Immuno- and correlative light microscopy-electron tomography methods for 3D protein localization in yeast.

Abstract

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.

DOI: 10.1111/tra.12192

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@article{Mari2014ImmunoAC, title={Immuno- and correlative light microscopy-electron tomography methods for 3D protein localization in yeast.}, author={Muriel C Mari and Willie J C Geerts and Fulvio Reggiori}, journal={Traffic}, year={2014}, volume={15 10}, pages={1164-78} }