrations (1 mM), the − 20-nm
lever-arm swing reversal was more frequently observed (Fig. 3a,b).
That we observed fewer backward steps at low ATP concentrations could be because the post-lever-arm swing conformation ismore stable in the nucleotide-free state than in the ADP-boundstate18,33,36, which increases with rising ATP concentrations, therefore reducing the likelihood of lever-arm swing reversals.
To estimate the probability of a nucleotide-free or ADP-boundstate in the lead head, we calculated the dissociation rate of bothheads.
According to Oguchi et al.37, ADP dissociations from the leadand rear heads are given by−
kADP_leading(F ) = 13 exp(−6.2F / kBT )−kADP_trailing(F ) = 13 exp(1.25F / kBT )respectively, where kADP − is the rate of ADP dissociation; F, force;and kBT, thermal energy. Assuming the internal load is 1.5 pN when
the lead head is in the pre-powerstroke state33, the ADP dissociation rate from the lead head is 1.3 s − 1, which corresponds to a time
constant of 743 ms, and from the rear head is 21 s − 1, which corresponds to a time constant of 49 ms. At 3 µM ATP and 1 mM ATP,the dwell time of the rigour state for the rear head is 417 ms andonly a few milliseconds, respectively28. Therefore, at low ATP concentration (3 µM), the time before ADP dissociation from the leadhead (743 ms) is comparable to the time before ATP binding to therear head (417 + 49 = 466 ms). On the other hand, at physiological
ATP concentration (1 mM), the total dwell time of the rear head isonly about 50 ms, which suggests that for much of the stepping time
the lead head is in the ADP bound state. Thus, the populationof the ADP bound state for the lead head increases with ATP
concentration.
Load-dependent kinetics of the lever-arm swing and reversal.
The durations of state 3 and 2 in Fig. 2c, τ1 and τ2, respectively,
were load-dependent (Fig. 4a) and fit to a single exponential
curve. The inverse of the durations describes the transition rates(1/τ1 = ktotal1 = kreversal + kcatch + kdetach1 and 1/τ2 = ktotal2 = kswing+ kinchworm-like + kdetach2). The both durations (τ1 and τ2) are affected
not only by kreversal or kswing. Especially, τ1 increase at higher load
because kcatch rapidly decreases depending on load. The ratios of therates correspond to the frequency of the observed events (kreversal:
kcatch: kdetach1 = Nreversal: Ncatch: Ndetach1 and kswing: kinchworm-like:kdetach2 = Nswing: Ninchworm-like: Ndetach2; where Ni is the numberof observations for state i). In general, the above relationships
include the rate constant for 40 nm forward inchworm-like steps.
However, because these were negligible (Fig. 2b,c), we can assume
kinchworm-like = 0, simplifying the above relationships.
Although we only observed 9 reversal events among the 68 events
at low load (Fig. 4b), we cannot completely rule out a possibility
that our experimental system poorly detects the lever-arm swing
at low load, presumably due to the restricted time resolution or
rotation of the bead. This means the step-size histogram may
include unresolved 77 nm steps. If so, we cannot determine whether
detachment occurs from the pre- or post-state at low load. This
becomes a problem when analysing τ2, because the size of τ2 depends
on the state from which detachment occurs (Fig. 4c). Therefore, we
exclude dwell times before detachment events for our analysis of
τ2 at low load, instead only analysing dwell times before lever-arm
swings.
Figure 4b shows the normalized frequencies of the subsequenttransition state from states 2 and 3 used to estimate the transition
rates in Table 1 and Fig. 4c.
Figure 5a shows the load dependent transition rate for the leverarm swing and the lever-arm reversal. The lever-arm swing involves
a structural change that is opposed by the load. Therefore, the reaction rate decreases with increasing load in a manner that obeys an
Arrhenius-type transition (Methods; equation (2)). In contrast,
load should promote lever-arm reversal, which is consistent with
our observation that the rate slightly increases with increasing load.
The fitting parameter, d, which relates to the force dependencyof the reaction, indicates the lever-arm reversal is less sensitive toload than the lever-arm swing (Table 1). Therefore, once the leverarm has swung, it robustly maintains the post-lever-arm swing
conformation when sensing load.
Distance parameter of the lever-arm swing. The distance parameter for the swing (dswing) plus the distance parameter for the reversal
swing (dreversal) should equal the total swing distance. However,
because the bead displacement by the swing peaks at 13 ± 5 nm
(mean ± s.d.) in the histogram without correction (data not shown),
experimentally the total distance parameter dswing + dreversal should

nature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1934

a

b
80

14
12
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8
6
4
2
0

ber

Num

Number

60

40

rd
wa

ck
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– 20

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(
ize

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)
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ard
rw

