Processive acceleration of actin barbed-end assembly by N-WASP

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is crucial for actin polymerization driving invadopodia and podosome formation.
• N-WASP activates Arp2/3 and binds actin filament barbed ends, concentrating them in invasive cellular structures.
• Understanding N-WASP's role in Arp2/3-independent actin assembly is key to cell invasion mechanisms.

Purpose of the Study:

• To investigate how clustered N-WASP influences Arp2/3-independent actin barbed-end assembly using model cell surfaces.
• To quantify the growth rate of actin filaments under N-WASP influence at different densities and conditions.

Main Methods:

• Utilized nanofibers coated with N-WASP WWCA domains as artificial cell surfaces.
• Employed single-actin-filament imaging to observe and measure filament growth dynamics.
• Manipulated N-WASP constructs, profilin, and Mg(2+) concentrations to assess their effects on actin assembly.

Main Results:

• Individual barbed ends attached to N-WASP domains grew at or below the diffusion-limited rate.
• At high filament densities, overlapping filaments formed buckles that grew ~3.4-fold faster than unattached barbed ends.
• Profilin slowed barbed-end acceleration in N-WASP constructs with the polyproline region; increased Mg(2+) enhanced buckle formation.

Conclusions:

• N-WASP promotes Arp2/3-independent actin assembly through a novel mechanism involving filament buckling.
• This accelerated assembly provides force for nascent filament bundles to penetrate the basement layer during cell invasion.
• The findings reveal a new pathway for N-WASP-mediated cell motility and invasion.