Oocytes as an experimental system to analyze the ultrastructure of endogenous and ectopically expressed nuclear envelope components by field-emission scanning electron microscopy.
Xenopus oocytes provide a powerful model system for studying the structure and function of the nuclear envelope and its components. Firstly, the nuclear envelope is easily isolated by hand under gentle conditions that have little effect on its structural organization. They can then be prepared for several types of electron microscopy (EM) including field-emission scanning EM (feSEM) (described here) and cryo-EM. They can be immuno-gold labeled to determine the localization of individual proteins. There is also enough material to analyze biochemically. Secondly, they possess an efficient transcription and translation system so that proteins of interest can be ectopically expressed by injection of either mRNA into the cytoplasm or plasmids into the nucleus. Such proteins can be tagged and mutated. They are post-translationally modified and usually incorporate into the correct compartment. We describe here methods developed to analyze the structural organization of the nuclear envelope by feSEM including the structural organization of ectopically expressed nuclear envelope proteins.
Stick
Reimer
R
Department of Cell Biology, University of Bremen, Bremen, Germany. stick@uni-bremen.de
Goldberg
Martin W
MW
D000818:Animals; D054239:Blastodisc / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003584:Cytological Techniques; D007150:Immunohistochemistry / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008954:Models, Biological; D009685:Nuclear Envelope / Q000648:ultrastructure*; D022022:Nuclear Pore / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry* / Q000648:ultrastructure*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D014982:Xenopus laevis
D012333:RNA, Messenger