成像酵母核孔复合体：从经典电子显微镜到免疫扫描电子显微镜。

Imaging yeast NPCs: from classical electron microscopy to Immuno-SEM.

作者信息

Kiseleva Elena, Richardson A Christine, Fiserova Jindriska, Strunov Anton A, Spink Matthew C, Johnson Simeon R, Goldberg Martin W

机构信息

Laboratory of Morphology and Function of Cell Structure, Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia.

Department of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.

出版信息

Methods Cell Biol. 2014;122:59-79. doi: 10.1016/B978-0-12-417160-2.00003-5.

DOI:10.1016/B978-0-12-417160-2.00003-5

PMID:24857725

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.

摘要

电子显微镜（EM）已被广泛用于研究核运输以及核孔复合体（NPC）和核膜的结构。然而，在主要的模式生物之一——酿酒酵母中进行电子显微镜观察时，会面临一些特殊挑战。这些挑战源于细胞壁、液泡和密集堆积的细胞质的存在，对于透射电子显微镜（TEM）而言，这使得固定、包埋和成像变得困难。这些对于扫描电子显微镜（SEM）也存在问题，因为去除细胞壁和分离细胞核很容易损坏相对脆弱的NPC。我们展示了一些用于制备TEM和SEM样品的方案，以提供有关酵母NPC超微结构以及在该超微结构内通过免疫金标记确定核孔蛋白和转运因子位置的信息。