Effects of Macromolecular Crowding on Gelsolin-Mediated Actin Filament Mechanics and Severing Activities

Gelsolin is a Ca²⁺-dependent and pH-sensitive actin-binding protein that modulates actin filament dynamics within the actin cytoskeleton. Assembly and disassembly of actin structures and regulation of filament stiffness by gelsolin is crucial in cytoskeleton remodeling, force generation and cell motility. We have recently demonstrated how pH regulates gelsolin-induced filament severing, mechanics and conformations. While macromolecular crowding in the cell affects protein-protein interactions and functions, the effects of crowding on gelsolin-mediated filament mechanics and severing activities have not been well established. Utilizing Total Internal Reflection Fluorescence (TIRF) microscopy imaging, we show that thermal fluctuations of gelsolin-bound filaments are reduced in the presence of polyethylene glycol (PEG) or sucrose, and filament bending stiffness is enhanced with increasing concentrations of crowding molecules. Pyrene fluorescence assays were employed to monitor filament severing activities or depolymerization by gelsolin with crowding. Our results suggest that macromolecular crowding may play a role in controlling gelsolin’s ability to sever and stiffen filaments and may directly regulate actin-binding proteins for cytoskeleton dynamic remodeling necessary for cell mechanosensing.