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

Abstract

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.

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

“…The organization of the filaments (Gov, 2008) and the thickness of bundles (Lieleg et al, 2007) could be key factors in determining bundle nanomechanics-for example, actin bundles induced by depletion interactions were shown to increase in thickness as well as elastic modulus with an increase in the concentrations of PEG (Tharmann et al, 2006). A recent study has shown that crowding can tune the diameter of actin bundles crosslinked by actin binding proteins and possibly impact filament spacing (Park et al, 2021). Divalent cations were previously shown to alter the interfilament distance in bundles, with the greatest filament spacing at ∼7 nm for 30 mM Mg 2+ (Castaneda et al, 2018).…”

Regulation of Actin Bundle Mechanics and Structure by Intracellular Environmental Factors

“…In a living cell, actin bundles induced by ABPs are formed in a crowded cytoplasm; therefore, it is important to understand how crowding modulates ABP-induced bundling. Changes in filament bending stiffness and conformations in crowded environments [ 39 ] can influence interactions between filaments and ABPs, including actin-crosslinking proteins (e.g., fascin and α-actinin) [ 9 ] and severing proteins (e.g., gelsolin) [ 65 ]. Crowders with different sizes and concentrations [PEG and methylcellulose (MC)] have been shown to affect the organization patterns and potentially nucleation/growth of microtubule bundles crosslinked with microtubule-associated protein (MAP65) [ 66 ].…”

“…Crowders with different sizes and concentrations [PEG and methylcellulose (MC)] have been shown to affect the organization patterns and potentially nucleation/growth of microtubule bundles crosslinked with microtubule-associated protein (MAP65) [ 66 ]. Potential competitive interactions between crowding (PEG, sucrose, and Ficoll) and actin-crosslinking proteins, fascin, and α-actinin, have recently begun to be explored [ 9 ]. Macromolecular crowding influences the organization of either fascin or α-actinin bundles by reducing binding interactions between actin filaments and crosslinking proteins ( Figure 1C and Table 1 ), evidenced by fluorescence microscopy and atomic force microscopy imaging along with MD simulations [ 9 ].…”

“…Potential competitive interactions between crowding (PEG, sucrose, and Ficoll) and actin-crosslinking proteins, fascin, and α-actinin, have recently begun to be explored [ 9 ]. Macromolecular crowding influences the organization of either fascin or α-actinin bundles by reducing binding interactions between actin filaments and crosslinking proteins ( Figure 1C and Table 1 ), evidenced by fluorescence microscopy and atomic force microscopy imaging along with MD simulations [ 9 ]. MD simulations indicated that macromolecular crowding increases interaction energy between fascin or α-actinin and filaments, and reduces the number of hydrogen bonds [ 9 ].…”

“…Actin bundling proteins bind to actin filament (gray; unbound state, blue and green; bound state) during bundle formation with k on , which is slower than k off due to reduced bundling interactions between actin filament and bundling proteins. Compared with small crosslinkers (e.g., fascin; blue), longer crosslinkers (e.g., α-actinin; green) yield a significant decrease in D since their orientation switches from perpendicular to angled on filament, affecting bundle compactness [ 9 ]. (D) ABP-induced bundle formation in the presence of cations can result in modulations to association/dissociation constant k on and k off by cations influencing bundle organization.…”