Eat-Me Signaling Cascade in the Apoptotic Cell Engulfment

描述（由申请人提供）：由于缺乏合适的系统来研究这一过程，我们对efferocytosis或细胞尸体如何被移除的理解在很大程度上受到阻碍。从垂死细胞的角度来看，这个问题尤其棘手。我们最近开发了一种基于ph值的efferocytosis读数，可以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。我们新颖的基于细胞的检测方法能够进行高通量分析，以鉴定凋亡细胞中产生“吃我”信号所需的基因。除了寻求候选基因方法外，我们还将在果蝇细胞中实现高通量RNAi。通过整合几个强大的工具，包括全基因组RNAi文库、高通量高含量成像、计算图像分析、广泛的果蝇遗传RNAi工具包、经典果蝇遗传学和大量预编译的基因表达数据，我们将识别出与efferocytosis有关的新基因。额外的分析将帮助我们将鉴定的基因归类为大类，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的尸体负荷。对成年果蝇中神经胶质细胞去除损伤神经元的平行过程进行分析，将使我们能够识别出功能最广泛的信号。提出的研究将导致识别基因，促进efferocytosis和将形成我们的长期研究的基础

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.