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Christophe Leterrier 🔬Aug 15, 2023

I've not been here much lately, but here's a bit more about our paper just out in the Journal of Cell Biology, “Presynapses contain distinct actin nanostructures” (Twitter trained me to come up with short titles 🤏 and long threads 🧵, which are also doable in Mastodon I hope)
The article is available here: https://rupress.org/jcb/article-abstract/222/10/e202208110/276185/Presynapses-contain-distinct-actin
1/23

Presynapses contain distinct actin nanostructures | Journal of Cell Biology | Rockefeller University Press

Bingham et al. use bead-induced isolated presynapses and CRISPR-mediated tagging of endogenous actin in neurons coupled to super-resolution microscopy to reveal

Christophe Leterrier 🔬Aug 15, 2023

I want to highlight the work that went into the revision of the manuscript, crediting the contribution of lab members and collaborators, as well as give an overview of the Review Commons process we used (see https://www.reviewcommons.org/) 2/23

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Christophe Leterrier 🔬

We previously shared this work as a bioRxiv preprint in May last year, as the main work of Dominic Bingham who graduated in July 2022 (see Twitter thread: https://twitter.com/christlet/status/1527183879643201536?s=20) 3/23

Christophe Leterrier on Twitter

“🚌 Next stop on the NeuroCyto lab axonal journey: the presynapse🚏 Ever wondered how is presynaptic actin organized beyond the blobs seen by diffraction-limited microscopy? We certainly did! So why not do some STORM on it? https://t.co/0HFfmgQcm0 (1/6)”

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Christophe Leterrier 🔬Aug 15, 2023

In this work, we used beads to induce isolated presynapses along axons of cultured neurons, making it possible to directly visualize and quantify presynaptic actin without interference from the concentrated postsynaptic actin 4/23

Christophe Leterrier 🔬Aug 15, 2023

We directly visualized the selective enrichment of actin at presynapses, and could get good SMLM images of presynaptic actin at the nanoscale within bead-induced presynapses 5/23

Christophe Leterrier 🔬Aug 15, 2023

This led us to define three types of presynaptic actin nanostructures: a faint actin mesh at the active zone, actin rails within the presynapse, and dense perisynaptic actin corrals 6/23

Christophe Leterrier 🔬Aug 15, 2023

I was curious to try Review Commons and have our work openly reviewed upstream of journal submission so we submitted our preprint there, and the reviews were posted next to the preprint in July 2022 7/23

Christophe Leterrier 🔬Aug 15, 2023

The reviewers found the work interesting and important, but they wanted 1) more controls about the bead-induced presynapses, 2) better characterization of the actin structures at the nanoscale, and 3) confirmation in “real” synapses between two neurons 8/23

Christophe Leterrier 🔬Aug 15, 2023

We had 4 weeks to draft a detailed revision plan, then submitted the preprint together with reviews and revision plan to journals of the Review Commons consortium, with assurance they would reply within 10 days that they would consider a revised manuscript for publication 9/23

Christophe Leterrier 🔬Aug 15, 2023

I was over the moon when J Cell Biol editor Andrea Marat gave us a positive answer. So off we go in September to revise the manuscript and address the three points listed above - it was a lot of work! 10/23

Christophe Leterrier 🔬Aug 15, 2023

Marie-Jeanne Papandréou took care of all the bead-induced presynapse quantitative analyses with Fanny Boroni-Rueda, measuring hundreds more synapses to confirm that the bead-induced presynapses were indeed mature, and very similar to natural synapses 11/23

Christophe Leterrier 🔬Aug 15, 2023

On the SMLM front, we were lucky to have two great Master students joining us. Channa Jakobs from the Netherlands worked on STORM of actin (with DECODE processing), detailing how presynaptic actin is hidden by postsynaptic actin in excitatory and inhibitory natural synapses 12/23

Christophe Leterrier 🔬Aug 15, 2023

Channa also showed that the actin mesh, rails and corrals are present in all bead-induced presynapses, with variations in the size of the perisynaptic corrals. Mesh, rails and corrals are also found in both excitatory and inhibitory induced presynapses 13/23

Christophe Leterrier 🔬Aug 15, 2023

Meanwhile, Master student Eva Schentarra from Germany skillfully worked with Karoline Friedl on our Abbelight setup to obtain 2-color spectral STORM images of actin and Arp2/3, showing that the actin nucleator preferentially localizes to the mesh and corrals, but not rails 14/23

Christophe Leterrier 🔬Aug 15, 2023

Finally, visualizing these nanostructures in the presynaptic side of a natural synapse was a large project in itself. Simple transfection of actin probes like LifeAct cannot provide good-enough SMLM images, so we developed CRIPSR-based tagging of endogenous actin in neurons 15/23

Christophe Leterrier 🔬Aug 15, 2023

This was done by Nicolas Jullien in collaboration with Yuki Ogawa from Matt Rasband's lab, who developed an AAV-based variant of HiUGE for rat hippocampal neurons 16/23

Christophe Leterrier 🔬Aug 15, 2023

With these tools, Dominic Bingham last experiments in the lab showed that natural presynapses expressing HA-tagged actin had a selective enrichment in actin similar to bead-induced ones, which was also linked to a higher level of presynaptic components and vesicular cycling 17/23

Christophe Leterrier 🔬Aug 15, 2023

The key experiment was then to image these CRISPR-tagged natural synapses to confirm the presence of the presynaptic actin nanostructures. This required a lot of work for Nicolas to design various tags and for Channa to perform SMLM on tagged neurons 18/23

Christophe Leterrier 🔬Aug 15, 2023

Difficulty here was to get enough localizations to delineate the structures, but with a good enough precision to not blur things too much: small probe, with a lot of blinking events. We tested different tags labeled with antibodies or nanobodies, imaged by PAINT and STORM 19/23

Christophe Leterrier 🔬Aug 15, 2023

In the end the best results were obtained using an ALFA tag labeled with primary and secondary antibodies, or a triple ALFA tag with an anti-ALFA PAINT nanobody. In both cases, we could visualize the actin mesh, rails and corrals in tagged presynapses! 20/23

Christophe Leterrier 🔬Aug 15, 2023

So about a year after posting the preprint, we were ready to resubmit to @JCellBiol. Reviewers were happy, and things went swiftly after that. This journal is very special to me because of all the landmark neuronal cell biology papers it has published over the decades 21/23

Christophe Leterrier 🔬Aug 15, 2023

I liked the open @ReviewCommons process, and if you are interested, you can check the reviews, detailed revision plan and rebuttal which are available next to the article 22/23

Christophe Leterrier 🔬Aug 15, 2023

Finally, thanks to everyone who worked for months/years on this project, and to the funders that allowed to develop an ambitious new direction for the lab. A special thanks to Subhojit Roy for hooking me up with the bead-induced presynapses 🙌
https://rupress.org/jcb/article-abstract/222/10/e202208110/276185/Presynapses-contain-distinct-actin
23/23 - Thanks for reading the whole thread!

Presynapses contain distinct actin nanostructures | Journal of Cell Biology | Rockefeller University Press

Bingham et al. use bead-induced isolated presynapses and CRISPR-mediated tagging of endogenous actin in neurons coupled to super-resolution microscopy to reveal

Fillip Port Aug 16, 2023

@christlet Does this mean one can submit revcom reviews to several journals in parallel and than choose in case one gets more than one offer?

Christophe Leterrier 🔬Aug 16, 2023

@crisprflydesign Sorry I wasn't clear - you have to submit sequentially. It's quite fast though as journals are meant to reply within 10 days