Engulfment of Dying Cells in Drosophila Embryos

描述(由申请人提供)：程序性细胞死亡是一个基本的过程，通过这个过程，不需要的或有缺陷的细胞被有序地移除。因此，死亡细胞的吞噬必须是迅速和完整的，而不能释放死亡细胞的内容物。细胞死亡失败会导致畸形和癌症。未能吞噬死亡细胞也可能导致形态发生缺陷、构型错误、继发性坏死和炎症。细胞吞噬作用的研究主要集中在吞噬细胞上。已经确定了果蝇和线虫吞噬所需的几个基因。令人惊讶的是，在垂死的细胞中不需要专门的。我们对死亡细胞内促进其吞噬的细胞变化知之甚少。最近，我们在理解果蝇胚胎发生过程中死亡细胞的吞噬方面取得了重要进展，开发了一种使用荧光吞噬底物的活胚胎吞噬的灵敏方法，我们称之为VGAL。这种吞噬分析表明，吞噬的模式真实地反映了细胞死亡的模式。令人惊讶的是，在无法激活caspase的胚胎中，细胞吞噬的模式没有受到干扰，这表明细胞吞噬的信号独立于caspase激活级联。Caspase非依赖性细胞吞噬是一种新的、潜在的重要现象，但我们对此知之甚少。不依赖于caspase的吞噬作用挑战了caspase级联控制吞噬过程的教条。这项探索性的R21拨款提案旨在创造新的工具，用于可视化和操纵体内的吞噬。VGAL的一个重要限制是必须注射才能检测到吞噬，这限制了它在基因筛查中的使用。第一个目标是开发一种基于GFP的细胞吞噬报告，它将提供一种筛选吞噬缺陷的方法，并进一步证实caspase非依赖性的吞噬。这位基于GFP的记者也可以用来研究自噬。第二个目标将测试一个新的假设，即垂死的上皮细胞内的细胞骨架变化允许/促进垂死的细胞被健康的邻居吞噬。这个模型试图解释为什么吞噬细胞需要所有已知的吞噬突变，而死亡细胞不需要。这两个目标的成功实现将对发育调控的细胞死亡和吞噬的研究产生重大影响。

DESCRIPTION (provided by applicant): Programmed cell death is an essential process by which unwanted or defective cells are removed in an orderly fashion. The engulfment of dying cells, therefore, must be rapid and complete, without releasing the contents of the dying cells. Failures in cell death lead to deformities and cancer. Failure to engulf dying cells may also result in morphogenetic defects, patterning errors, secondary necrosis and inflammation. The study of cell engulfment has primarily focused on the phagocytic cells. Several Drosophila and C. elegans genes required for engulfment have been identified. Surprisingly, none are required exclusively in the dying cell. We have very little understanding of the cellular changes within the dying cell that promote its engulfment. We recently made an important advance to understanding the engulfment of dying cells during Drosophila embryogenesis by developing a sensitive assay for engulfment in living embryos using a fluorogenic, engulfment substrate, which we call VGAL. This engulfment assay showed that the pattern of engulfment faithfully mirrors the pattern of cell death. Surprisingly, the pattern of cell engulfment was unperturbed in embryos that were unable to activate their caspases, indicating that the signal for cell engulfment is independent of the caspase activation cascade. Caspase-independent cell engulfment is a new and potentially important phenomenon that we know very little about. Caspase-independent engulfment challenges the dogma that the caspase cascade controls the engulfment process. This exploratory, R21 grant proposal is intended to create new tools for visualizing and manipulating engulfment in vivo. A significant limitation of VGAL is that it must be injected in order to detect engulfment, which limits its use for genetic screening. The first aim is to develop a GFP-based cell engulfment reporter that will provide a screening method for engulfment defects and further confirm caspase- independent engulfment. This GFP-based reporter can also be used to study autophagy. The second aim will test a novel hypothesis that cytoskeletal changes within dying, epithelial cells permit/promote the engulfment of dying cells by their healthy neighbors. This model attempts to explain why all known engulfment mutations are required in the engulfing cells, but not the dying cells. Successful completion of both aims will have a significant impact on the study of developmentally-regulated cell death and engulfment.