Review pubs.acs.org/CR
Molecular Machines Like Myosin Use Randomness to Behave Predictably Peter Karagiannis,† Yoshiharu Ishii,‡ and Toshio Yanagida*,†,‡,§,∥ †
Quantitative Biology Center, Riken (QBiC), Furuedai 6-2-3, Suita, Osaka 565-0874, Japan Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan § Center for Information and Neural Networks (CiNet), Yamadaoka 1-4, Suita, Osaka 565-0871, Japan ∥ Immunology Frontier Research Center (IFReC), Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan machines. They work needing very little input, as energy levels not far from average thermal energy (kBT) are sufficient for a given task.1 This property too contrasts with artificial machines, which work much more rapidly, accurately, and deterministically, but with higher energy demands and less adaptability. To elucidate how molecular machines operate, single molecule techniques have been developed to measure the dynamic behavior of individual biomolecules with an accuracy that can correlate thermal effects on machine function. Understanding the uniqueness of how molecular machines operate and exploiting this mechanism will reveal the strategies used by nature to build its machines and, therefore, new paradigms for CONTENTS how we can build ours. At their most rudimentary, molecular machines are 1. Introduction A tranducers that convert chemical energy into mechanical 2. Early Single Molecule Imaging A energy to move ions past a membrane, propel a cell through 2.1. Before Single Molecule Imaging A a solution, and build DNA. Some of the most studied machines 2.2. Beginnings of Single Myosin Molecule by single molecule imaging techniques are translational motors, Imaging B which include myosin, kinesin, and dynein. Although each has 2.3. Fundamental Single Molecule Techniques C its own distinct properties, for the most part they serve an 3. Processive Myosin E identical purpose: the moving of an object, or cargo, from one 3.1. Myosin V E location of the cell to another. They do so using a chemo3.2. Myosin VI G mechanical coupling mechanism that, after exhausting one unit 4. Brownian Motion I of an energy source, which happens to be ATP for all three, 4.1. Brownian Steps I begins a new cycle that repeats the transduction automatically 4.2. Substeps J until no more energy is available. Although a great deal of 4.3. Alternative Steps L literature has been devoted to these motors, this review focuses 5. From Single Molecules to Muscle M on myosin, the first molecular motor to be studied, to explain 6. Concluding Remarks M how this conversion is done. By including descriptions of single Author Information N molecule technologies, we explain how myosin movement Corresponding Author N depends on the hand-over-hand mechanism, which involves a Notes N reliable set of ordered movements in the myosin structure. We Biographies N put particular emphasis on evidence that shows this orderliness Acknowledgments N is not the result of a deterministic process, but rather depends References N on a strong Brownian component that is biased and enables reliable function. ‡
1. INTRODUCTION We use machines to move, to lift, and to build. In a similar way, cells use machines to grow, to reproduce, and to live. These machines play essential roles in cellular functions such as cell signaling, energy transduction, and motion, to name just a few. Because they operate inside a cell, they are tiny and operate on a physical scale that makes them very different from the manmade, macroscopic objects we normally imagine when we hear the word “machine”. Further, their size and soft structure allows them to be much more dynamic and robust than artificial © XXXX American Chemical Society
2. EARLY SINGLE MOLECULE IMAGING 2.1. Before Single Molecule Imaging
Perhaps no molecular machine has garnered more scientific study than myosin. One reason is that myosin II assembles to Special Issue: 2014 Single Molecule Mechanisms and Imaging Received: June 27, 2013
A
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
form muscle, making it the rare molecular motor that can be studied at the macroscopic level. In fact, it was muscle studies that first led to the realization that ATP is the energy source for the force generated by molecular motors.2 More than half a century ago, it was proposed based on optical and electron microscopy observations that muscle contraction is caused by sliding movement between myosin and actin filaments. A. F. Huxley proposed a simple yet elegant model for muscle contraction that still remains relevant today.3 The model assumes that (1) the thermal motion of myosin around an equilibrium point acts as the motive force for the sliding movement on actin filaments and (2) the attachment and detachment of myosin to an actin filament occur in an asymmetric manner. Soon thereafter, experimental evidence that supported this model emerged. Electron microscopy images and low-angle X-ray diffraction pattern data showed cross-bridges, or myosin heads, protruding from the myosin filaments and interacting with the actin filament (Figure 1). H.
