Single-Molecule Biology: What Is It and How Does It Work?

Molecular Cell 24, 317–329, November 3, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2006.10.017

Single-Molecule Biology: What Is It and How Does It Work? Jordanka Zlatanova1,* and Kensal van Holde2 1 Department of Molecular Biology University of Wyoming Laramie, Wyoming 82071 2 Department of Biochemistry and Biophysics Oregon State University Corvallis, Oregon 97331 Biochemistry and structural biology are undergoing a dramatic revolution. Until now, we have tried to study subtle and complex biological processes by crude in vitro techniques, looking at average behaviors of vast numbers of molecules under conditions usually remote from those existing in the cell. Researchers have realized the limitations of this approach, but none other has been available. Now, we can not only observe the nuances of the behaviors of individual molecules but prod and probe them as well. Perhaps most important is the emerging ability to carry out such observations and manipulations within the living cell. The long-awaited leap to an in vivo biochemistry is at last underway.

Single-Molecule Approaches: General Introduction Recent years have witnessed the emergence of an entirely new field of science, often referred to as single-molecule (SM) biology (or biochemistry) (Science, 1999; Journal of Biological Chemistry, 1999; Leuba and Zlatanova, 2001; Chapter 16 in van Holde et al. [2006]). SM methods represent a truly novel approach to biochemical/biological problems. All classical structural and biochemistry/biophysics methods describe the behavior of enormous ensembles of molecules, averaging the measured parameters over the entire molecular population. How any one molecule may behave over time cannot be revealed by such studies; neither can the behavior of individual molecules having different conformations and properties. It is important to realize that seemingly homogenous populations of macromolecules that have no chemical differences do possess intermolecular variations (often referred to as ‘‘static disorder,’’ Kelley et al. [2001]). SM methods provide the only available way to study their functional differences, by recording the behavior of individual members of a certain population of molecules. In addition, SM approaches reveal fluctuations in the observable parameters of a single molecule over time, often with very high temporal resolution, usually on the order of milliseconds. Assessing this ‘‘dynamic disorder’’ (Kelley et al., 2001) is beyond the capabilities of population methods. SM methodology provides a powerful tool for measuring important kinetic parameters. For example, if a macromolecule exists in two states and a SM signal, such as fluorescence, can distinguish between them, one can follow the behavior of the molecule in real time and determine a distribution of ‘‘dwell times’’ for each state. The average dwell time t in a given state can simply be *Correspondence: [email protected]

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determined by constructing a histogram of the measured dwell times and fitting its envelope to a single exponential function. This should work for a simple (random) process, for which the dwell times should follow a Poisson distribution (see Colquhoun and Hawkes [1995]). In cases in which a single exponential fit does not work, the process is either more complex than two state or may involve some kind of memory effect (deviates from randomness). The dwell times of both states can be obtained in a single experiment, without perturbing the system (which is not possible in bulk experiments), and can be converted into the familiar first-order rate constants (the value of t in each state is the inverse of the rate constant for the process that leads to transition to the alternative state). The SM methods do have, at present, limitations in determining what is happening during the change between the two states: this is simply due to present limits of time resolution. In other words, we are able to determine how long a molecule waits to do something, but not how it does it. A major advance in time resolution at the SM level would open a whole new universe of understanding. Another major and unique advantage that SM methodology provides is that it circumvents the need for synchronization of large numbers of molecules at a certain stage of a biochemical process. Such synchronization is difficult, often impossible, to attain. Even if the molecular population can be synchronized (usually by blocking all molecules at a certain stage of a process by, for example, using drugs or withdrawing certain components from the mixture or by perturbing physical parameters), the release from the block quickly leads to loss of synchrony because of the intrinsically stochastic nature of individual reaction steps and the multistep character of biochemical pathways. SM methodology relies on the use of two general types of approaches: those that allow observation of single molecules under thermodynamic equilibrium or nonequilibrium conditions (without externally imposed perturbations of the system under study), and those that study molecular behavior under applied force. Examples of the first type of approaches include microscopic observation of fluorescently labeled single molecules and the so-called tethered-particle motion (TPM) method, introduced in J. Gelles’s laboratory to follow motions of a particle attached to a macromolecule, which in turn is tethered to a surface (Schafer et al., 1991; Yin et al., 1994). The second class of approaches provides unique opportunities to investigate the mechanical responses of biological systems to applied tension and/or torsion (Khan and Sheetz, 1997; Leckband and Israelachvili, 2001; for a brief overview, see Zlatanova and Leuba [2003a]). Forces can be applied by the use of bendable beams (atomic force microscope [AFM], optical or glass fibers) or by the so-called external-field manipulators (optical tweezers [OT], magnetic tweezers [MT]) (Bustamante et al., 2000a). We begin with a brief description of the principles of action of the four most widely used SM methods. This will be followed by selected examples of how these methods can