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ize

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tep

)
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s

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nt

ue

eq

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�

ADP

Head trajectory1

ATP
ATP waiting state

Inchworm-like

ADP.PiPre-state

Detachment
ADP2

k detach2

5

20 nm
swing

20 nm
k swing

Brownian searchk reversal

ADP.Pi
Post-state

Detachment

3

ADP

58 nm

58 nm
Pi

ATP waiting state

k catchHand-over-hand

ADP

catch

k detach1

4

Figure 2 | Step size and scheme for myosin-V motion. (a) Distribution of step sizes at 3 µM ATP concentrations (N = 796). The peak positions of athree Gaussian function fit were − 20 ± 11 nm (blue line), 20 ± 8 nm (red line) and 65 ± 24 nm (orange line) (mean ± s.d.). (b) Correlation of step size forbackward steps and the subsequent forward step size. The size of the backward step was plotted against the size of the subsequent step, creating a threedimensional histogram of the data set (N = 168). Most backward steps couple with 20 nm forward steps. This strong correlation suggests that ~40 nm
inchworm-like steps (see (c)) are negligible. (c) Schematic diagram for myosin-V motility under load. The rear head binds ATP and detaches from actin(state 1→2 transition). A 20 nm sub-step results from a lever-arm swing (state 2→3 transition). The free head undergoes a Brownian search-and-catchto bind to the next actin-binding site, resulting in a 57-nm sub-step (state 3→4 transition). For the − 20 nm peak, the lever-arm reverses its motion (state
3→2 transition). Detachment occurs only during the one-headed bound state (states 2 or 3). Inchworm-like steps are negligible (state 2→5 transition).
The right schematic shows a head trajectory that traces a single motor domain during one ATP cycle.

actually equal this displacement 13 ± 5 nm, not 20 nm. Our findingthat dswing + dreversal = 8.8 ± 1.7 nm (Supplementary Table S1) is inagreement with the experimental 13 ± 5 nm value.
Free energy difference between the pre-and post-states. Given theindividual rate constants, we can estimate the free-energy difference(∆G) between the pre- and post-lever-arm swing states of the leadnature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1934

Detach200

Detach 1.5

Detach

1.0
100
0.5

Force (pN)

Bead displacement (nm)a00100 ms per divisionb

100

Number

80
60
40
20
0
–100

– 50

0

50

100

150

Step size (nm)Figure 3 | Detection of 20 nm and − 20 nm lever-arm swings. (a) Myosin
motility at saturating ATP concentrations (1 mM). Grey traces, data lowpass filtered at 400 Hz. Individual steps (black line) were detected by arunning t-test (Methods). Blue arrowheads indicate − 20 nm lever-arm
reversals; black arrows indicate detachment events. (b) Distributions ofstep sizes at 1 mM ATP concentrations. Peak positions of a three Gaussianfunction fit show steps at − 17 ± 8 nm (blue line), 20 ± 6 nm (red line), and
56 ± 25 nm (orange line) (mean ± s.d.). N = 1,056.