Figure 2. ATPase cycle based on the Lymn and Taylor model.7 Myosin hydrolyzes ATP and uses the released energy to conform its structure, which allows it to walk along an actin filament. When ATP attaches to a myosin molecule, the myosin detaches from the filament (bottom right). Myosin will then hydrolyze the ATP and rebind to the actin filament at a different location via its head domain (orange). While the hydrolysis products, ADP and Pi, remain bound to myosin, the attachment to actin will be weak (top left). Releasing the hydrolysis products results in a strong attachment, which generates force (top right). Whether the force is generated by a lever arm swing (or power stroke) in the neck domain (green) or some other mechanism like a Brownian motion remains a topic of contention. When another ATP binds to myosin, the cycle repeats. This chemomechanical process is responsible for muscle contraction.
which interact with actin via the head domain.9,10 When ATP binds to myosin, it causes the latter to dissociate from the actin filament and take the detached state. As ATP is hydrolyzed, the myosin conformation changes to bind to the actin filament weakly. This step is followed by one that generates forces and leads to the release of the hydrolysis products, Pi and ADP, in that order. Although force has been normally associated with the power stroke, which describes a tilting of the neck domain, there remains argument on whether an alternative mechanism like Brownian motion is responsible. Upon releasing from actin, a new ATP molecule can bind to myosin and recommence the cycle.
Figure 1. Early electron micrograph and X-ray diffraction pattern of the acto-myosin filament. In the electron micrograph (left), dark shadows indicate actin filaments. The superimposed light images are myosin filaments, marking cross-bridges. Cross-bridges are seen as repeated horizontal layers in the X-ray diffraction (right). From ref 4. Reprinted with permission from AAAS.
2.2. Beginnings of Single Myosin Molecule Imaging
No experimental assay for monitoring the sliding movement of purified myosin and actin was available until the 1980s. Videoenhanced differential interference contrast microscopy successfully observed molecular machines in action by imaging the transport of organelles along microtubules.11 It was later discovered that these organelles were being transported by the until-then unknown molecular motor kinesin.12 Around the same time, the first observations of single actin filaments came from video images of HMM-coated beads sliding along actin cables consisting of hundreds of filaments prepared from Nitella cells.13,14 It was further demonstrated using single molecule techniques that the sliding movements were the result of myosin dynamics.15 During this same time period, individual actin filaments were visualized by attaching to them fluorescently labeled phalloidin.16 The phalloidin stabilized the filamentous actin form, as otherwise the low actin concentration needed for microscopy would result in depolymerization. Microscopic images showed the filaments had innate thermal motions that were enhanced upon myosin binding in the presence of ATP, demonstrating that the energy released from ATP hydrolysis was dissipated as thermal energy in the solution.
E. Huxley found structural changes of cross-bridges between the states of contraction and relaxation, offering the first structural evidence that myosin conformations were responsible for the tensions of the actin filaments and, by extension, muscle.4 Shortly thereafter, A. F. Huxley and Simmons discovered that dynamic changes in muscle arise with tension recovery after a sudden alteration in muscle length during contraction, an observation that caused them to alter their original model to include multiple cross-bridge states.5,6 Before single molecule detection techniques, data describing myosin dynamics came from biochemical experiments. These studies gave insight on the ATPase done by myosin ensembles, not individuals, providing information on the average behavior of purified soluble myosin molecules, subfragment-1 (S1) and heavy-meromyosin (HMM), in solution. Interactions between actin and myosin are normally described by the ATPase cycle, which dates back to the H. E. Huxley model and experiments from Lymn and Taylor (Figure 2).7 In its most basic form, the cycle can be reduced to four steps where actin and myosin interactions are regulated by the state of the ATP bound to the myosin. Myosin II is just one of 35 classes of myosin,8 all of B
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
Figure 3. Total internal reflection fluorescence microscopy (TIRFM): (a) A laser path and objective lens are positioned such that all light is reflected away from the glass slide and never enters the aqueous solution. However, an evanescent field penetrates the solution and illuminates a small region near the glass surface (green rectangle in water). (inset) The region near the glass surface is relatively small so that single molecules like a fluorescent molecule (star) can be observed. (b) ATP hydrolysis can be observed by labeling myosin with one fluorescent marker (GFP) and ATP with another (Cy3). Myosin are adhered to the glass slide and visualized as green dots (bottom left). ATP will be visualized only when it enters the evanescent field as a red spot and binds to myosin, which causes it to stop diffusing (bottom middle). When the ATP is hydrolyzed, the Cy3 will remain bound to the ADP, which is expelled from the myosin (bottom right).
