Identification of new components of the Toll receptor signaling pathway in Drosophila

点击翻译按钮获取中文摘要

First discovered for its role in embryonic dorsal/ventral (DV) patterning, studies of Toll receptor signaling in Drosophila have led to major advances in our understanding of innate immunity and inflammation in humans, and of the regulation of eukaryotic gene expression. In the early single-cell fly embryo, Toll is distributed throughout the plasma membrane, but only activated ventrally. This is mediated by three sequentially-acting extracellular serine proteases, the last of which, Easter, cleaves a precursor form of the Toll ligand on the ventral side of the embryo. Ventral activation of Easter is controlled by Pipe, a Golgi- localized sulfotransferase that is expressed in ventral cells of the follicular epithelium that surrounds the developing oocyte. Pipe generates a sulfated cue that becomes embedded ventrally in the eggshell. Many aspects of this pathway remain to be elucidated, including the precise molecular nature of the direct substrate of Pipe (protein, carbohydrate, or lipid?), and the mechanism through which the Pipe target directs the ventral activation of Easter. In addition to the ventral restriction of pipe expression, uniform low levels of pipe expression around the developing oocyte results in embryos with residual DV polarity, indicating that there exists another, previously undetected mechanism that can define embryonic DV polarity. This application requests support for a pilot project, the objective of which is to utilize a novel approach, proximity labeling by in vivo biotinylation, to identify new components of the Toll receptor signaling pathway. The rationale is that because most of the known components were identified in screens for strict maternal effect mutants, genes encoding pathway components required for viability of females to fertile adulthood are likely to have been overlooked. Proximity labeling identifies factors that interact or colocalize with proteins-of- interest and is not limited to proteins that are dispensable for survival. The following specific aims will be pursued: 1. To identify new regulators and effectors of the serine proteases operating in the perivitelline space we will express them in the female germline as fusions to newly-developed, highly active biotin ligases, then perform proximity labeling in progeny embryos. 2. To identify proteins that interact with or colocalize in the Golgi with Pipe we will perform proximity labeling with Pipe-biotin ligase fusions expressed in ovarian follicle cells. For both specific aims, a subset of the identified presumed interactors will be tested for effects upon embryonic DV patterning. The identity of previously unappreciated pathway components will provide further insight into the control of Toll activity and stimulate new avenues of research. In addition, this project will also serve as a feasibility study of the extent to which proximity labeling can effectively identify bona fide protein/protein interactions in developing embryos and the extent to which the availability of useful genetic backgrounds, for example mutants that alter the subcellular localization of Easter or Pipe, can enhance the effectiveness of this approach.