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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中具有关键和保守的作用。如何