Cutting edge technology reveals cell structures that were previously only possible

New methods allow Austrian IST Austrian scientists to look inside cells. High-resolution images of deep-frozen cells show structures that were previously only conceivable.

The cells of our body are in motion. Some move from A to B to heal wounds or fight pathogens. They do this with the help of small “legs” on the leading edge of the migratory cells, the so-called lamelipodia. These thin extensions are pushed forward և tied to the surface while the rest of the cell is stretched lengthwise. Inside these legs is a dense web of intertwined protein fibers called actin fibers that make up the cell’s cytoskeleton. Until now, it was unclear how the Arp2 / 3 complex, which is a collection of seven central proteins for cell motility, originated from pre-existing new actin fibers, thus creating dense, branched networks that provide the cell with the necessary propulsive forces.

Difficult choices

Until now, scientists had to decide when they want to analyze the structure of the Arp2 / 3 complex. One option is to study it separately, where the protein complex is in an inactive structure և, thus, does not allow to understand what the network is like. composed of: However, in order to be fully active, the Arp2 / 3 complex must be bound to actin filaments. This requires the use of a method called electron tomography, which costs significantly lower resolution. “Previous electron tomography data of Arp2 / 3 complexes were too inaccurate to bind actin filaments in an in vitro environment. It was impossible to say exactly where the individual elements of the complex should be located,” explains Florian Fոլhler, a postdoctoral fellow. IST Austrian Professor Florian Schur Group.

Protein complex Arp2 / 3

The Arp2 / 3 protein complex, with its seven subunits (color), simultaneously binds to actin fibers (gray). Loan © Florian Fոլhler, IST Austria

For more than two years, he has been looking for a way to visualize a protein complex in its natural environment so that individual structures can be accurately analyzed. Now he has succeeded. He depicted the complex inside the lamellipodia of mouse cells with its active actin-binding. “We told ourselves. Well, we enter a cell where the environment is much more complex, because there are not only protein complexes – actin fibers, but also all sorts of other things. “But that was the only way we could maintain this network so that we could determine its structure,” said Florian Schur, a molecular biologist.

Shock-frozen cells

This became possible due to the temperature of minus 196 degrees Celsius, Within milliseconds, the researchers froze the samples too fast to allow ice crystals to break down, destroying the cell’s delicate structures. They then used one of the most powerful cryoelectron microscopes, the only one of its kind in Austria, to image cells from different angles using cryoelectron tomography. With this, the team gathered enough data for 3D reconstruction of more than 10,000 Arp2 / 3 complexes in their active state. Together with advanced image processing, they later developed the 3D model of the Arp2 / 3 complex with a resolution of less than one nanometer. For comparison. The thickness of human hair is about 50,000 nanometers. “We are now able to describe relatively accurately the structure of the protein complex, its subdivisions, and how they form the fibrous network of actin inside the lamelipodium of formerly living cells,” says Florian Fյhler. “Five years ago, probably no one would have thought it could be done,” Shur added.

To the border!

With advanced technology, the team could refute the earlier model, which assumed much larger areas of Arp2 / 3 complex “actin filaments”. However, scientists have confirmed how this complex is regulated, creating new actin filaments. With this knowledge, other scientists can now better understand the regulation of this important protein complex և its activity beyond cell motility ից its development outside of disease in its many roles. “What we have done is to go as far as possible in terms of methodology and solution with such complex samples. With the current formula we have gained new biological insights, but it also showed methodological progress. It is possible, ”Shur says excitedly. Florian Fոլhler now wants to further refine the method by imagining other proteins to study the extent to which the method allows us to see inside a cell. “We are just beginning to realize the full potential of cryo-electron tomography,” says Shur.

Loan “Cryoelectric tomographic structure of Arp2 / 3 complex in cells reveals new ideas of the branch node” Florian F Ֆhler, Georgi Dim Դ, Victor-Valentin Hodirnau, William Wani և Florian K.M. Shuri, December 22, 2020 Nature communications,
DOI: 10.1038 / s41467-020-20286-x:

Funding. Austrian Science Foundation (FWF), Georgy Dimch, Florian Schur.

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