2.3. Fundamental Single Molecule Techniques
Very quickly thereafter, a number of reports were published on the actomyosin dynamics. Using fluorescently labeled actin filaments, Kron and Spudich observed the sliding movement of individual actin filaments on myosin filaments fixed to a slide glass.17 Yanagida and his colleagues observed the sliding movement of individual actin filaments on single-headed myosin filaments, suggesting that the myosin can function as a monomer and, therefore, the head domain is sufficient for chemo-mechanical coupling.18 In fact, the sliding velocity of one-headed myosin was the same as that of two-headed myosin and even consistent with the velocity of intact muscle.19 In these early studies, the mechanism of the sliding activity was determined by measuring the average sliding distance during the hydrolysis of single ATP molecules, which was estimated by simultaneously measuring the velocity of the sliding and the ATPase, using the in vitro motility assay.20−23 This assay quickly became popular, as it is easy to implement and provides good fluorescent images of the transport along actin filaments. There was little consensus among researchers when interpreting the results, however, mainly because parameters such as the working stroke time in a single ATPase cycle could not be directly measured and therefore required an estimate that varied between groups. Additionally, although in principle single molecule measurements can be achieved by the in vitro motility assay, the reality has made this method obsolete.
No technology has had a greater impact on the study of molecular machines than single molecule detection techniques. Before single molecule measurements, information on individual molecules was extrapolated by taking the average value from the large number of molecules observed in ensemble measurements. Fundamentally, single molecule techniques derive from two technologies: single molecule imaging, which has made it possible to detect chemical reactions like ATP hydrolysis and motions at the single molecule level, and single molecule nanomanipulation, which has made it possible to measure the mechanical reactions of single molecules. For imaging, myosin is commonly labeled with a fluorescent probe and visualized using fluorescence microscopy. Single fluorophores were first observed in a solid matrix at liquid helium temperature by Moerner and Kador.24 A number of reports later demonstrated the imaging of single fluorophores with nanometer spatial resolution.25−28 It was not until 1995, however, when the Yanagida group used total internal reflection fluorescence microscopy (TIRFM) and succeeded in observing single fluorophores in aqueous solution, opening the door to a brand new field of biological study, as single molecules could be now observed in real time (Figure 3a).29 One obstacle to visualizing single fluorophores in aqueous solution was the background noise generated from the Raman scattering of water molecules, background fluorescence, and dust, among other factors. At the same time, another problem was that the fluorescence signal was both extremely weak and unstable due C
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
Figure 4. Scanning probe nanomicroscopy: Scanning probe microscopy is a nanometry system that can control myosin movement. The setup is very much like that for the TIRFM system seen in Figure 3 except for the addition of a glass microneedle and probe, to which the myosin is attached, and a photodiode for detecting movements in the microneedle. Myosin movement along an actin filament adhered to a glass slide will pull the microneedle (inset), which changes the number of photons reaching the photodiode and thus reveals mechanical events.
