ARTICLE

doi:10.1038/nature09450

Video imaging of walking myosin V by
high-speed atomic force microscopy
Noriyuki Kodera1,2, Daisuke Yamamoto1,2, Ryoki Ishikawa3 & Toshio Ando1,2

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical
microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate wasthereby demonstrated. However, the protein molecules themselves are invisible in the observations and havetherefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structureand dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize
myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution
movies not only provide corroborative ‘visual evidence’ for previously speculated or demonstrated molecularbehaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to acomprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a
powerful new approach to studying the structure and dynamics of biomolecules in action.

Proteins are dynamic in nature and work at the single-molecule level.
Reflecting this fact, single-molecule fluorescence microscopy has been
widely exploited to understand how proteins operate (reviewed in ref. 1).
However, what we can observe thereby is the dynamic behaviour ofindividual fluorescent spots (each being emitted from a fluorophore
attached to a selected locus of the molecule), not of the protein molecules
themselves. The structure of proteins has been studied by electron
microscopy, X-ray crystallography and NMR, but the obtained structures are essentially static. To overcome this long-standing problem andmake it possible to record the structure and dynamics of functioning
biomolecules simultaneously, high-speed atomic force microscopy (HSAFM) has been developed2–5. The recent significant improvement in
its performance has been demonstrated in a few imaging studies ofproteins6–8.
Myosin V is a two-headed processive motor and functions as a cargotransporter in cells (reviewed in ref. 9). Facilitated by the processivity ofthe molecule10,11, numerous single-molecule studies have demonstrated that it moves hand over hand with a ,36-nm advance12,13 forevery adenosine triphosphate (ATP) hydrolysis14. However, a comprehensive description of the molecule in action has not yet been attained.
We applied HS-AFM to this highly dynamic protein to capture its
minute dynamic behaviours. In addition to known and speculated
behaviours, previously unappreciated ones clearly appeared in themolecular movies: ‘stomping-like’ brief detachment and rebinding ofeither head; occasional unwinding of the coiled-coil tail of the twoheaded bound molecule followed by the leading lever-arm swing, indicating the presence of intramolecular tension responsible for the powerstroke; and repeated conformational switching of the leading head byadenosine diphosphate (ADP) binding and release. These findings leadto a better understanding of how myosin V operates for its motor
function.

Unidirectional processive movement
Partially biotinylated actin filaments were immobilized using streptavidin with a low surface density on biotin-containing lipid bilayers
formed on a mica surface (Supplementary Fig. 1). To facilitate theweak sideways adsorption of tail-truncated myosin V (M5-HMM)
onto the bilayer surface, a positively charged lipid (1,2-dipalmitoyl3-trimethylammonium-propane (DPTAP), 5%) was included in thebilayer unless otherwise stated. This condition was necessary toclearly visualize M5-HMM molecules.
Figure 1a shows images of M5-HMM moving processively withdiscrete ,36-nm steps (Supplementary Movie 1). A processive run
almost always continued to the full extent of each imaging range used(that is, 4–16 steps). Typical long runs tracked by shifting the scan
area are shown in Supplementary Movie 1c, d. In the two-headed
bound M5-HMM (where by head we mean the motor domain andthe neck domain), the neck–motor domain junction appears smoothin the leading head (L-head) but is V-shaped in the trailing head
(T-head) without exception (Fig. 1b), because the neck regions
emerge from different parts of the motor domain. The short coiledcoil tail was mostly tilted towards the minus end of actin (Fig. 1a,arrows). These features are consistent with electron microscopy
observations15–17 and can be used to determine the actin polarity whenbound M5-HMM is stationary.
The average translocation velocity (V) as a function of ATP concentration ([ATP]) was well fitted by V 5 ds(1/k1[ATP] 1 1/k2)21, where
ds is the step size, k1 is the second-order ATP binding rate constant andk2 is the first-order ADP dissociation rate constant (Supplementary
Fig. 2). The maximum velocity observed without DPTAP was similar
to that measured by fluorescence microscopy13,18, indicating the negligible effects of the tip–sample and surface–sample interactions on
motor activity. When 5% DPTAP was included in the substrate surface, the values k1 5 0.9 6 0.3 mM21 s21 and k2 5 7.2 6 1.5 s21 wereobtained (Supplementary Table 1). The former agrees with previous
reports, but the latter is about 70% of previously reported values13,18(,11 s21), suggesting that the surface–sample interaction slightly
reduces the rate of ADP release from the T-head.

