Dr.Lydia Danglot: Decifering organelles & synaptic molecular assemblies ...

abbelight

Decifering organelles and synaptic molecular assemblies using quantitative super-resolving 3D microscopy

About the speakerDr Danglot received her PhD from Pierre &amp; Marie Curie University in Paris in the lab of Antoine Triller at Ecole Normale Supérieure Paris where she studied the formation and maturation of inhibitory synapses in hippocampal neurons. Her post-doctoral training at the Institut Jacques Monod in the lab of Thierry Galli, focused on EGF receptors and transport in cancer cells. She joined in 2010, as a permanent researcher the french national health institute INSERM. Beyond its fondamental research in Neuroscience and Cell Biology, she is the Scientific Director of the NeurImag imaging facility at the Institut de Psychiatrie et Neurosciences de Paris (IPNP). She received awards from the Brain Research Fondation in 2018, and from INSERM in 2020 with the national Research and Education Excellence award. She is a board member of the National Research Group ImaBio on Advanced Microscopy for Biology (&gt;900 Members), board member of the Club Exocytosis Endocytosis gathering cell biologist around trafficking and fusion (200 membres), and scientific advisory board member of the ICON-Europe nanoscopy that focused on fluorescence-based super-resolution microscopy.AbstractElucidating molecular organization and synapse functions requires localizing precisely single or aggregated molecules and analyzing their spatial distributions. We develop a statistical method SODA (Statistical Object Distance Analysis) that uses either micro- or nanoscopy to significantly improve standard co-localization techniques (Lagache et al, Nature Comm 2018). Our method considers neuronal geometry to provide statistical maps of isolated and associated (coupled) molecules. SODA can be used with three-color structured-illumination microscopy (SIM) images to statistically characterize spatial organization of thousands of protein clusters or vesicles. As this methods do not rely on physical overlaps, SODA can be used to detect indirect molecular association in conventional microscopy or even in super resolution microscopy where higher resolution prevent the use of conventional overlay methods.
We used SODA with three-color SIM images of hippocampal neurons, and statistically characterized spatial organization of thousands of synapses. We show that presynaptic synapsin is arranged in asymmetric triangle with the 2 post-synaptic markers homer and PSD95 indicating a deeper localization of homer. As a proof of concept, we then imaged presynaptic glutamatergic terminals with 3D-STORM microscopy and analyzed the coupling between more than 180,000 localizations of vesicular Glutamate Transporter (VGLUT) and Synapsin molecules inside synaptic boutons. We evaluated with SODA that each Synapsin or VGLUT localization is at a mean distance of 52 ± 0,04 nm. We will show how our plugin SODA can also be useful to decipher molecular distance between protein within specific organelles like ER using 3D STED (Gallo et al, JCS 2020) or golgi apparatus in STORM (Sikora et al. Biology Open 2021).
 To reconstitute molecular maps within cellular shape, we recently developed new membrane probes call the MemBright family (Collot 2019, on the cover of Cell Chemical Biololgy - April). MemBright is a family of six cyanine based fluorescent turn-on PM probes that emit from orange to near infrared when reaching the PM. These probes are compatible with long-term live-cell imaging and immunostaining both in culture and tissue without any use of transfection or transgenic animals. We will show how MemBright probes can be used in 3D multicolor dSTORM imaging to unravel the plasma membrane structures of synaptic terminals or tiny dendritic spines protrusions in combination with immunostaining of synaptic proteins.

Membrane morphology of neuronal cells can be deciphered with conventional (fire LUT) or 3D STORM microscopy (blue spheres corresponding to single molecule lipids) thanks to incorporation and blinking of MemBright probes within dendritic plasma membrane. Dendritic spine shape can be reconstructed in 3D mesh volume (in light cyan on the right) thanks to single molecule localization of the molecular probes.

Dr.Lydia Danglot: Decifering organelles & synaptic molecular assemblies ...

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