Dissecting Fat cadherin function in vivo

脂肪钙粘附素在协调组织生长和组织方面具有重要和保守的作用。多么 这些细胞黏附分子协调地控制生长和图案形成仍然不是很清楚。至 针对这一需求，我们启动了基于CRISPR的果蝇体内诱变，并 用GFP标记的内源性Ft进行免疫共沉淀蛋白质的质谱分析。 对内源性FT的精细突变表明，Ft在控制双侧对称性方面起着关键作用。 对内源性复合体的蛋白质组学研究发现，核蛋白与Ft相互作用，提示了新的 Ft在转录调控中的作用。为了研究ft的这些作用，我们提出了三个具体目标。 1.勾画生长和PCP结构域，并确定体内的蛋白质相互作用。我们将使用体内基因 定义Ft结构域的编辑方法，该结构域调节平面细胞极性(PCP)组织结构和 成长。利用内源性Ft-GFP，我们确定了新的体内Ft结合伙伴。我们会确认这些 并调查它们在功能上的相关性。 2.定义了Ft在波动不对称(FA)中的作用。高度肥胖的区域的缺失 在果蝇和人类身上保存下来的结果是，存活的果蝇失去了对两侧对称性的精细控制。 这意味着Ft在双侧对称性的基础机制中起作用，并具有感觉或执行功能 精细的器官检查站。我们将测试Ft(PCP、河马或线粒体)已知输出的变化 功能)负责FA，并测试FA中涉及的基因是否受Ft影响。我们将确定 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.明确Ft在核内的作用。我们发现Ft的胞质结构域可以定位于 并与染色质重塑复合体相互作用。我们将定义NLS和NES序列，以及 确定删除内源性基因座中的那些序列的后果。我们将确定是否 核Ft特异性地影响河马、PCP信号或代谢，并研究Ft转录调控 目标基因。RNASEQ、CHIP-SEQ和体内报告分析将被用来确定 核堡垒。 如果成功，这些研究将阐明Ft在控制两侧对称性和 转录，并提供了对体内Ft不同功能的调节和整合的机械性洞察

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