Eat-Me Signaling Cascade in the Apoptotic Cell Engulfment

描述(由申请人提供)：由于缺乏研究这一过程的系统，我们对胞吐作用或细胞身体是如何被移除的理解在很大程度上受到了阻碍。从濒临死亡的细胞的角度来看，这个问题特别棘手。我们最近开发了一种基于pH的泡沫化读数，允许对果蝇细胞培养和体内发育的图像进行可靠的可视化和定量测量。我们新的基于细胞的分析方法使高通量分析能够识别在凋亡细胞中产生“Eat-Me”信号所必需的基因。除了寻求候选基因方法外，我们还将在果蝇细胞中实施高通量RNAi。通过整合几种强大的工具，包括全基因组RNAi文库、高通量高含量成像、计算图像分析、广泛的果蝇先前存在的遗传RNAi工具包、经典果蝇遗传学和大量预先汇编的基因表达数据，我们将发现与胞吐作用有关的新基因。额外的分析将帮助我们将识别的基因归入广泛的类别，如促凋亡基因或磷脂酰丝氨酸暴露所需的基因。为了评估候选基因在体内的功能，我们将测量注射RNAi的果蝇胚胎巨噬细胞中的身体负载。利用神经元中遗传驱动的RNAi对成年果蝇神经胶质细胞移除受损神经元的平行过程进行分析，将使我们能够识别最广泛的功能信号。这项拟议的研究将导致识别促进胞吐作用的基因，并将成为我们长期研究的基础。 在苍蝇和脊椎动物系统中的研究，以更好地了解人类疾病中的胞吐和细胞凋亡，并调节潜在的途径。在我们的研究中获得的知识可以用于在外周血细胞活动减弱的疾病中增加“Eat-Me”信号，例如癌症、动脉粥样硬化、一些呼吸系统疾病以及包括系统性红斑狼疮和类风湿性关节炎在内的自身免疫性疾病。我们对胞泡吞噬的理解对于包括神经变性在内的疾病也很重要，在这种疾病中，这一过程可能被上调。

DESCRIPTION (provided by applicant): Our understanding of efferocytosis or how the cell corpses are removed has been largely impeded by lack of amenable systems to study this process. This problem has been particularly intractable from the perspective of the dying cell. We recently developed a pH-based readout of efferocytosis that allows for reliable visualization and quantitative measurements of images from Drosophila cell culture and in vivo development. Our novel cell- based assay enables high-throughput analysis to identify genes necessary to produce "eat-me" signals in apoptotic cells. In addition to pursuing a candidate gene-approach, we will implement high-throughput RNAi in Drosophila cells. By integrating several powerful tools including the whole-genome RNAi libraries, high- throughput high-content imaging, computational image analysis, the extensive preexisting genetic RNAi toolkit in flies, classical Drosophila genetics, and a vast amount of pre-compiled gene expression data, we will identify novel genes involved in efferocytosis. Additional assays will help us place the identified genes into broad categories such as pro-apoptotic genes or genes required for phosphatidylserine exposure. To assess the function of gene candidates in vivo, we will measure the corpse load in the macrophages of RNAi-injected Drosophila embryos. An analysis in the parallel process of the removal of injured neurons by glia in adult flies with genetically driven RNAi in neurons will enable us to identify the most broadly-functional signals. The proposed research will lead to identification of genes that promote efferocytosis and will form the basis of our long-term studies in flies and vertebrate systems to better understand efferocytosis and apoptosis in human diseases and to modulate the underlying pathways. The knowledge gained in our studies could be applied to increase the "eat-me" signaling in diseases where efferocytic activity is diminished, such as cancer, atherosclerosis, a number of respiratory diseases, and autoimmune disorders that include systemic lupus erythematous and rheumatoid arthritis. Our understanding of efferocytosis is also important for disorders that include neurodegeneration, where this process may be up-regulated.