FESEM IMAGING OF NUCLEAR STRUCT IN TRANSCRIPTION SPLICING & RNA TRANSPORT

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We are interested in understanding the functional and structural organization of the mammalian cell nucleus. Due to the highly compact nature of the nucleus, it is diff'cult to analyze the insoluble portion of the nucleus by examining optimally fixed cells in resinless sections using high resolution scanning electron microscopy. The resinless preparation of the specimen may reveal otherwise indistinguishable structural features. Immunolabeling of these structures will further our understanding of the functional organization of the nucleus. Dr. Spector and Ms Huang are investigating the structural and functional organization of the mammalian cell nucleus. The goal is to determine the macromolecular domains in the nucleus associated with transcription by RNA polymerase I and II, sites of assembly of splicing factor, sites of pre-mRNA splicing, and of transport of mRNA to the nuclear envelope. After either chemical fixation and epon embedding, or rapid cryo-fixation followed by freeze-substitution and embedding, thin sections were imaged by transmission EM. Unfortunately micrographs of thin sections are often difficult to interpret. We shall therefore prepare thicker sections (200 nary), then remove the epon with the method developed by Ris and Malecki (1993), and prepare stereo micrographs using low voltage-FESEM (Hitachi S-900). Progress repon: The blocks received so far from Ms Huang suffered from unsatisfactory preservation of cellular structure. In July 1997 Ms Huang will assume a faculty position at Northwestem University Medical School in Chicago. We shall then continue this project.