Understanding dual filament regulation in muscle using single molecule imaging in vitro and in myofibrils

Muscle is essential for human survival, enabling processes such as locomotion and heart contraction. Even with many decades of investigation, much still needs to be understood about the underlying molecular mechanisms that both enable and control contraction. Perhaps the biggest open question is how force is regulated in contraction, here we seek to directly observe regulation using sensitive imaging technologies.

Muscle is highly organised into two types of protein strands: thick and thin filaments. Contraction occurs when motor proteins called myosin from the thick filament use energy in the form of ATP to pull on actin in the thin filament. This is regulated by calcium binding to regularly spaced control proteins on the thin filament, which helps regulate access of myosin to actin. The force at which contraction occurs depends on the number of myosins available.

If left uncontrolled, muscle would constantly contract and use up all the organism's available energy. Therefore, regulating the timing and force of muscle contraction is crucial for survival. Our over-arching goal in this proposal is to provide a clear understanding of this process both in the test-tube (in vitro) and in muscle tissue extracts (in vivo). We will use cutting-edge single molecule imaging to study regulation directly on both thick and thin filaments, which work together to control contraction in a process known as 'dual filament regulation'.

To study dual filament regulation, we will use two unique assays that our lab possesses. Firstly, an in vitro 'tightrope' assay, which involves suspending thin filaments above a microscope coverslip surface between glass beads. Using this assay, we can watch activation and its relaxation directly by following where fluorescently tagged myosins bind. To trigger myosin binding we can add calcium, but because it binds to any control protein along the thin filament, we cannot be sure where the thin filament is activated. This is important because from the point of calcium binding, activation spreads along the thin filament forming a 'cooperative unit'. Therefore, to control the point of activation we will engineer a fluorescent control protein to be active in the absence of calcium. As the myosin binds it reports on the spatial range over which the thin filament is turned on relative to the point of activation. This will enable a precise measurement of the physical size of the cooperative unit to be made, which would be an important breakthrough.

The second (in vivo) assay is to study regulation in myofibrils, which are extracted from muscle tissue. Since myosin uses ATP during contraction we can detect where this occurs by following labelled ATP. Using this assay, we will discover how the rules we learned from the in vitro studies translate in vivo. We also intend to adapt our imaging system to perform a series of systematic studies that will reveal how the thin and thick filaments communicate in the dense, complex 3-dimensional matrix of a myofibril. No-one has made such direct measurements to date, which are highly valuable for understanding the molecular basis of dual filament regulation. These studies lie at the current frontier of muscle biology research and are important for understanding significant diseases such as cardiomyopathies and skeletal myopathies.

Muscle contraction requires the coordinated sliding of thin over thick filaments powered by ATP. Thin filaments contain actin and the control proteins troponin (Tn) and tropomyosin (Tm). Thick filaments are mostly comprised of myosin II and myosin binding protein-C (MyBP-C). Calcium binding to Tn initiates contraction by enabling Tm to move over actin and expose myosin-binding sites. The sites exposed, along with the number of myosins available to interact with them, determine the force of contraction. This is the underlying principle of dual filament regulation, for which it is imperative to know the physical size of the active region. Here, we aim to directly visualise how muscle contraction is regulated using single molecule imaging both in vitro and in intact myofibrils (in vivo).

To map the physical size of the cooperative unit, we will constitutively activate a single location on the thin filament and image the proximal binding of fluorescent-myosins. This will provide a full spatial measurement of the cooperative unit induced by myosin or calcium. In parallel, we will investigate regulation in the native milieu of a myofibril (the contractile organelle of muscle). By exchanging into myofibrils a fluorescently-tagged mutant Tn (active in the absence of calcium), we can detect actin-activated myosins by measuring their consumption of fluorescent ATP. This will provide the first physical measure of cooperative unit size within a myofibril. The availability of myosin is driven by its distribution between on and off detached states. We will modulate this equilibrium using calcium, phosphorylation and drugs to determine how myosin availability affects cooperative unit size.

By the end of this project, we will have a new and unprecedented level of understanding for how the thin and thick filaments together contribute to dual filament regulation, both in vitro and in vivo. We will work with collaborators to make this available in an open-source model.