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重要的过程，通过该过程，以有序的方式去除不需要的或有缺陷的细胞。因此，死亡细胞的吞噬必须迅速而完全，而不释放死亡细胞的内容物。细胞死亡的失败导致畸形和癌症。未能吞噬垂死的细胞也可能导致形态发生缺陷、图案错误、继发性坏死和炎症。细胞吞噬的研究主要集中在吞噬细胞。几种果蝇和C.已经鉴定了线虫吞噬所需的基因。令人惊讶的是，在垂死的细胞中不需要专门的。我们对垂死细胞内促进其吞噬的细胞变化知之甚少。我们最近取得了一个重要的进展，了解死亡细胞的吞噬在果蝇胚胎发育过程中，通过开发一个灵敏的检测吞噬活胚胎使用荧光，吞噬基板，我们称之为VGAL。该吞噬测定表明，吞噬模式忠实地反映了细胞死亡模式。令人惊讶的是，细胞吞噬的模式在不能激活其半胱天冬酶的胚胎中未受干扰，表明细胞吞噬的信号独立于半胱天冬酶激活级联反应。半胱天冬酶非依赖性细胞吞噬是一个新的和潜在的重要现象，我们知道的很少。半胱天冬酶非依赖性吞噬挑战了半胱天冬酶级联控制吞噬过程的教条。这项探索性的R21拨款提案旨在创建用于可视化和操纵体内吞噬的新工具。VGAL的一个重要限制是它必须被注射以检测吞噬，这限制了它在遗传筛查中的应用。第一个目标是开发基于GFP的细胞吞噬报告基因，其将提供用于吞噬缺陷的筛选方法并进一步证实半胱天冬酶非依赖性吞噬。这种基于GFP的报告基因也可用于研究自噬。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/促进其健康邻居吞噬垂死的细胞。这个模型试图解释为什么所有已知的吞噬突变都需要吞噬细胞，而不是垂死的细胞。这两个目标的成功完成将对发育调节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.