The dramatic and predictable changes in external and internal structures during molting and metamorphosis make insects ideal systems for studying the mechanisms underlying animal development. During the developmental process, tissues are restructured and obsolete cells are destroyed by a process called programmed cell death (PCD), which destroys specific cells in a manner that does not damage neighboring cells. It is now clear from studies on mammalian cells that mitochondria, organelles that provide most of the energy (ATP) for the cell, play a key role in the early steps of PCD. It is not known, however, if these organelles play a role in this process in insect cells. The proposed research will reveal changes in insect mitochondrial function during PCD by studying the mitochondria isolated from the midgut epithelium of the tobacco hornworm (Manduca sexta). This epithelium is an ideal system for studying mitochondrial function during PCD because the larval midgut dies at a predictable time in development and the epithelium's large size allows for biochemical studies not possible on small tissues or single cells. If insect mitochondria are involved in PCD in a manner similar to that in mammalian cells, then early in the PCD process the mitochondrial membrane potential (DY) will depolarize (dissipate) and the organelle will release a small protein, cytochrome c, into the cytoplasm. To test this hypothesis, mitochondria will be isolated from the midguts of larvae at different stages of metamorphosis, before and after the process of PCD begins. The DY of isolated mitochondria will be determined by monitoring the transmembrane distribution of a lipophilic cation. In addition, the cytochrome c content of the mitochondria as well as the cytoplasm will be measured to determine if release of this protein into the cytosol is an early event in insect PCD. The fact that changes in mitochondrial function occur early in PCD presents an interesting dilemma for cells. The process of PCD requires ATP, yet the organelle that generates ATP is being altered during the cell death process. In order to understand how cells deal with this problem, the proposed research will employ metabolic control analysis to quantify how oxidative phosphorylation, the process by which mitochondria make ATP, is altered during PCD. In addition to studying mitochondrial function, the proposed studies will use electron microscopy to reveal how the structure of midgut mitochondria changes during metamorphosis and PCD. Finally it is hypothesized that the molting hormones, the ecdysteroids, are ultimately responsible for initiating PCD in the midgut epithelium. This hypothesis will be tested by studying whether an ecdysteroid agonist effects structural and functional changes in midgut mitochondria similar to those seen during metamorphosis.
