Cholesterol oxides mediated changes in cytoskeletal organisation involves Rho GTPases

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• The actin cytoskeleton, regulated by small GTPases Rho, Rac, and Cdc42, is crucial for eukaryotic cell function.
• Cholesterol oxides are known to affect cellular processes, but their precise impact on cytoskeletal organization and cell migration requires further elucidation.

Purpose of the Study:

• To investigate the effects of cholesterol oxides, specifically 7-ketocholesterol and 25-hydroxycholesterol, on cell migration, adhesion, and cytoskeletal organization in lens epithelial cells (LEC).
• To elucidate the role of small GTPases (Rho, Rac, Cdc42) in mediating the observed cellular responses to cholesterol oxides.

Main Methods:

• Immunofluorescence confocal microscopy was used to visualize cytoskeletal changes.
• Overexpression of dominant-negative forms of Cdc42, RhoA, and Rac1 was employed to determine the involvement of specific GTPases.
• Cell migration assays were performed to quantify the impact of cholesterol oxides on cell motility.

Main Results:

• 7-ketocholesterol induced cell arborization, vimentin and tubulin bundling, filopodia, and stress fiber formation.
• 25-hydroxycholesterol caused vimentin collapse towards the nucleus and lamellipodia formation.
• Both cholesterol oxides significantly decreased cell migration.
• The cytoskeletal alterations were linked to the activation of Rho, Rac, and Cdc42 GTPases, as evidenced by inhibition experiments.

Conclusions:

• Cholesterol oxides significantly disorganize the actin cytoskeleton and impair cell migration in LEC.
• The effects are mediated through the differential activation of Rho, Rac, and Cdc42 GTPases.
• These findings suggest a general mechanism by which cholesterol oxides induce cytoskeletal disorganization across different cell types.