Hand-over-hand movement
As observed above, the ,36-nm advance was completed within one
frame (146.7 ms); therefore, the molecular process occurring during a

1

Department of Physics, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. 2CREST, JST, Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. 3Department of Molecular and Cellular
Pharmacology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.

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ARTICLE RESEARCHab

L-headPlus
end

T-headMinus
end
293 ms 2,201 ms+

c

1,027 ms 2,347 ms

Colour code of images
Straight L-head
T-head foot stomp
T-head translocation Unspecified

Moving direction

147 ms 1,614 ms

147 msStreptavidin
11,174 ms 2,494 msed

147 ms++

293 ms

293 ms

440 ms

440 ms

587 ms

587 ms

734 ms

734 ms2
1,320 ms 3,521 ms

1,467 ms

3,814 ms

3

Minus
end

Plus
end

Figure 1 | Directly visualized walking M5-HMM. a, Successive AFM images
showing processive movement of M5-HMM in 1 mM ATP (Supplementary
Movie 1). Arrowhead, streptavidin molecule; arrows, coiled-coil tail of M5HMM tilted towards the minus end of actin. Scan area, 130 3 65 nm2; scale bar,
30 nm. b, Schematic of two-headed bound M5-HMM. c, Schematic explaining
the images in d and e. d, e, Successive AFM images showing hand-over-handmovement in 1 mM ATP (d; scan area, 150 3 75 nm2; scale bar, 50 nm) and in
2 mM ATP (e; scan area, 130 3 65 nm2; scale bar, 30 nm) (Supplementary
Movie 2). The swinging lever is highlighted with a thin white line. Verticaldashed lines in a, d and e show the centres of mass of the motor domains, and aplus sign is used to indicate the plus end of actin. All images were taken at a146.7 ms per frame.

step could not be resolved. However, additional streptavidin molecules placed on the substrate surface as moderate obstacles to theadvance allowed the visualization of this process (Fig. 1c–e). After
T-head detachment, the nearly straight leading neck swung from thereverse arrowhead (R-ARH) orientation to the arrowhead (ARH)orientation (Supplementary Movie 2), confirming the swinging
lever-arm motion initially proposed for muscle myosin19. The
detached T-head rotationally diffused around the advancing neck–
neck junction (no translational diffusion on the actin occurs;
Supplementary Movie 2c) and then bound to a forward site on theactin filament, completing one step. Thus, the hand-over-hand movement12,13, including the intermediate process, was directly visualized
at high resolution. We did not observe other behaviours of M5-HMM
predicted by the ‘inchworm’20 and ‘biased diffusion’21 models, at least
with the time resolution used. The captured images show that theforward movement is driven not by bending but by rotation of theL-head. The rotation seems to occur spontaneously after T-headdetachment, suggesting that intramolecular tension driving theL-head swing exists in the two-headed bound molecules.

two-headed bound M5-HMM, and that the tension release causedby the unwinding results in the L-head rotation, similar to theL-head behaviour in the hand-over-hand movement. These observations provide the following insights into the motor mechanism: first,
the neck–motor domain junction of L-head–ADP is sufficiently flexible to allow the head to bind strongly to actin in the R-ARH orientation, even without transitioning through a weak-binding ADP–Pibound state (Pi, inorganic phosphate); second, this strong binding
forces the L-head into a strained ‘pre-stroke’ state; and third, therotation of the L-head is likely to occur spontaneously when theconstraint holding it in the strained state is removed by T-headdetachment, suggesting that the lever swing by the L-head is notaccompanied by chemical transitions there.

