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 aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in cells of 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 complete. 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 the nucleus has to acquire and maintain a spherical shape of a volume 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 focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a conserved regulator of phospholipids synthesis;in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER). In yeast, this is also accompanied by expansion of certain regions of the nucleus. In particular, we were able to show that only the NE 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 NE expansion when phospholipid levels increase. Our subsequent studied revealed that vesicle trafficking is involved in determining nuclear shape under conditions of excess membrane (e.g. in the absence of Spo7 function). The mechanism by which this happens is currently under investigation. To gain a better understanding of how the different domains of the NE are determined and to uncover additional proteins that affect nuclear shape, we conducted a high throughput screen in collaboration with Dr. Brenda Anderws'lab (University of Toronto), in which we screened through the yeast deletion collection for mutations that cause abnormal nuclear shape. Of the 5000 mutants tested, mutations in nearly one hundred genes resulted in altered nuclear morphology. We are currently screening through this subset to determine functional relationships between these genes and how they affect nuclear shape. Among the mutations that led to abnormal nuclear shape were roughly 40 mutants that caused a cell cycle delay in mitosis, prior to chromosome segregation. In all of these mutants the nucleus developed an extension that coincided with the NE associated with the nucleolus, much like in the spo7 mutant. We showed that the altered nuclear morphology in mutants that cause a cell cycle delay was not due to a direct involvement of these genes in nuclear architecture, but rather that a mitotic delay, in and of itself, leads to altered nuclear shape. Delay in other cell cycle stages does not result in changes to nuclear morphology. We also found that during a mitotic delay, phospholipids continue to accumulate at the same rate as in wild type cycling cells. Our data suggest that during a mitotic delay, cell continue to add membrane to the NE despite the block to chromosome segregation, and that under these circumstances the added membrane is not distributed evenly around the entire nucleus, but rather the NE expands only the region adjacent to the nucleolus. The mechanism that regulates this process is currently under investigation. To understand what regulated nuclear envelope expansion and how the nuclear envelope extension is confined to the nucleolus in mitotically arrested cells, we are in the process of identifying conditional (temperature sensitive) mutants that either fail to form an extension upon arresting in mitosis, or form multiple extensions at the non-permissive temperature. Thus far we have identified several mutants in the first class. These fall into two groups: mutants that expands the nuclear envelope isometrically and those that fail to expand altogether. We have also identified several mutants that exhibit abnormal nuclear shape upon gene inactivation. The identify of these mutants and the mechanisms by which they affect nuclear morphology are under investigation.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$489,421
Indirect Cost
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State
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Heald, Rebecca; Cohen-Fix, Orna (2014) Morphology and function of membrane-bound organelles. Curr Opin Cell Biol 26:79-86
Walters, Alison D; May, Christopher K; Dauster, Emma S et al. (2014) The yeast polo kinase Cdc5 regulates the shape of the mitotic nucleus. Curr Biol 24:2861-7
Cinquin, Bertrand P; Do, Myan; McDermott, Gerry et al. (2014) Putting molecules in their place. J Cell Biochem 115:209-16
Arnone, James T; Walters, Alison D; Cohen-Fix, Orna (2013) The dynamic nature of the nuclear envelope: Lessons from closed mitosis. Nucleus 4:
Webster, Micah; Witkin, Keren L; Cohen-Fix, Orna (2009) Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly. J Cell Sci 122:1477-86