Chain-like supramolecular assemblies of inactivated actin oligomers reveal a multistage assembly pathway

Besides the conventional monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can adopt a thermodynamically stable inactivated oligomeric state (I-actin) when it loses bound nucleotide and coordinating divalent cation under mild denaturing or stress conditions. However, the supramolecular organization of these assemblies remains poorly understood. Here, using size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) and negative-stain transmission electron microscopy (NS-TEM), we show that heat-inactivated actin (heat-I-actin) forms flat, disk-like oligomers ("beads") that further assemble into linear, unbranched chains. SEC-SAXS reveals strongly elongated particles whose cross-sectional parameters closely match those previously established for I-actin oligomers by hydrodynamic measurements, while NS-TEM directly visualizes bead-like particles of ∼165 Å in diameter that occasionally align into unbranched chains, structures not seen for G- or F-actin. Together with previously published structural insights, these data support a multistage assembly pathway in which I-actin subunits first form dimers, then condense into flat oligomeric beads, which in turn connect into one-dimensional supramolecular chains. We discuss how such assemblies may relate to poorly characterized short oligomers of nuclear actin.