freezing technology shows structures in cells that were only only guessed at.

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New methods allow scientists at IST Austria to look into the innermost sections of cells, including structures that were previously just guessed at.

The cells in our organism are actively moving. Some do shifts to treat wounds or battle disease.

The cell’s feet help it move, and are located at the leading edge of the cell’s migration.

These long projections are forced forward and stretch into the surface, while the rest of the cell is pulled along with them.

Within the internal DNA are numerous interwoven protein filaments which form the cell’s cytoskeleton. Until now, how the Arp2/3 complex, a series of seven proteins essential to cell motility, created new actin filaments without having to take apart the existing ones to create thick, branching networks.

Significant decisions.
Until now, scientists have had to choose whether to study the structure of the Arp2/3 complex when it’s in the inactive form and thus unable to demonstrate how the complex works.

However, once the Arp2/3 complex is bound to actin filaments, it is fully activated.

This involves electron-microscope tomography, which limits the amount of detail looked at in the image. “Previous electron tomography data of Arp2/3 complexes bound to actin filaments in a test tube environment were too imprecise and made it impossible to say unambiguously where the individual elements of the complex must be located,” explains Fäßler.

For over two years he has been searching for the structural units that make up the protein complex so that it can be studied one at a time. Now he has been popular. He observed the complex in mouse cell lamellipodia during the activated, active conformation. We wanted to go inside the cell, where there are not just the actin proteins and the actin filaments, but also all of the rest.

But it was the only way we could maintain the network in such a manner that it could be explored by experimentation.

Frozen cells.
It was realized by generating a minimum of 196 degrees Celsius.

Within microseconds, the researchers froze the samples in an attempt to maintain the integrity of the cell structures.

Then, the team used a state-of-the-art cryo-electron microscope – and the only one of its kind in Austria – to obtain 3D images of the cells from different angles using cryo-electron tomography.

Through their study, the team gathered enough data to recreate 10,000 active Arp2/3 complexes during the yeast cell cycle.

To provide better resolution, the researchers used advanced image processing and combined this with a model of the Arp2/3 complex at a resolution of less than one nanometer.

Compared to human hairs, human fibres are around 50,000 nanometers thick. “We can now describe in relative detail how the protein complex and its subunits are constructed and how they form the actin filament network inside the lamellipodium of previously living cells,” “Five years ago, probably no one would have thought this was possible,” adds Schur.

To the max.
Thanks to the improved approach used, the team was able to prove that the Arp2/3 complex binds to the actin filaments in thinner filaments than previously thought. The scientists proved that the complex is also regulated in other ways, as well as forming new actin filaments.

Now that we understand the importance of this protein complex, we can better understand its functions beyond cell motility and disease growth. We’ve gone as far as is possible with these complex samples, using state-of-the-art analytical methods and techniques.

Due to the resolution’s new biological insights, the study also gained methodological insight by showing: it is possible.

Dr. Fäßler will develop his method, and he will also be testing the extent of what is possible with the method. We are just at the start of discovering the possibilities of cryo-electron tomography.

Credit for the electron micrography study is as follows:

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