Unfolding of the coiled-coil tailIn a saturating amount of ADP, both heads were bound to a single
actin filament and the L-head always appeared nearly straight (curved
slightly outwards; Fig. 2a, Supplementary Movie 3 and Supplementary
Fig. 3b), as observed for M5-HMM moving in 0.1 mM and 1 mM ATP.
In this nearly straight conformation, the neck domain appears to
emerge from the rear of the motor domain17. During imaging of thetwo-headed bound M5-HMM, we sometimes encountered unwinding of the short coiled-coil tail. After unwinding, the monomerized
L-head immediately rotated towards the ARH orientation (Fig. 2b andSupplementary Movie 4). This head separation was rarely reversed(Supplementary Movie 4c, d). This unwinding is unlikely to be causedby the tip–sample interaction because monomeric heads weredetected in the earliest frames in the successive movies in new scan
areas (see the latter half of Supplementary Movie 4b), and because aprevious study showed the presence of a single-head fragment with anintact coiled-coil region in an expressed M5-HMM sample, although
it was interpreted to be due to partial proteolysis22. Rather, this finding directly demonstrates that intramolecular tension exists in theFoot stomp in ATP
In general, during the actomyosin ATPase cycle, the strained prestroke state is always formed after Pi is released from an ADP–Pibound head weakly interacting with actin23. Thus, it may be consideredthat the strained pre-stroke state, which is formed directly by thebinding of L-head–ADP to actin, does not participate in the forward
step. However, we observed molecular behaviour in ATP that indicatesthat it does participate. In two-headed bound M5-HMM, both of the
motor domains frequently exhibited brief dissociation and reassociation on the same actin filament (or a brief translocation by around
65 nm along the actin filament), whereas M5-HMM remained at
approximately the same position on the filament (Fig. 1, Supplementary Fig. 3a, Supplementary Movies 1 and 2, and Methods). We havetermed this behaviour ‘foot stomp’. Note that when the T-head dissociates, it rarely rebinds to actin without stepping forward; thus, theT-head foot stomp is usually observed as a brief translocation along theactin filament. The foot stomp was more frequently observed at
the L-head than at the T-head (by a ratio of approximately 3:1;
Fig. 3a). The briefly detached L-head does not carry bound Pi because
Pi is rapidly released from an ADP–Pi-bound head when it attaches toactin24. Nevertheless, the detached L-head with only ADP boundrebinds to actin, still in the R-ARH orientation, and then swings
forward following T-head detachment.
At each step, the L-head foot stomp occurred about twice in 0.1 mM
ATP and 0.4 times in 1 mM ATP on average. These high frequencies of
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RESEARCH ARTICLE
a

a

+

{76}
{339}
{468}
<207> <256> <170>
[38]
[34]
[196]
(7,307) (5,538) (4,279)

0.33 s12.99 s8.33 s

13.33 s9.00 s

14.66 sFoot stomp events (%)

100

9.33 s<1,568>[88](30,889)<873> <456>[137](32,897)50

14.99 s00.1 μM

1 μM

1 μM
Long runADP(1 mM)

{76}
{339}
{468}
[196]
[38]
[34]
(7,307) (5,538) (4,279)[137](32,897)

All

ATP

b

b

26.32 s26.66 s27.66 scd17.33 s+++0.33 s0.33 s
17.66 s

Proportion of conformations (%)

