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，组织被重组，过时的细胞被称为程序性细胞死亡(PCD)的过程破坏，该过程以不损害邻近细胞的方式破坏特定的细胞。现在，对哺乳动物细胞的研究清楚地表明，线粒体是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，目前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。这项拟议的研究将通过研究从烟草角虫(Manduca Sexta)中肠上皮分离的线粒体来揭示PCD期间昆虫线粒体功能的变化。这种上皮细胞是研究PCD期间线粒体功能的理想系统，因为幼虫中肠在可预测的发育时间死亡，而且上皮的巨大尺寸允许进行小组织或单细胞无法进行的生化研究。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)将去极化(消散)，细胞器将释放一种小的蛋白质，细胞色素c，进入细胞质。为了验证这一假设，在PCD过程开始之前和之后，将从变态不同阶段的幼虫中肠中分离出线粒体。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，还将测量线粒体和细胞质的细胞色素c含量，以确定这种蛋白释放到胞浆中是否是昆虫PCD的早期事件。线粒体功能的改变发生在PCD的早期，这一事实给细胞带来了一个有趣的两难境地。PCD的过程需要三磷酸腺苷，而产生三磷酸腺苷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，拟议的研究将使用代谢控制分析来量化氧化磷酸化-线粒体制造ATP的过程-在PCD期间是如何改变的。除了研究线粒体的功能外，拟议的研究还将使用电子显微镜来揭示中肠线粒体在变态和PCD期间的结构变化。最后，假设蜕皮激素，即蜕皮类固醇，是启动中肠上皮PCD的最终原因。这一假说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来检验，这些变化类似于变态过程中看到的变化。

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.