Within animal tissues, the shape of cells and intracellular organelles is carefully sculpted to serve their various functions. Membrane morphogenetic proteins are key drivers of cell and organelle shape, and alterations in these proteins lead to so-far incurable human diseases which, in many cases, affect the peripheral nervous system. However, until recently the study of nanoscale membrane architecture within native nervous tissue remained technically challenging. In the cryoNERVE project, we employ cryo-electron tomography (cryo-ET) to investigate the fine morphology of cellular membranes within native peripheral nervous tissues, as well as their alterations in pathological conditions. We aim to understand the structural basis of membrane shaping in situ to reveal molecular mechanisms of diseases affecting some of the longest axons in the body, thereby enabling future developments of diagnostic and therapeutic approaches.
The challenge, however, is to image cell membranes within native tissues by high-resolution cryo-ET. Peripheral nerves are formed by close interactions between multiple cell types, such as neurons, glia and epithelial cells, and mapping these interactions is critical to understand nerve physiology. On the other hand, vitrification of such complex samples for cryo-ET imaging is difficult, as these specimens are much thicker than the cultured cells commonly used in cryo-ET studies. To overcome this challenge, we devised a cryo-protection strategy that enables vitrification of peripheral nerves by plunge freezing. This in turn allows high-quality cryo-ET imaging of these tissues with a throughput comparable to studies on cultured cells.
These technical advances enable us to study the molecular architecture and membrane-shaping processes within native peripheral nerves with deep structural detail. Furthermore, the methods established here are not limited to nerve tissue, and may allow researchers in other fields to address a broad variety of questions within various native tissue specimens.
