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.

The cellular environment is crowded with high concentrations of macromolecules that significantly reduce the accessible volume limiting biomolecule 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 conformations, dynamics, and mechanical properties is unknown. In this study, we investigate the effects of solution crowding on the modulations of filament mechanics and structure both in vitro and in silico. Direct visualization of filaments in the presence of crowding agents is achieved by fluorescence microscopy imaging, allowing for the quantification of filament thermal bending dynamics and mechanics. Biophysical analysis indicate that molecular crowding modulates thermal fluctuations, enhances filament's effective bending stiffness, and reduces average filament lengths. Utilizing the explicit molecular dynamics simulations, we demonstrate that molecular crowding alters filament conformations and structural properties indicating overall compaction and stabilization that are directly coupled to filament mechanics. Taken together, our study suggests the interplay between excluded volume effects and non‐specific interactions raised from molecular crowding may modulate actin filament mechanics and structure.Support or Funding InformationThis study was supported by the UCF start‐up fund and In‐House grant for H.K. The authors would like to thank the National Science Foundation for support of this work through REU site EEC 1560007. We acknowledge the computational time from UCF stokes cluster.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Actin polymerization drives cell movement and provides cells with structural integrity. Intracellular environments contain high concentrations of solutes, including organic compounds, macromolecules, and proteins. Macromolecular crowding has been shown to affect actin filament stability and bulk polymerization kinetics. However, the molecular mechanisms behind how crowding influences individual actin filament assembly are not well understood. In this study, we investigated how crowding modulates filament assembly kinetics using total internal reflection fluorescence (TIRF) microscopy imaging and pyrene fluorescence assays. The elongation rates of individual actin filaments analyzed from TIRF imaging depended on the type of crowding agent (polyethylene glycol, bovine serum albumin, and sucrose) as well as their concentrations. Further, we utilized all-atom molecular dynamics (MD) simulations to evaluate the effects of crowding molecules on the diffusion of actin monomers during filament assembly. Taken together, our data suggest that solution crowding can regulate actin assembly kinetics at the molecular level.