Actin Filament Mechanics and Structure in Crowded Environments

Abstract

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce accessible volume for biomolecular interactions. Reductions in cellular volume can generate depletion forces that affect protein assembly and stability. The mechanical and structural properties of actin filaments play critical roles in various cellular functions, including structural support, cell movement, division, and intracellular transport. Although the effects of molecular crowding on actin polymerization have been shown, how crowded environments affect filament mechanics and structure is unknown. In this study, we investigate the effects of solution crowding on the modulations of actin filament bending stiffness and conformations both in vitro and in silico. Direct visualization of thermally fluctuating filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging. Biophysical analysis indicates that molecular crowding enhances filament's effective bending stiffness and reduces average filament lengths. Utilizing the all-atom molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and intersubunit contacts that are directly coupled to the mechanical properties of filaments. Taken together, our study suggests that the interplay between excluded volume effects and nonspecific interactions raised from molecular crowding may modulate actin filament mechanics and structure.

Crowding tunes the organization and mechanics of actin bundles formed by crosslinking proteins

“…6). Altered stiffness and helical twist of filaments in the presence of crowding [49] potentially modulate binding interactions offascin and α‐actinin, resulting in different organizations and mechanics of crosslinked bundles. In the case of fascin, solution crowding results in densely packed bundles and disrupts binding of fascin to actin filaments (Fig.…”

“…Actin was purified from rabbit skeletal muscle acetone powder (Pel‐Freez Biologicals Inc., Rogers, AR, USA) and gel‐filtered over Sephacryl S‐300 equilibrated in buffer A (2 m m Tris/HCl, 0.2 m m CaCl 2 , 1 m m NaN 3 , 0.2 m m ATP, 0.5 m m DTT, pH 8.0) as described in Ref. [49]. Rhodamine rabbit skeletal muscle actin (> 99% purity) was purchased from Cytoskeleton, Inc. (Denver, CO, USA).…”

“…Ca 2+ ‐actin monomers were converted into Mg 2+ ‐actin monomers by the addition of 0.2 m m EGTA and MgCl 2 , equal to the initial concentration of G‐actin plus 10 μ m for 5 min; then, 1/10th volume of 10× polymerization buffer (500 m m KCl, 20 m m MgCl 2 , 100 m m imidazole, pH 7.0, 10 m m ATP, and 10 m m DTT) was added and incubated at room temperature ( T ~ 22 °C) for 1 h, as described in Ref. [49]. Bundle formation was induced by adding human recombinant protein fascin with a His‐tag (Novus Biologicals, Littleton, CO, USA) or rabbit skeletal muscle α‐actinin (Cytoskeleton, Inc.) in the polymerization buffer containing crowding agents: polyethylene glycol (PEG) (MW = 8000 Da) (Fisher, Waltham MA, USA), sucrose (MW = 342 Da) (Sigma, Saint Louis, MO, USA), or Ficoll 70 (MW = 70000 Da) (GE Healthcare, Chicago, IL, USA) at varying concentrations.…”

“…The concentrations of crowding agents were chosen to emulate the estimated concentrations of macromolecules within the cytoplasm. In particular, we chose the concentrations for PEG and sucrose based on our recent study [49], which demonstrated the effect of crowding on actin filament mechanics and structure. The concentrations of Ficoll were selected following a previous study [41] that showed how macromolecular crowding affects actin polymerization kinetics.…”

“…Although many studies have explored the effects of crowding on actin, the way crowding affects interactions between actin-binding proteins (ABPs) and actin filaments is not well understood. Castaneda et al [49] recently demonstrated that crowding modulates filament bending mechanics and conformational changes, including helical twists of filaments. We hypothesize that these changes in filament mechanics and conformations by crowded environments may also potentially affect fascin or α-actinin binding to filaments and subsequent bundling activities.…”