New article out: Mapping the axon initial segment components by mass spectrometry

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!

Check all these new AIS proteins!

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.

Mical3 is present along the AIS but does not associate with the periodic actin/spectrin scaffold.

Our paper is out! The ultrastructure of the axonal actin rings revealed

Our work on the ultrastructure of the periodic actin/spectrin scaffold along axons is out in Nature Communications. It’s a collaboration with platinum-replica electro microscopy specialist Stephane Vassilopoulos from the Myologie Institute in Paris.

It’s easier to go there to read it (and it’s open access!)

In this work that was made available as a preprint back in May, we used ultrasonic unroofing to expose the submembrane cytoskeleton along axons in neuronal cultures. This allowed to observe it both by optical super-resolution microscopy and by platinum-replica electron microscopy, zooming down to individual proteins and actin filaments.

We could visualize for the first time by EM the periodic submembrane scaffold along axons, formed of actin rings connected by spectrin tetramers. Moreover, we discovered that actin rings are not made of small actin filaments bundled together as previously assumed, but by braids of long filaments that are likely to result in their stability and flexibility. Finally, we directly visualized elements of the periodic scaffold (actin, spectrins, myosin, ankyrin) using correlative super-resolution microscopy and platinum-replica electron microscopy.

Zooming from a cultured neuron to single ankyrins along the axon initial segment by correlative STORM/PREM

A press release from CNRS is available here in English and here in French for more details about this work. We are very happy to see it out!

Just out: commentary on how ß2-spectrin drives axonal transport

Christophe just published a commentary about the latest article from Damaris Lorenzo and Van Bennet’s labs in PNAS. This work shows how ß2-spectrin is not only a crucial component of the periodic actin/spectrin scaffold along axons, but also directly participates in axonal transport by associating with intra-axonal vesicles.

Check it out in more details here:
Leterrier C, A dual role for βII-spectrin in axons. Proceedings of the National Academy of Sciences, 2019 Jul 30;116(31):15324-15326. doi: 10.1073/pnas.1909789116

Article out: tips and tricks for Single Molecule Localization Microscopy

Our Methods article about our tips and tricks for optimized Single Molecule Localization Microscopy (SMLM, previously announced here when we preprinted it) has just been published in its final form. This is part of a special issue on super-resolution microscopy, thanks to Jan Tønnesen for the invitation to contribute. If you want to optimize sample preparation and imaging for STORM and DNA-PAINT of classic celular targets (microtubules, actin, clathrin-coated pits), check it out!

STORM imaging of clathrin-coated pits (top) and actin (bottom)

Pumpy is out! Perform advanced microscopy experiments thanks to NanoJ-Fluidics

The LEGO Pumpy (or more officially NanoJ-Fluidics) paper is out ! A joint venture with the Henriques lab, this details how to build a fully open-source multi-channel syringe pumps with LEGO and Arduino. We provide examples on how to use it directly on the microscope for complex imaging protocols: live-to-fixed correlative acquisitions, image-analysis triggered fixation, sequential imaging… Check the video we put together showing the possibilities:

In the lab, we used Pumpy to perform complex STORM/PAINT multiplexed acquisitions. Here it’s a 5-color imaging of actin, mitochondria, intermediate filaments, microtubules and clathrin. It’s made with 1 single-color STORM and two 2-color PAINT sequential acquisitions:

Imaging was a breeze thanks to Pumpy, so the main challenge was to optimize the fixation and immunolabeling for 5 distinct targets. Great work from Ghislaine Caillol and Fanny Boroni-Rueda! Check the full article here for more.

Just out: overview of the NanoJ framework for open-source super-resolution

The Henriques lab and its collaborators have a new paper out in the Journal of Physics D: Applied Physics. This is an overview of the NanoJ framework they are developing for open-source super-resolution in ImageJ/Fiji. In includes SRRF, SQUIRREL, NanoJ-Fluidics aka Pumpy, but also utilities for drift correction, chromatic aberration registration and single-article averaging. Have a look at the accepted manuscript here!

New paper out: slow axonal transport of actin via hotspots and trails

Our latest work (previously on bioRxiv) is now published in the Journal of Cell Biology. We collaborated with the Roy lab to reveal a new mechanism of slow axonal transport, based on the previous discovery of actin hotspots and trails. Hotspots are static actin clusters that appear and disappear within minutes every 3-4 µm along the axon. They generate the assembly of trails, long actin filaments that polymerize along the axon and collapse within seconds. Our new article first shows that trails polymerize at their barbed ends, located at the surface of hotspots. Each trail is thus pushed away from the hotspot as as it grows, resulting in a net displacement of actin monomers after trail collapse. In addition, trails grow in both directions (anterograde and retrograde), but with a small bias toward the tip of the axon (58% anterograde vs 42% retrograde).

The combination of these two processes (displacement of actin by trails and anterograde bias) results in the slow progress of actin along the axon. Modeling from the Jung lab allowed to determine the overall actin transport speed resulting from the hotspots and trails dynamics. Strikingly, this slow anterograde transport speed of actin (0.4 mm/day) precisely matches the values obtained by classic radio-labeling studies. This is a fundamentally new mechanism of slow axonal transport for cytoskeletal components, based on a biased assembly/disassembly mechanism rather than processive transport by motor proteins.

In this work, we used STORM imaging of axonal actin to pinpoint the architecture of hotspots, showing that the multi-directional growth of trails make them appear as asters when the axon is thicker (see Figure). Furthermore, we imaged hundreds of hotspots by STORM and quantified their diameter to ~200 nm. This is a first step toward elucidating the molecular organization of hotspots and trails, which will be crucial to understand their cellular functions.

Details of hotspots seen by STORM
Quantification of hotspots diameter from STORM images

Just published: our review on the architecture of axonal actin

Marie-Jeanne and Christophe wrote a review detailing how recent discoveries renewed the understanding of axonal actin organization. In the axon shaft itself, new nano-structures such as rings, hotspots and trails have been described, but their function remains to be elucidated. At presynapses, the precise architecture of actin is still elusive, and contradicting findings have been reported regarding its function. This is an exciting time to study actin in axons!

The review is now published in Molecular and Cellular Neuroscience, and will be part of a special issue on “Membrane Trafficking and Cytoskeletal Dynamics in Neuronal Function”. If you don’t have access to the review, a preprint manuscript is available on Zenodo.

Fig.2 from Papandreou & Leterrier 2018