Nebulin no longer nebulous! Scientists obtain first high-resolution 3D
image of muscle protein

Date:
February 18, 2022
Source:
Max Planck Institute of Molecular Physiology
Summary:
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 has brought to
light the chance to better understand the role of nebulin, as its
functions have remained largely nebulous due to its large size
and the difficulty in extracting nebulin in a native state from
muscle. The team used electron cryo-tomography to decipher the
structure of nebulin in impressive detail. Their findings could
lead to novel therapeutic approaches to treat muscular diseases,
as genetic mutations in nebulin are accompanied by a dramatic loss
in muscle force known as nemaline myopathy.



FULL STORY ========================================================================== 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 has brought to light the chance to better
understand the role of nebulin, as its functions have remained largely
nebulous due to its large size and the difficulty in extracting nebulin
in a native state from muscle. The team of Max Planck researchers, led
by Stefan Raunser, Director at the Max Planck Institute of Molecular
Physiology in Dortmund, in collaboration with Mathias Gautel at King's
College London, used electron cryo-tomography to decipher the structure
of nebulin in impressive detail. Their findings could lead to novel
therapeutic approaches to treat muscular diseases, as genetic mutations
in nebulin are accompanied by a dramatic loss in muscle force known as
nemaline myopathy.


==========================================================================
An elusive protein Skeletal and heart muscles contract and relax upon
sliding of parallel filaments of the proteins myosin and actin. Nebulin, another long slender protein, which is present only in skeletal muscle,
pairs up with actin, stabilising and regulating it. Mutations in the gene encoding nebulin can produce an abnormal nebulin that causes nemaline
myopathy, an incurable neuromuscular disorder with various degrees of
severity, from muscle weakness to speech impediments and respiratory
problems.

Knowing the structure of nebulin and how it interacts with actin
could be pivotal to the development of new treatments. But traditional experimental approaches that reconstitute nebulin in vitro have failed
because of the size of the protein, its flexibility, and the fact that it
is intertwined with actin. Raunser and his team take a different approach:
they visualise these proteins directly in their native environment,
the muscle, by using a powerful microscopy technique called electron cryo-tomography (cryo-ET). A cryo-ET experiment in the Raunser lab begins
with flash-freezing muscle samples. Then, scientists apply a gallium-based
ion beam to the sample to shave away extra material from it and reach an
ideal thickness of around 100 nanometres for the transmission electron microscope. This powerful tool then acquires multiple images of the
sample tilting along an axis. Finally, computational methods render a three-dimensional image at an impressively high resolution.

Pushing the limits of cryo-ET In a 2021 publication, the Max Planck
researchers produced the first detailed 3D image of the sarcomere, the
basic contractile unit of skeletal and heart muscle cell that contains
actin, myosin and, eventually, the nebulin protein.

The resolution of one nanometre (a millionth of a millimetre) was good
enough to image actin and myosin but too low for visualising nebulin. This time, the team improved their data acquisition and processing pipeline to obtain a 3D picture of skeletal muscle filaments at near atomic resolution (0.45 nanometres). By comparing the images of the skeletal muscle with the nebulin- free cardiac muscle, the structure of the long nebulin protein
became distinct and the researchers were able to build an atomic model of nebulin. "This is the first high-resolution structure using FIB-milling
and cryo-ET and it proves that we can reach atomic models in a reliable
way. It's a quantum leap!," says Raunser.

The findings reveal that each nebulin repeat binds with an actin
subunit, demonstrating nebulin's role as a ruler that dictates the
length of the actin filament. Besides, each nebulin repeat interacts
with every neighbouring actin subunit, which explains its role as a
stabiliser. Finally, the scientists propose that nebulin regulates the
binding of actin and myosin, and hence muscle contraction, by interacting
with another protein called troponin.

Experiments were done on mouse muscles that are very similar to the
human ones -- and were isolated at King's College London.

"We obtained a detailed in situ 3D structure of nebulin, actin and myosin
heads that can be used to pinpoint the mutations leading to myopathies,"
notes Raunser. Pharmaceutical developers can then take advantage of
this new structure to locate binding sites for small molecules of pharmaceutical interest, he adds. Driven by their recent success,
the group will now concentrate on unveiling the structural details of
myosin, the other sliding filament. Such findings could finally help
paint the complete picture of the intricate details behind skeletal
muscle contraction.

========================================================================== Story Source: Materials provided by Max_Planck_Institute_of_Molecular_Physiology. Note: Content may be edited
for style and length.


========================================================================== Related Multimedia:
* 3D-Structure_of_Nebulin ========================================================================== Journal Reference:
1. Zhexin Wang, Michael Grange, Sabrina Pospich, Thorsten Wagner,
Ay Lin
Kho, Mathias Gautel, Stefan Raunser. Structures from intact
myofibrils reveal mechanism of thin filament regulation through
nebulin. Science, 2022; 375 (6582) DOI: 10.1126/science.abn1934 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/02/220218100707.htm

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