head by using the formula kreversal/kswing = exp((∆G − F∆x)/kBT),
where F, ∆x, kB and T denote force exerted by the optical trap, thecharacteristic distance, Boltzmann’s constant and absolute temperature (297 ± 1 K), respectively (Fig. 5b). An electron microscopy study
has found that 93% of lead heads are in the pre-lever-arm swing state
when both myosin heads are bound to actin36. This corresponds toa ∆G = − kBT ln(0.07/0.93) =  + 2.5 kBT, which when extrapolatingfrom Fig. 5b results in a 3.0-pN load (2.6–3.6 pN with a parameter
error). This value agrees with other reports that found the intramolecular load in the two-headed bound state to be 1.5–3.5 pN (refs33,37). At no load, which describes the maximum energy bias of thelever-arm swing, ∆G was estimated to be − 3.3 kBT (Supplementary
Table S1). Because the ATP concentration was saturating, myosin-V
should be predominantly in the ADP-bound state.
In a conventional optical tweezers assay4,7,8, where myosin-V
is loaded via the tail domain (Fig. 6a, inset), myosin-V produces
18 kBT (2 pN×36 nm = 72 pNnm) of work near stall force (2–3 pN).
The work by a single lever-arm swing apparently seems to reach
10 kBT (2 pN×20 nm lever-arm swing), which is inconsistent
with our estimate of the bias energy (3.3 kBT). The discrepancy isreconciled in the discussion.
Maximum work done by the Brownian search-and-catch. The work
done by a myosin dimer can be divided into two parts, work done bystructural changes in the lever-arm and work done by the Brownian
search-and-catch17,20. We showed hand-over-hand stepping is triggered by a 3.3 kBT lever-arm swing in the lead head. The Browniansearch-and-forward-catch contributes work to this system as load
is exerted onto the myosin molecule. During physiological vesicletransport, myosin-V apparently distributes the load from a cargobound to the tail domain completely onto the lead head. However,when the rear head undergoes Brownian motion, the lever-arm in
the lead head should bend 10–15 nm backwards when a 2–3 pN

load is exerted (bending stiffness = ~0.2 pN/nm (ref. 38)). In thesecases, the Brownian head will conduct work when it catches an actin
target 72 nm forward. Therefore, estimation of how large work the
Brownian search-and-catch can potentially generate has significant
meaning. We found that the majority of myosin-V molecules completed the Brownian search-and-catch against 0.93 pN on average(data not shown). This equates to a Brownian search-and-catch contribution of 13 kBT (57 nm×0.9 pN) of work, which is similar to theenergy released from the Pi release (12 kBT) (ref. 34) (Fig. 2c).

Discussion
Here we analysed the lever-arm swing and Brownian searchand-forward-catch done by the lead and rear heads, respectively,
during myosin-V hand-over-hand steps. Attaching a 200-nm bead
and applying a load to one myosin head sufficiently slowed the stepping process, allowing us to estimate the energetic contributionsof these two processes. Furthermore, we could observe lever-arm
reversals during hand-over-hand steps, which could not be seenin previous nano-imaging studies that used fluorescent dyes16 andgold nanoparticles17 or high-speed AFM observations18, because
these used unloaded conditions. Surprisingly, the reversal occurredunder 1 pN load (less than half of stall force (2–3 pN as a dimer))and the estimated free-energy difference between the pre- and postlever-arm swing states was 3.3 kBT, which is much less than the total
work near stall force (18 kBT).
Is the 3.3 kBT energy bias of lever-arm swing sufficient to produce
2–3 pN force and 18 kBT total work at high load? On the basis of ourresults, the rates for the pre- and post-lever-arm swing states areequal at 1.7 pN (Fig. 5a). However, using the load dependency of theenergy difference between the pre- and post-lever-arm swing states
described in Fig. 5b, we can show that when 2.5 pN force is applied
to the tail, ∆G is + 1.5 kBT. This translates into only 19% of the leadheads swinging forward or being in the post-lever-arm swing state(Fig. 2c, state 2→3). However, because kcatch is sufficiently fast, this
19% is almost assured of advancing to state 4 to complete the step
(Supplementary Fig. S5). Thus, while the lever-arm swing seems togenerate > 10 kBT of work ( > 2 pN ×20 nm lever-arm swing), themajority of this actually comes from the forward catch.
In conventional optical trapping nanometry (tail loading),
myosin-V showed ~ ± 20 nm half-steps within 36 nm regular steps
at near stall forces of 1.7–3.0 pN (refs 4,7,8). Our results show thatthe 20 nm forward and backward steps originate from lever-arm
swings and reversals by the lead head during the one-headed boundstate (Fig. 2c, state 2–3). Using our measurement system (head loading), we could observe − 20 nm lever-arm swing reversals at lessthan 1 pN, because the geometry of our system slowed the time for
the Brownian head to catch forward. In a conventional tail-loading
condition, the Brownian head diffuses relatively freely, and rapidly
catches forward actin before lever-arm swing reversal occurs suchthat ~20 nm steps are only observed at high load (see arrows in
Supplementary Fig. S5b).
Electron microscopy results have suggested that the lever-arm
in the lead head is fully populated in the pre-lever-arm swing
state (93%) because of an intramolecular load36 that is consistent
with our results. On the other hand, Sellers and Veigel reported
that the probability between the lever arm-swing and reversal statesin single heads are equal at ~4 pN load33. This difference is likely
because their definition of the lever arm-swing and reversal rate isdifferent from ours. Whereas they defined the lever arm-swing andreversal rate as the reciprocal of the average dwell time in the prestate and post-state, respectively, we obtained the lever arm-swing
and reversal rate by subtracting other rates such as the detachment
and catch rate from the reciprocals of the average dwell times (seeResults and Supplementary Discussion). Additionally, the difference in the force application geometry may partially explain thediscrepancy.

