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). 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). RNAi against a group of 10 genes resulted in uneven distribution of NPCs (Joseph-Strauss et al 2012). Six out of these genes coded for Sm proteins, which are core components of the spliceosome. Down-regulation of other splicing factors did not affect NPC distribution, suggesting that Sm proteins may have a function that is independent of splicing. Down-regulation of Sm proteins also led to incomplete disassembly of NPCs during mitosis, but had no effect on lamina disassembly, suggesting that the defect in NPC disassembly was not due to a general defect in nuclear envelope breakdown. These mitotic NPC remnants persisted on an ER membrane that juxtaposes the mitotic spindle. At the end of mitosis, the remnant NPCs moved toward the chromatin and the reforming NE, where they ultimately clustered by forming membrane stacks perforated by NPCs. Our results suggest a novel, splicing-independent, role for Sm proteins in NPC disassembly, and point to a possible link between NPC disassembly in mitosis and NPC distribution in the subsequent interphase.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$520,688
Indirect Cost
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Rahman, Mohammad M; Munzig, Mandy; Kaneshiro, Kiyomi et al. (2015) Caenorhabditis elegans polo-like kinase PLK-1 is required for merging parental genomes into a single nucleus. Mol Biol Cell 26:4718-35
Rahman, Mohammad M; Rosu, Simona; Joseph-Strauss, Daphna et al. (2014) Down-regulation of tricarboxylic acid (TCA) cycle genes blocks progression through the first mitotic division in Caenorhabditis elegans embryos. Proc Natl Acad Sci U S A 111:2602-7
Heald, Rebecca; Cohen-Fix, Orna (2014) Morphology and function of membrane-bound organelles. Curr Opin Cell Biol 26:79-86
Joseph-Strauss, Daphna; Gorjánácz, Mátyás; Santarella-Mellwig, Rachel et al. (2012) Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos. Dev Biol 365:445-57
Cohen-Fix, Orna (2010) Cell biology: Import and nuclear size. Nature 468:513-6
Golden, Andy; Liu, Jun; Cohen-Fix, Orna (2009) Inactivation of the C. elegans lipin homolog leads to ER disorganization and to defects in the breakdown and reassembly of the nuclear envelope. J Cell Sci 122:1970-8