Immuno- and correlative light microscopy-electron tomography methods for 3D protein localization in yeastMari, M., Geerts, W. J. C. & Reggiori, F., Oct-2014, In : Traffic. 15, 10, p. 1164-1178 15 p.
Research output: Contribution to journal › Article › Academic › peer-review
Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three-dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno-electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy-electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.
|Number of pages||15|
|Publication status||Published - Oct-2014|