Using a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron crytomography, researchers identified a dense, dynamic and disorganized actin filament nanoscaffold that is induced in response to a molecular signal. This is the first time researchers have directly visualized, at the molecular level, a structure that is triggered in response to a cellular signal, according to a team from Sanford Burnham Prebys and UNC-Chapel Hill. Their study was published in the Proceedings of the National Academy of Sciences yesterday.
"Cryo-electron microscopy is revolutionizing our understanding of the inner workings of cells," says Dorit Hanein, Ph.D., senior author of the paper and professor at SBP. "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 visualize 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 nanometers) —took years to develop to its current level of robustness and accuracy.
The images revealed a densely packed, disorganized, scaffold-like structure comprised of short actin rods. These structures 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. This dynamic scaffold contrasted sharply with various other actin assemblies in areas of low Rac1 activation—some comprised of long, aligned rods of actin, and others comprised of 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," says Niels Volkmann, Ph.D., a co-corresponding author of the paper. "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."
"This study is only the beginning. Now that we developed this quantitative nanoscale workflow that correlates dynamic signaling behavior with the nano-scale 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," says Hanein.