Mechanism of protocadherin-mediated self-avoidance

项目总结 树突自我回避是许多神经系统发育的一个关键方面，而且现在才刚刚发现。 开始因其在自闭症等神经发育障碍中的重要性而受到重视。自我- 回避是由神经元表面的感受器介导的，这些感受器传递信号给接触的 两个突起来自同一细胞的同一细胞树突。在这两种脊椎动物以及 果蝇，这种密码是由细胞黏附受体的随机选择性剪接产生的，它给出了一个 每个细胞都有独特的亲同性粘附性标识。在果蝇中，这是由Ig超家族黏附介导的 Dscam分子，而在脊椎动物中，它是由聚集的原钙粘附素(Pcdhs)介导的，这两个分子类似 粘附性很强的经典钙粘附素。有缺陷的自我回避反映在异常的 果蝇姊妹树突间“树突桥”的持久性。我们的数据显示，Pcdh是 精确定位于哺乳动物神经元的树突桥接触点。已经取得了重大进展 关于这些分子家族的识别及其敲除或扰动对神经系统的影响 体内发育。尽管如此，尚未得到回答的是：表面上类似粘合剂的物质是如何 当分子结合时，会导致膜的避免吗？答案可能会对以下问题产生重大影响 自我回避如何在神经发育障碍中出错。我们已经研究了细胞生物学。 委员会的活动目的是回答这一问题。很明显，Pcdh在以下方面非常不同 它们从相关的强黏附经典的细胞内转运到内溶酶系统的条件 钙粘附素，它缺乏这种贩运。我们已发表的工作和新的初步数据表明， 在最初的粘附性聚集之后，可能通过触发 粘连部位的内吞作用。我们发现一种新的内吞调节因子FCHSD2富含PcdH 可能是Pcdh黏附时的内吞作用的触发因素。这种内吞作用可能针对其他 用于降解的亲粘性分子。在这个R15提案中，我们计划开发一种新的自我检测方法 在培养中避免，以便许多突变体Pcdhs和FCHSD2可以用结构-功能方法进行评估。 我们将使用体内具有全功能的转基因Pcdh-GFP构建物来标记树突桥 联系并研究他们的动态和命运。我们还会拆除Pcdhs和FCHSD2，并研究 树突桥和自我回避。一旦这些化验结果成立，我们就会改变内吞作用 和/或Pcdhs中的运输基序，并确定这些突变对自我回避和树突的影响 大桥。Pcdh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 斯坦顿岛学院细胞和分子神经生物学专业的本科生。

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.