Date of Award
Doctor of Philosophy (PhD)
Molecular, Cellular, and Developmental Biology
In cell division, chromosomes align and attach to the mitotic spindle with high fidelity in order to limit missegregation of chromosomes that form individual nuclei, termed micronuclei. During cell division, membrane-bound organelles are cleared to the periphery of the cell; lack of clearance of membranes from chromosomes leads to chromosome missegregation. Cells regulate the biogenesis of their membranes throughout the cell cycle. Cancer cells frequently have upregulation of membrane lipid synthesis and micronuclei, but a connection between membrane biogenesis and chromosome missegregation leading to formation of micronuclei has not been established. In my thesis work, I show that the protein phosphatase CTDNEP1 regulates membrane biogenesis and formation of micronuclei in human cell lines. I elucidate how CTDNEP1 controls synthesis of ER membranes through its dephosphorylation and activation of the phosphatidic acid phosphatase lipin 1. I show that ER membrane abundance is increased in mitotic cells lacking CTDNEP1, and endoplasmic reticulum (ER) membranes are less cleared in prometaphase to metaphase. I show that CTDNEP1 has conserved functions for restricting membranes to the surface of the nuclear envelope during nuclear envelope assembly and for maintaining nuclear morphology. Using quantification of mitotic cells in a fixed asynchronous population, I corroborate the results of previous studies showing that CTDNEP1 is necessary for correct timing of mitotic progression. Errors in attachment to the mitotic spindle (that may or not be surveilled by the spindle assembly checkpoint) lead to chromosome missegregation that results in formation of micronuclei. Inhibition of the spindle assembly checkpoint in synchronized cells leads to a small increase in micronuclei in CTDNEP1-depleted cells. In contrast, transient spindle disassembly that causes unbalanced attachment errors not sensed by the spindle assembly checkpoint results in severely micronucleated nuclei in CTDNEP1-depleted cells, showing that micronuclei in CTDNEP1-depleted cells form through decreased mitotic error correction. Lipidomic analysis of total cellular lipids in CTDNEP1-depleted cells reveals that phosphatidylcholine/phosphatidylethanolamine are increased with loss of CTDNEP1. I show that inhibition of fatty acid synthesis suppresses ER membrane expansion in CTDNEP1-depleted cells. I observe that depletion of the fatty acid transcriptional regulators sterol regulatory element binding proteins 1 and 2 and stearoyl Co-A desaturase partially suppress ER membrane expansion, illuminating the role of fatty acid synthesis gene upregulation in expansion of ER membranes in CTDNEP1-depleted cells. I show that severe micronucleation after transient spindle disassembly and the incidence of micronuclei in untreated CTDNEP1-depleted cells is rescued with inhibition of fatty acid synthesis. These data elucidate the mechanism for how CTDNEP1 controls ER lipid synthesis in human cells. Together, these data support the conclusion that increased fatty acid synthesis leads to excess ER membranes that interfere with chromosome segregation in mitosis, leading to formation of micronuclei. This study provides the first connection to misregulation of lipid synthesis to formation of micronuclei, two events that are common in cancer cells. This study thus provides a link between regulation of lipid synthesis and chromosome segregation and informing our understanding of how they are altered in cancer.
Merta, Holly Elizabeth, "Analyzing the role of ER membrane biogenesis in mitotic fidelity" (2021). Yale Graduate School of Arts and Sciences Dissertations. 196.