Mechanism of protocadherin-mediated self-avoidance

原钙粘蛋白介导的自我回避机制

• 批准号: 10291761
• 负责人: GREG R PHILLIPS
• 金额: $ 44.88万
• 依托单位: COLLEGE OF STATEN ISLAND
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291761
• 关键词: Adhesions; Adhesives; Alternative Splicing; Animal Model; Antibodies; Binding; Biological; Biological Assay; Brain; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell Line; Cell membrane; Cells; Cellular Neurobiology; Code; Complex; Cytoplasmic Tail; Data; Dendrites; Development; Down Syndrome Cell Adhesion Molecule; Drosophila genus; Electron Microscopy; Endocytosis; Endosomes; Ephrins; Exhibits; Extracellular Domain; Family; Functional disorder; Future; Goals; Image; Immunoglobulins; In Vitro; Invertebrates; Island; Knock-out; Length; Light; Link; Maps; Mediating; Membrane; Methods; Molecular; Molecular Neurobiology; Morphology; Mutate; Mutation; Nervous system structure; Neurodevelopmental Disorder; Neurons; PTK2 gene; Phospholipids; Postdoctoral Fellow; Process; Proteins; Proteomics; Publishing; Signal Pathway; Signal Transduction; Sister; Site; Specificity; Structure; Surface; System; Testing; Training; Variant; Vertebrates; Work; adhesion receptor; autism spectrum disorder; base; college; design; experimental study; in vivo; insight; knock-down; light microscopy; mutant; nervous system development; novel; protein protein interaction; receptor; recruit; trafficking; training opportunity; undergraduate student; uptake

Project summary Dendrite self-avoidance is a critical aspect of development in many neuronal systems and is just now beginning to be appreciated for its significance in neurodevelopmental disorders such as autism. Self- avoidance is mediated by receptors on the neuronal surface that convey a “code” that signals to a contacting same-cell dendrite that the two processes originate from the same cell. In both vertebrates as well as Drosophilia, the code is generated by stochastic alternative splicing of cell adhesion receptors that give a unique homophilic adhesive identity to each cell. In Drosophila, this is mediated by the Ig superfamily adhesion molecule Dscam, while in vertebrates, it is mediated by the clustered protocadherins (Pcdhs), which are similar to the strongly adhesive classical cadherins. Defective self-avoidance was reflected in the abnormal persistence of “dendritic bridges” between sister dendrites in Drosophila. Our data show that Pcdhs are precisely located at dendritic bridge contact points in mammalian neurons. Significant progress has been made on identifying these families of molecules and effects of their knockout or perturbations on nervous system development in vivo. Despite this, what has not yet been answered is: how do apparently adhesive-like molecules cause the avoidance of membranes when they bind? The answer could shed significant light on how self-avoidance might go wrong in neurodevelopmental disorders. We have studied the cell biological activity of the Pcdhs with the goal of answering this question. It is clear that the Pcdhs are very different in terms of their intracellular trafficking to the endolysosome system from the related strongly adhesive classical cadherins, which lack this trafficking. Our published work and new preliminary data suggest that the Pcdhs could cause the detachment of same-cell membranes, after initial adhesive clustering, by triggering endocytosis at the adhesive site. We found that a novel endocytosis regulator, FCHSD2, is enriched in Pcdh complexes and could be the trigger for endocytosis upon Pcdh adhesion. Such endocytosis might target other pro-adhesive molecules for degradation. In this R15 proposal, we plan to develop a novel assay for self- avoidance in culture, so that many mutant Pcdhs and FCHSD2 can evaluated in a structure-function approach. We will use transfected Pcdh-GFP constructs, which are fully functional in vivo, to mark dendritic bridge contacts and study their dynamics and fate. We will also knock down Pcdhs and FCHSD2 and study effects on dendritic bridges and self-avoidance. Once these assays are established, we will mutate the endocytosis and/or trafficking motifs in Pcdhs and determine the effects of these mutations on self-avoidance and dendritic bridges. Alternative signaling pathways for Pcdhs in self-avoidance will be considered as well. These studies will shed new light on the cellular mechanism of self-avoidance. They will also expand the capacity to train undergraduate students in cellular and molecular neurobiology at the College of Staten Island.

项目摘要 树突自我避免是许多神经元系统发展的关键方面，而现在 开始因其在自闭症等神经发育障碍中的意义而受到赞赏。自己- 避免是由神经元表面的接收器介导的，该神经元表面传达了“代码”，该“代码”发出信号 这两个过程源自同一单元的同一细胞树突。在两个脊椎动物中 果蝇，该代码是由细胞粘合剂受体的随机替代剪接生成的 对每个细胞的独特同质粘附身份。在果蝇中，这是由IG超家族粘合剂介导的 分子DSCAM，而在脊椎动物中，它是由簇的协议蛋白（PCDH）介导的，它们是相似的 到强烈的经典钙粘蛋白。自我避免有缺陷反映在异常中 果蝇姐妹树突之间的“树突桥”的持久性。我们的数据表明PCDH是 精确地位于哺乳动物神经元中的树突桥接触点。取得了重大进展 确定这些分子家族以及其敲除或对神经系统的扰动的影响 体内发展。尽管如此，尚未回答的是：显然如何像粘合剂一样 分子在结合时会避免膜的避免？答案可能会阐明 在神经发育障碍中，自我避免如何出错。我们研究了细胞生物学 PCDH的活动是为了回答这个问题。显然，PCDH在 相关粘合剂经典的细胞内贩运术语 钙粘蛋白，缺乏这种贩运。我们发表的工作和新的初步数据表明PCDHS 在初始粘合聚类之后，可能导致同一细胞膜的脱离，通过触发 粘附部位的内吞作用。我们发现一种新型的内吞作用调节剂FCHSD2富含PCDH 复合物，可能是PCDH粘合剂后内吞作用的触发因素。这种内吞作用可能针对其他 促粘性分子降解。在此R15建议中，我们计划为自我开发一种新颖的测定 在培养中避免，因此许多突变的PCDH和FCHSD2都可以通过结构功能方法进行评估。 我们将使用翻译的PCDH-GFP构建体，该构建体在体内功能完全实用，以标记树突状桥 接触并研究他们的动态和命运。我们还将击倒PCDH和FCHSD2以及对研究的影响 树突桥和自我避免。一旦建立了这些测定，我们将突变内吞作用 和/或PCDH中的贩运图案，并确定这些突变对自我避免和树突状的影响 桥梁。也将考虑PCDH自避免的替代信号通路。这些研究 将向自我避免的细胞机理开发新的启示。他们还将扩大培训的能力 史坦顿岛学院的细胞和分子神经生物学的本科生。