Single-Molecule Nanobiotechnology
Kazuo Kitamura, Akihiko Ishijima, Makio Tokunaga and Toshio Yanagida

Abstract

Kazuo Kitamura

Akihiko Ishijima
Department of Applied Physics,
School of Engineering, Nagoya
University, Chikusa-ku, Nagoya,
Aichi 464-8603, Japan

Makio Tokunaga
Structural Biology Center, National
Institute of Genetics, Mishima,
Shizuoka 411-8540, Japan

1. Introduction
Actomyosin, a complex of actin filaments
and myosin motor proteins, is responsible formuscle contraction. The sliding movement of
actin filaments relative to myosin molecules isdriven by the chemical energy of ATP hydrolysis. A long-held model for this process hypoth-

Toshio Yanagida
Department of Physiology &Biosignaling, Osaka University
Medical School, 2-2 Yamadaoka,
Suita, Osaka 565-0871, Japan

Fig. 1 Direct capture and manipulation of asingle S1 molecule by a scanning probe.
(a) Schematic drawing of the experiment. A
single S1 molecule, biotinylated andfluorescently-labeled with Cy3, was attached(at its tail end, through the biotin-streptavidin
system) to a scanning probe and observed by
objective-type TIRFM. The displacement
produced when the S1 molecule was brought
into contact with an actin bundle bound to a
glass surface was determined by measuring
the position of the needle with nanometer
accuracy.
(b) Fluorescence images of single S1 molecules.
The micrograph shows superimposed imagesof single S1 molecules either captured by theprobe (arrowhead) or bound to actin bundles
on the surface of the coverslip. The red andyellow spots respectively represent those seenin images before and after the stage was
moved by a piezoelectric actuator. The
captured S1 molecule (arrowhead) did not
move with the stage but could be moved
independently by piezoelectric scanners
holding the needle. Bar: 5 µm.

eses that one ATP molecule is hydrolyzed by amyosin head, causing the myosin head tochange its structure and pull the actin filament
by one step.1) This model is analogous to theprinciple of man-made machines that operate
deterministically at energies much higher than
the thermal noise. The myosin molecule, however, is nanometers in size and has a flexible
structure, and it operates at an energy as small
as the average energy of thermal noise. It istherefore very prone to thermal agitation. Actomyosin motors can thus operate under thestrong influence of thermal noise, with highchemo-mechanical energy conversion (40%maximum). The mechanism underlying theactomyosin motor must therefore be essentiallydifferent from the mechanism one would predict by analogy to man-made machines. The
working principle of the actomyosin motor can-Epoch-making techniques for manipulating a single biological macromolecule havebeen developed recently and used to measure
directly the chemo-mechanical reactions of asingle molecule of actomyosin, the molecularmotor of muscle. The dynamic properties andthe unique operation of actomyosin molecules,
which are different from those of man-mademachines, suggest that the mechanisms of molecular machines are flexible and effective.

Single-Molecule Processes Project,
JST, 2-4-14 Senba-Higashi, Mino,
Osaka 562-0035, Japan

(a)
Prob

Water
Glass
Oil

e

Evanescent Field

Nanometryθc=61.0oθa=67.1oObjective Lens N.A.=1.4

Biotinylated probe

ManipulationStreptavidin
S1
Displacement

Evanescent field(b)

Actin bundle

Laser

Glass
Laser in

Laser out

Single-molecule imaging

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(a)not be elucidated without resolving the intrinsic characteristics of these molecular machines.2)
To explore the mechanism of the actomyosin motor, we developed a subtle techniqueusing a scanning probe to manipulate a single
myosin molecule.3) This technique has been able
to resolve the process generating the displacements produced by a single myosin head. We
have also demonstrated recently that single
fluorophores in aqueous solution can be observed in real time by total-internal-reflection
fluorescence microscopy (TIRFM) refined so thatthe background noise is very low4,5) and haveused this technique to detect individual ATP
turnovers of a myosin molecule by observing
the single association/dissociation events of afluorescent ATP analog, Cy3-ATP. Furthermore,by combining techniques for single-molecule
imaging and nanomanipulation using an optical trap, we have developed an assay for simultaneously measuring individual ATPase andmechanical reactions of single myosin molecules while they are generating force.6) This
assay shows how the ATPase reaction corresponds to the mechanical events in the actomyosin motor.