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be applied to study the mechanical properties of macromolecules or their complexes, the forces that govern intra- and intermolecular interactions, and the functioning of biomolecules in various biochemical processes. Finally, we will review some applications of these methods to macromolecules in cells during their functioning in an in vivo context. Physical Principles of SM Methods Fluorescence Fluorescence methods have been used for biological research for decades. Recent technical advances have extended the use of fluorescent tags attached to macromolecules to the realm of SM biology. To that end, a single dye molecule is attached to a specific location in the molecule of interest and then followed in a number of different ways to report on the behavior of that molecule. Alternatively, fluorescent protein probes, like the green fluorescent protein (GFP) or its variants (Tsien, 1998), can be attached through recombinant DNA technology to any protein of interest and used as fluorescent tags in vitro or in vivo. The observables in SM fluorescence studies include dye localization, fluorescence quenching by a nearby nonfluorescent quencher, fluorescence anisotropy, and, most frequently, fluorescence (Fo¨rster) resonance energy transfer (FRET) between two fluorophores. The theory and instrumentation behind these methods have been reviewed in numerous publications (Weiss, 1999, 2000; Lilley and Wilson, 2000; Selvin, 2000; Ha, 2001a, 2001b; Deniz et al., 2001; Axelrod, 2001; Michalet et al., 2003; Peterman et al., 2004; Neuweiler and Sauer, 2004; Haustein and Schwille, 2003, 2004); for some more application-oriented reviews, the reader is also referred to Ha (2004) and McKinney et al. (2004). A brief statement concerning criteria for fluorophores is warranted here. They must (1) be bright (have high extinction coefficients and high quantum yield, i.e., be able to transform a large proportion of the incident energy into emitted light), (2) absorb and emit light in the visible region of the spectrum, (3) show little fluctuation in the emission intensity for the duration of the experiment, (4) be relatively small so as not to perturb the molecule under investigation, (5) be available in a form suitable for covalent conjugation to the molecule, and (6) be photostable (Ha, 2001b). After w105 rounds of excitation and emission, dyes lose their ability to be further excited, i.e., they undergo photobleaching. The lifetime of a fluorophore strongly depends on the intensity of excitation, so if a prolonged (more than 5–10 s) period of observation is required, it is advisable to reduce the excitation power to the possible minimum or to intermittently switch the incident light on and off. Another property that needs to be considered in interpreting SM fluorescence data is the ‘‘blinking’’ behavior, spontaneous transitions between bright (emitting) and dark (nonemitting) periods (Ambrose et al., 1994; Xie and Dunn, 1994) that occur before photobleaching. The exact events causing blinking are unclear, but the possibility of blinking should be taken into account when interpreting spFRET data (see Sabanayagam et al. [2005]). The fluorescence SM detection methods fall into two broad categories: wide-field and confocal. The widefield methods follow the behavior of immobilized mole-

cules over time by using two-dimensional detectors such as CCD cameras. The biggest advantage of these methods is the simultaneous observation of hundreds of molecules in parallel, allowing the investigation of both reversible and irreversible reactions and the detection of rare events. In the confocal method, the excitation beam is focused to a diffraction-limited spot through an objective, which is also used to collect the emitted fluorescence. The out-of-focus background fluorescence is removed with a pinhole that is positioned at the back aperture of the objective. The detection of the fluorescent spot is done with photomultiplier tubes or avalanche photodiodes (APDs) (for specific usage of these detectors, see Ha [2001b]). The confocal setups allow observation of surface-immobilized molecules, as well as of molecules traversing (by diffusion or flow) the small volume of liquid excited by the laser. Confocal detection is also used in a couple of related fluorescence techniques that study the behavior of a small number of molecules (fluorescence correlation spectroscopy and two-color crosscorrelation spectroscopy, see Haustein and Schwille [2003, 2004]). Wide-field instrumentation uses two different approaches for excitation: epifluorescence and total internal reflection (TIR). In standard epifluorescence, the light is sent through the epi-illumination port of conventional fluorescence microscopes; thus, both the microscope optics and the sample contribute to out-of-focus background fluorescence, reducing the signal-to-noise ratio. In TIR, the excitation light is directed toward an interface between two media of different refractive indices (i.e., from an optically denser medium, such as a glass slide, into a less-dense medium, such as water) (Axelrod, 2001). The incident angle of the beam is set larger than a certain critical angle, defined by the properties of the two media; all the light is reflected off the glass and does not penetrate into the solution (Figure 1A). However, an electromagnetic field that oscillates with the same frequency as the incident light does form in the less-dense medium (the water on the other side). Because this electromagnetic field (also called evanescent field or wave) decays exponentially from the glass surface, it is capable of exciting fluorophores only in a very small volume close to the surface, thus effectively preventing out-of-focus fluorescence background. The excitation light itself is cleanly removed from the observation chamber, reducing the background even further. The two types of TIR fluorescence microscopy— prism-based and objective-based—are described in an excellent recent tutorial (Knight et al., 2005) (see also Peterman et al. [2004]; Zheng et al. [2005]). Of all SM fluorescence methods, we will briefly introduce only FRET, as this method is being most widely used. FRET relies on nonradiative transfer of electronic excitation energy between two fluorophores, a donor and an acceptor. The theory developed by Fo¨rster (1959) predicts that the efficiency of energy transfer is a function of the distance (R) between the two dyes: EFRET = 1/[1 + (R/Ro)6], where Ro is the interdye distance at which half of the energy is transferred. Ro depends on a number of characteristics of the dye pair (see Selvin [2000]; Ha [2001b]; Neuweiler and Sauer [2004]). Following the experimental verification of the theory (Stryer and Haugland, 1967), EFRET has been widely used as

