The actin/spectrin scaffold shapes axonal physiology – ASHA

Advanced optical microscopy

Partner 1 is a recognized specialist of the application of super-resolution microscopy to studying the neuronal cytoskeleton. We deployed a range of advanced techniques: Single Molecule Localization Microscopie (SMLM: Stochastic Optical Reconstruction Microscopy STORM, DNA-Point Accumulation in Nanoscale Topography DNA-PAINT) to resolve structural details down to ~20 nm in fixed cells; structured illumination microscopy (SIM) that reaches ~100 nm resolution in living cells; Total Internal Reflection (TIRF) microscopy to visualize the exocytosis of single vesicle along the axon.

Electron microscopy

Partner 2 is an internationally-renowned specialist of platinum-replica electron microscopy (PREM), which allows to interrogate the ultrastructural architecture of the membrane-associated cytoskeleton in cultured cells. Only this approach has been able to visualize the axonal actin/spectrin scaffold by electron microscopy so far, showing that actin rings were made of two intertwined long filaments (Vassilopoulos et al., 2019 10.1038/s41467-019-13835-6).

Correlative approaches

We developed several approaches that maximize insight by combining different microscopy approaches on the same sample: correlative PREM/STORM and PREM/SIM of immunolabeled samples allowed to identify the molecular identity of ultrastructural details; correlative live-cell TIRF microscopy/STORM made it possible to map the nanoscale environment of exocytic sites.

• Regulation of axonal endocytosis by the actin/spectrin scaffold

A central hypothesis of the project was that the actin/spectrin scaffold functions as an insulation layer that restricts membrane trafficking along the axon. This hypothesis was directly tested by analyzing clathrin-mediated endocytosis at the axon initial segment using correlative super-resolution microscopy and platinum-replica electron microscopy. The project demonstrated that clathrin-coated pits are not randomly distributed along proximal axons but form within discrete circular clearings that are devoid of actin/spectrin mesh. These clathrin-coated pits are unusually stable and long-lived, resulting in very low endocytic activity at steady state along the axon initial segment. Genetic or pharmacological disruption of the spectrin scaffold increased the formation of clathrin-coated pits, demonstrating that the scaffold physically restricts endocytosis. Importantly, endocytosis at the axon initial segment can be actively triggered by physiological stimulation. Neuronal activity induces the polymerization of branched actin structures around pre-existing clathrin-coated pits, promoting their scission and internalization. These results reveal a two-level regulation of axonal endocytosis: a structural restriction imposed by the actin/spectrin scaffold and an activity-dependent mechanism that unlocks endocytosis when required. This work revises the long-standing view that axonal endocytosis is absent or negligible and identifies a regulated reservoir of endocytic sites embedded within the axonal scaffold.

These results were preprinted in December 2023 (10.1101/2023.12.19.572337), and published in Science in August 2024 (Wernert, Moparthi et al. 10.1126/science.ado2032). This work was distinguished by the “Grandes Avancées Française en Biologie” prize from the Académie des Sciences in 2025.

• Existence of non-synaptic exocytosis along the axon shaft and regulation by the MPS

In the second main part of this project, we focused on exocytosis rather than endocytosis along the axonal shaft. We developed fast and sensitive live-cell TIRF to image the exocytosis on single vesicles along the axon, revealing a the existence of a small fraction of exocytic events outside of presynapses in resting neurons. This non-synaptic exocytosis along the axon shaft is regulated by the MPS, which ensures its low occurence. At the nanoscale level, correlative live-cell TIRF/multicolor STORM showed the like endocytosis, exocytosis occurs in holes of the actin/spectrin mesh, but these holes are distinct from the clearings that contain clathrin-coated pits.

These results have been preprinted in September 2025 (Wiesner et al. 10.1101/2025.09.17.676728).

This project has been tremendously successful, leading to a profound shift in how we understand the cellular roles of the axonal actin/spectrin scaffold and the physiology of axons. It has also opened new insights in the mechanism of clathrin-mediated endocytosis: its unique characteristics in axons can be comparatively used to understand how it works in all cells. The importance of our results, and the beauty of the images obtained by our microscopy approaches have helped reaching beyond the community of specialists. This contributes to reaffirming the role of science and discovery in a world where knowledge and expertise are under attack.

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.