Mitochondrial Function During Insect Programmed Cell Death

昆虫在蜕皮和变态过程中外部和内部结构的戏剧性和可预测的变化使昆虫成为研究动物发育机制的理想系统。在发育过程中，组织被重建，过时的细胞被称为程序性细胞死亡（PCD）的过程破坏，该过程以不损害相邻细胞的方式破坏特定细胞。现在从对哺乳动物细胞的研究中可以清楚地看到，线粒体，为细胞提供大部分能量（ATP）的细胞器，在PCD的早期步骤中发挥着关键作用。然而，尚不清楚这些细胞器是否在昆虫细胞的这一过程中发挥作用。该研究将通过研究烟草天蛾（Manducasexta）中肠上皮细胞分离的线粒体，揭示昆虫线粒体功能在PCD过程中的变化。这种上皮是研究PCD期间线粒体功能的理想系统，因为幼虫中肠在发育中的可预测时间死亡，并且上皮的大尺寸允许对小组织或单细胞进行不可能的生化研究。如果昆虫线粒体以类似于哺乳动物细胞的方式参与PCD，那么在PCD过程的早期，线粒体膜电位（DY）将降低（消散），细胞器将释放一种小蛋白质，细胞色素c，进入细胞质。为了验证这一假设，线粒体将被分离从幼虫中肠在不同阶段的变态，之前和之后的过程中的PCD开始。分离的线粒体的DY将通过监测亲脂性阳离子的跨膜分布来确定。此外，将测量线粒体以及细胞质的细胞色素c含量，以确定该蛋白质释放到胞质溶胶中是否是昆虫PCD中的早期事件。事实上，线粒体功能的变化发生在PCD的早期，这对细胞来说是一个有趣的困境。PCD的过程需要ATP，但产生ATP的细胞器在细胞死亡过程中被改变。为了了解细胞如何处理这个问题，拟议的研究将采用代谢控制分析来量化氧化磷酸化，即线粒体制造ATP的过程，在PCD期间如何改变。除了研究线粒体功能外，拟议的研究还将使用电子显微镜来揭示中肠线粒体的结构在变态和PCD期间如何变化。最后，据推测，蜕皮激素，蜕皮激素，最终负责启动PCD中肠上皮细胞。这一假设将通过研究蜕皮激素激动剂是否影响中肠线粒体的结构和功能变化进行测试，类似于变态过程中所看到的。

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.