Dissecting Fat cadherin function in vivo

剖析脂肪钙粘蛋白的体内功能

基本信息

批准号：
10225527
负责人：
Helen McNeill
金额：
$ 45.54万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10225527
关键词：
Address Affect Bacterial Artificial Chromosomes Bilateral Binding Binding Proteins Biochemical Biological Cadherins Cell Adhesion Molecules Cell Line Cell Nucleus Cell-Cell Adhesion ChIP-seq Chromatin Remodeling Factor Clustered Regularly Interspaced Short Palindromic Repeats Communication Complex Craniofacial Abnormalities Cytoplasmic Tail Data Defect Development Disease Drosophila genus Ecdysone Embryo Fatty acid glycerol esters Genes Genetic Genetic Models Genetic Transcription Goals Growth Human Impairment Kidney Lead Left Limb structure Malignant Neoplasms Mass Spectrum Analysis Mediating Metabolism Mitochondria Molecular Mus Mutagenesis Mutation Nuclear Nuclear Proteins Nucleic Acid Regulatory Sequences Organ Organ Size Output Pathway interactions Pattern Phenotype Process Proteins Proteomics Regulation Reporter Role Sense Organs Signal Transduction Specificity Structure Testing Tissues Transcription Coactivator Transcriptional Regulation Transgenes Van Maldergem syndrome Wing Yeasts base experimental study fly human disease in vivo insight malformation man mutant novel organ growth overexpression planar cell polarity response transcriptome sequencing yeast two hybrid system

项目摘要

Fat cadherins have critical and conserved roles in coordinating tissue growth and tissue organization. How these cell-adhesion molecules coordinately control growth and patterning is still not well understood. To address this need, we have initiated comprehensive CRISPR-based mutagenesis of Drosophila ft in vivo, and used endogenous Ft tagged with GFP to identify co-immunoprecipitating proteins with mass spectrometry. Fine-scale mutagenesis of endogenous ft indicated Ft has critical roles in control of bilateral symmetry. Proteomic studies of endogenous complexes revealed nuclear proteins that interact with Ft, suggesting novel functions of Ft in transcriptional regulation. To investigate these roles of Ft we propose three specific Aims. 1. Delineate growth and PCP domains and define protein interactors in vivo. We will use in vivo gene editing approaches to define domains of Ft that regulate planar cell polarity (PCP) tissue organization and growth. Using endogenous Ft-GFP, we identified novel in vivo Ft binding partners. We will confirm these interactors and investigate their functional relevance. 2. Define the function of Ft in fluctuating asymmetry (FA). Deletion of a region of Fat that is highly conserved in Drosophila and humans results in viable flies that have lost fine control of bilateral symmetry. This implies Ft has a role in mechanisms underlying bilateral symmetry, and functions to sense or implement fine-scale organ checkpoints. We will test if alterations in known outputs of Ft (PCP, Hippo or mitochondrial function) are responsible for FA, and test if genes implicated in FA are affected by Ft. We will determine the tissue specificity of Ft in restricting FA, and investigate proteins that bind the D region for function in FA. 3. Define the function of Ft in the nucleus. We found the cytoplasmic domain of Ft can localize to the nucleus and interact with chromatin remodeling complexes. We will define NLS and NES sequences, and determine the consequences of deleting those sequences in the endogenous locus. We will determine if nuclear Ft specifically affects Hippo, PCP signaling or metabolism, and investigate Ft transcriptional regulation of target genes. RNAseq, ChIP-seq and in vivo reporter analyses will be used to determine the function of nuclear Ft. If successful, these studies will illuminate novel functions of Ft in control of bilateral symmetry and transcription, and provide mechanistic insight into regulation and integration of Ft’s diverse functions in vivo

脂肪钙粘蛋白在协调组织生长和组织组织方面具有关键且保守的作用。这些细胞粘附分子协调控制生长和图案化仍不清楚。为了满足这一需求，我们已经启动了果蝇体内基于 CRISPR 的全面诱变，并且使用带有 GFP 标记的内源 Ft 通过质谱法鉴定免疫共沉淀蛋白。内源 ft 的精细诱变表明 Ft 在控制双侧对称性中具有关键作用。内源复合物的蛋白质组学研究揭示了与 Ft 相互作用的核蛋白，这表明新的 Ft 在转录调控中的功能为了研究 Ft 的这些作用，我们提出了三个具体目标。 1. 描绘生长和 PCP 结构域并定义体内蛋白质相互作用因子我们将使用体内基因。编辑方法来定义调节平面细胞极性（PCP）组织组织的 Ft 域使用内源性 Ft-GFP，我们鉴定了新的体内 Ft 结合伙伴。交互者并研究它们的功能相关性。 2. 定义 Ft 在波动不对称性 (FA) 删除高度脂肪区域中的函数。在果蝇和人类中保守的果蝇会导致存活的果蝇失去对双边对称性的精细控制。这意味着 Ft 在双边对称性的机制中发挥着作用，并且具有感知或实施的功能我们将测试 Ft（PCP、Hippo 或线粒体）的已知输出是否发生变化。功能）负责 FA，并测试与 FA 相关的基因是否受 Ft 影响。 Ft 在限制 FA 中的组织特异性，并研究结合 D 区的蛋白质在 FA 中的功能。 3.定义Ft在细胞核中的功能我们发现Ft的细胞质结构域可以定位于细胞核。我们将定义 NLS 和 NES 序列，并与细胞核相互作用。确定删除内源基因座中的这些序列的后果我们将确定是否。核 Ft 特异性影响 Hippo、PCP 信号传导或代谢，并研究 Ft 转录调控 RNAseq、ChIP-seq 和体内报告基因分析将用于确定目标基因的功能。核英尺。如果成功，这些研究将阐明 Ft 在控制双边对称性和转录，并为 Ft 体内多种功能的调节和整合提供机制见解