Changepoint Analysis for Single-Molecule Polarized Total Internal Reflection Fluorescence Microscopy Experiments

The experimental study of individual macromolecules has opened a door to determining the details of their mechanochemical operation. Motor enzymes such as the myosin family have been particularly attractive targets for such study, in part because some of them are highly processive and their “product” is spatial motion. But single-molecule resolution comes with its own costs and limitations. Often, the observations rest on single fluorescent dye molecules, which emit a limited number of photons before photobleaching and are subject to complex internal dynamics. Thus, it is important to develop methods that extract the maximum useful information from a finite set of detected photons. We have extended an experimental technique, multiple polarization illumination in total internal reflection fluorescence microscopy (polTIRF), to record the arrival time and polarization state of each individual detected photon. We also extended an analysis technique, previously applied to FRET experiments, that optimally determines times of changes in photon emission rates. Combining these improvements allows us to identify the structural dynamics of a molecular motor (myosin V) with unprecedented detail and temporal resolution

Using electrical and optical tweezers to facilitate studies of molecular motors

Dielectrophoresis was used to stretch and suspend actin filaments across a trench etched between two electrodes patterned on a glass slide. Optical tweezers were used to bring a motor protein-coated bead into close proximity to a pre-selected, suspended actin filament, facilitating the attachment of the myosin-coated bead to the filament. The clearance beneath the filament allowed the bead to move freely along and around its filamentous track, unhindered by solid surfaces. Using defocused images, the three-dimensional position of the bead was tracked as a function of time to obtain its trajectory. Experiments were carried out with myosin V and myosin X. Both motor proteins followed left-handed helical paths with the myosin X motor exhibiting a shorter pitch than the myosin V. The combined use of electrostatic and optical tweezers facilitates the preparation of motility assays with suspended tracks. Variants of this technique will enable higher complexity experiments in vitro to better understand the behavior of motors in cells

The mechanics of short rod-like molecules in tension

The rapid development of single molecule experimental techniques in the last two decades has made it possible to accurately measure the force-extension response as well as the transverse fluctuations of individual rod-like macromolecules. This information is used in conjunction with a statistical mechanical model based on the treatment of the molecule as a fluctuating elastic rod to extract its bending and extension moduli. The models most commonly used to interpret the experimental data assume that the magnitude of the Brownian fluctuations are independent of the length of the macromolecule, an assumption that holds only in the asymptotic limit of infinitely long rods, and is violated in most experiments. As an alternative, we present a theoretical treatment of a finite length, fluctuating rod and determine its mechanical behavior by measuring the transverse Brownian fluctuations under the action of large stretching forces. to validate of our theory, we have applied our methods to an experiment on short actin filaments whose force-extension relation is difficult to measure, but whose transverse deflections can be captured by current microscopy techniques. An important consequence of the short contour lengths is that the boundary conditions applied in the experiment affect the fluctuations and can no longer be neglected as is commonly done when interpreting data from force-extension measurements. Our theoretical methods account for boundary conditions and can therefore be deployed in conjunction with force extension measurements to obtain detailed information about the mechanical response of rod-like macromolecules

Self-Assembled Charged Hydrogels Control the Alignment of Filamentous Actin

We demonstrate a novel route to control attachment of filamentous actin (F-actin) on hydrogel films. By incorporating an amine-terminated silane, the hydrogel surface charge and surface topography are varied. With increasing silane content, F-actin reorients from perpendicular to parallel to the hydrogel surface, ceases to wobble, and forms mainly elongated or cyclic structures. F-Actin coverage reaches a maximum at 2.5 vol% silane and declines at higher silane content. This biphasic behavior is explained by the simultaneous increase in surface charge and the self-assembly of a micron scale pattern of positively charged islands. Our approach provides guidelines for constructing nanoscale tracks to guide motor proteins underlying nano-engineered devices such as molecular shuttles

Twirling of Actin by Myosins II and V Observed via Polarized TIRF in a Modified Gliding Assay

The force generated between actin and myosin acts predominantly along the direction of the actin filament, resulting in relative sliding of the thick and thin filaments in muscle or transport of myosin cargos along actin tracks. Previous studies have also detected lateral forces or torques that are generated between actin and myosin, but the origin and biological role of these sideways forces is not known. Here we adapt an actin gliding filament assay in order to measure the rotation of an actin filament about its axis (“twirling”) as it is translocated by myosin. We quantify the rotation by determining the orientation of sparsely incorporated rhodamine-labeledactin monomers, using polarized total internal reflection (polTIRF) microscopy. In order to determine the handedness of the filament rotation, linear incident polarizations in between the standard s- and p-polarizations were generated, decreasing the ambiguity of our probe orientation measurement four-fold. We found that whole myosin II and myosin V both twirl actin with a relatively long (~ µm), left-handed pitch that is insensitive to myosin concentration, filament length and filament velocity

Electron Tomography of Cryofixed, Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

BACKGROUND: Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ. METHODOLOGY: We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the "target zone", situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening. CONCLUSION: We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments

Structural Changes in Isometrically Contracting Insect Flight Muscle Trapped following a Mechanical Perturbation

The application of rapidly applied length steps to actively contracting muscle is a classic method for synchronizing the response of myosin cross-bridges so that the average response of the ensemble can be measured. Alternatively, electron tomography (ET) is a technique that can report the structure of the individual members of the ensemble. We probed the structure of active myosin motors (cross-bridges) by applying 0.5% changes in length (either a stretch or a release) within 2 ms to isometrically contracting insect flight muscle (IFM) fibers followed after 5–6 ms by rapid freezing against a liquid helium cooled copper mirror. ET of freeze-substituted fibers, embedded and thin-sectioned, provides 3-D cross-bridge images, sorted by multivariate data analysis into ∼40 classes, distinct in average structure, population size and lattice distribution. Individual actin subunits are resolved facilitating quasi-atomic modeling of each class average to determine its binding strength (weak or strong) to actin. ∼98% of strong-binding acto-myosin attachments present after a length perturbation are confined to “target zones” of only two actin subunits located exactly midway between successive troponin complexes along each long-pitch helical repeat of actin. Significant changes in the types, distribution and structure of actin-myosin attachments occurred in a manner consistent with the mechanical transients. Most dramatic is near disappearance, after either length perturbation, of a class of weak-binding cross-bridges, attached within the target zone, that are highly likely to be precursors of strong-binding cross-bridges. These weak-binding cross-bridges were originally observed in isometrically contracting IFM. Their disappearance following a quick stretch or release can be explained by a recent kinetic model for muscle contraction, as behaviour consistent with their identification as precursors of strong-binding cross-bridges. The results provide a detailed model for contraction in IFM that may be applicable to contraction in other types of muscle