Membrane-associated Periodic Skeleton: A developmental perspective

Abstract

The Membrane-associated Periodic Skeleton (MPS) is a critical cytoskeletal structure observed in diverse neuronal subtypes across multiple species. Since its identification in 2013, it has been demonstrated to be essential for neuronal stability, function, and circuit formation; however, the mechanisms underlying its assembly have remained elusive. This study examined the developmental patterns and regulatory processes of MPS in embryonic chick dorsal root ganglia (DRG) neurons and in differentiating SH-SY5Y cells. Using STED, a super-resolution microscopy technique, we observed that MPS formation began as early as day-in-vitro-1 (DIV1) and progressed over time, as indicated by the enhanced autocorrelation and reduced dispersal in periodicity (spacing). Acute pharmacological treatments that disrupt actin and microtubule lead to MPS disruption. Chronic interference with microtubule dynamics hindered MPS formation at DIV2 and DIV4. Interestingly, MPS formation was found to be independent on chronic F-actin stabilization and myosin inhibition. Blocking the actin nucleators, Arp2/3 and Formins impeded MPS stability at DIV2 and DIV4 with a diminishing effect, indicating ongoing F-actin renewal and scaffold instability. However, MPS showed resistance to these inhibitors at DIV6, suggesting a more stable scaffold. FRAP analysis of βII-spectrin revealed reduced mobility during development, indicating its integration into the MPS scaffold. Latrunculin-A treatment increased βII-spectrin mobility in early stages but had no effect at DIV4, demonstrating that its reliance on cortical F-actin interaction is limited to early developmental phases. FRAP-based analysis of βII-spectrin domain mutants in differentiated SH-SY5Y βII-spectrin KO neuron showed that its plasma membrane and actin-binding activities are essential for its mobility and incorporation into the MPS, supporting the DRGs Lat-A-treated FRAP results. To figure out the functional aspect of a forming MPS scaffold, we ablated axons and found a positive correlation between MPS assembly and axonal tension in DRGs. When βII-spectrin KO SH-SY5Y neurons were ablated, we observed a modest decrease in axonal tension, highlighting potential role in maintaining axonal tension. These discoveries offer new perspectives on the developmental assembly, regulatory mechanisms, and functional significance of MPS, underscoring the importance of cytoskeletal interactions in its formation and maintenance.