disappear from the image. However, although TIRFM was a major advancement in the imaging of chemical events, it does not allow for the imaging of mechanical events and, therefore, fails to provide information on the energy transduction when implemented alone. These mechanical measurements can be done by nanomanipulation, like laser traps and scanning probe microscopy, which can capture and manipulate molecular machines.37 Scanning probe microscopy uses glass needles that are labeled with probes such that one probe interacts with one molecule (Figure 4).38 For example, upon attaching a probe to a myosin molecule, the system can then be manipulated to force a single myosin to engage with actin filaments sparsely coating a glass slide. The force exerted by the myosin on the actin is measured by detecting the bend in the microneedle, which operates like a spring. By recording differences in the intensity of the projected image onto two photodiodes, the displacement of the tip of the microneedle can be detected with a spatiotemporal accuracy of nm and ms.39,40 However, while this method offers some of the best resolutions to date, it is laborious, as a new scanning probe must be prepared for each biomolecule studied. The laser trap especially has become an indispensible method for studying basic properties of molecular machines, like the force a machine exerts and the distance a machine moves (Figure 5a). A laser trap functions by capturing dielectric particles, often polystyrene beads, that range in nm to mm in diameter via the force exerted from the radiation pressure of a focused laser beam. Dielectric particles are necessary because the molecular machines of interest are too small to trap themselves. In its simplest form, the optical trap uses the momentum of photons to create a force field. A high NA objective focuses the beam onto and traps the bead. A molecular machine can be controlled and evaluated by conjugating it to the bead, which is then manipulated by the position and stiffness of the trap, which like the scanning probe acts like a spring. Consequently, the displacements and forces
to collisions with water molecules, contaminating radical ions, and photobleaching effects. TIRFM reduces the noise by locally illuminating only areas near the glass surface. The light source reaches the objective lens at angles less than the critical angle so that no light is transmitted between the two media. However, electromagnetic waves cause an evanescent field to emanate along the media interface. This field propagates perpendicular to the interface in the form of an evanescent wave with an infinitely long period. The depth of the wave depends on a number of basic properties, including the angle of the light path, the wavelength of the light, and the refractive index difference between the two media, but is normally
Molecular Machines Like Myosin Use Randomness to Behave Predictably Peter Karagiannis,† Yoshiharu Ishii,‡ and Toshio Yanagida*,†,‡,§,∥ †
Quantitative Biology Center, Riken (QBiC), Furuedai 6-2-3, Suita, Osaka 565-0874, Japan Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan § Center for Information and Neural Networks (CiNet), Yamadaoka 1-4, Suita, Osaka 565-0871, Japan ∥ Immunology Frontier Research Center (IFReC), Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan machines. They work needing very little input, as energy levels not far from average thermal energy (kBT) are sufficient for a given task.1 This property too contrasts with artificial machines, which work much more rapidly, accurately, and deterministically, but with higher energy demands and less adaptability. To elucidate how molecular machines operate, single molecule techniques have been developed to measure the dynamic behavior of individual biomolecules with an accuracy that can correlate thermal effects on machine function. Understanding the uniqueness of how molecular machines operate and exploiting this mechanism will reveal the strategies used by nature to build its machines and, therefore, new paradigms for CONTENTS how we can build ours. At their most rudimentary, molecular machines are 1. Introduction A tranducers that convert chemical energy into mechanical 2. Early Single Molecule Imaging A energy to move ions past a membrane, propel a cell through 2.1. Before Single Molecule Imaging A a solution, and build DNA. Some of the most studied machines 2.2. Beginnings of Single Myosin Molecule by single molecule imaging techniques are translational motors, Imaging B which include myosin, kinesin, and dynein. Although each has 2.3. Fundamental Single Molecule Techniques C its own distinct properties, for the most part they serve an 3. Processive Myosin E identical purpose: the moving of an object, or cargo, from one 3.1. Myosin V E location of the cell to another. They do so using a chemo3.2. Myosin VI G mechanical coupling mechanism that, after exhausting one unit 4. Brownian Motion I