BetaII-Spectrin Gaps and Patches Emerge from the Patterned Assembly of the Actin/Spectrin Membrane Skeleton in Human Motor Neuron Axons

The membrane-associated periodic skeleton (MPS), composed of F-actin and spectrin, is a cytoskeletal structure that supports axonal integrity and organization. Using high-resolution imaging, we characterized the spatial distribution and assembly dynamics of βII-spectrin in human motor neuron (MN) axons derived from induced pluripotent stem cells (iPSCs). We discovered a striking “gap-and-patch” pattern in the medial axon, where sharply demarcated βII-spectrin gaps alternate with patches containing a well-organized MPS. These gaps lack F-actin and αII-spectrin and do not reflect axonal degeneration or spectrin cleavage. The pattern increases with culture time and is acutely induced by the kinase inhibitor staurosporine. Notably, pharmacological inhibition of actin polymerization prevents patch formation, indicating a requirement for actin nucleation in MPS assembly. Our data supports a model in which spectrin incorporation into nascent MPS patches depletes neighboring regions, producing periodic interruptions. This study provides the first detailed nanoscale analysis of MPS organization in human MNs, offering new insights into cytoskeletal remodeling in axons and its potential relevance to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Key words: iPSCs, actin, spectrin, cytoskeleton, motor neurons, staurosporine.