Pushing with actin: from cells to pathogens

Cells use actin polymerization to form lamellipodia, which are essential for cell movement. This process involves the assembly of actin filaments into branched networks. While prior research has shown how actin structures support cell motility, the detailed architecture of these networks remained unclear. Pathogens such as Listeria and baculovirus also use actin to move within host cells, but the structural specifics of this process are less understood. No prior work had resolved the three-dimensional organization of actin filaments during both cell protrusion and pathogen movement. That uncertainty drove this investigation into actin-driven propulsion. The application of advanced imaging techniques was necessary to capture these dynamic structures in situ. This gap motivated the use of electron tomography to study actin filament arrangements in both cellular and pathogenic contexts. The study aimed to bridge the knowledge between lamellipodia formation and pathogen invasion through structural analysis.

The purpose of this research was to investigate the structural basis of actin-driven propulsion in both cell movement and pathogen invasion. The specific problem addressed was the lack of high-resolution, three-dimensional data on actin filament organization during these processes. The motivation came from the need to understand how actin networks are assembled and remodeled during protrusion and pathogen dissemination. The researchers proposed to use electron tomography to capture detailed images of actin structures in situ. This approach allows for the visualization of actin filament arrangements in their native context. The study also aimed to compare lamellipodia formation in cells with the actin comet tails used by pathogens. By examining both systems, the authors sought to identify structural similarities and differences in actin-driven motility. The ultimate goal was to provide a comprehensive structure-function analysis of actin propulsion mechanisms.

The researchers employed electron tomography (ET) to visualize actin filament structures in situ. This method enabled them to capture three-dimensional images of actin networks within lamellipodia and pathogen-infected cells. To achieve high-resolution imaging, they used image averaging techniques to generate a detailed model of branch junctions. The ET data was complemented by live-cell imaging to track actin network dynamics during protrusion and inhibition. For pathogen studies, the team applied ET to analyze actin comet tails formed by Listeria, Rickettsia, and baculovirus. They used negatively stained cytoskeletons to enhance filament resolution and cryo-ET to preserve the overall three-dimensional morphology. The combination of these techniques allowed for a complete structure-function analysis of actin-driven propulsion. The data was analyzed to determine how actin networks are created and remodeled during both cell movement and pathogen dissemination.

The study revealed that lamellipodia are formed by subsets of actin filaments joined by branch junctions. Image averaging produced a 2.9 nm resolution model of these junctions, which closely matched the electron density map of the Arp2/3-actin complex in vitro. Correlated live-cell imaging and ET showed how actin networks are created and remodeled during protrusion initiation and inhibition. Baculovirus was found to generate a fishbone-like array of branched actin filaments at its rear. On average, only four filaments were engaged in pushing at any one time. The application of ET with negatively stained cytoskeletons improved filament resolution. Cryo-ET preserved the overall three-dimensional morphology of the structures. These findings provided a detailed structure-function analysis of actin-driven propulsion in both cells and pathogens.

The authors concluded that actin-driven propulsion in both cell movement and pathogen invasion relies on the assembly of branched actin filament networks. The study confirmed that lamellipodia formation involves subsets of actin filaments joined by branch junctions. The researchers proposed that the Arp2/3 complex plays a central role in this process. The application of ET was crucial for capturing high-resolution images of these structures in situ. The findings suggest that pathogens such as baculovirus exploit the same actin machinery as host cells. The fishbone-like arrangement of actin filaments at the rear of baculovirus indicates a specific mechanism for propulsion. The combination of negatively stained cytoskeletons and cryo-ET was essential for a complete structural analysis. These results support the hypothesis that actin-driven propulsion is a conserved mechanism across both cellular and pathogenic systems.