The actin/spectrin scaffold shapes axonal physiology – ASHA

In neurons, the axon propagates action potentials, transmitting signals to target cells. A unique cytoskeletal organization allows its architecture to be both robust and adaptable. A periodic submembrane actin/spectrin scaffold has recently been discovered along axons using optical super-resolution microscopy, but its functions remain elusive. We have recently revealed the ultrastructure of this axonal actin/spectrin scaffold by combining super-resolution microscopy and metal-replica electron microscopy. Our objective is now to determine the role of the submembrane periodic scaffold in shaping the axon morphology and physiology. We want to show how this scaffold restrict endo/exocytosis along the axon shaft, ensuring targeted transport across long distance to synapses. We will dissect the poorly known processes of axonal endocytosis and exocytosis using our unique combination of live-cell imaging, super-resolution and electron microscopy, including innovative correlative approaches. We will knockdown axonal spectrins using validated viral vectors that disassemble the actin/spectrin scaffold in the proximal and/or distal axon. We will assess clathrin organization, endocytosis and exocytosis in these spectrin-depleted axons from dynamics to ultrastructural detail to reveal the role of the submembrane periodic scaffold in regulating access to the axonal plasma membrane. We will demonstrate how this regulation of endo/exocytosis is determinant for proper axonal arborization as well as functional organization of presynapses. The physiological relevance of our findings will be explored by studying how pathological mutations in axonal spectrins impair these processes in human neurons. Spectrin mutations previously identified in human patients will be engineered into human induced pluripotent stem cells by CRISPR/Cas9 genome editing, and we will differentiate these cells into mature neurons to study the organization of the axonal periodic scaffold and the potential dysregulation in endo/exocytosis caused by these mutations.