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Figure 1. Detecting Fluorescence from Individual Fluorophores by Total Internal Reflection Fluorescence Microscopy and Principle of Single-Pair FRET for Studying Biological Macromolecules (A) In total internal reflection fluorescence (TIRF) microscopes, the sample is excited by the intense electromagnetic field that is created by the totally reflected laser beam at the interface of two media of different refractive indexes (usually quartz and water). This field, also known as an evanescent wave, decays exponentially with the distance from the interface and thus excites only the fluorophores present in a small volume in close proximity to the quartz. The figure illustrates the more commonly used prism-based setup; the advantages of the alternative, objective-type TIRFM are discussed in Knight et al. (2005). (B) Principle of spFRET (see text).

a spectroscopic ruler. Because Ro for many dye pairs lies in the vicinity of 5–6 nm, FRET is best used to measure distances in the range of 2–8 nm (see illustration of principle in Figure 1B). This distance range is not covered by any other solution technique. One should keep in mind that the distances determined are not to be taken as absolute values but rather used for comparative purposes because Ro may depend on dye environment. Since measuring FRET requires recording of the donor and acceptor emissions in different channels, dye pairs for spFRET should have a large spectral separation so as to avoid leakage of the donor signal into the acceptor channel and direct excitation of the acceptor by the laser. It is also highly desirable to choose pairs with comparable emission quantum yields so that the occurrence of FRET is clearly recordable as anticorrelated changes in the fluorescence intensities of both dyes: if the intensity of the donor emission falls, the intensity of the acceptor emission should simultaneously increase, and vice versa. Finally, one has to be aware of blinking effects (see above). Dark periods of the donor do not pose problems in interpretations, since no

fluorescent signal is available during these periods. If the acceptor, on the other hand, blinks, a concomitant increase in donor emission will be observed and possibly misinterpreted as a loss of FRET due to conformational changes or biochemical processes. Since the emission ‘‘on’’ time is dependent on the excitation intensity, and the efficiency of FRET is not, blinking can be distinguished from actual distance changes in the molecules by varying the intensity of the exciting radiation. Atomic Force Microscope The AFM (Binnig et al., 1986) has opened new horizons in both imaging and force measurements. In the AFM, a sharp probe (tip) is mounted at the end of a flexible cantilever and allowed to interact with a surface-immobilized sample. Precise lateral and vertical displacement of the sample with respect to the probe is achieved by a computer-controlled piezoceramics stage holding the sample (Figure 2A) or, conversely, the cantilever holder. Interaction forces between atoms on the sample and atoms on the tip cause deflection of the cantilever that is registered by a laser reflected off its back into a photodiode detector. These deflections can be converted into a topographic image of the sample (when the probe is raster scanned in the x-y plane) or used to produce force-distance curves (when the probe is moved in the z direction only). AFM imaging has become routine and has led to significant advances in our understanding of how biological molecules function. Although the resolution of AFM is comparable (for soft biological samples) to that of the electron microscope, AFM has the enormous advantage in that it can deal with moist or even buffer-immersed samples, allowing imaging under physiologically relevant conditions. The application of AFM for force spectroscopy has also significantly contributed to our understanding of intra- and intermolecular interaction forces, especially in the range of 50 pN to 1–2 nN (for some earlier examples, see Table 1 in Zlatanova et al. [2000]). Force-distance curves are obtained upon moving the probe upwards (retraction curve) after it has been pushed into the sample (approach curve); the signature of the retraction curve can be used to measure interactive forces (a more detailed description of the types of interaction forces that are ‘‘felt’’ by the cantilever during the approach and retraction portions of the force curve can be found in Zlatanova et al. [2000]). An illustration of a force curve obtained upon stretching of proteins is presented in Figure 2B. Optical Tweezers The OT technique has been extensively reviewed both in terms of theory and instrumentation (e.g., Visscher and Block, 1998; Smith et al., 2003; Leuba et al., 2003; Bennink et al., 2005; Knight et al., 2005) (for a fascinating account of the history of the development of optical trapping, see Ashkin [1997]). Light can exert forces on small transparent beads in such a way that the bead is kept suspended at a point close to the waist of a focused laser beam (Figure 3A). If such a ‘‘trapped’’ bead is pulled out of its equilibrium position by whatever external force, a net restoring force resulting from the bead’s interaction with light will effectively pull it back toward the equilibrium position. To a first approximation, the trap can be considered as a Hookean spring, with the force determined by F = kDx, where k is the spring

