A light-inducible protein trapping system for studying cellular dynamics in Drosophila

项目摘要/摘要 细胞动力学的时空调节是动物发育的基础。一次彻底的 对发育机制的理解需要允许体内扰动的方法 内源蛋白质以空间和时间受控的方式。这种方法尤其是 对于了解神经元的形态发生和神经性疾病的病因学很重要 通常占据广阔的空间域，并在不同的树突和轴突上表现出不同的生长动力学 树枝。然而，现有的在动物模型中操纵内源性基因功能的技术，如 基因敲除、RNAi和蛋白质降解影响整个细胞，需要时间才能生效，以及 因此缺乏解剖神经元动态生长行为所需的空间和时间分辨率 在亚细胞水平上。我们建议在果蝇中开发一种光遗传系统来实现快速抑制 在精确定义的细胞区域内的内源蛋白质。这将通过光诱导陷阱来实现 大蛋白质簇中GFP标记的内源蛋白质。这样的战略应该有效地抑制 其功能需要特定的亚细胞位置的蛋白质。我们的系统将在感觉神经元上进行测试 和果蝇幼虫的表皮上皮细胞，这两种细胞类型与广泛的人类 疾病。几种大小、亚细胞定位和功能不同的内源性标记蛋白质将被 首先在蛋白质捕获试验中进行了测试。为了验证光诱导蛋白质抑制的有效性，这些作用 利用GFP技术研究Rab5和Fry在果蝇感觉神经元树突形态发生中的作用 标记的内源蛋白。Rab5和Fry对于树枝状图案的形成很重要。然而，它是未知的 它们是通过局部调控树突状细胞的动力学还是通过整体调控基因来控制树突状细胞的生长 表情。通过局部抑制单个树突分支中的Rab5和Fry，将确定是否 它们调节局部树突的动态。这个项目的主要目标是建立第一个果蝇光- 用于研究内源性局部和力矩函数的可感生功能损失系统 体内的蛋白质。果蝇体内GFP标记的内源蛋白数量的增加 CRISPR/Cas9介导的基因组编辑的方便性使我们的方法适用于广泛的 各种蛋白质、生物过程和人类疾病。我们的方法也应该适用于其他 模型生物。

PROJECT SUMMARY/ABSTRACT Spatiotemporal regulation of cellular dynamics is fundamental to animal development. A thorough understanding of developmental mechanisms requires approaches that permit in vivo perturbation of endogenous proteins in a spatially and temporally controlled manner. Such approaches are particularly important for understanding neuronal morphogenesis and the etiology of neurological disorders, as neurons usually occupy broad spatial domains and exhibit diverse growth dynamics at different dendritic and axonal branches. However, existing techniques for manipulating endogenous gene function in animal models, such as gene knockout, RNAi, and protein degradation, affect the whole cell and require time to take effect, and therefore lack the spatial and temporal resolution needed for dissecting dynamic growth behaviors of neurons at the subcellular level. We propose to develop an optogenetic system in Drosophila to enable rapid inhibition of endogenous proteins in precisely defined regions of cells. This will be achieved by light-inducible trapping of GFP-tagged endogenous proteins in large protein clusters. Such a strategy should be effective for inhibiting proteins whose functions require specific subcellular locations. Our system will be tested in sensory neurons and epidermal epithelial cells of Drosophila larvae, two cell types that are relevant to a broad range of human diseases. Several endogenously tagged proteins of diverse size, subcellular localization, and function will be first tested in a protein trapping assay. To validate the effectiveness of light-induced protein inhibition, the roles of Rab5 and Fry in dendrite morphogenesis of Drosophila sensory neurons will be investigated using GFP- tagged endogenous proteins. Rab5 and Fry are important for dendritic patterning. However, it is unknown whether they control dendritic growth by locally regulating dendritic dynamics or by globally modulating gene expression. By locally inhibiting Rab5 and Fry in individual dendritic branches, it will be determined whether they regulate local dendritic dynamics. The primary goal of this project is to establish the first Drosophila light- inducible loss-of-function system for investigating the local and moment-to-moment function of endogenous proteins in vivo. The increasing number of endogenous proteins tagged by GFP in Drosophila and the convenience of CRISPR/Cas9-mediated genome editing make our approach applicable to the study of a wide variety of proteins, biological processes, and human diseases. Our approach should also be applicable in other model organisms.