Engulfment of Dying Cells in Drosophila Embryos

果蝇胚胎中死亡细胞的吞噬

• 批准号: 7031123
• 负责人: JONATHAN S MINDEN
• 金额: $ 16.45万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031123
• 关键词: Drosophilidae; actins; apoptosis; arthropod genetics; autophagy; beta galactosidase; bioassay; cell death; cell growth regulation; clinical research; cytoskeleton; developmental genetics; embryogenesis; gene mutation; green fluorescent proteins; invertebrate embryology; lysosomes; reporter genes; technology /technique development

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.

描述（由申请人提供）：程序性细胞死亡是一个重要的过程，通过它以有序的方式去除不需要的或有缺陷的细胞。因此，垂死细胞的吞噬必须快速且完全，且不能释放垂死细胞的内容物。细胞死亡失败会导致畸形和癌症。未能吞噬垂死细胞还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。已经鉴定出果蝇和线虫吞噬所需的几种基因。令人惊讶的是，死亡细胞中没有一个是专门需要的。我们对垂死细胞内促进其吞噬的细胞变化知之甚少。最近，我们通过使用荧光吞噬底物（我们称之为 VGAL）开发了一种灵敏的活体胚胎吞噬测定方法，在了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。这种吞噬测定表明吞噬模式忠实地反映了细胞死亡的模式。令人惊讶的是，在无法激活半胱天冬酶的胚胎中，细胞吞噬的模式不受干扰，这表明细胞吞噬的信号独立于半胱天冬酶激活级联。不依赖半胱天冬酶的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。不依赖半胱天冬酶的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 资助提案旨在创建用于可视化和操纵体内吞噬的新工具。 VGAL 的一个显着限制是必须注射才能检测吞噬，这限制了其在基因筛查中的应用。第一个目标是开发基于 GFP 的细胞吞噬报告基因，为吞噬缺陷提供筛选方法，并进一步确认不依赖 caspase 的吞噬。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，即死亡上皮细胞内的细胞骨架变化允许/促进健康邻居吞噬死亡细胞。该模型试图解释为什么吞噬细胞需要所有​​已知的吞噬突变，而不是垂死细胞。这两个目标的成功完成将对发育调控细胞死亡和吞噬的研究产生重大影响。