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examples of using OT are presented below (Figures 3C, 3D, 5C, 5D, 6C, and 6D). Magnetic Tweezers MT is another force-field manipulation technique in which the macromolecule is acted upon from a distance, this time through a controllable magnetic field (Figure 4A) (Strick et al., 1996, 2000; Zlatanova and Leuba, 2003b; Leuba et al., 2003; Revyakin et al., 2005). The macromolecule is attached between a surface and a superparamagnetic bead. Manipulation of the external magnetic field allows application of stretching forces to the tethered molecule, the magnitude of the force being precisely controlled by the distance between the cuvette and the external magnet(s). The force on the bead, and thus the tether, is measured using the equipartition theorem, with two observables: the extent of Brownian motion in the x-y plane and the tether length. A major advantage of this instrument is the ability to introduce controlled levels of torsional stress in the molecule by simply rotating the magnetic field clockwise or counterclockwise, depending on the sign of supercoiling one wants to achieve. Due to space limitations, we have only illustrated this powerful technique in Figure 4B, but the reader is referred to the recent review by Charvin et al. (2005) that focuses on the application of MT to the study of topoisomerases; Zlatanova et al. (2006) have discussed the application of MT (among other methods) to the study of transcription initiation and elongation.

Figure 2. Use of the Atomic Force Microscope for Imaging and Force Spectroscopy (A) Schematic of the principle of action of the AFM (see text). (B) A schematic representation showing the structural transitions in a multidomain protein upon mechanical stretching using the AFM. The sawtooth pattern of the force-extension curves results from strings of successive enthalpic and entropic portions, reflecting the unfolding of individual domains, followed by entropic stretching of the unfolded domain. The unfolding of each domain adds significant length to the chain and relaxes the stress on the cantilever, which returns to its nondeflected state. The unfolded portion of the polypeptide chain can now undergo entropic stretching, which is accompanied by gradual deflection of the cantilever (adapted from Zlatanova et al. [2000]).

constant of the trap and Dx is the displacement of the bead from the focus of the trap. In OT applications, the molecule is tethered between the optically trapped bead and a movable platform (another bead, usually held in a micropipette, or a cover slip) (Figure 3B). The platform can be moved in a controllable way to apply tension (and, recently, torsion) to the tether to record force-extension curves. Some specific

Applications of SM Methodology to Biological Problems Mechanical Properties of Biological Macromolecules and Their Complexes A conceptual framework for these kinds of studies has been provided by Bustamante et al. (2004). DNA was the first biomacromolecule to be investigated under applied mechanical force by a variety of SM methods (Smith et al., 1992, 1996; reviewed in Bustamante et al. [2000b, 2003]; Zlatanova and Leuba, 2002, 2003a, 2004). An example stretching-relaxation curve obtained with OT is presented in Figure 3C. The stretching curve reveals different behavior of the molecule under different force regimes: (1) up to 10 pN, the molecule behaves as a flexible polymer, accurately described by the wormlike chain model; (2) above 10 pN, the chain has been extended, and the DNA now behaves as a stretchable solid, lengthening beyond its B form contour length; and (3) at forces exceeding 65 pN, the molecule suddenly yields, changing conformation to an overstretched form (w1.7 times its contour length). The nature of the overstretching transition, frequently referred to as B-to-S (stretched) transition, is still debated. OT have been also used to stretch individual chromatin fibers or nucleosomal arrays, either directly reconstituted in the flow cell of the instrument (Bennink et al., 2001; Figure 4D) or preassembled from defined arrays of nucleosome positioning sequences and purified core histones (Brower-Toland et al., 2002). The high temporal and spatial resolution achieved in these experiments led to defining the force (20–40 pN) needed to unravel the DNA from individual nucleosomal particles (for further discussion, see Zlatanova and Leuba [2002, 2003a, 2004]; Zlatanova [2003]).

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Figure 3. Optical Trapping and Its Application for Stretching DNA and Chromatin Fibers (A) The behavior of a dielectric bead in a focused laser beam (see text). (B) OT can be used to study macromolecules by attaching them to the optically trapped bead at one end and to a movable platform (in the illustration, another bead held in a micropipette) at the other end. Moving the platform away from the trapped bead will apply stretching forces to the macromolecule, causing displacement of the trapped bead from its equilibrium position. This displacement can be measured and used to estimate the forces applied to the molecule. (C) Stretching of l-DNA. A typical DNA force-extension curve exhibits three portions: entropic stretching of the worm-like chain at the beginning, enthalpic (elastic) stretching of the structure at lengths of the molecule approaching or slightly exceeding the contour length (in this case w16 mm), and finally, at w65 pN force, a transition from the B to the so-called S (stretched) form. The physical nature of the S form has been controversial (for detailed discussion, see Bustamante et al. [2000b, 2003]; Zlatanova and Leuba [2002, 2003a, 2004]). The DNA was stretched to w20 mm (red curve) and then relaxed (blue curve). (D) Stretching of a chromatin fiber assembled on naked l-DNA molecule by the addition of X. laevis egg extract directly into the flow cell of the instrument. The extract contains core histones and protein factors needed for assembly (assembly is manifested by shortening of the distance between the two beads with time). Note the sharp discontinuities in the force-extension curve reflecting the unraveling of the DNA from around the histone octamer that forms the core of the nucleosomal particles. Nucleosomes can unwrap either individually or in groups of two, three, or four. At high extension, when all histones have been forced off, the curve approaches that of naked DNA. Note that the two force-extension curves for DNA (C) and chromatin (D) are aligned with respect to the length of the structure during stretching, so that a direct comparison of the behavior of DNA and of chromatin is possible. (C) and (D) are modified with author permission (Bennink, 2001).

