The long term objective of the proposed research is to understand the early protein changes that occur during developmentally regulated apoptosis in Drosophila embryos. Developmentally regulated cell death is a complex and dynamic process, with many input signals and many downstream cellular responses. These cell death inputs funnel through a highly conserved caspase activation cascade that leads to the systematic dismantling of the cell. Using a proteomics screening method developed in his laboratory, the investigator discovered that a specific isoform of the ubiquitous small heat shock protein, HSP27, increases in embryos with elevated levels of cell death. This HSP27 change, which is most likely a change in phosphorylation, occurs early in apoptosis, before any overt signs of nuclear decay. Removal of Hsp27 by mutation, or reduction by RNA interference, causes a dramatic loss of cell death in embryos and adult tissues, as evidenced by a lack of acridine orange staining of apoptotic nuclei in embryos and the rescue of defects caused by over-expression of pro-apoptotic genes in adult tissue. In mammalian systems, heat shock proteins, including Hsp27, have been shown to serve both pro- and anti-apoptotic functions. An important difference between Drosophila and mammalian apoptosis is that release of cytochrome c and the involvement of the Bcl-family of proteins, which does not appear to play as central a role in triggering cell death in Drosophila as in mammalian cells. Thus the cell death pathway in Drosophila appears to be more streamlined than in mammals. The investigators propose to take advantage of Drosophila's amenability to genetic, cell biological, and biochemical dissection to establish where in the cell death pathway HSP27 functions and how it is regulated. Moreover, analysis of cell death in Drosophila allows one to assess apoptosis in the context of an intact, developing organism. Three sets of aims are described to address the following questions: (1) How does Hsp27 function in embryonic cell death? Genetic rescue and replacement methods will be used to determine if phosphomimetic and non-phosphorylatable forms of Hsp27 are able to rescue the loss-of-cell-death phenotype of a null Hsp27 mutation. (2) How does Hsp27 function in the apoptotic pathway in eye and wing development? Dominant suppression assays and epistasis experiments will be used to determine where in the apoptotic pathway Hsp27 operates. (3) What is the molecular basis for Hsp27 function in cell death? Co-immunoprecipitation and protein-folding assays as well as proteomic analysis will be used to study the molecular mechanisms of Hsp27's role in apoptosis. These aims are intended to lay the foundation for studying Hsp27's role in Drosophila cell death. These studies will lead to a better understanding of the molecular events and factors required for this vitally important process. RELEVANCE: Human development, as well as continued health, depends on the removal of excess and defective cells by a cell death process called apoptosis. The molecular mechanisms of apoptosis are highly conserved from fruit flies to man. Failures in apoptosis during development lead to severe birth defects and death;while failures in cell death in adults lead to diseases such as cancer, where cells resist apoptosis, or diabetes and Alzheimer's disease, where cells die prematurely. In this application, the investigators intend to use genetics, cell biology, and biochemistry to study the role Hsp27, a conserved, ubiquitous protein, plays in fruit fly apoptosis.