Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Actin depolymerizing factor (ADF)/cofilin proteins regulate actin dynamics in a pH-dependent manner.
• ADF/cofilin binding to F-actin causes changes in helical twist and fragmentation, promoting depolymerization.
• Previous studies identified villin headpiece competition with ADF for F-actin binding.

Purpose of the Study:

• To characterize the two distinct actin-binding sites on human cofilin using site-directed mutagenesis.
• To elucidate the independent and combined roles of these sites in actin monomer and filament interactions.
• To investigate the functional consequences of specific cofilin mutations on actin binding and filament dynamics.

Main Methods:

• Site-directed mutagenesis of human cofilin to target specific actin-binding sites.
• Biochemical assays to assess cofilin binding to actin monomers and filaments.
• Analysis of cofilin-induced changes in actin filament structure (helical twist, fragmentation) and dynamics (subunit dissociation).

Main Results:

• A mutation in the loop region abolished cofilin binding to filaments but not monomers.
• A mutation mimicking phosphorylated cofilin (S3D) inhibited filament binding at physiological ionic strength.
• The S3D mutant induced filament twist and fragmentation at low ionic strength but not subunit dissociation.
• Evidence suggests a two-site binding model: loop site for initial filament association, followed by helix site interaction.

Conclusions:

• Human cofilin utilizes two distinct sites for actin binding: a loop site for filament association and a helix site for monomer interaction.
• The sequential binding through these sites induces actin filament twist and fragmentation.
• The observed twist change is not the direct cause of accelerated actin subunit dissociation from filaments.