nature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1934

1.0

1.0

0.5

Cumulative frequency

Cumulative frequencya�1
0.6 pN0.9 pN
1.2 pN

20 nm

0.5

0
0

b

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2.0

Figure 4 | Dwell time and frequency of the 20 nm and − 20 nm lever-arm swings. (a) Cumulative frequency of dwell times for post- (left) andpre- (right) lever-arm swing states at different loads, respectively. The inverse of the durations equals the sum of each transition rate for each state(1/τ1 = ktotal1 = kreversal + kcatch + kdetach1 and 1/τ2 = ktotal2 = kswing + kdetach2). Dwell times were fit to a single exponential function (see Methods andequation (1)) after removing data < 60 ms. Inset: Schematic diagram of the transition pathways from the post- (left) and pre- (right) lever-arm swing
states. Following a lever-arm swing, myosin-V takes one of the reversal, catch, or detach1 pathway. Assuming reversal, it next takes either the swing
or detach2 pathway. (b) The normalized frequency of each transition state from the post- (left) and pre- (right) lever-arm swing states. The frequencyof each transition was experimentally obtained by counting the number of reactions. Error bars show s.e.m. The frequencies were 67 at low load, 65
at middle load, and 66 at high load for the post-lever-arm swing state (left), and 27 at low load, 27 at middle load, and 28 at high load for the prelever-arm swing state (right). (c) Load dependencies of the reaction rates kcatch, kdetach1, kdetach2, kreversal, and kswing were described by a single
exponential (see Methods and equation (2)). Each plot was estimated by the load-dependency of the durations in Fig. 4a and the frequency ofeach transition state in Fig. 4b. Error bars show the s.e.m.

Table 1 | Kinetic parameters obtained from the model analysis.
Transition
Reversal
Catch
Detach1
Swing
Inchworm-like
Detach2± indicates standard deviation.

kj(F)=k0 exp( − Fd/kBT)
k0 (s − 1)d (nm)

1.1 ± 0.5
65.6 ± 38
4.9 ± 3.9
39.1 ± 3.6
NA1.3 ± 2.9

− 2.9 ± 1.6
12.5 ± 3.5− 1.6 ± 3.2
5.9 ± 0.5
NA
− 1.9 ± 10.0

Figure 6a shows the contribution of the lever-arm swing and
Brownian search-and-forward-catch for total myosin-V work at
various loads when assuming conventional nanometry (tail loading) or physiological vesicle transport geometry. When 0.5 pN force
is applied to the tail end, the lever-arm swing conducts 2.4 kBT(20 nm×0.5 pN) of work, whereas the total work done by the
myosin-V is 4.4 kBT (36 nm×0.5 pN), meaning the Brownian searchand-catch component is 2.0 kBT. Although the amount of work
done by the lever-arm swing increases with load, it never exceeds
3.3 kBT at saturating ATP concentrations (ADP-bound state).
Any residual work then done by the Brownian search-and-catch
actually dominates at high loads. Thus, our results argue the myosinV is a lever-arm-driven motor under low loads, but a Brownian

