title: 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.
authors.affiliation: Department of Cell Biology, Institute for Molecular Medicine, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: Muriel
authors.initials: M
authors.lastname: Mari
authors.forename: Willie J C
authors.initials: WJ
authors.lastname: Geerts
authors.forename: Fulvio
authors.initials: F
authors.lastname: Reggiori
mesh_terms: D055032:Electron Microscope Tomography / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
keywords: Tokuyasu cryosectioning; correlative microscopy; electron microscopy; electron tomography; fluorescence microscopy; immunogold labelling; yeast