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

Abstract

Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures—such as those mandated by epithelial−mesenchymal transition, for example—or resetting the region by completely dissipating.

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

“…Cryogenic electron tomography (cryo-ET) enables structural characterization of macromolecular complexes in intact vitrified cells in their native state [14][15][16][17][18][19]. Correlative light and electron microscopy (CLEM) technologies can directly tie high-resolution structural information from cryo-ET to spatiotemporal dynamics of cellular events at spatially defined regions of the cell [20][21][22][23]. A prerequisite to using CLEM technology for the study of mechanotransduction is the availability of cell culture substrates amenable to cryo-ET that can also constrain the morphology of the cells to be imaged.…”

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies

“…In preparation for cell seeding on EM grids patterned with ECM proteins, MDCKII or PtK1 cells from cultures in logarithmic phase were trypsinized and resuspended in culture medium containing 10% serum to inactivate trypsin. Cells were seeded onto EM grids and allowed to adhere for 12-16 hr, prior to fixation and imaging as described previously (22,42). HFF cells were plated on patterned grids, incubated for 1 hr, imaged live for approximately 1 hr and fixed after another incubation period of 0.5 hr.…”

“…Cryogenic electron tomography (cryo-ET) enables structural characterization of macromolecular complexes in intact vitrified cells in their native state (14)(15)(16)(17)(18)(19). Correlative light and electron microscopy (CLEM) technologies can directly tie high-resolution structural information from cryo-ET to spatio-temporal dynamics of cellular events at spatially defined regions of the cell (20)(21)(22)(23). A prerequisite to using CLEM technology for the study of mechanotransduction is the availability of cell culture substrates amenable to cryo-ET that can also constrain the morphology of the cells to be imaged.…”

Stochastic Methods for Inferring States of Cell Migration

“…Rac1, the family member studied here, produces cell protrusions by interacting with effector proteins that modulate actin polymerization, including formins and Paks. A prevailing hypothesis is that Rac1 induces localized actin polymerization to trap random, thermal driven outward movements of the cell edge (Ridley, 2015;Marston et al, 2019;Schaks et al, 2019).…”