Max Planck Institute researchers are using cryogenic tomography to reveal new molecular details for skeletal sarcomeres
An old technique that flexes his muscles
Sarcomers are small, repeated subunits of myofibrils, which are the long cylinders that come together to form muscle fibers. Within sarcomas, myosin and actin protein filaments interact to generate muscle contraction and relaxation. To date, conventional experimental methods have been performed to investigate the structure and function of muscle tissue on protein complexes reconstructed or suffered from low resolution. Instead, “cryogenic tomography allows for detailed, artifact-free 3D images of frozen muscles,” says Raunser.
Cryo-ET has long been a well-established but specialized methodology. But recent technical advances in electron microscopy (cryo-EM) as well as the new development of cryo-concentrated ion beam (FIB) milling are driving the cryo-ET decision. Similar to cryo-EM, researchers freeze the biological sample at a very low temperature (- 175 ° C). Through this process, the sample maintains its hydration and delicate structure, and remains close to its original state. FIB grinding is then applied to remove the excess material and obtain an ideal thickness of about 100 nm for the transmission electron microscope, which acquires multiple images while tilting the sample along the axis. Finally, computational methods reconstruct a 3D image with high resolution.
Ronser’s team performed cryo-ET on isolated mouse muscle fibers at King’s College, and obtained a resolution of one nanometer (a millionth of a millimeter, which is enough to see the fine structures within the protein): “We can now look at the myofibrils in detail. Imagine only four years ago. It’s cool! ”Says Ronser.
Fibers in their natural course
The calculated reconstructions of myofibrils revealed the three-dimensional organization of the myofibrils, including the M-, A- and I-bands sub-regions, and the Z-disk, which unexpectedly forms a more regular network and adopts a different symmetry. The scientists used a sample of myosin that is tightly bound to actin, which represents a stage of muscle contraction called the stern state. Indeed, they were able to visualize for the first time in the original cell how two heads of the same myosin connect to an actin filament. They also discovered that the double head not only interacts with the same actin filament but was also found split between two actin strands. This has never been seen before and it shows that the proximity to the following actin filaments is stronger than the cooperative effect between the adjacent vertices.
“This is only the beginning.” “Cryo-ET is moving from specialized technology to widespread technology in skeletal biology,” says Ronser. “Soon we will be able to examine muscle diseases at the molecular to atomic level.” Mouse muscles are similar to human muscles, yet the scientists are planning To examine muscle tissue from biopsies or pluripotent stem cells.