25.99 s1000.67 s50

015.33 sc

0.1 μM

6
Average lifetime (s)20.33 s2.33 s

1 μM

1 μM
Long runNF

0.1 μM 0.5 μM

50 μM

1 mM

<131>
<161>
<147>

4

<336>
2

k+[ADP]
ST

k–

kS–B

Foot stomp events
L-head foot stomp
T-head foot stomp

STADP

Molecular conformations

k–Sharply bent L-headBNStraight L-head

2 μM
ADP

20.66 sColour code of images

Sharply Straight
bent
NF

[112]
[81]
[134]
[88]
[84]
(21,807) (17,367) (39,617) (23,142) (30,889)

ATP+
14.99 s<417>

Straight L-head

0
0

1.0

2.0

ADP concentration (μM)

Sharply bent L-head
15.66 s4.00 sL-head foot stomp

Figure 2 | AFM images of M5-HMM in ADP and under the nucleotide-free
condition. a, Successive AFM images taken in 50 mM ADP (Supplementary
Movie 3). b, Successive AFM images showing unwinding of the short coiledcoil tail of two-headed bound M5-HMM observed in 50 mM ADP(Supplementary Movie 4). c, Successive AFM images made under the
nucleotide-free condition (Supplementary Movie 5). d, AFM image andillustration of nucleotide-free M5-HMM with heads bound to adjacent actin
subunits. A plus sign is used to indicate the plus end of actin. All images weretaken at 333.2 ms per frame with a scan size of 90 3 90 nm2. Scale bars, 30 nm.

occurrence indicate that a foot stomp at the L-head occurs while it isbound to ADP, not while it is bound to ADP–Pi. If Pi release, the rateof which is independent of [ATP], were significantly delayed by theinteraction of M5-HMM with the substrate surface containing 5%
DPTAP, M5-HMM in high [ATP] would walk much more slowly
than observed. Moreover, the foot stomp also occurs when DPTAP
is absent from the substrate. A foot-stomp-like behaviour was previously suggested in a fluorescence microscopy observation of walking
myosin V molecules25. Thus, the foot stomp is an inherent behaviourof myosin V. As will be described later, the foot stomp also occurs in
ADP and under the nucleotide-free condition.

Conformational transition in the L-head
Even under the nucleotide-free condition, M5-HMM was bound toactin through the two heads (Fig. 2c, Supplementary Movie 5 andSupplementary Fig. 3c). However, unlike in the presence of nucleotides,

Figure 3 | Foot stomp and L-head conformations in different nucleotide
conditions. a, b, Proportion of foot stomp frequencies observed at the L- andT-heads (a) and of the detected straight and sharply bent conformations of theL-head (b). The numbers of steps, events, molecules and frames examinedusing images within which both heads were fully located are enclosed in {curly},,angle., [square] and (round) brackets, respectively. ‘Long run’ represents
data obtained using wide imaging areas. NF, nucleotide free. c, Average
lifetimes of the L-head with a straight conformation for various [ADP] values.
The total numbers of straight-to-bent transition events observed with ,50
different molecules under the respective [ADP] values are shown in angle
brackets. Inset, reaction scheme (ST, nucleotide-free, straight L-head; STADP,
ADP-bound, straight L-head; BN, sharply bent, nucleotide-free L-head). Error
bars, 6s.e.m.; kS–B 5 0.32 6 0.02 s21 (best-fit value 6 s.d.).

the leading neck frequently had a sharply bent conformation and alternated back and forth between this conformation and the nearly straightconformation (Fig. 2c and Supplementary Fig. 3c), indicating that they
are in equilibrium. The sharply bent conformation at the L-head was
also observed recently by negative-stain electron microscopy of actoM5-HMM, although the chemical state was unspecified17. In the sharply
bent conformation, both heads have the ARH orientation around the
motor domain; thus, both necks appear to emerge from the front of therespective motor domains. As will be clarified later, this sharp bending
has no relevance to power-stroke generation but provides a useful indicator of whether or not the L-head contains ADP.
The proportions of the straight L-head detected (r) were 0.98 in 1 mM
ADP and 0.36 under the nucleotide-free condition (Fig. 3b). This finding
is consistent with the ARH orientation distributions of single-headed