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nature communications | DOI: 10.1038/ncomms1934

b

a

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Reaction cordinate–3Pre-state–4

k reversal

5

Parameter error

0

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1.2

0Figure 5 | Lever arm stability under load. (a) Reaction rates, kreversal andkswing plotted versus load. The load dependency can be described as asingle exponential (see Methods and equation (2)). The characteristic
distances, dreversal and dswing, are − 2.9 nm and 5.9 nm, respectively(Table 1), meaning the energy barrier between the two states isasymmetrical. Thus, the lever-arm reversal is more robust to load
than the lever-arm swing. The intersection of the two lines (1.7 pN)
indicates the force at which pre- and post-lever-arm swing conformations
occur at equal probability. The number of replicates studied is 26 at low
load, 19 at middle load, and 15 at high load for kswing, and 9 at low load,
6 at middle load, and 17 at high load for kreversal. (b) Load dependencyof the energy difference between the pre- and post-lever-arm swing
states. Log(kreversal/kswing) is plotted versus load (see Methods andequation (4)). Each plot was estimated using plot data from Fig. 5a.
∆G was estimated to be − 3.3 kBT at no load. This value defines the
maximum energy contribution of the lever-arm swing. Error bars show
the standard error of the mean. Inset: Energy landscape of the two statelever-arm model.

search-and-catch driven motor under high ones (Fig. 6b). This suggests that myosin-V can change its force-generating mechanism
depending on the external force applied to the tail.
Knowing that the vast majority of work done by myosin-V athigh load is a stochastic process (Brownian search-and-catch)and that the proportion of work done by the deterministic leverarm swing and Brownian search-and-catch varies with load offers
important insights into the mechanisms used by myosin-V for its
function. The hand-over-hand steps caused by deterministic leverarm swings would be advantageous for smooth rapid movement at
near-zero load when no obstacles are present (Supplementary Movie
1). However, cells contain a cytoskeleton meshwork and a numberof dynamic molecules and vesicles that risk disturbing transport39.
In these cases, the Brownian search-and-catch mechanism should
be advantageous for avoiding such obstructions (Supplementary
Movie 2). Thus, our findings indicate that myosin-V is an optimized
nanomachine that is highly adaptable to its intracellular environment. These results should have significant implications on thedesign of artificial nanomotors.
That the Brownian search-and-catch significantly contributes toforce generation in myosin-V challenges the theory that biologicalmolecular motors, such as myosin, kinesin and dynein, generate
force primarily from a structural change14,15,40,41 (in the case of
myosin-V, this is the lever-arm swing). More likely, the work distribution between the structural change and the Brownian searchand-catch would vary with the molecular motor. The Brownian
search-and-catch would be favourable for myosin-V, because it
robustly moves long distances (several micrometers) through adiverse cellular environment. Other long distant transporters likekinesin and dynein might have similar features. In fact, a previous
report has estimated that the contribution of the structural change
in kinesin-1, called neck linker docking, is ~1.2 kBT (ref. 42). On
the other hand, muscle myosin (myosin-II) may depend more on

Limit (3.3 k BT )0.9 kBT
0.1

0.5

1

1.5

2

Load applied to Myosin V tail (pN)b

Low load
Lever-arm driven

High load by a obstacle
Brownian search-and-catch driven

Load

Deterministic
Rapid transport

Highly stochastic
Slow, but adaptable to obstacles

Figure 6 | Switching the mode of force generation. (a) Work doneby myosin-V depends on the load applied to the tail (inset). Whereas
the work done by the Brownian search-and-catch increases with load,that done by the lever-arm is constrained by the energy difference
between the pre- and post-lever-arm swing states. (b) Model formyosin-V force generation. At low load, the contribution of the leverarm swing to myosin-V movement is greater, making it a deterministic
and rapid transporter. At high load, the contribution of the Brownian
search-and-catch is greater, making myosin-V a stochastic andadaptable transporter.

the structural change component for force as the region that muscle
myosin functions is more constrained.