Protein unfolding as a result of mechanical stretching has been one of the major applications of AFM. The groundbreaking papers of Rief et al. (1997) and Oberhauser et al. (1998) showed that unfolding of proteins that contain numerous individually folded domains produces a sawtooth pattern in the force-extension curves reflecting successive unfolding of each individual domain (the appearance of a typical force-extension curve and its interpretation are illustrated on the example of a recombinant polyprotein consisting of tandemly repeated monomeric domains; see Figure 2B). A series of publications, mainly from J. Fernandez’s laboratory, revealed (1) the existence of unfolding intermediates in which each domain abruptly extends by a certain length

before the actual full unfolding (Marszalek et al., 1999a), (2) the occurrence of misfolding events that can be detected upon repeated extension/relaxation cycles (Oberhauser et al., 1999), (3) domain length polymorphism at a resolution comparable to that of structural methods, such as NMR and X-ray crystallography (Carrion-Vazquez et al., 1999), and (4) folding trajectories of single proteins upon force relaxation (Fernandez and Li, 2004; note that the interpretations of this work have been questioned, Sosnick [2004]; Best and Hummer [2005]). An interesting technical development allowed the use of AFM to calibrate the distance-dependent decay of an evanescent wave (see above); simultaneous measurements during stretching of fluorescence

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Figure 4. Magnetic Tweezers and Their Application for Studying the Response of DNA to Torsion (A) Schematic of the principle of action of MT (see text). (B) Relative extension of a DNA molecule as a function of the degree of imposed supercoiling (reproduced from Strick et al. [1998], with permission from the Biophysical Society; the figure has been slightly modified in terms of the labeling of the lines). MT were used to introduce a controlled level of supercoiling density in a single molecule of DNA tethered between a surface and a superparamagnetic bead in a topologically constrained way. The changes in tether length (extension) were followed as the external magnets were rotated either clockwise or counterclockwise, to create negative or positive supercoiling tension, respectively. Force-extension curves taken at different levels of stretching forces reveal intriguing differences in the behavior of positively versus negatively supercoiled DNA. The linear shortening of the molecules results from plectoneme formation, whereas the portions of the curves that remain parallel to the x axis despite the continuous pumping in of superhelical tension reflect structural transitions in the molecules that are not accompanied by shortening. In the case of negative superhelical tension, the helix undergoes local denaturation (Strick et al., 1998). In the case of positive superhelical tension, the B-DNA undergoes a structural transition to a new phase called P-DNA (Pauling-DNA, Allemand et al. [1998]); in this conformation, the phosphate-sugar backbones of the two strands are wound inside the structure, with the bases exposed to the solution.

intensity and protein length from a single ubiquitin polyprotein resolved w20 nm steps in the unfolding of ubiquitin (Sarkar et al., 2004). Finally, a recent paper identified a folding intermediate of individual ribonuclease molecules stretched and relaxed with OT and defined

the energy landscape of this enzyme (Cecconi et al., 2005). A striking example of the resolution power of SM methods to investigate the mechanical properties of biomacromolecules under tensile stress is the identification of chair-boat transitions in the glucopyranose ring of polysaccharides as the basis for the molecule’s elasticity (Marszalek et al., 1998). Further, a two-step conversion from chair to boat and then to an inverted chair has been reported; it was postulated that chair inversion results from torque generated by force application to axial (but not equatorial) glycosidic bonds (Marszalek et al., 1999b). SM methods have also been used to study the behavior of double-helical DNA upon twisting. Croquette and Bensimon pioneered the use of MT to investigate the structural transitions of dsDNA under superhelical tension (Strick et al., 1996; Allemand et al., 1998; reviewed in Strick et al. [2000]; Zlatanova and Leuba [2003b]). These investigators topologically constrained a single DNA molecule between a glass surface and a magnetic bead by attaching all four ends of the molecule through multiple points to the respective surfaces. Such an attachment prevented DNA from swiveling about its anchoring points, allowing the introduction of superhelical tension in the molecule by precisely controlled rotation of the external magnetic field. By recording the relative extension of the molecule as a function of superhelical density at fixed force values, differences in the behavior of positively versus negatively supercoiled molecules were revealed (Figure 4B) and structural transitions (denaturation or formation of Pauling-like DNA structures) deduced. Understanding Transcription The wealth of information already available from biochemistry and high-resolution structural studies has been recently supplemented by SM data. All stages of transcription have been investigated, beginning with the search for and binding to the promoter region, the unwinding of the DNA double helix at the promoter, the initial synthesis of abortive transcripts, the transition to elongation, elongation itself, and termination. It is beyond the scope of this tutorial to cover the SM transcription field, but a comprehensive review has been recently published (Zlatanova et al., 2006). Here, we will only illustrate how two methods, TPM and OT, have been used to study elongation, providing unprecedented insights into the ‘‘static’’ heterogeneity of individual molecules in a RNAP population and into the different kinds of pauses that intersperse the overall monotonic movement of the enzyme along the DNA template. Yin et al. (1994) made use of the TPM method designed earlier (Schafer et al., 1991) (Figure 5A) to obtain real-time trajectories of the longitudinal movement of the DNA template past the catalytic center of individual immobilized RNAPs (Figure 5B). The statistical evaluation of such trajectories led them to conclude that the kinetic properties of individual RNAP molecules in a putatively homogeneous population varied to a larger degree than that expected on the basis of the experimental uncertainty of the rate measurements. Recently, the TPM method was modified to avoid the use of immobilized enzymes and thus a potential artifactual source of enzyme heterogeneity (Tolic´-Nørrelykke et al., 2004). The same

