Nuclear architecture and nuclear function appear to go hand in hand: defects in nuclear organization are associated with aging and diseases such as cancer. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in cells of metazoans. There is also accumulating evidence to suggest that lamins contribute to various nuclear processes. (2) The yeast nuclear membrane remains intact throughout the cell cycle, unlike nuclear membrane of higher eukaryotes, which breaks down during mitosis. Nonetheless, in an earlier study from our lab (Campbell et al, 2006), we showed that the shape of the yeast nucleus is determined by three factors: the composition of the nuclear membrane, the shape of the chromatin, and the presence of an unidentified nuclear structure that tethers the nuclear membrane to the chromatin, akin to nuclear lamins of higher eukaryotes. Our previous study focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a regulator of phospholipids synthesis;in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER) and certain regions of the nucleus. In particular, we were able to show that only the membrane associated with the nucleolus (a sub-compartment of the nucleus) expands, whereas the rest of the nuclear membrane remains juxtaposed to the bulk of the chromatin. This led to the hypothesis that in yeast there is a nuclear tether that associates the nuclear membrane to the chromatin and resists membrane expansion when phospholipid levels increase. Based on this hypothesis, we assume that inactivating this tether will further alter nuclear shape, to the point where nuclear functions will be severely compromised, and this will be reflected in reduced cell viability. Thus, we sought mutations whose combination with a spo7 mutation leads to cell death or severe growth defects. We conducted a screen for randomly induced mutations that cause cell death in a yeast background lacking Spo7 function (known as a yeast synthetic lethal screen). This was followed by a secondary screen for mutants that have an altered nuclear shape. So far we have identified over a dozen mutations that lead to an altered nuclear shape. We are in the process of studying how these proteins affect nuclear shape and what role they play in coordinating nuclear architecture with nuclear function. We also combined mutations in candidate genes with the spo7 mutation, looking for reduced growth in the double mutant. The candidate genes were selected based on the function and localization of the protein for which they code;most were involved in processes that take place near or at the nuclear membrane. Through this screen we found a mutant that was known to affect spindle pole body function. The yeast spindle pole body is equivalent to the metazoan centrosome and it acts in nucleating spindle microtubules during chromosome segregation and in nucleating cytoplasmic microtubules that are needed for nuclear movement. The mechanism by which the spindle pole body is inserted and maintained in the nuclear membrane is not known, but this is a question of immense importance because the mechanism of nuclear membrane insertion could apply to other nuclear membrane structures, such as nuclear pore complexes. We found that additional mutations known to abrogate spindle pole body assembly also relay on the Spo7 pathway for viability. These findings suggest that the composition of the nuclear membrane affects the function of nuclear membrane associated structures. Our studies are now focused on understanding the nature of this defect, using cell biology and genetic methodologies. We are also collaborating with Dr. Sue Jaspersen in order to look at spindle pole bodies by electron microscopy and to determine the relationship between nuclear membrane composition and the assembly of integral membrane structures. Finally, in collaboration with Dr. Brenda Anderws'lab, we screened through the yeast deletion collection for mutations that cause abnormal nuclear shape. Of the 5000 mutants tested, mutations in over two hundred genes resulted in altered nuclear morphology. We are currently screening through this subset to determine functional relationships between these genes. This subset contains a wide range of functional groups but is enriched in genes involved in certain processes, including DNA repair and protein synthesis. Our goal is to determine which of these genes contribute directly to nuclear architecture and which do so in an indirect manner.