The neuronal cytoskeleton in physiology and disease
Good and busy time at the 13th Single Molecule Localization Microscopy Symposium SMLMS2024 in Lisbon! Great organization by Ricardo Henriques and Pedro Matos Pereira, great venue at ITQB NOVA, great science from all participants with talk and roundtable with Christophe, posters by Christopher on hihs spectral STORM workflow.
We’re over the moon to see our latest work with Stéphane Vassilopoulos and his team published in Science. Check out how we reveal the unique architecture of clathrin-coated pits and endocytosis at the axon initial segment!
Don’t have access? Just email me or use this invited link to access the pdf without a subscription.
What did we discover? When we started using super-resolution microscopy and platinum-replica electron microscopy (PREM) to reveal the ultrastructure of the periodic actin-spectrin scaffold along proximal axons, we were surprised to see many clathrin-coated pits along the plasma membrane of the AIS on PREM images. Clathrin-coated pits form at the center of unique circular area of the periodic actin-spectrin scaffold that expose the bare plasma membrane: we named these exclusion area “clearings”, like in a forest.
We characterize these clearings using Structured Illumination Microscopy (SIM), Single Molecule Localization Microscopy (SMLM), PREM, and their correlative combination. Messing with axonal spectrins (using RNA interference or a drug called diamide) disrupts the periodic scaffold and results in more pits along the AIS, demonstrating that the spectrin mesh regulates pit formation. This is in line with previous findings that the spectrin scaffold can negatively define the localization of endocytic activity in epithelial cells and fibroblasts, as well as negatively regulate the endocytosis of cannabinoid receptors along axons.
When we tried to monitor endocytosis from these pits, there was another surprise! Dextran feeding resulted in most dextran cluster being present at the surface in clathrin-coated pits rather than inside the axon. This suggests that the pits are super stable and indeed, two-color TIRF-SIM of spectrin and clathrin along the AIS showed clathrin-coated pits staying for tens of minutes inside clearings:
So, why would these clathrin-coated pits form and just stay there? We show that they provide “on-demand” endocytosis: long-term depression-like stimulation with NMDA results in the scission of pits via the polymerization of “actin nests” within clearings, triggering endocytosis. This novel, regulated endocytosis mechanism makes a lot of sense at the AIS, as it allows to trigger rapid endocytosis within the otherwise super-stable submembrane scaffold. This might be how sodium channels are endocytosed from the AIS in plasticity situations to adjust neuronal excitability.
This was a blast to work on this with Stéphane, PhD student Florian Wernert and the rest of the NeuroCyto team (Florence Pelletier, Eline Simons, Fanny Boroni-Rueda, Nicolas Jullien, Marie-Jeanne Papandréou), post-doc Satish Moparthi, Jeanne Lainé, Gilles Moulay and Sofia Benkhelifa-Ziyyat in Stéphane’s team! It was mainly funded by the ANR “ASHA” we have together with Stéphane, as well as equipments grants that helped set up our Nikon Center of Excellence for Neuro-NanoImaging. Looking for a summary of our findings in French? Check the CNRS Biologie website!
Sorry for the lack of updates in the news… But this is definitely on the to-do list! Meanwhile, you can hear about the lab news (and more!) on Christophe’s twitter feed.
Two French scientific magazines have highlighted our recent article about the ultrastructure of axonal actin rings, both in their March issue: Science et Vie (bottom on the pic below) made a short story about it while Pour la Science (top) featured a PREM image of an axon as a beautiful double-page spread.
On the other side of the Atlantic, our work was featured in the March-April issue of the American Scientist magazine for their “Sightings” column. Happy to see our work getting attention in the press!
Christophe was in Finland this week, first in Turku invited by Guillaume Jacquemet, then in Helsinki to serve as an opponent for Amr Abouelezz, a brillant PhD student from Pirta Hotulainen’s lab. The PhD defense is a serious affair in Finland and Amr passed with flying colors! Thanks to Pirta for this opportunity.
You can read the PhD work of Amr in two published article: one here on the resistance of the axon initial segment actin rings to actin depolymerizing drugs, and his main work here on the presence and role of tropomyosin at the AIS. Congrats Amr!
Our latest preprint is out, a collaboration with our long-standing collaborator Subhojit Roy and his lab, in particular the talented postdoc Archan Ganguly. We uncover how clathrin is transported along axons as assembled structures that are unrelated to endocytosis: the “transport packets”. In this work, we pushed DNA-PAINT to image these axonal clathrin packets. Here they are by EM of APEX-clathrin on top and by DNA-PAINT of endogenous clathrin at the bottom. We also repurposed the ChimeraX software to render PAINT data and see inside axons!
Where are the packets going? Inside presynapses! We visualized clathrin in presynapses – 3D DNA-PAINT could clearly separate presynaptic and postsynaptic clusters. With ChimeraX we rendered and measured these presynaptic clathrin packets – similar in size to the ones along axons.
Check the preprint for the whole story – there’s so much more in there. Great work from Florian and Ghislaine in the team, congrats everyone!
Ganguly A, Wernert F, Phan S, Boassa D, Das U, Sharma R, Caillol G, Han X, Yates JR, Ellisman M, Leterrier C, Roy S.
Mechanistic Determinants of Slow Axonal Transport and Presynaptic Targeting of Clathrin Packets.
bioRxiv, 2020 Feb 20.
doi: 10.1101/2020.02.20.958140
We have a new preprint out! This is a collaboration led by the lab of Steven F. Lee in Cambridge. They developed a new platform, vLUME (short for VisuaLization of the Universe in a Micro Environment) that allows to interact with localization-based microscopy data in a virtual reality environment. Learn more about it in this video:
If you are interested, have a look at the movies highlighting the functions of vLUME and head over to GitHub to try the 1.0 version of the software.
Spark A, Kitching A, Esteban-Ferrer D, Handa A, Carr A, Needham L, Ponjavic A, Santos M, McColl J, Leterrier C, Davis S, Henriques R, Lee S.
vLUME: 3D Virtual Reality for Single-molecule Localization Microscopy
bioRxiv, 2020 Jan 21.
doi: 10.1101/2020.01.20.912733
Local newspaper La Provence ran an article about the NeuroCyto team in its January 17 edition. Thanks to journalist Delphine Tanguy for her visit and interest in our work!
Christophe wrote a Spotlight in the Journal of Cell Biology highlighting a nice recent paper from the group of Pei-Lin Cheng in Taiwan. In this article, Lee et al. showed how degradation of the chloride transporter NKCC1 by proteasomes anchored at the AIS have a key role in lowering the intracellular chloride concentration, leading to the perinatal reversal of GABA effect from excitatory to inhibitory.
First work of 2020 work is out! A collaboration with Matt Rasband’s lab in Nature Communications. This is a significant paper for the axon initial segment field. Matt’s lab used BioID of key AIS proteins for mapping AIS components. Dozens of new candidates for future studies!
We performed super-resolution microscopy of several of the newly identified AIS components. IN particular, we showed that Mical3, a protein linking microtubules and actin, forms clusters along the AIS that are not periodically organized along the actin/spectrin scaffold.
