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Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model

Sofia Tumminia, Louisa Mezache, Theresa Wiesner, Benoit Vianay, Manuel Théry, Marie-Jeanne Papandréou, Christophe Leterrier

Posted on: 18 October 2024 , updated on: 19 October 2024

Preprint posted on 5 September 2024

An arranged bumpy road to study the presynapse: a novel setup to explore neuronal architecture

Selected by Felipe Del Valle Batalla

Categories: cell biology, neuroscience

 

Graphical abstract. The fabrication micropatterned surfaces with ngnl spots allows for detailed study of the presynapse in cultured neurons. Using super-resolution microscopy and live-cell imaging it is possible to dissect the distribution of actin nanostructures at the presynapse (Corrals, Mesh, Rails) together with the detection of synaptic activity markers (Exocytic events). This application helped to understand the spatial distribution of actin with respect to other structural components of the presynapse (Bassoon). Illustration by Antonia P. Berger.

Background

Neuronal communication depends on synapses, with cytoskeletal actin playing a crucial role in neuronal structure and synaptic function (McAllister, 2007). Actin dynamics modulate synaptic transmission, but the complex organization of actin in the presynapse has only recently been studied with greater precision and resolution, thanks to newer tools and microscopy techniques.

Single-molecule localization microscopy (SMLM) and other super-resolution techniques, offer better molecular specificity, enabling detailed investigation of structures within neurons, particularly the presynapse.

One of the main challenges in analyzing distinct subcellular nanostructures is having an optical setup that allows precise spatiotemporal control over the processes and structures of interest. In this work, the authors refine and improve upon previous research from the same group (Bingham et al., 2023), which used a model of isolated bead-induced synapses to characterize presynaptic actin nanostructures.

This new study addresses the limitations of the previous model by improving optical accessibility and presynapse orientation with an elegant setup. Employing SMLM, the authors explore in high detail the distinct conformations of actin at the presynapse, along with functional markers of synaptic activity. 

Key Findings

The study introduces a presynaptic model on glass to induce synapse formation in cultured neurons using neuroligin-1 (nlgn)-stamped spots. This model enabled the authors to:

  • Validate the model: The authors demonstrated that nlgn spots induce presynapse formation with clusters of presynaptic markers (bassoon, synaptophysin), synaptotagmin uptake (indicating vesicle cycling activity), and actin enrichment, similar to natural synapses.
  • Confirm previous findings: The authors corroborated earlier observations of two categories of presynapses—actin-enriched (A+) and non-actin-enriched (A-)—and found that A+ presynapses exhibited higher concentrations of presynaptic components and greater cycling activity.

Fig.2A &2B from the preprint: Actin-enriched (A+) and non-actin-enriched (A-) structures.

  • Visualize actin nanostructures: Using STORM, the authors identified and confirmed three distinct actin nanostructures within nlgn-induced presynapses: corrals (large, branched structures on the periphery), rails (small linear structures within the bouton), and a mesh (a weak cluster of nanostructures in the active zone).
  • Expand on actin spatial arrangement: Multicolor localization microscopy revealed that the actin mesh is situated above and between bassoon nanoclusters, suggesting a role in organizing nanoclusters within the active zone.

Fig.3B from the preprint: Distribution of presynapse components related to distinct actin nanostructures.

  • Correlate exocytosis sites with nanostructures: Using correlative live-cell/STORM imaging, the authors observed that synaptic vesicle exocytosis occurs in presynaptic regions devoid of actin, suggesting that actin may help define vesicle release hotspots.

Fig.5A & 5B from the preprint: Correlative live-cell microscopy / STORM to show location and co-ocurrence of synaptic activity by exocytosis and Munc13-1 at ngnl spots.

Why This Work is Important

This preprint is significant not only because it provides deeper insights into the architecture and function of presynaptic actin, but also because it serves as proof of concept for the potential use of patterned surfaces (or other iterations of this setup in the future) to induce synapse formation and study specific phenotypes in controlled conditions.

Future Directions and Questions for the Authors

  • The authors present an elegant approach to studying presynapses with nlgn stamps. An interesting alternative could be coating the stamps with other postsynaptic components, such as PSD95 or EphB. What do the authors think of this approach?
  • The study primarily focuses on actin. It would be fascinating to explore the organization and interactions of other cytoskeletal components within this model, such as tubulin and spectrin, which have been shown to form a periodic interrupted network in presynapses. Have the authors considered investigating other cytoskeletal components?
  • Given previous evidence suggesting contradictory roles for actin—acting either as a mesh that gathers vesicles before exocytosis or as a physical barrier within presynaptic subdomains—it would be interesting to know what other experiments the authors would conduct to further test this hypothesis.
  • The correlative live-cell/STORM approach provided valuable insights into the spatial relationship between exocytosis events and actin nanostructures. Expanding this approach to include real-time tracking of actin dynamics during exocytosis could further enhance our understanding of actin’s role in shaping vesicle release.