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Figure 5. Studying Transcription Elongation (A) Schematic of the tethered particle motion (TPM) method, based on the dependence of the extent of Brownian motion of a bead (readily measured by light microscopy) on the length of the DNA template tethering it to the surface. In transcription experiments, the polymerase is immobilized on a glass surface and a bead is attached to either the upstream or the downstream end of the DNA template; elongation will result in lengthening or shortening of the tether (and a change in the Brownian motion of the bead), depending on the particular geometry used. (B) Transcription elongation as measured by the TPM method. Example tether-length/ time courses with the bead at the upstream (left traces) or downstream (right traces) end of the template (reproduced from Yin et al. [1994] with permission from the Biophysical Society). (C) Concept of recent experiments from S. Block’s laboratory using the novel passive, all-optical force clamp that takes advantage of the existence of a region in the trap where the force is approximately constant for small bead displacements (Greenleaf et al., 2005; Herbert et al., 2006). Two beads are held in separate optical traps: the transcribing polymerase is attached to one of the beads and the upstream end of DNA to the other bead, creating a bead-DNA-RNAP-bead dumbbell geometry. In the configuration used, the trap applies an assisting load. This instrument achieves angstrom-level resolution. (D) Representative records of transcription along a repetitive DNA template containing eight repeats of an w230 bp sequence that possesses previously identified pause sites. Most records display distinct pauses at the expected pause sites; in addition, other pause sites are identified that display sequence similarities (reprinted from Herbert et al. [2006] with permission from Cell Press).

conclusion was reached, recently confirmed by Herbert et al. (2006), using OT. Block’s group has recently developed a passive, alloptical force clamp OT (Greenleaf et al., 2005) that is highly stable, allowing lateral resolution in the angstrom range. Using a dumbbell geometry—bead-DNA-RNAPbead—the group has shown that the polymerase advances in steps of w3.4 A˚, i.e., a translocation step of one base pair follows each catalytic step of adding one base to the nascent transcript (Abbondanzieri et al., 2005). Using a similar geometry (but rather different instrumentation) (Figure 5C), they have also extensively studied the nature of the pauses experienced by the elongating enzyme. It was convincingly demonstrated that the short, frequent, ubiquitous pauses (Figure 5D) are sequence dependent (Herbert et al., 2006) and, as earlier work has shown, are characterized by sharp transitions into and out of the pause (Neuman et al., 2003). During the long (>20 s) pauses, the RNAP backtracks along the template with extrusion of an already synthesized portion of the RNA transcript from the catalytic center (Shaevitz et al., 2003). Sophisticated averaging procedures allowed probing details of polymerase motion that could have remained masked by noise in records of individual RNAP molecules. Three successive phases of the long pause were discerned: backtracking at the beginning, pausing per se, and slow downstream recovery. The duration and frequency of the backtracking pauses are strongly dependent on application of either opposing or assisting force, in contrast to the short pauses, which are load independent.

Understanding Molecular Motors Translocating on Linear Tracks Molecular motors are enzymes that use the energy from NTP hydrolysis to translocate along tracks (DNA in the case of polymerases, helicases, and nucleosome remodeling factors, etc., or actin filaments or microtubules in the case of cargo transporters, such as myosin, kinesin, and dynein). SM studies of RNA polymerases have been described above. We now consider as an example the application of SM techniques to the study of kinesin motion on microtubules. There have been two categories of mutually excusive models constructed on the basis of structural and biochemical studies. Each of the models has specific predictions on the movement of the two heads and on the rotation of the stalk during translocation. The SM studies illustrated in Figure 6 show the variety of methodologies used and the evolution of our knowledge on the motion mechanism of kinesin (for details, see the figure legend and the original papers cited there; reviewed in Yildiz and Selvin [2005b]). Gelles and coauthors performed a SM microtubule gliding assay, which involved immobilization of the kinesin tail regions onto a glass surface; the translocation of microtubules was followed by microscopy (Hua et al., 2002; Figure 6A). No rotation of the microtubules was observed (Figure 6B); the data were interpreted in terms of inchworming motion of the kinesin along the microtubule but were also consistent with an asymmetric, handover-hand motion. Two groups, using slightly different approaches, observed ‘‘limping’’ of kinesin during

