Dissecting Fat cadherin function 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 这些细胞粘附分子协调控制生长和图案化仍然没有很好地理解。到 为了满足这一需求，我们已经启动了果蝇体内全面的基于CRISPR的诱变， 使用GFP标记的内源性Ft，用质谱法鉴定共免疫沉淀蛋白。 内源性ft的精细突变表明，Ft在控制双侧对称性中具有关键作用。 内源性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的目标。 1.描绘生长和PCP结构域并定义体内蛋白质相互作用物。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）组织的Ft结构域， 增长使用内源性Ft-GFP，我们确定了新的体内Ft结合伴侣。我们将确认这些 相互作用，并研究其功能相关性。 2.定义Ft在波动不对称性（FA）中的作用。删除一个区域的脂肪，这是高度 在果蝇和人类中保守的基因导致失去对两侧对称性的精细控制的可存活的果蝇。 这意味着Ft在双侧对称性的机制中发挥作用，并具有感知或实施功能。 精细的器官检查站我们将测试已知的Ft输出（PCP、Hippo或线粒体）是否发生改变， 功能）负责FA，并测试涉及FA的基因是否受到Ft的影响。康贝特人将以 组织特异性的Ft限制FA，并研究蛋白质结合D区的功能在FA。 3.定义Ft在原子核中的作用。我们发现Ft的胞质结构域可以定位于 细胞核和染色质重塑复合物相互作用。我们将定义NLS和内斯序列， 确定在内源基因座中缺失那些序列的后果。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 目标基因。将使用RNAseq、ChIP-seq和体内报告基因分析来确定 核子堡 如果成功的话，这些研究将阐明Ft在控制双侧对称性方面的新功能， 转录，并提供机制洞察调控和整合Ft的多种功能在体内

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