References

Bingham, D., Jakobs, C. E., Wernert, F., Boroni-Rueda, F., Jullien, N., Schentarra, E. M. et al. (2023). Presynapses contain distinct actin nanostructures. J Cell Biol, 222(10), e202208110.

McAllister, A. K. (2007). Dynamic aspects of CNS synapse formation. Annu Rev Neurosci, 30, 425-450.

Tags: axon, microscopy, neuron, synapse

doi: https://doi.org/10.1242/prelights.38714

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Author's response

The authors shared

  • The authors present an elegant approach to studying presynapses with nlgn stamps. An interesting alternative could be coating the stamps with other postsynaptic components, such as PSD95 or EphB. What do the authors think of this approach?

We chose neuroligin-1 as it was the protein initially used by Funahashi et al. to induce presynapses by coupling it the glass over the whole coverslip (Funahashi et al., 2018). One advantage of using micropatterned neuroligin dots is that it can induce presynapses along the axon of neurons without having to overexpress their partner neurexin. Another protein that has been used to induce presynapses on micropatterns is SynCAM1 (Czöndör et al., 2013). Other candidates could be used, but they must be proteins expressed at the surface of the postsynapse: patterning PSD-95 is not likely to work, as it is an intracellular scaffold that does not have direct interactions with presynaptic surface proteins. EphB is a postsynaptic surface receptor that could be used, leveraging its interaction with Ephrin-B along axons to induce presynapses (Hruska & Dalva, 2012), but to our knowledge it has not been tried so far.

  • The study primarily focuses on actin. It would be fascinating to explore the organization and interactions of other cytoskeletal components within this model, such as tubulin and spectrin, which have been shown to form a periodic interrupted network in presynapses. Have the authors considered investigating other cytoskeletal components?

We have used this model to look at actin as well as to visualize presynaptic components such as Bassoon and Munc13-1 at the nanoscale (Fig. 5 of our preprint). Indeed, it would be interesting to visualize the interruption of the spectrin rings and the membrane-associated periodic scaffold (MPS) at induced presynapses, something we visualized previously for bead-induced presynapses (Bingham et al., 2013). Of note, actin on our STORM images of pattern-induced presynapses show not only the presynaptic nanostructures (corral, mesh and rails), but also the actin rings that are part of the MPS along the shaft next to the induced presynapse, confirming the interruption of the MPS at presynapses.

  • Given previous evidence suggesting contradictory roles for actin—acting either as a mesh that gathers vesicles before exocytosis or as a physical barrier within presynaptic subdomains—it would be interesting to know what other experiments the authors would conduct to further test this hypothesis.

We have tried to address this by imaging exocytosis and correlate the exocytic location with the nanostructures of actin (Fig. 5F our preprint). Results from these experiments suggest that exocytosis occurs away from active zone structures at the nanoscale, although the complexity and low throughput preclude a more quantitative conclusion at this time. Beyond correlative imaging, being able to selectively perturb active zone actin would be a great way to pinpoint its functions, but there is not tool at this time that is selective enough biochemically or spatially. The best we could do at this point would be to use inhibitors of Arp2/3 or formins, which we have shown to preferentially target the corral/mesh and rails, respectively (Bingham et al., 2023).

  • The correlative live-cell/STORM approach provided valuable insights into the spatial relationship between exocytosis events and actin nanostructures. Expanding this approach to include real-time tracking of actin dynamics during exocytosis could further enhance our understanding of actin’s role in shaping vesicle release.

We could image actin dynamics in parallel to VAMP2-pHluorin, however there are a few caveats to consider. Firstly, most actin-binding probes (such as LifeAct or SiR-actin) have a stabilizing effect on actin dynamics itself that would have to be minimized. Secondly, live-cell imaging would have to be done at the diffraction-limited resolution, so we would not be able to visualize the nanostructures beyond the overall intensity at the presynapse, which we have shown correlate with the size of the actin corral (Fig. 3C of our preprint). Nevertheless, even at diffraction-limited resolution, live imaging of actin at induced presynapses together with exocytosis could provide an interesting insight on the relationship between actin enrichments and overall activity of individual presynapses.

References

Bingham D, Jakobs CE, Wernert F, Boroni-Rueda F, Jullien N, Schentarra EM, Friedl K, Moura JDC, Bommel DM van, Caillol G, Ogawa Y, Papandréou MJ, Leterrier C. Presynapses contain distinct actin nanostructures. J Cell Biol. 2023;222(10):e202208110.

Czöndör K, Garcia M, Argento A, Constals A, Breillat C, Tessier B, Thoumine O. Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation. Nat Commun. 2013 Aug 12;4(1):2252.

Funahashi J, Tanaka H, Hirano T. Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface. Front Cell Neurosci. 2018;12:140

Hruska M, Dalva MB. Ephrin regulation of synapse formation, function and plasticity. Mol Cell Neurosci. 2012;50(1):35–44.

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