Methods

Myosin construct. Human Myosin-V-HMM heavy chain construct: pFB1flag-HT-hMyosinVaHMM-Myc. The 3′ end [bp 3714 to 5484] of the Human
Myosin-Va complementary DNA (ORK07567) was deleted to obtain a MyosinVa-HMM cDNA fragment (encoding a.a. Met1-Thr1238). This fragment includes
the motor, neck, and coiled-coil domains. Flag-tag and HaloTag (DHA, Promega)fragments were attached at the 5′ end of the myosin-Va cDNA fragment for
purification and biotin labelling; Myc-tag was attached at the 3′ end for detection.
The Flag-HT-hMyosin-VaHMM-Myc fragment was introduced into the
Baculovirus expression vector pFastBac1 between the BamHI and HindIII sites.
Human calmodulin construct: pFB-hCaM. The calmodulin coding sequence(Kazusa, ORK11793) was also inserted into pFastBac1 between the BamHIand HindIII sites.
Protein expression and purification. Recombinant viruses for myosin-V heavy
chain and calmodulin were produced by homologous recombination using the
Bac-to-Bac Baculovirus Expression System (Invitrogen). Sf9 insect cells weremaintained in a monolayer culture in ventilated 175 cm2 flasks at 28 °C with 10%
FBS (Invitrogen) and 1×Antibiotic-Antimycotic liquid (Invitrogen) /EX-Cell 420(SAFC Biosciences). After co-infection at an multiplicity of infection of 1 and
0.5, respectively, and incubation for 60 h, cells were collected by centrifugation at
6,000 g for 5 min and stored at − 80 °C. Frozen cells were suspended and sonicatedin 10 ml per g cells of Lysis solution (30 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM
EGTA, 5 mM MgCl2, 5 mM ATP, and 0.05 mM DTT) containing Complete EDTAfree (Roche Diagnostics). After ultra centrifugation at 10,000 g for 20 min, soluble
fractions were mixed with pre-activated Flag-affinity resin (ANTI-FLAG M2
Affinity Gel, Sigma) for 1 h. After removal of the unbound protein, 1 ml Lysis
solution and 1.25 µl HaloTag PEG-Biotin Ligand (Promega) were added at anature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1934

