Nuclear architecture in budding yeast

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Nuclear architecture and nuclear function appear to go hand in hand, as 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 regards: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in metazoans, and (2) the yeast nuclear envelope (NE) remains intact throughout the cell cycle, unlike the NE of higher eukaryotes, which breaks down during mitosis and reassembles after chromosome segregation is completed. Nonetheless, the yeast nucleus shares important features with nuclei of higher eukaryotes: the NE has to expand during the course of the cells cycle, and during vegetative growth the nucleus maintains a spherical shape, with a volume that is proportional to cell volume. How the NE expands and what determine nuclear size and shape are questions that remain to be resolved in all systems. Our previous studies identified a number of conditions that lead to abnormal nuclear morphology, most notably elevated phospholipid synthesis and a cell cycle delay in mitosis. In both cases the alteration to nuclear shape is confined to the NE region adjacent to the nucleolus, and our studies showed that this confinement is dependent on the conserved polo-like kinase Cdc5. To further understand what affects nuclear morphology we sought additional proteins whose activity is required for normal nuclear size and shape. One of these turned out to be in the conserved topoisomerase 2 gene, TOP2. Cells with mutations in the TOP2 genes display multiple projection of the nuclear envelope that are not confined to the region adjacent to the nucleolus. Interestingly, only certain top2 mutants confer an abnormal nuclear shape, and this phenotype is correlated with abnormal DNA structure as determined by reduced DAPI staining. This suggests that DNA structure can affect nuclear morphology. This project is carried out in collaboration with Jeff Bachant's lab and should be submitted for publication in the coming year. To gain a better understanding of how nuclear envelope expansion is regulated, we are currently examining conditions, mutations and developmental states that affect nuclear expansion. One such state is exposure to mating pheromone, which causes the nuclear envelope to expand predominantly in the region adjacent to the nucleolus, much like in a mitotic delay. Using this system we are investigating whether expansion depends on phospholipid synthesis, and what signals are generated leading to nuclear envelope expansion. We will then examine whether principles that apply to nuclear envelope expansion upon exposure to pheromone also apply to mitosis nuclear envelope expansion.