High Rac1 activity is functionally translated into cytosolic structures with unique nanoscale cytoskeletal architecture

Significance We have correlated signaling behaviors seen with biosensors (conformational changes of Rac1) with the nanoscale resolution of electron cryotomography. The workflow and approaches we describe bridge five orders of magnitude (tens of microns to nanometers) to identify, in the crowded environment within eukaryotic cells, distinct nanoarchitectures induced locally by Rac1 as it mediates cell motility, mechanosensing, and invasion. Our studies reveal a dynamic switching mechanism that generates a nanoscaffold system that can either propagate to highly complex actin filament architectures mandated by biological processes or completely dissipate. The workflow implemented here offers a powerful analytical tool not only for deciphering the function of Rho GTPase molecular switches but for the dynamics of many other macromolecular machines as well.