molecular ratio of 1:3 to the resin, which was lightly incubated on ice for 10 min toachieve N-terminal biotinylation. Afterward, Wash solution (30 mM Tris–HCl pH
8.0, 200 mM NaCl, 0.1 mM EGTA, 2 mM MgCl2, 2 mM ATP, and 0.05 mM DTT)
was added to remove any unreacted reagent. Biotinylated protein was eluted by
Elution solution (30 mM Tris–HCl pH 8.0, 200 mM NaCl, 0.1 mM EGTA, 2 mM
MgCl2, 2 mM ATP, and 0.05 mM DTT) including 0.15 mM Flag peptide. The
obtained myosin-V recombinant (~0.3 mg ml − 1) was aliquoted and stored at− 80 °C until use. Biotinylation was confirmed by using a streptavidin-alkaline
phosphatase conjugate and bromochloroindolyl phosphate/nitro blue tetrazolium
as the substrate. All steps were performed at less than 4 °C.
Biotinylated and DIG-labelled DNA linker preparation. A DNA linker (174 bp)
was obtained from a pBluescript II KS( − ) Plasmid using PCR amplification and a
DNA fragment purification system (Wizard PCR Preps DNA Purification System,
Promega). Primers for the PCR amplification were as follows.
Biotinylated T7 primer, 5′ Biotin-CGCGTAATACGACTCACTATAG-3′;
and DIG-T3 primer, 5′ AminoC6 + Digoxigenin-GCGCAATTAACCCTCACTAAAG-3′.
DNA-bead labelling. A 174-base-pair double-stranded DNA oligomer was
synthesized with digoxigenin at the 5′ end of one strand and biotin at the
5′ end of the other. A solution of double-stranded DNA oligomers was mixed at
1,000-hold molar excess with neutravidin. For all incubations and experiments,
buffers contained 25 mM KCl, 20 mM HEPES-KOH (pH 7.8), 5 mM MgCl2
and 1 mM EGTA. Carboxylate modified polystyrene beads (Invitrogen; 0.2 µm
in diameter) were crosslinked to polyclonal anti-digoxigenin (Roche) and coatedwith 10 mg ml − 1 BSA. The beads were incubated with a DNA solution thatwas previously mixed with neutravidin for 30 min at 4 °C. After incubation,
the beads were linked to DNA via anti-DIG antibodies and centrifuged toremove excess neutravidin. Myosin-V was then mixed with the DNAlinked beads at an adjusted ratio such that 30% of the beads displayed
movement.
Single-molecule assay. Fluorescently labelled actin filaments were quickly flowed
into sample chambers in which α-actinin was directly adhered to a glass slide. The
glass surface was then coated with 5 mg ml − 1 casein. Single-molecule assays were
performed at 24 ± 1 °C in buffer containing 0.05% tween, ATP (1 mM or 3 µM) andATP regenerase (0.1 mg ml − 1 creatine kinase and 2 mM creatine phosphate) andan oxygen scavenger system (0.11 mg ml − 1 glucose oxidase, 18 µg ml − 1 catalase,
2.3 mg ml − 1 glucose and 0.5% 2-mercaptoethanol).
Instrumentation and data analysis. Myosin-V tagged to a bead via the DNAhandle was optically trapped and brought to single actin filaments on the coverglass by observing its fluorescent image. Nanometry of the optically trapped bead
was done by projecting the dark-field image onto a quadrant-photodetector witha response time of ~50 µs (ref. 22). Data were sampled at 24 kHz with a bandwidthof 10 kHz. Step sizes and dwell times of sub-steps were analysed automatically bya custom software (LABVIEW 9.0) or by eye. There was no significant difference between the two methods. Data were 400 Hz low-pass filtered before step
detection. t-test values were calculated from low-pass filtered time series of themean displacement and variance using a running time window of five data points.
To determine step sizes, we calculated the time-averaged displacement between
preceding step changes. Amplitudes were calculated relative to the preceding step.
Bead displacement was corrected by an attenuation factor43 such that step sizes
include bending in the lever arm and stretching of the DNA-handle. The initial
step of processive movement and its corresponding dwell time were not includedin the analysis.
Dwell-time distributions of pre- and post-states were plotted as cumulative andnormalized distributions. The transition rate, ktotal, was obtained by least-squares
fitting the model function:

P (F ) = 1 − exp(−ktotal ,it )(1)where t is time, F is force, P is probability, and i = 1 or 2. Load dependency of thereaction rates was described by an Arrhenius-type transition:

 Fd 
k j (F ) = k0 exp  − kBT (2)where k0 is the transition rate in the absence of load, F is force, d is the characteristic distance, kBT is the thermal energy, and j = [swing, catch, detach1, detach2,
or reversal].
Load dependency of the energy difference between the pre- and post-lever-arm
swing states was also described by an Arrhenius-type transition: ∆G − Fd 
kreversal= exp kswing
 kBT 

(3)

Taking the natural logarithm of equation (equation (3)) gives,k∆G Fdln( reversal ) =−kswingkBT kBT(4)

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Acknowledgements

We thank Tetsuya Shimokawa for valuable discussion, Peter Karagiannis for reading themanuscript, and colleagues of the Yanagida laboratory for comments and discussion.
The work was supported by the Grant-in-Aid for Scientific Research on Innovative
Areas of the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
and RIKEN.

Author contributions

M.I. conceived the idea and protocol for the experiments, developed and constructed themeasurement apparatus. K.F. carried out the experiments under the directions of M.I.
and wrote software to analyse the data. K.F. and M.I. prepared the reagents and samples.
A.I. constructed and purified the myosin-V constructs and DNA handle. L.M. carriedout the numerical simulations. M.I. supervised the work and wrote the majority of thepaper. K.F. and L.M. prepared supplementary data and T.Y. provided suggestions.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.
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How to cite this article: Fujita, K. et al. Switching of myosin-V motion between
the lever-arm swing and Brownian search-and-catch. Nat. Commun. 3:956
doi: 10.1038/ncomms1934 (2012).
License: This work is licensed under a Creative Commons Attribution-NonCommercialShare Alike 3.0 Unported License. To view a copy of this license, visit http://
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nature communications | 3:956 | DOI: 10.1038/ncomms1934 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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