of an energy source, which happens to be ATP for all three, 4.1. Brownian Steps I begins a new cycle that repeats the transduction automatically 4.2. Substeps J until no more energy is available. Although a great deal of 4.3. Alternative Steps L literature has been devoted to these motors, this review focuses 5. From Single Molecules to Muscle M on myosin, the first molecular motor to be studied, to explain 6. Concluding Remarks M how this conversion is done. By including descriptions of single Author Information N molecule technologies, we explain how myosin movement Corresponding Author N depends on the hand-over-hand mechanism, which involves a Notes N reliable set of ordered movements in the myosin structure. We Biographies N put particular emphasis on evidence that shows this orderliness Acknowledgments N is not the result of a deterministic process, but rather depends References N on a strong Brownian component that is biased and enables reliable function. ‡
1. INTRODUCTION We use machines to move, to lift, and to build. In a similar way, cells use machines to grow, to reproduce, and to live. These machines play essential roles in cellular functions such as cell signaling, energy transduction, and motion, to name just a few. Because they operate inside a cell, they are tiny and operate on a physical scale that makes them very different from the manmade, macroscopic objects we normally imagine when we hear the word “machine”. Further, their size and soft structure allows them to be much more dynamic and robust than artificial © XXXX American Chemical Society
2. EARLY SINGLE MOLECULE IMAGING 2.1. Before Single Molecule Imaging
Perhaps no molecular machine has garnered more scientific study than myosin. One reason is that myosin II assembles to Special Issue: 2014 Single Molecule Mechanisms and Imaging Received: June 27, 2013
A
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
form muscle, making it the rare molecular motor that can be studied at the macroscopic level. In fact, it was muscle studies that first led to the realization that ATP is the energy source for the force generated by molecular motors.2 More than half a century ago, it was proposed based on optical and electron microscopy observations that muscle contraction is caused by sliding movement between myosin and actin filaments. A. F. Huxley proposed a simple yet elegant model for muscle contraction that still remains relevant today.3 The model assumes that (1) the thermal motion of myosin around an equilibrium point acts as the motive force for the sliding movement on actin filaments and (2) the attachment and detachment of myosin to an actin filament occur in an asymmetric manner. Soon thereafter, experimental evidence that supported this model emerged. Electron microscopy images and low-angle X-ray diffraction pattern data showed cross-bridges, or myosin heads, protruding from the myosin filaments and interacting with the actin filament (Figure 1). H.
Figure 2. ATPase cycle based on the Lymn and Taylor model.7 Myosin hydrolyzes ATP and uses the released energy to conform its structure, which allows it to walk along an actin filament. When ATP attaches to a myosin molecule, the myosin detaches from the filament (bottom right). Myosin will then hydrolyze the ATP and rebind to the actin filament at a different location via its head domain (orange). While the hydrolysis products, ADP and Pi, remain bound to myosin, the attachment to actin will be weak (top left). Releasing the hydrolysis products results in a strong attachment, which generates force (top right). Whether the force is generated by a lever arm swing (or power stroke) in the neck domain (green) or some other mechanism like a Brownian motion remains a topic of contention. When another ATP binds to myosin, the cycle repeats. This chemomechanical process is responsible for muscle contraction.
which interact with actin via the head domain.9,10 When ATP binds to myosin, it causes the latter to dissociate from the actin filament and take the detached state. As ATP is hydrolyzed, the myosin conformation changes to bind to the actin filament weakly. This step is followed by one that generates forces and leads to the release of the hydrolysis products, Pi and ADP, in that order. Although force has been normally associated with the power stroke, which describes a tilting of the neck domain, there remains argument on whether an alternative mechanism like Brownian motion is responsible. Upon releasing from actin, a new ATP molecule can bind to myosin and recommence the cycle.
Figure 1. Early electron micrograph and X-ray diffraction pattern of the acto-myosin filament. In the electron micrograph (left), dark shadows indicate actin filaments. The superimposed light images are myosin filaments, marking cross-bridges. Cross-bridges are seen as repeated horizontal layers in the X-ray diffraction (right). From ref 4. Reprinted with permission from AAAS.
