Establishment of stable human fibroblast cell lines constitutively expressing active Rho-GTPases

Cells respond to mechanical forces through cytoskeletal reorganization. Rho-GTPases regulate cytoskeletal structures and adhesion. These proteins are key in translating external forces into cellular responses. Prior research has shown that Rho-GTPases influence cell shape and movement. However, their role in gravity perception remains unclear. This gap motivated the need for a stable model system. No prior work had resolved how Rho-GTPases might respond to microgravity. Establishing such a system could clarify their function in cellular mechanics.

The aim was to create a cellular model for studying Rho-GTPase activity in gravity perception. Researchers focused on RhoA, Cdc42, and Rac1, which are known to regulate cytoskeletal dynamics. The study sought to determine if these proteins could influence cellular responses to gravity. A stable cell line was needed to allow long-term observation. The team hypothesized that Rho-GTPases might mediate microgravity effects. They aimed to test this hypothesis using constitutively active mutants. The model would help explore molecular mechanisms of gravity perception. This approach could provide insights into cytoskeletal adaptation.

Researchers generated stable fibroblast cell lines expressing active Rho-GTPases. The cell lines expressed either RhoA, Cdc42, or Rac1 in their active forms. These proteins were constitutively expressed to ensure consistent activity. Morphological changes were observed using microscopy techniques. The presence of filopodia and lamellipodia was assessed. Actin stress fiber organization was analyzed in each line. Cell viability was monitored to confirm no toxicity from overexpression. Population doubling time was measured to assess growth impact.

The three cell lines showed distinct morphologies and cytoskeletal structures. RhoA-expressing cells formed actin stress fibers and filopodia. Cdc42-expressing cells exhibited increased filopodia formation. Rac1-expressing cells displayed more lamellipodia and spread morphology. Overexpression did not reduce cell viability in any line. Cell doubling time remained unchanged across all conditions. These findings suggest Rho-GTPases can be overexpressed safely. The model is suitable for studying cytoskeletal responses to forces. The results support using these lines for gravity perception research.

The cell lines are viable and suitable for investigating Rho-GTPase roles in gravity perception. The authors suggest these models could help clarify cytoskeletal adaptation to forces. The findings indicate that Rho-GTPases influence cell morphology and structure. The study supports the hypothesis that Rho-GTPases may mediate microgravity effects. The model allows for controlled experiments on cytoskeletal dynamics. The results show no adverse effects from overexpression. These lines provide a stable platform for future studies. The authors propose using them to explore cellular gravity perception mechanisms.