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ARTICLE RESEARCH
myosin V under the nucleotide-free and ADP-bound conditions (Supplementary Fig. 4). Under the nucleotide-free condition, the orientation
angle relative to the long axis of the actin filament was 34 6 10u (mean 6s.d.) (although a double-Gaussian distribution is somewhat significant:33 6 9u and 51 6 7u; F-test, P , 0.05). In ADP, however, the angle wasdistributed more widely (29 6 11u and 51 6 15u; F-test, P , 1026),
which may be relevant to two different ADP-bound states in equilibrium23. Therefore, the hinge around the neck–motor domain junction of
the ADP-bound head is more flexible than that of the nucleotide-freehead. Because of the relatively rigid hinge of the nucleotide-free head, theleading neck in the R-ARH orientation tends to be sharply bent to release
the large strain accumulated in the neck. This rigid hinge is also indicatedby the observation that both heads are rarely bound to adjacent actin
subunits predominantly in the nucleotide-free condition (Fig. 2d).

ADP release from the L-head
In 1 mM ATP and even in 0.1 mM ATP, the leading neck was mostly
straight (r < 0.98) (Fig. 3b), which suggests that the L-head retains
ADP until the T-head binds to ATP and detaches from actin, consistent with previous reports14,26–30. From the proportion (Fig. 3b) andlifetime (Fig. 3c and Supplementary Movie 6) of the straight L-head asa function of [ADP], we estimated the kinetic parameters for ADP
binding/dissociation on the L-head: the ADP dissociation constant is
Kd 5 k2/k1 5 0.075 6 0.013 mM; the ADP dissociation rate constantis k2 5 0.100 6 0.004 s21; and the second-order ADP binding rate
constant is k1 5 1.3 6 0.3 mM-1s-1. The value of k2 is ,70 times
smaller than the corresponding rate for the T-head estimated above(k2 5 7.2 6 1.5 s21). This degree of asymmetry between the two heads
is similar to that previously reported26. The value of k1 is ,10 times
smaller than the corresponding value, 12.6 mM21 s21, previously
measured in a solution study on single-headed myosin V24. The
ADP dissociation rate constant of 0.100 s21 means that, on average,
one ADP is released from the L-head every 10 s. However, M5-HMM
walks many steps during 10 s. Thus, we confirm the consensus viewthat the sequential events of ADP release, the subsequent ATP binding and the resulting head dissociation take place solely at the T-head,
which is the basis for the processivity and hand-over-hand stroke
generation14,26–30. Accordingly, the sharp bending observed at thenucleotide-free L-head has no relevance to power-stroke generation.

Foot stomp in ADP and nucleotide-free conditions
The foot stomp observed in ATP also occurred with higher frequencies
at the L-head under all the nucleotide conditions examined here
(Fig. 3a, Supplementary Fig. 5 and Supplementary Movies 3 and 5).
Note that in ADP and under the nucleotide-free condition, complete
detachment of the T-head was only sometimes observed. This detachment was followed by a forward step. This event was also counted as afoot stomp. The foot stomp at the T-head mostly occurred as brieftranslocation along actin. The frequency difference between the twoheads became smaller when the L-head assumed a sharply bent conformation (L-head/T-head frequency ratio, ,3:2). Thus, the frequencyof foot stomping strongly correlates with the orientation of the boundhead. Both heads would by nature preferentially take the ARH orientation. However, the nucleotide-bound L-head is forced to bind to actinin the R-ARH orientation and hence tends to detach from actin, leading to more frequent foot stomping. This implies that the L-head would
be less susceptible to catalytic activation by actin, possibly contributing
to some extent to the asymmetry in the kinetic constants between thetwo heads.