2. Nanomanipulation of single
myosin heads by a scanningprobe
We first investigated the process generating the movement of single actomyosin molecules by using new technologies for the imaging and nanomanipulation of single
biomolecules.3) By using scanning-probe-based
technology (Fig. 1), we measured the tiny force
that an individual myosin head exerts on actin- just piconewtons - and the tiny displacement
it causes - just nanometers. The scanningprobe, consisting of a fine glass needle (50100 µm long and 0.3 µm in diameter) and a 5to 7-µm-long ZnO crystal whisker with a very
sharp tip (one whose radius of curvature wasabout 15 nm), was mounted on a three-dimen-(b)
Split photodiodePL

Objective lensFrequency doubled
Nd:YAG laser

Probe
Samplestage
Objectivelens NA1.4

DM

A

λ/4

L
M
IISIT

BF

PL

Fig. 2 (a) Experimental setup for manipulating a single myosin head by using a scanning probe.
(b) Schematic of the system. λ/4, quarter-wave plate; A, diaphragm; L, focusing lens; M, mirror; DM, dichroic
mirror; PL, projection lens; BF, bandpass filter; IISIT, image-intensified SIT camera.

sional piezoelectric scanner. The stiffness ofthe needles was low (0.01 - 0.03 pN/nm), andthe forces and displacements produced by asingle myosin head could thus be clearly detected.
Myosin head molecules were fluorescently
labeled and biotinylated at the tail end, awayfrom the ATP- and actin-binding sites on the
myosin head, thereby avoiding damage due tolabeling and interactions with the attached surface. The tip of the scanning probe was coatedwith streptavidin so that single myosin heads
could be captured specifically at thebiotinylation site. As seen in Fig. 1(b), single
myosin head molecules captured on the tip of
the scanning probe were visualized clearly, at ahigh fluorescence-to-background ratio, by anevanescent field produced in a simple way by
objective-type TIRFM.5) In this system, the illumination mode is easily switching from epi-fluo-

rescence microscopy to TIRFM by moving a mirror. Objective-type TIRFM has the advantage
of providing a free space above the coverslip,
so it is easy to combine with scanning probe
microscopy.
The fluorescence of the myosin head captured on the tip of the scanning probe was characterized by a single, approximately Gaussian,intensity distribution and single-step
photobleaching (data not shown), strongly indicating that only a single myosin head was
captured. One was captured and manipulatedby the fine scanning probe under a TIRFM and,in the presence of ATP, brought into contact
with an actin filament bound to a glass surface. The displacements due to interactions
between the myosin head and actin molecule
were detected by using a differential pair ofphotodiodes to measure deflections of theneedle with sub-nanometer accuracy.

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Fig. 3 Displacement caused by single
S1 molecules.
(a) Upper: typical recording of thedisplacements made by an S1
molecule. Lower: stiffness calculated
from the variance of the probe
position.
(b) An example of a single
displacement event and its risingphase on an expanded time scale.
(c) Records of the rising phase ofdisplacements under various
conditions: (i) 1 µM ATP, 20ºC; (ii) 0.1µM ATP, 20ºC; (iii) 1 µM ATP, 27ºC.
Horizontal gridlines have beendrawn at a spacing of 5.5 nm. In thefalling phase of the displacements,
stepwise movement was notobserved (last traces in (i) and (iii)).

Stiffness Displacement(pN/nm)
(nm)

(a)
40
0

-40
1.5
1.0
0.5
0.0
1

0

3

2

5

4

Time (s)

(c)

(b)

(i)

5.5 nm
5 ms

10 nm
0.1 s

detachment(ii)

5.5 nm5 ms(iii)

5.5 nm10 ms
5.5 nm5 ms

3. Stepping motion
When the myosin head was not associated with an actin filament, large thermal fluctuations of the probe were apparent. Theirr.m.s. amplitude was about 13 nm. When the
myosin head bonded to actin the r.m.s. amplitude of the fluctuations fell to less than 4.5 nm(Fig. 3(a), upper trace). Probe motion causedby actomyosin interactions could be clearly distinguished from thermal noise by monitoring
the increase in stiffness, calculated as the reciprocal of the variance of the fluctuating probe
position (Fig. 3(a), lower trace). The concentration of ATP was low in these experiments

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detachment(0.1 or 1 µM), leading to prolonged actomyosin interactions and thus enabling individual
mechanical events to be identified easily. Whenthe myosin head was attached to an actin molecule, the stiffness of the probe-myosin-actinolinkage was (at 20 C) 0.2-1.5 pN/nm, more thanten times the stiffness of the probe. The highdegree of stiffness during the generation of displacements greatly improved the temporal resolution and signal-to-noise ratio, allowing us toresolve the elementary processes generating thedisplacements.
Displacements observed with a low temporal resolution took place abruptly (Fig. 3(a)),
which appeared to be consistent with the con-ventional model. On an expanded timescale,
however, we found that the displacements were
not actually abrupt but instead developed in multiple steps (Fig. 3(b)). The time course of therising and falling phases of displacements in thepresence of ATP at different concentrations andtemperatures is shown on an expandedotimescale in Fig. 3(c). The steps at 20 C areoclearer than those at 27 C because the dwelltime between them was longer. In the fallingphase of the displacements (detachment of the
myosin head from actin following ATP binding),
the probe returned to the zero position with a
1/e time of 2-5 ms (Fig. 3(c), parts (i) and (iii),
last traces), which was similar to the settling timeof the free needle. No regular steps were evident during the falling phase. The size of steps
seen during the rising phase was regular andconsistent with the periodicity of adjacent actin
molecules in an actin filament, 5.5 nm (Fig. 4(a)).
The number of steps per displacement event
varied stochastically from one to five (Fig. 4(b)),
yielding overall displacements with sizes between
5 and 30 nm. Steps were not always forward
but were sometimes backward (10% of the total number of steps). The size of the backward
steps was also 5.5 nm. These patterns of steps
indicate that the myosin head steps back andforth along the actin molecules in a filament byBrownian motion rather than by changing shape(Fig. 4(c)). Note that the individual displacementsof single myosin molecules could not be determined directly by optical trapping measurements
because the variance in start positions was large(〜30 nm) and the signal-to-noise ratio was low
because of the series compliance. As a result,only a mean displacement could be determined.2)

