The neuronal cytoskeleton in physiology and disease
Christophe was at the 10-year anniversary retreat of the Cytomorpho lab right in front of the sea in Les Goudes, Marseille. Thanks for the invitation Manuel and Laurent, it was great discussing with actin and microtubules specialists, and hearing the various paths taken by the lab alumni.
Marseille hosted the 2019 French neuroscience meeting from May 22 to May 24. The INP institute was present with numerous talks and posters showcasing the work of our labs (see here for a complete list). The NeuroCyto team was of course there, with Dominic presenting his first poster on the role of presynaptic actin, as well as a talk from Christophe on the ultrastructure of axonal actin rings that he also presented the day before at the satellite meeting on the spinal chord .
An exciting project from the lab is now out on bioRxiv. This is a collaboration with electron microscopy guru Stéphane Vassilopoulos from the Myologie Institute in Paris. One of the most striking discovery of super-resolution microscopy so far has been the periodic actin/spectrin scaffold along axons, with actin rings spaced every 190 nm by spectrin tetramers. Since its discovery in 2013, a number of labs, including ours, have used various super-resolution microscopy techniques (SMLM, SIM, STED) to refine our knowledge about this structure. However, actin rings and the axonal periodic scaffold had not been observed by EM until now.
Using mechanical unroofing and platinum-replica electron microscopy (PREM), we were able to visualize the axonal actin rings regularly spaced under the exposed plasma membrane of axons, and to determine their ultrastructure and molecular organization. And we had a big surprise: the actin rings are not made of short, capped actin filaments as it was assumed since their discovery, by analogy with actin inside the erythrocyte submembrane cytoskeleton. Instead, they are braids made of two long (~0.5 to 1 µm) actin filaments!
These actin braids are connected by a dense mesh of spectrins, which we unambiguously identified using immunogold labeling and PREM. Moreover, we probed the stability of the axonal periodic scaffold using actin-targeting drugs, and found similar results by super-resolution and electron microscopy. Finally, we directly demonstrated the identity and organization of the scaffold components (actin, spectrins) by performing correlative SMLM/PREM, visualizing the same sample by super-resolution and electron microscopy.
It was a great pleasure to work with Stéphane! The project benefited from the magic unroofing touch of our Master 2 student Solène, and from important ground work by Angélique and Ghislaine from the team. To read more, please see our bioRxiv preprint and let us know what you think!
Christophe spoke at the Fluorescence Microscopy Workshop IV on cutting-edge technologies at Institut Pasteur on May 13. A nice day devoted to super-resolution microscopy with academic speakers mixed with technological presentation from companies. The workshop continued for the whole week with the possibility to test various microscopes, such as the new N-SIM S fast structured illumination microscopy from Nikon.
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!
Christophe participated to the Emerging Concepts in the Neuronal Cytoskeleton meeting in Villarica, Chile. He was the first speaker of the meeting in the “Super-resolution microscopy of the neuronal cytoskeleton, straight after three flights and 30 hours of travel! He presented new results on the visualization of axonal actin rings using super-resolution and electron microscopy (see the preprint here). The meeting was excellent with a stunning location by the lake, lots of amazing talks and interesting discussions. Read more on Twitter.
Check also the beautiful photographs of the participants taken by Rodolfo Carvajal (full slideshow here):
We will co-organize the next edition in 2021 with Stephanie Gupton (UNC, North Carolina, USA) and Carlos Wilson (IMMF, Cordoba, Argentina), so stay tuned!
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.
We have a new preprint out! Want to do good super-resolution images? We have put together all our SMLM tips and tricks. This is a methods paper that describes our SMLM workflow, using benchmark samples such as microtubules and clathrin-coated pits.
A good image starts with a good sample… that’s why the first part is about how to our fixation and immunolabeling procedures. Then it’s “just” a matter of imaging and processing. Tips for acquiring 3D-STORM images, as well as multi-color DNA-PAINT. Congrats to Angélique and Karoline for their beautiful images. Check our preprint to know everything, and let us know what you think!
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!
Today we welcomed a new team member, Karoline Friedl. She will be in the lab thanks to a collaboration with French startup Abbelight, and will help develop and test new super-resolution modalities for our projects. Welcome Karoline!