Discussion
In general, the conformational change in myosin upon the hydrolysisof bound ATP to ADP–Pi is the recovery stroke (that is, the poststroke-to-pre-stroke transition), and the actin-activated Pi releasefrom ADP–Pi reverses the head conformation to produce a forward
force23. However, a forward force (or the strained pre-stroke state) can

also be produced by the direct binding of the ADP-bound L-head toactin. In ATP, the force that is produced in this way after a foot stomp
at the L-head drives lever-arm swing. The number of foot stomp
events at the L-head per step is around one only for low [ATP].
However, this type of force generation is likely to take place also forhigh [ATP] under a loaded condition. Moreover, this force generation
mechanism suggests that the ADP-bound head has the potential toproduce a force repeatedly when an appropriate backward force isapplied. This potential was recently indicated in an optical-trap-based
experiment on a single myosin V head interacting with actin under acertain backward load31. The power stroke is reversed and actinbound pre- and post-stroke conformations are repeated before one
ATPase cycle is completed. In Supplementary Fig. 6, we propose amodel for the chemomechanical cycle of myosin V based on previous
studies and the new findings obtained here.
As demonstrated here, previously known and unknown behavioursof M5-HMM clearly appear in the molecular movies. Unlike previous
methods, high-resolution HS-AFM imaging unselectively provides
comprehensive information on the structure and dynamics of a functioning molecule. Thus, the HS-AFM imaging of functioning biomolecules has the potential to transform the fields of structural
biology and single-molecule biology.

METHODS SUMMARY
We prepared M5-HMM from chick brain, and prepared partially biotinylated
actin filaments and stabilized them with phalloidin11. Biotin-containing lipid
bilayers were formed on a mica surface8. We immobilized the biotinylated actin
filaments on the bilayer surface and then deposited a solution of M5-HMM on thesurface. All imaging experiments were performed in the tapping mode using alaboratory-built HS-AFM apparatus2,3.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 3 June; accepted 24 August 2010.
Published online 10 October 2010.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank T. Uchihashi and H. Yamashita for technical assistanceand discussion, and J. Sellers for critical reading of the draft and for comments. This
work was supported by CREST/JST, Special Coordination Funds for Promoting Scienceand Technology (Effective Promotion of Joint Research with Industry, Academia, andGovernment) from JST, a Grant-in-Aid for Basic Research (S) from JSPS, andKnowledge Cluster/MEXT–Japan.
Author Contributions N.K. performed the HS-AFM experiments and data analysis andwrote the first draft of the manuscript. D.Y. and N.K. developed the lipid-bilayer-based
assay system. R.I. participated in the sample preparations in the early stage of thisstudy. T.A. and N.K. developed the HS-AFM apparatus. T.A. designed the experimentand prepared the final manuscript.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should be
addressed to T.A. (tando@kenroku.kanazawa-u.ac.jp).

