Student Theses and Dissertations

Date of Award

2012

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Steller Laboratory

Abstract

The survival and development of a multicellular organism is reliant upon proper cell and organelle function, and the tight regulation of cell number. The endoplasmic reticulum (ER) is the cellular compartment where membrane and secretory pathway proteins are folded and modified for transport within or outside of the cell. When the ER becomes overwhelmed by the accumulation of unfolded or misfolded proteins, it enters a state of “ER stress” and the organelle activates the unfolded protein response (UPR) to alleviate the strain. One prominent event during ER stress and the UPR is the Ire1-mediated splicing of unspliced xbp1 (xbp1U) into the potent transcription factor spliced Xbp1 (Xbp1S). If the ER cannot return to homeostasis, the apoptotic program is activated. Programmed cell death (apoptosis) is an evolutionarily conserved, genetically controlled program which eliminates excess or irreparably damaged cells and it is necessary to maintain tissue integrity. The failure to properly activate or suppress cell death can greatly impact the health and survival of an organism. Cancer is caused by the failure to remove compromised cells leading to the generation of highly-proliferative and renegade cells which are harmful the animal. In contrast, excessive cell death in neuronal tissues is associated with a variety of neurodegenerative diseases. Research conducted by several groups has led to the identification of the primary regulators of the UPR and apoptosis in Drosophila. However, limited studies have taken place to isolate genes which specifically affect both the proximal event of the UPR and the distal event of apoptosis. In order to identify novel regulators of ER stress-induced apoptosis, an ex vivo assay was developed to screen 452 p-element lethal FRT lines generated by the UCLA Undergraduate Consortium in Functional Genomics. Drosophila is amenable for a modifier screen using the FLP/FRT technique, in which mitotic recombination is used to generate loss-of-function clones in specific tissues. In particular, the use of the FLP/FRT system allows clonal analysis of mutant genes in dispensable tissues of the fly when a gene cannot be easily studied in the homozygous condition. Eye imaginal discs, dissected from the progeny of selected p-element lethal FRT allele males mated to females with GFP-marked wild-type chromosomes were cultured in ER stress inducing conditions and analyzed for differential cleaved caspase 3 activity. CG8108 was identified in the screen as a strong loss-of-function suppressor of ER stress-induced apoptosis and the gene was selected for further study. This thesis shows that CG8108 is necessary for efficient splicing of xbp1 during the early phase of the UPR and for the activation of caspases at the final stages of ER stress-induced apoptosis. The function and protein expression pattern of CG8108 is currently unknown and this work provides the initial characterization of CG8108 localization during development.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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