Myosin is a molecular motor that steps along actin tracks. This motor’s ability to generate force and motion relies on its capacity to hydrolyze ATP and to convert chemical energy into mechanical work. The myosin superfamily now consists of nearly 30 different classes of myosin associated with biological functions as diverse as muscle contraction, organelle transport, cell motility, and cell division. Our laboratory and collaborators focus on an extremely diverse group of myosins:

Class II: Skeletal, smooth, and cardiac myosins responsible for muscle contraction.

Class V: A long-necked processive myosin associated with vesicular transport directed towards the cell periphery (collaborators: Trybus Lab, Walcott Lab).

Class XIV: A single-headed myosin involved in Toxoplasma gondii parasite locomotion and host cell invasion (collaborator: Ward Lab).

These myosins share significant structural and functional capacities, i.e. they possess a catalytic domain that has hydrolytic, actin-binding, and motor capacities. Emerging from the motor domain is a light-chain and/or calmodulin-binding domain that serves as a mechanical lever to amplify small conformational changes that originate within the motor domain’s active site. The length of this lever can vary depending on the myosin class. The differences in both structure and function among the various myosin classes can provide a model system to help probe the molecular structure and function of myosin as a chemomechanical enzyme.

MyBP-C is a striated muscle contractile protein, mutations to which have been linked to genetic forms of hypertrophic and dilated cardiomyopathies as well as skeletal muscle myopathies (i.e. distal arthrogryposis). MyBP-C is expressed as 3 isoforms; fast skeletal, slow skeletal, and cardiac. It’s an elongated protein with at least 10 globular domains. MyBP-C associates with the myosin thick filament at one end and both the myosin head region and actin at its other end.

\

Striated muscles are composed of repeating contractile units called sarcomeres in which the thin, actin-containing and thick, myosin-containing filaments overlap and slide past each other during muscle shortening. The myosin thick filament is composed of 300 myosin molecules that form a bipolar filament in which the myosin heads on each half of the thick filament are oriented to move the thin filament towards the center of the sarcomere. Interestingly, MyBP-C is localized to two regions (C-zones) near the center of the thick filament and thus the sarcomere. Why is MyBP-C localized to only these discrete zones is a major lab interest?

Although MyBP-C affects actomyosin function, the molecular mechanism by which this occurs is far from certain, with multiple models proposed for its action. The specific model depends on MyBP-C’s binding partner. For example, by binding to the myosin head region, MyBP-C may limit myosin’s interaction with actin and maintain myosin in a super-relaxed state (SRX). Alternatively, MyBP-C may bind to actin and act as an internal load to shortening or activate the thin filament in a calcium-independent manner.

Our laboratory uses molecular biophysics to unravel the key interactions between MyBP-C and both actin and myosin that allow this unique protein to modulate actomyosin function in the heart and in skeletal muscles.

Myosin Va is a double-headed molecular motor that transports intracellular cargo by taking numerous ~36 nm steps along its actin track (see animation). Although a single motor can carry cargo, normally teams of myosin Va motors share this responsibility. Complicating matters is that the intracellular highway these motors travel on is a 3-dimensional meshwork that presents both a physical barrier and a directional challenge to the delivery of cargo to its destination. To address this dilemma we use both simplified in vitro model systems of myosin Va transport within biomimetic actin cytoskeletons and in vivo transport in tissue cultured cells (see movie of insulin granule transport in INS-1 cells). Using the laser trap, single molecule fluorescence detection techniques, and super-resolution microscopy (STORM), we are in a position to define the molecular mechanisms underlying myosin Va’s capacity as a single motor or within teams to maneuver cargo through Mother Nature’s 3-dimensional actin filament maze. These studies require expertise in molecular biophysics, protein expression (in collaboration with Dr. Kathleen Trybus). and biological systems modeling (in collaboration with Dr. Sam Walcott).

This assay represents a simplified model of muscle force and motion generation.  In this assay, myosin, a molecular motor, is adhered to a nitrocellulose-coated microscope coverslip. The coverslip is one surface of a 20 µl experimental microchamber. Fluorescently labeled actin filaments in a solution containing MgATP are perfused into the chamber and then the movement of single actin filaments over the myosin surface is visualized in a Total Internal Reflectance Fluorescence (TIRF) microscope. Variations of this assay include the use of native myosin thick filaments and native actin thin filaments.

The laser trap has the capacity to capture microscopic particles, such as 1 µm polystyrene beads, in solution. A trap is created by focusing a laser through a high numerical aperture objective, forming a trap at the focal point of the objective. This technique can be used to measure the molecular forces (piconewtons) and displacements (nanometers) generated by a single myosin molecular motor as it interacts with a single actin filament. For these measurements, two traps are formed by a computer-controlled acousto-optic deflector that timeshares the laser between two positions at 10 kHz. Then, a single NEM-modified myosin-coated bead is captured in each trap. With the ability to manipulate the position of the traps through software, the investigator attaches a bead to each end of an actin filament. The actin filament is then lowered towards the motility surface where larger beads serve as a myosin platform. As the myosin pulls on the actin filament, the displacement of the actin filament is detected through the motion of one or both of the beads, the image of which is projected onto a quadrant photodiode detector. we have developed a computer-based force clamp feedback system allowing us to characterize the effect of load on the mechanics and kinetics of a single myosin molecule

Super-Resolution STORM Microscopy

STochastic Optical Reconstruction Microscopy (STORM) uses the ability to identify single fluorophores with ~20nm spatial resolution in order to effectively improve the resolution of the light microscope 10-fold. As an example, compare the STORM and TIRF images of the same field of actin filaments, labeled with fluorescent phalloidin and adhered to a glass surface. To reconstruct the super-resolution image, fluorophores are forced into the dark state from which they recover stochastically and fluoresce. As they fluoresce, their positions (i.e. center of point spread functions) are identified with sub-pixel resolution and the positions for all fluorophores on a single actin filament are then used to reconstruct the super-resolution image.

By placing a cylindrical lens in the optical path, an optically-induced astigmatism can be used to resolve the Z-position of a fluorophore within the focal plane. As an example, the height of fluorescently-labeled actin filaments suspended from 3µm beads can be resolved with 50nm resolution.