βII and βIII spectrin paralogues define robustness and specialization of the neuronal membrane periodic skeleton

Abstract Neuron connectivity and signal processing rely on a complex, dynamic morphology regulated by the cytoskeleton. The membrane-associated periodic skeleton (MPS), a 190-nm periodic actin-spectrin lattice, is a conserved feature of neurons. Here, we show that the dendritic MPS is built from a dual β-spectrin system in which βII- and βIII-spectrins are co-expressed and interleaved at the nanoscale in shafts and spine necks. 3D MINFLUX nanoscopy suggests that these paralogues form both homotypic (βII- or βIII-only) and heterotypic (βII/βIII) tetramers with an approximately 100-nm radial periodicity. Either paralogue alone is sufficient to maintain lattice architecture, whereas their combined loss disrupts MPS integrity. Using targeted mutagenesis, we show that actin binding is required to stabilize both paralogues within the MPS, whereas βIII-spectrin additionally depends on phosphoinositide interactions. Our findings reveal that, unlike the axonal MPS, the dendritic MPS is a composite scaffold in which structural redundancy coexists with paralogue-specific regulatory mechanisms, with potential consequences for synaptic function.