Endogenous and Exogenous Actin Modifications Alter F-Actin Network Architecture and Mechanics by Charge Redistribution.

Mammalian actin comprises six highly conserved isoforms that, despite their near-identical amino acid sequences, form filament networks with remarkably diverse structural and viscoelastic properties. This diversity primarily originates from minor variations at the protein N-termini. To investigate the mechanisms driving these differences, we used microrheology to examine β -actin networks with modified N-termini. We focused on the effects of surface-charge alterations, which can occur naturally in cells or be introduced artificially via fluorescent labelling. Specifically, we studied post-translationally arginylated β -actin (Arg-actin), where multiple negative N-terminal charges are replaced by positive ones alongside naturally occurring intermediate variants. Our findings reveal that N-terminal charges act as localized filament-bundling agents, altering structural and viscoelastic properties in a manner distinct from traditional cross-linkers or chelators such as divalent cations. By intrinsically reducing the filament's net negative surface charge, Arg-actin lowers the thermodynamic threshold for Manning counter-ion condensation. Consequently, Mg2+ drives premature, localized bundling at significantly lower concentrations, shifting the network from a purely entangled state to a nonlinear, stiffened architecture. Beyond advancing our understanding of cellular network mechanics, this study highlights critical implications for the use of fluorescent probes and other experimental protein modifications, demonstrating that such alterations can inadvertently shift network properties by orders of magnitude.