Tension-Regulated Actin Severing Revealed by Surface-Free Single-Molecule Force Spectroscopy

Abstract

Actin-severing proteins, such as gelsolin, are key regulators of actin filament turnover. Studies suggest that the severing process is regulated by mechanical forces in actin filaments. However, the specific types and levels of force involved are unknown because the existing methods for applying force on actin filaments require surface attachment. These attachments introduce a combination of tension, compression, torsion and shear. We apply a surface-free force spectroscopy method that uses a high-speed cross-slot hydrodynamic trap to generate pure tension in actin filaments. Buffer containing actin filaments or severing proteins flows in from two opposite directions and exits via the two orthogonal outlets to create an elongational flow field with a stagnation point in the center. As a result, filaments near the stagnation point are stretched by the viscous drag from the flow. In addition, the pressure in one of the outlet reservoirs is electronically controlled with a high-speed feedback algorithm to stabilize the actin filament at the stagnation point. This allows us to measure the severing activity of actin filaments under pure tension. We found that the severing rate by gelsolin was independent of tension up to 10pN. In comparison, a previous magnetic tweezers experiment that applies a mixture of tension and bending forces shows a positive correlation between severing rate and pulling force for gelsolin over the range of 0.1 to 4 pN. Our result suggests that changes at actin monomer interfaces due to bending rather than tension enhances the severing rate by gelsolin. By enabling us to applying pure tension rather than a mixture of forces, our method provides a powerful tool for understand and differentiating the driving forces of structural changes in filamentous biomolecules.