Marine Laporte
Univ-Lyon, France
Marine Laporte is a principal investigator at the MeLiS Institute (University of Lyon, France), a recently established department bringing together cell and developmental biologists who study the molecular mechanisms across various cellular compartments—ranging from cilia, membranes, and axons to synapses and neural circuits—with a focus on neuronal and muscular cells. Her primary research interest centers on membrane remodeling mechanisms. She did her PhD at the Institute of Neuroscience in Grenoble (France), working on the contribution of membrane deformation on cerebral development and neuronal excitability. Then, she moved to the university of Geneva (Switzerland) to study centrioles/cilia biogenesis. During her post-doc she developed an expansion microscopy protocol adapted to the observation of membranous organelles as well as cytoskeleton.
Now, combining this technique to genetic manipulation, she investigates how membrane composition and dynamics contribute to ciliary homeostasis and their role in brain-related ciliopathies.
Visualizing the native cellular organization by coupling cryo-fixation with expansion microscopy (Cryo-ExM)
Abstract
Super-resolution fluorescent microscopy (SRM), encompassing expansion microscopy (ExM) since few years now, allows to locate proteins with nanometer resolution in a cellular context. However, SRM often requires cell fixation with aldehyde-based chemical crosslinkers, such as paraformaldehyde, or protein precipitation with cold methanol which potentially alter the native cellular state and the following interpretations. Cryo-fixation has proven to be the gold standard for efficient preservation of the native cell ultrastructure compared to chemical fixation, however it is not widely used in fluorescence microscopy owing to implementation. We recently developed a method combining cryo-fixation to ExM (Cryo-ExM), which allow nanoscale observation of a wide cellular compartment in their native state. We could demonstrate that Cryo-ExM allows the native preservation of membrane-based organelles such as mitochondria, endoplasmic reticulum, golgi and lysosomes together with the cytoskeleton component actin and microtubules and preserve all the structure in the same way, contrary to chemical fixations. Moreover, direct comparison with the gold-standard chemical fixation PFA-GA for the preservation of cellular structure, demonstrate that cryo-fixation bypassed drawbacks associated with this chemical fixation such as antigen accessibility due to strong protein-protein crosslinking. Overall.
In summary, we introduce a new method to perform super-resolution expansion microscopy by coupling cryo-fixation of a biological specimen with ExM, providing a universal framework to visualize subcellular compartments without chemical fixation artefacts. Importantly, this method also demonstrates that the classical cryo-substitution protocols developed for electron microscopy are compatible with expansion microscopy by replacing the EM resin with hydrogel monomer solutions. Therefore, this approach may also be applicable on tissues cryo-fixed by high-pressure freezing as well as in hydrogel-based tissue clearing. Finally, as expansion microscopy is also compatible with SIM, STED or dSTORM 18–20, our method now allows all these microscopy modalities to image cells in their native state, paving the way for further studies of complex and rapid dynamic cellular processes.