CaMKII generates actin bundle morphology and mechanics distinct from canonical bivalent cross-linkers

Actin cross-linkers are essential modulators of the actin cytoskeleton, enabling structural diversity and dynamic remodeling. Among them, calcium/calmodulin-dependent protein kinase II (CaMKII) is unique in serving a dual role as a kinase and as a multivalent structural binder of actin in dendritic spines, where it plays a major role in supporting dendritic spine structure via its β subunit. To understand the basis of the structural function of CaMKII, we quantified the morphology and mechanics of actin bundles formed by CaMKII and directly compared them with bundles assembled by the canonical bivalent cross-linkers α-actinin and fascin. Using fluorescence microscopy, we measured contour length, straightness ratio, and persistence length across different cross-linker concentrations. Fascin generated progressively shorter, straighter, and stiffer bundles with increasing concentration, whereas α-actinin bundles remained largely unchanged. In contrast, increasing CaMKII concentration reduced both bundle straightness and persistence length, revealing a unique concentration-dependent increase in bundle flexibility. To investigate the structural origins of these mechanical behaviors, we complemented the experiments with coarse-grained simulations of cross-linked actin bundles. Simulations revealed that CaMKII-generated bundles retained a substantially larger fraction of their curvature in their time-averaged configuration than bundles formed by α-actinin or fascin, indicating that bundle mechanics are strongly influenced by organizational features beyond thermal bending fluctuations alone. CaMKII bundles displayed behavior consistent with additional structural complexity arising from their multivalent architecture and flexible linker domains. Together, our experimental and computational results identify CaMKII as a structurally and mechanically distinct actin cross-linker. We propose that the combination of multivalency and linker flexibility enables CaMKII to function as a molecular structural pivot, promoting flexible, adaptable actin assemblies that are mechanically compatible with the dynamic remodeling required for dendritic spine plasticity.

Actin cross-linkers play a central role in cytoskeletal architecture and mechanics, yet how multivalent cross-linkers differ from canonical bivalent proteins remains incompletely understood. CaMKII is unique in serving both as a kinase and a multivalent actin cross-linker in dendritic spines. By directly comparing CaMKII with fascin and α-actinin, we show that increasing CaMKII density uniquely reduces actin bundle stiffness, while simulations reveal enhanced curvature retention within CaMKII bundles. Together, these results suggest that CaMKII multivalency and flexible linker domains enable organizational modes and mechanical responses inaccessible to conventional bivalent cross-linkers. This work provides a biophysical basis for understanding how CaMKII supports the dynamic actin remodeling required for dendritic spine plasticity.