The nucleus is arguably one of the most important organelles in the cell, and yet surprisingly little is known about how its shape is maintained and what determines its size. The importance of nuclear shape is underscored by the fact that various diseases, including cancer and premature aging, are associated with changes in nuclear shape, and yet the relationship between nuclear shape and nuclear function is poorly understood. In most metazoan cells, the nuclear envelope (NE) undergoes cycles of assembly and disassembly in each and every cell cycle, and one of the key outstanding questions in the field is which proteins facilitate these processes. To gain insight into NE dynamics, we initiated the C. elegans nuclear architecture project. This project has two main components: (a) to examine in C. elegans the role of proteins identified in a yeast screen as being involved in nuclear architecture (project DK057807-06);and (b) to screen for additional gene/proteins involved in determining nuclear shape and size, and in affecting NE breakdown and reassembly. In the past year we focused on the second goal, while the first goal awaits the discovery of additional yeast genes involved in nuclear morphology. To identify C. elegans genes involved in NE processes we constructed a C. elegans strain with which we can monitor nuclear dynamics. This strain was treated with RNAi's from an RNAi collection of roughly 2000 genes known to cause embryonic lethality when inactivated. This screen resulted in over 300 genes that when down regulated cause a defect in nuclear morphology. Two types of morphologies were pursued: paired nuclei, a phenotype that is similar to that caused by lipin inactivation (see Golden et al 2009), and uneven distribution of nuclear pore complexes (NPCs) (Joseph-Strauss et al 2012). The analysis of other genes whose down regulation affects nuclear morphology is ongoing. A subgroup of RNAi's that led to a paired-nuclei phenotype resulted in the formation of paired nuclei specifically in the one-cell embryo. These RNAi's all encoded genes involved in energy metabolism, and in particular genes coding for TCA cycle enzymes. We found that the defect was in progression into mitosis: these RNAi's did not affect the processes that occur immediately after fertilization, but they blocked NE break down that normally occurs after the maternal and paternal pronuclei meet. We found that under these RNAi conditions neither nuclear pore complexes, nor the nuclear lamina, disassembled. Additionally, centrosomes duplicated but did not separate. We found that the underlying defect is in the removal of cyclin-dependent kinase (Cdk) inhibitory phosphorylation. Thus, it appears that abrogating the TCA cycle inhibits progression through mitosis in the first embryonic division of C. elegans by preventing Cdk activation (manuscript in preparation).

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