Scientists Obtain First High-Resolution 3D Image of Muscle Protein

Scientists Zhexin Wang and Dr. Michael Grange in Molecular Physiology Cryo-ET-MPI Microscope – Published

Scientists have obtained the first high-resolution 3D image of nebulin, a giant actin-binding protein that is an essential component of skeletal muscle.

This discovery sheds light on the mysterious role of nebulin, a protein whose functions have remained unclear due to its large size and the difficulty of extracting it natively from the muscle.

Max Planck’s team of researchers used electron cryo-tomography to decipher the structure of nebulin in impressive detail. Their findings could lead to new therapeutic approaches to treat muscle diseases, as gene mutations in nebulin are accompanied by a dramatic loss of muscle strength, known as nemaline myopathy.

Knowing the structure of nebulin and its interaction with actin could be essential for the development of new treatments. But traditional experimental approaches that reconstitute nebulin in vitro have failed due to the protein’s size, flexibility, and the fact that it is intertwined with actin.

An elusive protein

Skeletal and cardiac muscles contract and relax during the sliding of parallel filaments of myosin and actin proteins. Nebulin, another long, thin protein found only in skeletal muscle, associates with actin, stabilizing and regulating it. Mutations in the gene encoding nebulin can produce abnormal nebulin which causes nemaline myopathy, an incurable neuromuscular disorder with varying degrees of severity, ranging from muscle weakness to speech impairment and breathing problems.

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Led by Stefan Raunser, director of the Max Planck Institute for Molecular Physiology in Dortmund, in collaboration with Mathias Gautel of King’s College London, the team took a different approach: they visualize these proteins directly in their native environment, the muscle, using a powerful microscopy technique called cryo-electron tomography (cryo-ET). A cryo-ET experiment in the Raunser lab begins with flash-freezing muscle samples. Next, scientists apply a beam of gallium-based ions to the sample to remove excess material and achieve an ideal thickness of around 100 nanometers for the transmission electron microscope.

This powerful tool then acquires multiple images of the sample tilted along an axis. Finally, computational methods render a three-dimensional image at an impressive resolution.

Pushing the boundaries of cryo-ET

In a 2021 publication, Max Planck researchers produced the first detailed 3D image of the sarcomere, the basic contractile unit of skeletal and cardiac muscle cells that contains actin, myosin and, possibly, the nebulin protein. One nanometer (one millionth of a millimeter) resolution was sufficient to image actin and myosin, but too low to visualize nebulin.

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This time, the team improved its data acquisition and processing pipeline to obtain a 3D image of skeletal muscle filaments at near-atomic resolution (0.45 nanometers). By comparing images of skeletal muscle with cardiac muscle without nebulin, the structure of the long nebulin protein became distinct and the researchers were able to construct an atomic model of nebulin. “This is the first high-resolution structure using FIB milling and cryo-ET, and it proves that we can reliably achieve atomic models.”

“It’s a quantum leap!” Raunser said.

The results reveal that each nebulin repeat binds to an actin subunit, demonstrating the role of nebulin as a rule that dictates actin filament length. In addition, each nebulin repeat interacts with each neighboring actin subunit, which explains its role as a stabilizer. Finally, the scientists propose that nebulin regulates actin and myosin binding, and therefore muscle contraction, by interacting with another protein called troponin. Experiments were done on mouse muscles very similar to those of humans – and were isolated at King’s College London.

“We obtained detailed in situ 3D structure of nebulin, actin and myosin heads that can be used to identify mutations leading to myopathies,” notes Raunser.

Researchers can then take advantage of this new structure to locate binding sites for targeting with small molecules of pharmaceutical interest, he adds.

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Driven by their recent success, the group will now focus on unveiling the structural details of myosin, the other sliding filament. Such findings could finally help paint a full picture of the intricate details behind skeletal muscle contraction.

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