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

用于研究果蝇细胞动力学的光诱导蛋白捕获系统

• 批准号: 9387859
• 负责人: Chun Han
• 金额: $ 18.73万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387859
• 关键词: Adaptor Signaling Protein; Affect; Afferent Neurons; Alpha Cell; Animal Model; Animals; Behavior; Biological Assay; Biological Models; Biological Process; CRISPR/Cas technology; Cells; Communities; Complex; Darkness; Dendrites; Development; Diffuse; Disease; Drosophila genus; Drosophila inturned protein; Effectiveness; Engineering; Environment; Epithelial Cells; Etiology; Exhibits; Family; Future; Gene Expression; Gene Expression Regulation; Goals; Growth; Human Development; Image; Individual; Intracellular Membranes; Knowledge; Larva; Light; Location; Measures; Mediating; Membrane; Methods; Molecular; Morphogenesis; Morphology; Neurodegenerative Disorders; Neurons; Outcome; Pattern; Problem Solving; Process; Protein Inhibition; Proteins; Publishing; RNA Interference; Reagent; Regulation; Research; Resources; Role; System; Tauopathies; Techniques; Testing; Thinness; Time; cell growth regulation; cell type; design; gene function; genome editing; human disease; in vivo; innovation; knockout gene; light gated; loss of function; nervous system disorder; novel; optogenetics; prevent; protein degradation; spatiotemporal; success; temporal measurement; tool; trafficking

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.

项目概要/摘要 细胞动力学的时空调节是动物发育的基础。彻底的 对发育机制的理解需要允许体内扰动的方法 以空间和时间控制的方式内源性蛋白质。此类方法特别 对于理解神经元形态发生和神经系统疾病的病因学很重要，因为神经元 通常占据广阔的空间域，并在不同的树突和轴突处表现出不同的生长动态 分支机构。然而，现有的在动物模型中操纵内源基因功能的技术，例如 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，并且 因此缺乏剖析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。我们建议在果蝇中开发光遗传学系统以实现快速抑制 精确定义的细胞区域中的内源蛋白质。这将通过光诱导捕获来实现 大型蛋白质簇中带有 GFP 标记的内源蛋白质。这样的策略应该能够有效抑制 其功能需要特定亚细胞位置的蛋白质。我们的系统将在感觉神经元中进行测试 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相关 疾病。几种具有不同大小、亚细胞定位和功能的内源标记蛋白将被 首先在蛋白质捕获测定中进行测试。为了验证光诱导蛋白抑制的有效性，作用 将使用 GFP- 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 对于树突图案化很重要。然而，尚不清楚 它们是通过局部调节树突动态还是通过全局调节基因来控制树突生长 表达。通过局部抑制单个树突分支中的 Rab5 和 Fry，将确定是否 它们调节局部树突动力学。该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即时功能的诱导性功能丧失系统 体内的蛋白质。果蝇中 GFP 标记的内源蛋白数量不断增加 CRISPR/Cas9 介导的基因组编辑的便利性使我们的方法适用于广泛的研究 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 模式生物。