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 树突的自我回避是许多神经系统发育的一个重要方面， 人们开始意识到它在神经发育障碍如自闭症中的重要性。自我- 回避是由神经元表面上的受体介导的，这些受体传递一种“代码”， 同细胞树突，这两个过程起源于同一个细胞。在两种脊椎动物以及 果蝇，该代码是由细胞粘附受体的随机选择性剪接产生的， 每个细胞都有独特的嗜同性粘附特性在果蝇中，这是由IG超家族粘附介导的 分子Dscam，而在脊椎动物中，它是由成簇的原钙粘蛋白（Pcdhs）介导的，这是类似的 到强粘附的经典钙粘蛋白。有缺陷的自我回避反映在异常的 果蝇姐妹树突之间的“树突桥”的持久性。我们的数据显示， 精确地位于哺乳动物神经元的树突桥接触点。取得了重大进展 识别这些分子家族及其敲除或干扰对神经系统的影响 体内发育尽管如此，至今尚未得到解答的是：如何做到表面上的胶粘剂状 分子在结合时会引起对膜的回避？这个答案可以让我们更清楚地了解 自我回避在神经发育障碍中是如何出错的。我们研究了细胞生物学 PCDHS的活动旨在回答这个问题。很明显，Pcdh在以下方面非常不同： 从相关的强粘附经典的细胞内运输到内溶酶体系统的术语 钙粘蛋白缺乏这种运输。我们发表的工作和新的初步数据表明，Pcdhs 在最初的粘附聚集后，通过触发 粘附部位的内吞作用。我们发现一种新的内吞调节因子FCHSD 2在Pcdh中富集， 复合物，并可能是触发Pcdh粘附后的内吞作用。这种内吞作用可能靶向其他 用于降解的前粘附分子。在这个R15提案中，我们计划开发一种新的自我检测方法， 避免在培养物中，因此许多突变体Pcdhs和FCHSD 2可以在结构-功能方法中评估。 我们将使用转染的Pcdh-GFP构建体，这是完全在体内功能，标记树突状桥 接触并研究他们的动态和命运。我们亦会敲除Pcdhs和FCHSD 2，并研究对 树突桥和自我回避。一旦这些检测建立起来，我们将改变内吞作用 和/或运输基序，并确定这些突变对自我回避和树突状细胞的影响。 桥梁. Pcdhs在自我回避中的替代信号通路也将被考虑。这些研究 将为自我回避的细胞机制提供新的线索他们还将扩大培训能力， 他是斯塔顿岛学院细胞和分子神经生物学专业的本科生。