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ARTICLE RESEARCHMETHODS
32

Protein purification and reagents. We prepared M5-HMM from chick brain .
Partially biotinylated (20%) actin filaments were prepared and then stabilizedwith phalloidin11. Streptavidin, synthetic lipids, apyrase and hexokinase werepurchased from Wako Pure Chemical, Avanti Polar Lipids, Sigma-Aldrich andRoche Diagnostics, respectively.
Preparation of samples for AFM observation. We formed biotin-containing lipid
bilayers on a mica surface8,33. A typical lipid composition was 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-3-trimethylammonium-propane(DPTAP) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)(biotin-cap-DPPE) in a weight ratio of 0.85:0.05:0.1. In some cases, the content of
the positively charged lipid DPTAP was reduced. After rinsing the lipid bilayer
substrate with buffer A (20 mM imidazole-HCl (pH 7.6), 25 mM KCl, 2 mM
MgCl2, 1 mM EGTA, 5 mM DTT), we deposited a drop (2 ml) of streptavidin in
buffer A (10 nM) on the substrate for 3 min. In some experiments, additional
streptavidin molecules (2 ml, 40 nM) were deposited on the surface for 2 min.
After rinsing with buffer A, we deposited a drop (2 ml) of partially biotinylated
actin filaments (1 mM) in buffer A on the lipid bilayers for 10 min. After rinsing
with a solution containing either 0.1 mM to 1 mM ATP, 0.1 mM to 1 mM ADP(1 U ml21 hexokinase and 10 mM glucose were added in the case of 1 mM ADP toremove contaminating ATP) or 1 U ml21 apyrase (to ensure the nucleotide-free
condition) in buffer A, we deposited a drop (2 ml) of the same solution plus M5HMM (0.1 to 1 nM) on the lipid bilayers for 3 min. Finally, the sample was attachedto the scanning stage of a HS-AFM apparatus and immersed in the same solution(,60 ml) without M5-HMM. When the lipid bilayers did not contain the positively
charged lipid DPTAP, we further added M5-HMM to the observation solution.
HS-AFM apparatus and cantilevers. We used a laboratory-built tapping-mode
HS-AFM apparatus2,3 together with small cantilevers designed for HS-AFM34(spring constant, 0.1–0.2 N m21; resonant frequency in water, 0.8–1.2 MHz; quality
factor in water, ,2). The probe tip was grown on the tip of a cantilever by electronbeam deposition and was further sharpened by argon plasma etching2. In the best
case, a tip apex radius of ,4 nm was achieved. To achieve a small tip–sample
loading force, the free-oscillation peak-to-peak amplitude of the cantilever (A0)was set to 1.5–2.5 nm and the amplitude set point was set at more than 0.9A0.
We note that the mechanical quantity that affects the sample is not the force itself
but the force impulse, that is, the product of force and the time over which the force
acts. In tapping-mode HS-AFM, the time of force action is short (,100 ns) and,
therefore, a relatively large peak force (,20 pN) would not affect the sample significantly. All the imaging experiments could be routinely conducted without difficulty except for the occasional deterioration of image quality owing to tip smear or
cracking, which was difficult to control. The tip smearing was caused by the attachment of molecules contained in the sample, but they were sometimes removed
during imaging. The cracking of the sharp tip yields a double image of every object,
as shown in Supplementary Movie 6.
HS-AFM imaging and processive run. All observations were performed at room
temperature (24–26 uC). To facilitate rapid imaging of moving M5-HMM molecules on actin, we chose sample areas in which actin filaments were aligned
approximately parallel to the x direction. Myosin V walks approximately straight
along actin filaments as the two heads span the filament’s helical pseudorepeat of,36 nm (refs 10, 12–15). This property and the high-precision feedback systemof the instrument2,35 facilitate the continuous imaging of walking M5-HMM
molecules weakly adsorbed sideways onto the DPTAP-containing substrate surface. In the absence of DPTAP in the substrate, M5-HMM molecules often
walked on the top surface of actin filaments. In this case, it was difficult to acquire
high-resolution images because AFM is inefficient for visualizing molecules notsupported on a surface.
High-resolution imaging of moving M5-HMM. To obtain high-resolution
images of M5-HMM molecules translocating along actin in 0.1–2 mM ATP, thescan area was narrowed (typically to about 130 3 65 nm2) and the number ofpixels was optimized (80 3 40 or 80 3 50) such that the phase delay in tracing thesample surface was minimized and the imaging rate (146.7 ms per frame) was
high enough to capture the moving molecules. When long processive runs (,15steps) in 0.1–2 mM ATP were observed, the molecules were tracked by shifting thenarrow scan area manually (mainly in the x direction). The tracking sometimes