2.2. Beginnings of Single Myosin Molecule Imaging
No experimental assay for monitoring the sliding movement of purified myosin and actin was available until the 1980s. Videoenhanced differential interference contrast microscopy successfully observed molecular machines in action by imaging the transport of organelles along microtubules.11 It was later discovered that these organelles were being transported by the until-then unknown molecular motor kinesin.12 Around the same time, the first observations of single actin filaments came from video images of HMM-coated beads sliding along actin cables consisting of hundreds of filaments prepared from Nitella cells.13,14 It was further demonstrated using single molecule techniques that the sliding movements were the result of myosin dynamics.15 During this same time period, individual actin filaments were visualized by attaching to them fluorescently labeled phalloidin.16 The phalloidin stabilized the filamentous actin form, as otherwise the low actin concentration needed for microscopy would result in depolymerization. Microscopic images showed the filaments had innate thermal motions that were enhanced upon myosin binding in the presence of ATP, demonstrating that the energy released from ATP hydrolysis was dissipated as thermal energy in the solution.
E. Huxley found structural changes of cross-bridges between the states of contraction and relaxation, offering the first structural evidence that myosin conformations were responsible for the tensions of the actin filaments and, by extension, muscle.4 Shortly thereafter, A. F. Huxley and Simmons discovered that dynamic changes in muscle arise with tension recovery after a sudden alteration in muscle length during contraction, an observation that caused them to alter their original model to include multiple cross-bridge states.5,6 Before single molecule detection techniques, data describing myosin dynamics came from biochemical experiments. These studies gave insight on the ATPase done by myosin ensembles, not individuals, providing information on the average behavior of purified soluble myosin molecules, subfragment-1 (S1) and heavy-meromyosin (HMM), in solution. Interactions between actin and myosin are normally described by the ATPase cycle, which dates back to the H. E. Huxley model and experiments from Lymn and Taylor (Figure 2).7 In its most basic form, the cycle can be reduced to four steps where actin and myosin interactions are regulated by the state of the ATP bound to the myosin. Myosin II is just one of 35 classes of myosin,8 all of B
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
Figure 3. Total internal reflection fluorescence microscopy (TIRFM): (a) A laser path and objective lens are positioned such that all light is reflected away from the glass slide and never enters the aqueous solution. However, an evanescent field penetrates the solution and illuminates a small region near the glass surface (green rectangle in water). (inset) The region near the glass surface is relatively small so that single molecules like a fluorescent molecule (star) can be observed. (b) ATP hydrolysis can be observed by labeling myosin with one fluorescent marker (GFP) and ATP with another (Cy3). Myosin are adhered to the glass slide and visualized as green dots (bottom left). ATP will be visualized only when it enters the evanescent field as a red spot and binds to myosin, which causes it to stop diffusing (bottom middle). When the ATP is hydrolyzed, the Cy3 will remain bound to the ADP, which is expelled from the myosin (bottom right).
2.3. Fundamental Single Molecule Techniques
Very quickly thereafter, a number of reports were published on the actomyosin dynamics. Using fluorescently labeled actin filaments, Kron and Spudich observed the sliding movement of individual actin filaments on myosin filaments fixed to a slide glass.17 Yanagida and his colleagues observed the sliding movement of individual actin filaments on single-headed myosin filaments, suggesting that the myosin can function as a monomer and, therefore, the head domain is sufficient for chemo-mechanical coupling.18 In fact, the sliding velocity of one-headed myosin was the same as that of two-headed myosin and even consistent with the velocity of intact muscle.19 In these early studies, the mechanism of the sliding activity was determined by measuring the average sliding distance during the hydrolysis of single ATP molecules, which was estimated by simultaneously measuring the velocity of the sliding and the ATPase, using the in vitro motility assay.20−23 This assay quickly became popular, as it is easy to implement and provides good fluorescent images of the transport along actin filaments. There was little consensus among researchers when interpreting the results, however, mainly because parameters such as the working stroke time in a single ATPase cycle could not be directly measured and therefore required an estimate that varied between groups. Additionally, although in principle single molecule measurements can be achieved by the in vitro motility assay, the reality has made this method obsolete.
