title: Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.
abstract: Electron microscopy (EM) has been used extensively for the study of nuclear transport as well as the structure of the nuclear pore complex (NPC) and nuclear envelope. However, there are specific challenges faced when carrying out EM in one of the main model organisms used: the yeast, Saccharomyces cerevisiae. These are due to the presence of a cell wall, vacuoles, and a densely packed cytoplasm which, for transmission EM (TEM), make fixation, embedding, and imaging difficult. These also present problems for scanning EM (SEM) because cell wall removal and isolation of nuclei can easily damage the relatively fragile NPCs. We present some of the protocols we use to prepare samples for TEM and SEM to provide information about yeast NPC ultrastructure and the location of nucleoporins and transport factors by immunogold labeling within that ultrastructure.
authors.affiliation: Laboratory of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia.
authors.forename: Elena
authors.initials: E
authors.lastname: Kiseleva
authors.affiliation: Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
authors.forename: A Christine
authors.initials: AC
authors.lastname: Richardson
authors.affiliation: Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
authors.forename: Jindriska
authors.initials: J
authors.lastname: Fiserova
authors.affiliation: Laboratory of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia.
authors.forename: Anton A
authors.initials: AA
authors.lastname: Strunov
authors.affiliation: Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Spink
authors.affiliation: Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
authors.forename: Simeon R
authors.initials: SR
authors.lastname: Johnson
authors.affiliation: Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
authors.forename: Martin W
authors.initials: MW
authors.lastname: Goldberg
mesh_terms: D002473:Cell Wall; D002474:Cell-Free System; D016616:Cryoultramicrotomy / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D005404:Fixatives; D006046:Gold / Q000737:chemistry; D021621:Imaging, Three-Dimensional; D007495:Iridium / Q000737:chemistry; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D022022:Nuclear Pore / Q000737:chemistry*; D028861:Nuclear Pore Complex Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013104:Spheroplasts / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D005404:Fixatives; D028861:Nuclear Pore Complex Proteins; D007495:Iridium; D006046:Gold
keywords: Electron microscopy; Immunogold labeling; Sacharomyces cerevisiae; Scanning; Transmission; Yeast