Studies in our laboratory are involved in evaluating the structural and functional organization of the mammalian cell nucleus. Since several major classes (U1, U2, U4/U6, U5) of small nuclear ribonucleoprotein particles (snRNPs) play a crucial role in the processing of pre-mRNA molecules, we have been interested in the localization of these particles within the cell nucleus. Using pre-embedding immunoperoxidase labeling combined with 3-dimensional reconstruction, we have recently shown that nuclear regions enriched in snRNPs form a reticular network within the nucleoplasm which extends between the nucleolar surface and the nuclear envelope. In the present study we were inte rested in extending these nuclear localizations using cell preparation techniques which avoid slow penetration of fixatives, chemical crosslinking of potential antigens and solvent extraction. CHOC 400 cells were cryofixed using a CF 100 ultra rapid cooling device (LifeCell Corp.). After cryofixation cells were molecular distillation dried, vapor osmicated, in filtra ted in 100% Spurr resin in vacuo and polymerized in molds a t 60°C. Using this procedure we were able to evaluate the distribution of snRNPs in resin embedded cells which had not been chemically fixed, incubated in cryoprotectants or extracted with solvents.
Faculty Opinions recommendation of The functional organization of axonal mRNA transport and translation.
