The outstanding preservation of Saccharomyces cerevisiae by high pressure freezing and freeze-substitution presents the opportunity to examine other cellular structures besides microtubules. The relatively small size of yeast cells makes it possible to reconstruct entire organelles from serial sections. We have turned our attention to nuclear envelope and nuclear pore complexes (NPCs). These essential organelles are required for macromolecular traffic between the nucleus and the cytoplasm. We have recently found that NPCs contain component proteins that appear to be shared with another nuclear envelope embedded organelle, the spindle pole body (SPB). These proteins were found to be the products of genes identified by mutant alleles defective in SPB assembly. We now wish to know if these genes are required for NPC assembly as well. No assay for NPC assembly in yeast has been devised. Furthermore, fundamental questions concerning NPCs in yeast are unanswered, such as the number of NPCs per cell, despite the expanding genetic and molecular analysis of this organelle. We have used 3D reconstruction of yeast nuclei from serial thin sections to determine the size and shape of the nucleus, and to determine the number of NPCs in individual nuclei from wild-type cells. Eighteen models of nuclei at different points of the cell cycle have been built and are being analyzed. Preliminary data suggests that nuclei in newly """"""""born"""""""" haploid cells contain approximately 60 to 70 NPCs. It appears that number of NPCs doubles before mitosis during which the NPC number remains stable. This data suggests that NPC assembly is limited to a specific part of the cell cycle in S. cerevisiae. Analysis of a mutant strain has begun. This mutant in a NPC component, NUP119 appears to affect the distribution of NPCs over the surface of the nucleus.
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