No technology has had a greater impact on the study of molecular machines than single molecule detection techniques. Before single molecule measurements, information on individual molecules was extrapolated by taking the average value from the large number of molecules observed in ensemble measurements. Fundamentally, single molecule techniques derive from two technologies: single molecule imaging, which has made it possible to detect chemical reactions like ATP hydrolysis and motions at the single molecule level, and single molecule nanomanipulation, which has made it possible to measure the mechanical reactions of single molecules. For imaging, myosin is commonly labeled with a fluorescent probe and visualized using fluorescence microscopy. Single fluorophores were first observed in a solid matrix at liquid helium temperature by Moerner and Kador.24 A number of reports later demonstrated the imaging of single fluorophores with nanometer spatial resolution.25−28 It was not until 1995, however, when the Yanagida group used total internal reflection fluorescence microscopy (TIRFM) and succeeded in observing single fluorophores in aqueous solution, opening the door to a brand new field of biological study, as single molecules could be now observed in real time (Figure 3a).29 One obstacle to visualizing single fluorophores in aqueous solution was the background noise generated from the Raman scattering of water molecules, background fluorescence, and dust, among other factors. At the same time, another problem was that the fluorescence signal was both extremely weak and unstable due C
dx.doi.org/10.1021/cr400344n | Chem. Rev. XXXX, XXX, XXX−XXX
Chemical Reviews
Review
Figure 4. Scanning probe nanomicroscopy: Scanning probe microscopy is a nanometry system that can control myosin movement. The setup is very much like that for the TIRFM system seen in Figure 3 except for the addition of a glass microneedle and probe, to which the myosin is attached, and a photodiode for detecting movements in the microneedle. Myosin movement along an actin filament adhered to a glass slide will pull the microneedle (inset), which changes the number of photons reaching the photodiode and thus reveals mechanical events.
disappear from the image. However, although TIRFM was a major advancement in the imaging of chemical events, it does not allow for the imaging of mechanical events and, therefore, fails to provide information on the energy transduction when implemented alone. These mechanical measurements can be done by nanomanipulation, like laser traps and scanning probe microscopy, which can capture and manipulate molecular machines.37 Scanning probe microscopy uses glass needles that are labeled with probes such that one probe interacts with one molecule (Figure 4).38 For example, upon attaching a probe to a myosin molecule, the system can then be manipulated to force a single myosin to engage with actin filaments sparsely coating a glass slide. The force exerted by the myosin on the actin is measured by detecting the bend in the microneedle, which operates like a spring. By recording differences in the intensity of the projected image onto two photodiodes, the displacement of the tip of the microneedle can be detected with a spatiotemporal accuracy of nm and ms.39,40 However, while this method offers some of the best resolutions to date, it is laborious, as a new scanning probe must be prepared for each biomolecule studied. The laser trap especially has become an indispensible method for studying basic properties of molecular machines, like the force a machine exerts and the distance a machine moves (Figure 5a). A laser trap functions by capturing dielectric particles, often polystyrene beads, that range in nm to mm in diameter via the force exerted from the radiation pressure of a focused laser beam. Dielectric particles are necessary because the molecular machines of interest are too small to trap themselves. In its simplest form, the optical trap uses the momentum of photons to create a force field. A high NA objective focuses the beam onto and traps the bead. A molecular machine can be controlled and evaluated by conjugating it to the bead, which is then manipulated by the position and stiffness of the trap, which like the scanning probe acts like a spring. Consequently, the displacements and forces
to collisions with water molecules, contaminating radical ions, and photobleaching effects. TIRFM reduces the noise by locally illuminating only areas near the glass surface. The light source reaches the objective lens at angles less than the critical angle so that no light is transmitted between the two media. However, electromagnetic waves cause an evanescent field to emanate along the media interface. This field propagates perpendicular to the interface in the form of an evanescent wave with an infinitely long period. The depth of the wave depends on a number of basic properties, including the angle of the light path, the wavelength of the light, and the refractive index difference between the two media, but is normally