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Figure 6. Representative SM Data on Kinesin Moving Along Microtubules (A) Experimental geometry for observing stalk rotation: a microtubule is bound to a single-surface-attached kinesin molecule. (B) In the presence of ATP, there is no rotation of the stalk, as seen by the overlaid traces (arrows) of a microtubule imaged at 1 s intervals. The right- and the left-hand traces show the same microtubule in two different time periods separated by 104 s (adapted from Hua et al. [2002] and reproduced with permission from AAAS). (C) Time course of the displacement of a single heterodimeric kinesin molecule, as visualized by OT nanometry (three left traces). This molecule contains two heads with very different mechanochemical cycle rates (wild-type and a single point mutant in the nucleotide-binding motif, with much-reduced microtubule-gliding speed). The traces indicate that in most cases the steps are 16 nm, but sometimes a shoulder at 8 nm is distinguishable. Thus, the observed 16 nm step is actually two successive steps of very different dwell times. The wild-type homodimer (two right traces) shows clear 8 nm steps of almost equal duration (reproduced from Kaseda et al. [2003] with permission from Nature Publishing Group, McMillan Publishers Ltd.). (D) Stepwise motion of recombinant kinesin homodimers as measured by OT: intrinsic stepping rate can alternate between two different values at each sequential step, causing the molecule to limp. The construct tested was DmK401, a truncated derivative of D. melanogaster kinesin containing two identical heads and a sufficient length of the neck coiled coil for dimerization. The dwell times between successive 8 nm steps alternate between slow and fast phases, causing steps to appear in pairs (red and blue). The dwell times in the record are numbered sequentially to make the point that the longer-lived dwell times tend to cluster systematically, in this specific trace, in the odd-numbered subset. The vertical bars indicate the occurrence of the steps (traces reproduced from Asbury et al. [2003], with permission from AAAS). (E) A typical image of surface-immobilized Cy3-DNA acquired within 0.5 s using objective-type TIRF. The image contains w14,000 photons and can be fitted to a two-dimensional Gaussian, which approximates the point spread function (PSF) of the Cy3; the center of the PSF yields w1 nm precision and accuracy in the center localization of the fluorescence spot (reproduced from Yildiz and Selvin [2005a] with permission from the American Chemical Society). This technique has been named FIONA (Yildiz et al., 2003; Yildiz and Selvin, 2005a). (F) Position versus time for kinesin motility as determined by FIONA: example traces for an E215C homodimer kinesin. Dots, experimental points; red lines show when steps occur (vertical jumps) and the average positions between steps (plateaus). Both the step sizes of an individual head of a kinesin dimer and dwell-time analysis support a hand-over-hand mechanism (reproduced from Yildiz et al. [2004] with permission from AAAS).

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Table 1. Selected Examples of Single-Molecule Studies In Vivo Object of Study and Technique

Main Observations/Conclusions

References

Lateral motion of fluorescently labeled lipids inserted into membranes of living cells; single-dye tracking by epifluorescence microscopy

A saturated lipid probe showed confined diffusion within small areas of w0.7 mm, whereas an unsaturated lipid diffused freely in the membrane; lipid domains (rafts) exist in cell membranes as areas with high local enrichment of saturated lipids One molecule of EGF interacts with an EGFR dimer, followed by binding of a second EGF molecule; formation of dimers of receptor molecules is directly visualized; EGFR becomes phosphorylated after dimerization The cAMP gradient in the medium (that causes the chemotactic movements) is converted to a spatial difference in the reaction state of the cAMP receptor, with cAMP signaling more active in the anterior region of the cell The distribution of diffusion constants within cells is broader than that obtained in glycerol solution, from simulations or theory; the intracellular motion cannot be described by simple diffusion Protein transport to the plasma membrane occurs in phases: docking of the Golgi carrier onto the membrane, its flattening, and diffusion of the protein within the membrane; both small spherical and large tubular carriers participate in the process Diffusion trajectories indicate complex motions, including free and anomalous diffusion, and directed motion by motor proteins both in the cytoplasm and nucleus; much faster infection than measured in bulk experiments Unsaturated lipids are confined within 230 nm f compartments for several milliseconds before hopping to adjacent compartments; the 230 nm compartments exist within larger w750 nm domains where lipids are confined for w0.33 s; diffusion in cellular membranes is slow because of compartmentalization Polymerization and depolymerization occur throughout lamellipodia with constant kinetics; polymerization occurs away from the tip of the lamellipodia The movement (before viral fusion with endosomes) occurs in three stages: (1) slow, actin-dependent movement in the cell periphery, (2) rapid unidirectional dyneindirected movement towards the nucleus, and (3) intermittent, often bidirectional movement on microtubules in the perinuclear region The diffusion of the lipid probe is blocked in the initial segment of the axon membrane by transmembrane proteins anchored to actinbased meshwork under the membrane

Schutz et al., 2000

Epidermal growth factor receptor (EGFR) signaling in membrane of living carcinoma cells; single-molecule tracking by objective-type TIRF; spFRET between Cy3-EGF and Cy5-EGF Association and dissociation of individual Cy3-cAMP molecules with the cell surface of Dictyostelium during chemotaxis; single-molecule tracking by objective-type TIRF Intracellular motion of R-phycoerythrin (autofluorescent protein containing w30 chromophores); single-molecule tracking by epifluorescence Final stages of constitutive exocytosis (fusion of single post-Golgi carriers with the plasma membrane); single-molecule tracking by objective-type TIRF