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(a)

(b)

800

100

0

400

5

15

10

Pairwise distance (nm)200

Number of events

Number

600

Number

(c)

200

20

10

5.5nm
0
-10

-5

0

5

10

15

20

Pairwise distance (nm)

0

0

1

2

3

4

5

1ATP

6

Number of steps / displacement

Fig. 4 (a) Histogram of the step sizes in the rising phase of displacements. Inset, histogram of the step sizes subtracting Gaussian distribution at 0 nm.
(b) Histogram of the number of steps per displacement.
(c) Model of an actomyosin motor.

We could determine the size of individual displacements directly in the present experiments,
though, because the myosin head was directly
attached to the working point and the series
stiffness during actomyosin interaction could beseveral times that in the previous experiments.
The signal-to-noise ratio in the present experiments was two to three times that in the previous experiments because the noise due to thermal fluctuations of the probe is inversely proportional to the square root of the stiffness.
Thus, the high stiffness during actomyosin interaction allowed us to resolve the elementary
process generating displacements.

in Fig. 7(b), changes in displacements wereaccompanied by changes in fluorescence intensity. Thus, each displacement corresponds toone biochemical cycle of ATP hydrolysis. Thatis, each 5.5-nm step in the rising phase of displacements was not coupled directly to a single
ATP hydrolysis reaction. This implies that themechanical reaction is loosely coupled to thebiochemical cycle of ATP hydrolysis.
A myosin head that had been detachedfrom an actin filament by the binding of Cy3ATP reattached to the actin filament and generated a displacement. The generation of displacement was associated with the release of
the bound Cy3-nucleotide, probably hydrolyzed
Cy3-ADP (Fig.7 (c)). The timing, however, wascomplex. In about half of the events, the nucleotide release coincided (to within the temporal
resolution of the system) with the generationof displacement (Fig. 8(a)). Surprisingly, in the

4. Chemo-mechanical coupling
How are the 5.5-nm steps coupled to thebiochemical cycle of ATP hydrolysis? To answer
this question we measured the ATP hydrolysis
reaction and the generation of displacement
simultaneously6) (Fig. 5). Individual ATP hydrolysis reactions of a single myosin head were measured by using TIRFM to monitor single
fluorescently labeled ATP molecules associating and dissociating with the myosin head, andthe individual mechanical events were measured by optical trapping nanometry. Measurement results are shown in Fig. 7, where part(a) shows the fluorescence image of individual
Cy3-ATP turnovers produced by a single myosin head during displacement generation. The
binding of a Cy3-ATP molecule to a myosin
head was clearly observed as a fluorescentspot. All of the fluorescence intensities weresimilar ( 〜 1000 photons per second), indicating that the fluorescent spots were indeed
due to single Cy3-ATP molecules. As shown

Fig. 5 Simultaneous measurement of individual ATP hydrolysis and the mechanical reaction of single myosin heads. A
single actin filament with beads attached to both ends was suspended in solution by optical tweezers. The suspended
actin filament was brought into contact with a single myosin head molecule bound to the surface of a coverslip.
Displacements due to actomyosin interactions were determined by measuring bead displacements with nanometer accuracy.
TIRFM was used to monitor individual ATP hydrolysis reactions as changes in fluorescence intensity due to association(hydrolysis) -dissociation events of a fluorescently labeled ATP molecule with the myosin head.

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other events the nucleotide dissociated fromfree myosin heads, which then reattached tothe actin filament and generated displacements
within 1 s (Figs. 8(b) and 8(c)). This indicatesthat the myosin heads could produce force andactive displacements after the release of thebound nucleotide.

