Identification of Novel Cell Death Regulators in C. elegans and Drosophila

Citation

Copeland, Jeffrey Michael (2005) Identification of Novel Cell Death Regulators in C. elegans and Drosophila. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/7T3M-DY09. https://resolver.caltech.edu/CaltechETD:etd-06012005-114127

Abstract

Apoptosis is a form of cell death executed by a class of cysteine proteases called caspases. Though caspases are well-conserved, the mechanisms by which caspases are regulated vary remarkably. This dissertation addresses three independent aspects of apoptosis and its regulation.

In the developing Drosophila eye, apoptosis is activated to remove extra cells that are initially present between ommatidia. Mutants for the gene echinus have a disorganized eye structure due to a failure of these cell deaths to occur. We demonstrate that echinus resembles a deubiquitinating enzyme, that it is expressed in the pupal eye during the time of cell death, and that echinus acts genetically upstream or independently of the death-inducing genes head involution defective, reaper, and grim. Based on in vitro assays and the fact that the Echinus enzyme lacks a catalytic cysteine residue, we propose that echinus and its orthologs constitute a novel class of inactive deubiquitinating enzymes, perhaps functioning in a dominant-negative manner to inhibit deubiquitination of specific substrates.

In C. elegans, the model for caspase inhibition is quite different from that in Drosophila and in mammals. To look for genes that directly inhibit the CED-3 caspase, we screened a C. elegans cDNA library for CED-3 suppressors in the yeast S. cerevisiae and found several suppressors. Experiments in yeast suggest that one of these genes, Y39B6A.12, requires the prodomain of CED-3 for suppression, and ectopic expression in the Drosophila eye shows that it can suppress apoptosis induced by the Bcl2 family member Debcl.

In Drosophila, DIAP1 is the focal point in the regulation of apoptosis. To identify novel regulators of DIAP1, deficiency chromosomes spanning the Drosophila genome were screened for dominant modifiers of a diap1 knockdown phenotype. Nine deficiencies were isolated that cover no known regulators, and two modifiers were mapped to small genomic regions. This screen has provided a starting point for identifying some of the many uncharacterized genes that are involved in regulating apoptosis.

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