Tomogram slice of an unroofed mouse fibroblast. Scale bar 100 nm
Cell unroofing for in situ cryo-electron tomography
Researcher: Dr. Jana Kroll, Freie Universität Berlin / Max-Delbrück-Centrum für Molekulare Medizin (MDC)
Measuring facility: CFcryoEM: Core Facility for Cryo Electron Microscopy (Charité)
Membrane rearrangements are required for a broad spectrum of cellular functions, ranging from general mechanisms like endocytosis or filopodia formation to more specialized functions like neurotransmitter release. The morphological changes going along with these processes have been characterized for decades using light and electron microscopy (EM) techniques. Studying the proteins that induce these membrane rearrangements is much more challenging, not only because their structure and interaction partners are typically transient, but also because they are ideally examined directly at their site of action. In situ cryo-electron tomography (ET), combined with subtomogram averaging, makes it possible to analyze the structure of macromolecules and molecular complexes within their cellular environment, meaning under native biophysical conditions and together with their interaction partners and/or at cellular membranes. One of the main challenges of in situ cryo-ET is the thickness and complexity of the cell, which limits the structurally interpretable resolution. In my project, I establish and optimize techniques to thin samples via cell unroofing. For cell unroofing, the cytoplasm and superior plasma membrane of cultured cells need to be detached using physical force. Patches of the inferior cell membrane, together with membrane-associated macromolecules, remain attached to the culturing substrate and can be utilized for direct plunge freezing and cryo-ET. As visible in the tomogram slice of an unroofed fibroblast, the remaining membrane is decorated with different kinds of cytoskeletal filaments, ribosomes, clathrin and putative caveolae. Within the funding period, I plan to optimize cell unroofing not only for fibroblasts but also for neuronal and neuroendocrine cells and combine it with protein labelling techniques to confirm the molecular identity of observed macromolecules.