Viral infection by adeno-associated viral particles; single viral particle tracking by epifluorescence

Movement of unsaturated phospholipids in the plasma membrane of rat kidney fibroblasts; single-molecule tracking by objective-type TIRF; single-particle tracking of a 40 nm gold bead attached to an unsaturated lipid probe by videomicroscopy

Polymerization and depolymerization of actin filaments in lamellipodia; single-molecule tracking of actin-GFP (SM speckle analysis) by epifluorescence Constitutive endocytosis of influenza virus; Single viral particle tracking by epifluorescence

Distribution of membrane proteins in different regions of polarized neuron membranes; single-molecule tracking by objective-type TIRF; single-particle tracking of a 40 nm gold bead attached to an unsaturated lipid probe by video-microscopy; dragging experiments on the gold-particle-labeled probe by OT DNA uptake during bacterial transformation; OT

Intracellular signal transduction pathway (activation of the oncoprotein Ras by epidermal growth factor); spFRET by objective-type TIRF

DNA molecules are transported at a linear rate without observable pausing, reversals, or slipping; the uptake rate is force independent to up to w40 pN of opposing force; proton motive force is used for DNA transport Activation of single Ras molecules leads to a considerable slowing down of their diffusion; the suppressed diffusion of Ras suggests the formation of large Ras-signaling complexes in the membrane

Sako et al., 2000b

Sako et al., 2000a; Ueda et al., 2001

Goulian and Simon, 2000

Schmoranzer et al., 2000

Seisenberger et al., 2001

Fujiwara et al., 2002

Watanabe and Mitchison, 2002

Lakadamyali et al., 2003

Nakada et al., 2003

Maier et al., 2004

Murakoshi et al., 2004

(Continued on next page)

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Table 1. Continued Object of Study and Technique

Main Observations/Conclusions

References

Protein translocation through nuclear pore complexes (NPC) by using a fluorescent model substrate (two identical GFP domains are linked together); single-molecule tracking by epifluorescence

Protein molecules spend w10 ms within NPC, moving rapidly in a random walk though the central pore; the rate-limiting step is the substrate exit from the pore; NPC must be capable of transporting w10 molecules simultaneously Single quantum dots diffused at characteristically different rates in different regions of the membrane or inside the cytoplasm T cell activation causes coclustering of several membrane proteins in discrete microdomains; two-color imaging reveals that these microdomains exclude or trap specific proteins; diffusional trapping through proteinprotein interactions participate in T cell signaling Peroxisomes move in steps in both anterograde (kinesin) and retrograde (dynein) directions, with step size of w8 nm; each peroxisome takes a few steps driven by dynein, then a couple by kinesin, etc.; dynein and kinesis work in coordination; several kinesins or dyneins can work in concert to move the cargo faster

Yang et al., 2004

Avidin-CD14 receptor movement in HeLa cells; single-particle tracking of biotinylated peptide-coated quantum dots by epifluorescence Signaling proteins in immortalized T cells upon T cell activation; combination of scanning confocal (population) methods and single-molecule tracking by objective-type TIRF

Organelle (peroxisone) movement in cultured Drosophila cells; FIONA

walking (Kaseda et al., 2003; Asbury et al., 2003; Figures 6C and 6D); such limping (alternating nonequivalent behavior of the two heads during successive translocation steps) excluded both the inchworm type of motion and a symmetric hand-over-hand motion. Finally, the fluorescence imaging with one nanometer accuracy (FIONA) studies of Selvin’s group (Yildiz et al., 2004; Figure 6E and F) indicated either a symmetric or asymmetric hand-over-hand mechanism. Thus, the collective SM data strongly support an asymmetric hand-over-hand mechanism. SM Studies of Living Cells The ultimate goal of all structural, biochemical, and SM research is to understand the function of biological macromolecules in their natural environments, i.e., in living cells. The challenge is enormous due to the complexity of living matter and the relative paucity of appropriate in vivo methods. SM methods, because of their ability to follow the behavior of one molecule at a time, seem to offer an entry into this problem. Thus far, the prevalent method for observing single molecules in living cells has been single-dye fluorescence or spFRET. Emission from single fluorophores is very difficult to detect at the significant background caused by cellular autofluorescent molecules, such as flavins, NADH, etc. To minimize such autofluorescence, fluorophores that are excited by long wavelength (such as YFP) are needed. The field is in its infancy, but already some remarkable successes have been accomplished. Some studies are summarized in Table 1 (see also Sako and Yanagida [2003] for an earlier review). Two recent papers from the laboratory of X.S. Xie demonstrate ingenious ways to investigate the stochastic expression of mRNA and protein molecules in individual cells. In one approach (Cai et al., 2006), bacteria trapped in microcavities exhibit bursts of fluorescent b-galactosidase synthesis:

Pinaud et al., 2004; for a review, see Michalet et al. (2005) Douglass and Vale, 2005

Kural et al., 2005

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