Actin Bundle Nanomechanics and Organization Are Modulated by Macromolecular Crowding and Electrostatic Interactions.

The structural and mechanical properties of actin bundles are essential to eukaryotic cells, aiding in cell motility and mechanical support of the plasma membrane. Bundle formation occurs in crowded intracellular environments composed of various ions and macromolecules. Although the roles of cations and macromolecular crowding in the mechanics and organization of actin bundles have been independently established, how changing both intracellular environmental conditions influence bundle mechanics at the nanoscale has yet to be established. Here we investigate how electrostatics and depletion interactions modulate the relative Young's modulus and height of actin bundles using atomic force microscopy. Our results demonstrate that cation- and depletion-induced bundles display an overall reduction of relative Young's modulus depending on either cation or crowding concentrations. Furthermore, we directly measure changes to cation- and depletion-induced bundle height, indicating that bundles experience alterations to filament packing supporting the reduction to relative Young's modulus. Taken together, our work suggests that electrostatic and depletion interactions may act counteractively, impacting actin bundle nanomechanics and organization.

Regulation of Actin Bundle Mechanics and Structure by Intracellular Environmental Factors.

The mechanical and structural properties of actin cytoskeleton drive various cellular processes, including structural support of the plasma membrane and cellular motility. Actin monomers assemble into double-stranded helical filaments as well as higher-ordered structures such as bundles and networks. Cells incorporate macromolecular crowding, cation interactions, and actin-crosslinking proteins to regulate the organization of actin bundles. Although the roles of each of these factors in actin bundling have been well-known individually, how combined factors contribute to actin bundle assembly, organization, and mechanics is not fully understood. Here, we describe recent studies that have investigated the mechanisms of how intracellular environmental factors influence actin bundling. This review highlights the effects of macromolecular crowding, cation interactions, and actin-crosslinking proteins on actin bundle organization, structure, and mechanics. Understanding these mechanisms is important in determining in vivo actin biophysics and providing insights into cell physiology.

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

Fascin and α-actinin form higher-ordered actin bundles that mediate numerous cellular processes including cell morphogenesis and movement. While it is understood crosslinked bundle formation occurs in crowded cytoplasm, how crowding affects the bundling activities of the two crosslinking proteins is not known. Here, we demonstrate how solution crowding modulates the organization and mechanical properties of fascin- and α-actinin-induced bundles, utilizing total internal reflection fluorescence and atomic force microscopy imaging. Molecular dynamics simulations support the inference that crowding reduces binding interaction between actin filaments and fascin or the calponin homology 1 domain of α-actinin evidenced by interaction energy and hydrogen bonding analysis. Based on our findings, we suggest a mechanism of crosslinked actin bundle assembly and mechanics in crowded intracellular environments.

Gelsolin-mediated actin filament severing in crowded environments.

Gelsolin is a calcium-regulated actin binding protein that severs and caps actin filaments. Gelsolin's severing activity is important for regulating actin filament assembly dynamics that are required for cell motility as well as survival. The majority of in vitro studies of gelsolin have been performed in dilute buffer conditions which do not simulate the molecular interactions occurring in the crowded intracellular environment. We hypothesize that crowding results in greater gelsolin severing activity due to induced conformational changes in actin filaments and/or gelsolin. In this study, we evaluated the effects of crowding on gelsolin-mediated actin filament severing and gelsolin binding to actin filaments in crowded solutions, utilizing total internal reflection fluorescence (TIRF) microscopy and co-sedimentation assays. Our data indicates that the presence of crowders causes a decrease in the rate of gelsolin severing as well as a decrease in the amount of gelsolin bound to actin filaments, with greater effects caused by the polymeric crowder. Despite the severing rate decrease, gelsolin-mediated filament severing is increased in the presence of crowders. Understanding the crowding effect on gelsolin-mediated actin filament severing offers insight into the interactions between gelsolin and actin that occur inside the crowded cytoplasm.

Actin Filament Mechanics and Structure in Crowded Environments.

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.

Cations Modulate Actin Bundle Mechanics, Assembly Dynamics, and Structure.

Actin bundles are key factors in the mechanical support and dynamic reorganization of the cytoskeleton. High concentrations of multivalent counterions promote bundle formation through electrostatic attraction between actin filaments that are negatively charged polyelectrolytes. In this study, we evaluate how physiologically relevant divalent cations affect the mechanical, dynamic, and structural properties of actin bundles. Using a combination of total internal reflection fluorescence microscopy, transmission electron microscopy, and dynamic light scattering, we demonstrate that divalent cations modulate bundle stiffness, length distribution, and lateral growth. Molecular dynamics simulations of an all-atom model of the actin bundle reveal specific actin residues coordinate cation-binding sites that promote the bundle formation. Our work suggests that specific cation interactions may play a fundamental role in the assembly, structure, and mechanical properties of actin bundles.