Powerful microscope captures 'haystack' nanoscaffold

In an effort to better understand how cells move throughout our bodies — and the rod-like actin filaments that drive the process — US scientists have used one of the most powerful microscopes in the world to identify a dense, dynamic and disorganised actin filament nanoscaffold — resembling a haystack — that is induced in response to a molecular signal.

This is the first time researchers have directly visualised, at the molecular level, a structure that is triggered in response to a cellular signal, and significantly expands our understanding of how cells move. The research was conducted by scientists from Sanford Burnham Prebys Medical Discovery Institute (SBP) and University of North Carolina at Chapel Hill (UNC-Chapel Hill), and published in the Proceedings of the National Academy of Sciences (PNAS).

“Cryo-electron microscopy is revolutionising our understanding of the inner workings of cells,” said Dorit Hanein, a professor at SBP and senior author on the paper. “This technology allowed us to collect robust, 3D images of regions of cells — similar to MRI, which creates detailed images of our body. We were able to visualise cells in their natural state, which revealed a never-before-seen actin nano-architecture within the cell.”

In the study, the scientists used SBP’s cryo-electron microscope (Titan Krios), artificial intelligence (AI) and tailor-made computational and cell imaging approaches to compare nanoscale images of mouse fibroblasts to time-stamped light images of fluorescent Rac1, a protein that regulates cell movement, response to force or strain (mechanosensing) and pathogen invasion.

This technically complex workflow — which bridged five orders of magnitude in scale (tens of microns to nanometres) — took years to develop to its current level of robustness and accuracy and was made possible through experimental and computational efforts of the structural biologist teams at SBP and the biosensors team at UNC-Chapel Hill.

The images revealed a densely packed, disorganised, scaffold-like structure comprising short actin rods. These structures sprang into view in defined regions where Rac1 was activated, and quickly dissipated when Rac1 signalling stopped — in as little as two and a half minutes.

This dynamic scaffold contrasted sharply with various other actin assemblies in areas of low Rac1 activation — some comprising long, aligned rods of actin and others comprising short actin rods branching from the sides of longer actin filaments. The volume encasing the actin scaffold was devoid of common cellular structures, such as ribosomes, microtubules, vesicles and more, likely due to the structure’s intense density.

“We were surprised that experiment after experiment revealed these unique hotspots of unaligned, densely packed actin rods in regions that correlated with Rac1 activation,” said Niels Volkmann, a professor at SBP who led the computational part of the study. “We believe this disorder is actually the scaffold’s strength — it grants the flexibility and versatility to build larger, complex actin filament architectures in response to additional local spatial cues.”

Next, the scientists would like to expand the protocol to visualise more structures that are created in response to other molecular signals and to further develop the technology to allow access to other regions of the cell.

“This study is only the beginning,” said Hanein. “Now that we developed this quantitative nanoscale workflow that correlates dynamic signalling behaviour with the nanoscale resolution of electron cryo-tomography, we and additional scientists can implement this powerful analytical tool not only for deciphering the inner workings of cell movement but also for elucidating the dynamics of many other macromolecular machines in an unperturbed cellular environment.

“Actin is a building-block protein; it interacts with more than 150 actin-binding proteins to generate diverse structures, each serving a unique function. We have a surplus of different signals that we would like to map, which could yield even more insights into how cells move.”

Image caption: Cryo-electron microscope images of actin assembly in the cell in the absence of a molecular signal (left) and the haystack-like actin filament nanoscaffold that was induced in response to a molecular signal (Rac1) and promotes cell movement (right). The structure sprang into view in defined regions where Rac1 was activated, and quickly dissipated when Rac1 signaling stopped — in as little as two and a half minutes. Image credit: Sanford Burnham Prebys Medical Discovery Institute (SBP).

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