Interferometric Scattering Microscopy for the Study of Molecular Motors.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Coupling between phosphate release and force generation in muscle actomyosin.

Energetic, kinetic and oxygen exchange experiments in the mid-1980s and early 1990s suggested that phosphate (Pi) release from actomyosin-adenosine diphosphate Pi (AM.ADP.Pi) in muscle fibres is linked to force generation and that Pi release is reversible. The transition leading to the force-generating state and subsequent Pi release were hypothesized to be separate, but closely linked steps. Pi shortens single force-generating actomyosin interactions in an isometric optical clamp only if the conditions enable them to last 20-40 ms, enough time for Pi to dissociate. Until 2003, the available crystal forms of myosin suggested a rigid coupling between movement of switch II and tilting of the lever arm to generate force, but they did not explain the reciprocal affinity myosin has for actin and nucleotides. Newer crystal forms and other structural data suggest that closing of the actin-binding cleft opens switch I (presumably decreasing nucleotide affinity). These data are all consistent with the order of events suggested before: myosin.ADP.Pi binds weakly, then strongly to actin, generating force. Then Pi dissociates, possibly further increasing force or sliding.

A small-molecule inhibitor of skeletal muscle myosin II.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

A maximum entropy analysis of protein orientations using fluorescence polarization data from multiple probes.

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a "figure of merit" is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

The ADP release step of the smooth muscle cross-bridge cycle is not directly associated with force generation.

When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a approximately 3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748-751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.

Trading force for speed: why superfast crossbridge kinetics leads to superlow forces.

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only approximately 1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.

Model-independent analysis of the orientation of fluorescent probes with restricted mobility in muscle fibers.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres.

Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, giving ten different probes. The labelled RLCs were exchanged into demembranated fibres from rabbit psoas muscle without significant effect on active force generation. Fluorescence polarisation measurements showed that nine out of the ten probe dipoles were more perpendicular to the fibre axis in the absence of ATP (in rigor) than in either relaxation or active contraction. The orientational distribution of the RLC region of the myosin head in active contraction is closer to the relaxed than to the rigor orientation, and is not equivalent to a linear combination of the relaxed and rigor orientations. Rapid length steps were applied to the fibres to synchronise the motions of myosin heads attached to actin. In active contraction the fluorescence polarisation changed both during the step, indicating elastic distortion of the RLC region of the myosin head, and during the subsequent rapid force recovery that is thought to signal the working stroke. The peak change in fluorescence polarisation produced by an active release of 5 nm per half sarcomere indicates an axial tilt of less than 5 degrees for all ten probes, if all the myosin heads in the fibre respond to the length step. This tilting was towards the rigor orientation for all ten probes, and could be explained by 14% of the heads moving to the rigor orientation. An active stretch tilted the heads away from the rigor conformation by a similar extent.

Fluorescence polarization transients from rhodamine isomers on the myosin regulatory light chain in skeletal muscle fibers.

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys108-5 or Cys108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of approximately 90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted approximately 30 degrees for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca2+. Polarization ratios for Cys108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys108-6 dipole were restricted to a cone of approximately 20 degrees half-angle, and those of Cys108-5 dipole to a cone of approximately 25 degrees half-angle. These values changed little between relaxation, active contraction, and rigor. Cys108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (approximately 60 degrees) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils.

Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.

Fluorescence polarization of skeletal muscle fibers labeled with rhodamine isomers on the myosin heavy chain.

Fluorescence polarization was used to examine orientational changes of Rhodamine probes in single, skinned muscle fibers from rabbit psoas muscle following either photolysis of caged nucleotides or rapid length changes. Fibers were extensively and predominantly labeled at SH1 (Cys-707) of the myosin heavy chain with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine. Results from spectroscopic experiments utilizing the two Rhodamine isomers were quite similar. Following photolysis of either caged ATP or caged ADP, probes promptly reoriented toward the muscle fiber axis. Changes in the fluorescence polarization signals with transients elicited by the photolysis of caged ATP in the presence of saturating Ca2+ greatly preceded active force generation. Photolysis of caged ADP caused only a small, rapid decrease in force but elicited changes in the fluorescence polarization signals with time course and amplitude similar to those following photolysis of caged ATP. Fluorescence polarization signals were virtually unchanged by rapid length steps in both rigor and active muscle fibers. These results indicate that structural changes monitored by Rhodamine probes at SH1 are not associated directly with the force-generating event of muscle contraction. However, the fluorescence polarization transients were slightly faster than the estimated rate of cross-bridge detachment following photolysis of caged ATP, suggesting that the observed structural changes at SH1 may be involved in the communication pathway between the nucleotide- and actin-binding sites of myosin.

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.

Mechanical characterization of skeletal muscle myofibrils.

A new instrument, based on a technique described previously, is presented for studying mechanics of micron-scale preparations of two to three myofibrils or single myofibrils from muscle. Forces in the nanonewton to micronewton range are measurable with 0.5-ms time resolution. Programmed quick (200-microseconds) steps or ramp length changes are applied to contracting myofibrils to test their mechanical properties. Individual striations can be monitored during force production and shortening. The active isometric force, force-velocity relationship, and force transients after rapid length steps were obtained from bundles of two to three myofibrils from rabbit psoas muscle. Contrary to some earlier reports on myofibrillar mechanics, these properties are generally similar to expectations from studies on intact and skinned muscle fibers. Our experiments provide strong evidence that the mechanical properties of a fiber result from a simple summation of the myofibrillar force and shortening of independently contracting sarcomeres.