Villin severing activity enhances actin-based motility in vivo

Area of Science:

• Cell Biology
• Biochemistry
• Biophysics

Background:

• Villin is an actin-binding protein crucial for cellular structures like microvilli.
• It exhibits multiple activities including bundling, capping, nucleating, and severing actin filaments in a calcium-dependent manner.
• The precise contribution of villin's severing activity to cell plasticity and motility remains unclear.

Purpose of the Study:

• To investigate the specific role of villin's actin-severing activity in enhancing cell plasticity and motility.
• To dissociate the severing function from other villin activities (bundling, nucleation, capping).
• To elucidate the mechanism by which villin influences actin dynamics in vivo and in vitro.

Main Methods:

• Utilized a loss-of-function strategy by introducing mutations in villin based on sequence comparison with CapG.
• Employed pyrene-actin assays to assess the severing, nucleation, and capping activities of the villin mutant.
• Analyzed villin's contribution to actin dynamics in vivo using the actin-based movement of Shigella flexneri in cells expressing wild-type and mutant villin.
• Reconstituted an in vitro actin-based bead movement assay to further examine the effects of villin and its mutant.

Main Results:

• The villin mutant exhibited significantly reduced actin-severing activity while retaining nucleation, capping, and bundling functions.
• Mutations in villin abolished the enhanced velocity of intracellular Shigella flexneri movement observed with wild-type villin.
• In vitro bead movement assays confirmed that villin's severing activity more than doubled bead velocity and reduced actin comet density.

Conclusions:

• Villin's actin-severing activity is a key determinant of its role in enhancing cell motility and bacterial propulsion.
• The study successfully isolated villin's severing function, demonstrating its distinct contribution to actin dynamics.
• A proposed model suggests that villin severing, coupled with capping, increases the pool of available actin monomers for polymerization, potentially driving cellular transitions like epithelial-mesenchymal transition.