(a)

5. Model of the movement of
the myosin head(b)

The size of the small steps making up thelarger displacements is the same as the distance
between adjacent actin monomers in one
strand of an actin filament (5.5 nm). Each step
takes place stochastically and some of the steps
are backwards. Multiple steps are produced
during a single biochemical cycle of ATP hydrolysis and the number of steps in each displacement is variable (loose-coupling).
All these results indicate that the movement of myosin relative to the actin monomers

BP1

Halogen lamp

Prism

Stage

Galvano scanners

Objective lensGreen laser

D1

He-Ne laser
Frosted glass filter

D2
YAG laser

BS

BS
D3

D4

BF1

I I SIT
Quadrant
photodiode

BF2

BP2

(a)

Avalanche photodiodeFig. 6 (a) Setup for simultaneous measurement of individual
ATPase and mechanical reactions.
(b) Schematic of the system. D, dichroic mirror; BF and BP,
bandpass filter; BS, beam splitter cube.

(b)
Displacement
(nm)

40

F. I.
(photons/s)

0
5
10

20
0
-20

4000

2000
0

10

20

15

Time (s)(c)

20

movement

25
ATP

30(s)
ATP

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30

ADP
Pi
ADP
Pi

Fig. 7 (a) Fluorescence imageof an association-(hydrolysis)dissociation event offluorescent ATP molecules witha myosin head during
generation of displacements.
( b ) Ti m e c o u r s e o f t h egeneration of displacements(upper trace) and of changes
in fluorescence intensity (lower
trace). Each displacement
event corresponds to one
biochemical cycle of ATPhydrolysis (c).

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6. Conclusion
Single-molecule imaging andnanomanipulation techniques have allowed us
to evaluate the dynamic nature of the actomyosin motor directly, and further investigations using these techniques will certainly reveal the effective and sophisticated mechanismof other molecular machines. As shown in thispaper, single-molecule detection techniques
unveil dynamic properties hidden from averaged ensemble measurements. These techniques are being used to study an expanding
range of life science, such as enzyme reactions,protein dynamics, DNA transcription, and cellsignaling.8)

F. I.
(photons/s) Displacement
(nm)

(a)
30
20
10
0
-10

4000

2000
0

1

2

3

4

5

4

5

1.5

2

Time (s)F. I.
(photons/s) Displacement
(nm)

(b)
20
10
0

Time lag

4000
3000
2000
0

1

2

3

Time (s)(c)
Number of eventsin an actin filament is a result of biased Brownian motion (Fig. 4(c)). Myosin heads are known
to bind two adjacent actin subunits in rigor,
and a myosin head may use these two bindingsites to walk along an actin filament without
detaching from the filament. Each step may
be produced by a mechanism such as the thermal ratchet.7) It is also possible that conformational changes within the actin filament play amajor role in the generation of force.
Multiple steps would be produced for eachmolecule of ATP split if the chemical energy
from ATP hydrolysis were stored in the myosin
head or the actin filament and released gradually during successive actomyosin interactions.
This idea challenges the widely accepted viewthat force generation is directly coupled to therelease of bound ligands. The simultaneous
measurements of ATPase and mechanical reactions show that the myosin head can attach
to actin and generate force for a considerable
time (>100 ms) after the bound nucleotide isreleased. The results presented here suggestthat the myosin head or actin filament, or both,
can store energy from ATP hydrolysis and release it productively in several packets of work.
If the movement of a myosin head is due
to Brownian motion, there must be somemechanism by which random Brownian motion is biased into directional movement. The
chemical energy from the ATP hydrolysis may
not be used to generate the movement of the
myosin directly but may instead be used to select unidirectional motions from Brownian motions. A mechanism by which myosin does notovercome but rather uses Brownian motion toeffectively gain a good distance with minuteenergy could explain how myosin can walk under the strong influence of thermal agitation,with high efficiency of energy conversion.

25
20
15
10
5
0
-1

-0.5

0

0.5

1

Time (s)Fig. 8 Timing of nucleotide dissociation and the generation of displacement.
(a) The generation of a displacement coincided with the release of Cy3-nucleotide.
(b) The displacement was generated after the release of Cy3-nucleotide.
(c) Histogram of the delay times from the release of Cy3-nucleatide to the generation of displacements.

References
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2)
3)
4)
5)
6)
7)
8)

R. Cooke, Physiol. Rev. 77, 671-697 (1997).
T. Yanagida et al., Curr. Opin. Cell Biol. 12, 20-25 (2000).
K. Kitamura et al., Nature (London) 397, 129-134 (1999).
T. Funatsu et al., Nature (London) 374, 555-559 (1995).
M. Tokunaga et al., Biochem. Biophys. Res. Commun. 235, 47-53 (1997).
A. Ishijima et al., Cell 92, 161-171 (1998).
R. D. Vale and F. Oosawa, Adv. Biophys. 26, 97-134 (1990).
Y. Ishii and T. Yanagida, Single Mol. 1, 5-16 (2000).

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