could not be continued when the target molecule collided with a molecule walking
ahead, when a portion of discontinuous substrate surface was encountered orwhen the manual tracking by an operator was not appropriately made. The
occasional failure of continual tracking is not due to the tip–sample interaction.
Velocity measurements. In the experiment to determine the translocation velocityof M5-HMM for various [ATP] values, we fixed the scan areas (400 3 125 nm2) andcaptured images at 146.3 ms per frame. To facilitate rapid imaging of this relatively
wide scan area, the number of pixels was reduced to 80 3 25 (thus, the scan velocity
in the x direction is only slightly higher than that in high-resolution imaging
mentioned above). The image quality was generally lowered but was high enoughto identify M5-HMM molecules on actin. When the velocity of a translocating
molecule was high, some of the dwell events could not be detected. In this case,
we determined it using at least four different dwell positions where the moleculebeing tracked was captured on video.
Imaging for detecting foot stomp and L-head conformations. In the experiment to analyse foot stomping and L-head conformations in ADP and under the
nucleotide-free conditions, we carried out the imaging at 333.2 ms per frame
using fixed imaging areas (typically 90 3 90 nm2 with 80 3 80 pixels). In theexperiment to analyse foot stomping and L-head conformations in ATP, theimaging was carried out at 146.7 ms per frame using two scan modes. In one
mode, the imaging areas were fixed (typically at 130 3 65 nm2 with 80 3 40
pixels). M5-HMM molecules that entered the imaging areas were captured onvideo. In the other mode, a translocating M5-HMM molecule was tracked byshifting the scan area (typically 130 3 65 nm2 with 80 3 40 pixels).
Image analysis. We applied to each AFM image a Gaussian filter for removing
spike noises and a flattening filter for removing the substrate-tilt effect. Eachposition of the motor domain of M5-HMM was determined by calculating thecentre of mass to measure its displacement. The following events were considered
as a foot stomp: (1) an entire head disappears and then reappears at approximately the same position; (2) either motor domain of a two-headed bound M5HMM molecule is translocated by around 65 nm along the actin filament; and(3) a motor domain disappears while its neck domain is visible (that is, the headdetaches while scanning the region where the motor domain was attached in theprevious frame scan, and then rebinds to actin). Moreover, when an entire T-headdisappeared in ADP and under the nucleotide-free condition, the molecule sometimes stepped forward. This event was also counted as a foot stomp. We note thata foot stomp was also observed even when the substrate surface did not contain
DPTAP, although we did not systematically analyse this. When the L-head is
sharply bent, the neck–neck junction becomes closer to the bound actin filament(and, hence, the space surrounded by the two heads and actin becomes narrower).
We identified the sharply bent conformation by visual observation of these features. For the quantitative analysis of foot stomp events and L-head conformations in ATP, we analysed only images within which both heads were fully locatedbefore and after a step. Therefore, we observed more steps than are shown in
Fig. 3a, b.
Kinetic analysis of conformational change at L-head. We analysed the proportion of the straight L-head conformation detected (r) as a function of [ADP](Fig. 3b) to estimate the ADP dissociation constant for the L-head (Kd) using
the relationshipr(½ADP)~rD ½ADPzrNF Kd½ADPzKd

where rD 5 0.98 and rNF 5 0.36 are the proportions of straight L-head detected ina saturating amount of ADP and under the nucleotide-free condition, respectively. Considering the reaction scheme (Fig. 3c, inset), the average lifetime of thestraight L-head, ÆtSTæ, is expressed approximately ask{ kz ½ADP {1k{ kSB {1znADP k{ zð1ÞhtST i ~nNF kSB zk{ zkSBk{ zkz ½ADP
where kS–B represents the spontaneous straight-to-bent transition rate for the
nucleotide-free L-head; nNF and nADP are the fractions of nucleotide-free
L-head and ADP-bound L-head for a given [ADP] value, respectively, which
are calculated using Kd 5 0.075 mM; and k2 and k1 are the ADP dissociation
rate constant and ADP association rate constant at the L-head, respectively. Using
equation (1), the observed ÆtSTæ as a function of [ADP] (Fig. 3c) was analysed toestimate kS–B, k2 and k1.
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tips. Jpn. J. Appl. Phys. 42, 4844–4847 (2003).
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