RUI:Protocadherin cytoplasmic interactions, trafficking and anti-adhesion

在从果蝇到人类的各种生物的大脑中，发育中的神经元通常通过特定类型的受体相互识别。众所周知，生长中的神经元表面可以通过称为细胞粘附分子的受体粘附到其他神经元和神经胶质细胞上。然而，来自同一神经元的轴突和树突必须相互避开，这样它们才不会交叉。原钙粘蛋白（Pcdhs）是一种与传统细胞粘附分子相关的蛋白质，但似乎介导了脊椎动物神经系统的自我回避过程。本项目旨在研究神经元实现自我回避的抗粘附过程的机制。本项目将招募本科研究人员，在首席研究员和经验丰富的研究生和/或研究助理的指导下，利用尖端的分子生物学和细胞成像技术研究Pcdh介导的细胞抗粘附。这个项目将有助于理解神经发育。这些本科生从这个项目中获得的经验将成为他们在教育、生物学研究、医学和牙科学校以及卫生保健相关领域的其他研究生课程的职业培训的重要组成部分。这个项目将研究一类有趣的神经细胞受体，集群原钙粘蛋白（Pcdhs），复杂脊椎动物大脑的标志，在发育过程中如何在连接神经元中起作用。Pcdhs类似于细胞表面粘附分子，但主要在包括核内体/溶酶体系统在内的内部区室中运输。据推测，这种细胞内运输可能与Pcdhs的抗粘附特性有关。一个控制细胞内运输的Pcdh家族成员的细胞质结构域的氨基酸片段（称为VCD基序）已经被绘制出来。该片段和其他Pcdh家族成员中类似片段的功能需要进一步阐述，Pcdh如何导致细胞膜分离需要进一步研究。在这个项目中，来自Pcdh亚家族代表的VCD基元将通过定点诱变和转运试验进行研究。这些基序的物理相互作用的能力也将被表征。VCD基序功能的关键残基将被识别并用于创建运输缺陷的Pcdh突变体，这些突变体将用于研究在抗粘附过程中正确的细胞内运输的要求。野生型和突变型Pcdhs从一个相互作用的细胞到另一个相互作用的细胞进行反内吞作用的能力，这是一种被提出的抗粘附机制，将通过相关的光学和电子显微镜来表征。最后，我们将描述Pcdh介导的细胞-细胞相互作用如何通过改变溶酶体降解的速率来影响其他细胞表面粘附分子的水平，并通过定量蛋白质组学确定Pcdh相互作用对蛋白质的上调或下调。这些实验的完成将有助于更好地理解Pcdhs如何影响神经发育中的相互作用细胞。

In the brains of organisms as diverse as fruit flies to humans, developing neurons generally recognize one another through specific types of receptors. It is well-known that The surfaces of growing neurons can adhere to other neurons and glial cells through receptors called cell adhesion molecules. However, axons and dendrites from the same neuron must avoid each other so that they do not cross. The protocadherins (Pcdhs) are proteins that are related to conventional cell adhesion molecules, but appear to mediate the self-avoidance process in the vertebrate nervous system. This project aims to study the mechanism of the anti-adhesive process by which neurons achieve self-avoidance. This project will recruit undergraduate researchers, under the guidance of the principal investigator and experienced graduate students and/or research associates, to study Pcdh mediated anti-adhesion in cells using cutting-edge molecular biological and cellular imaging techniques. The project will contribute to the understanding of neural development. The experience that these undergraduates receive from this project will be an essential part of their training for careers in education, biology research, as well as preparation for medical and dental schools and other postgraduate programs in health care-related fields. This project will study how an interesting class of neural cell receptors, the clustered protocadherins (Pcdhs), hallmarks of complex vertebrate brains, function in wiring neurons during development. The Pcdhs resemble cell surface adhesion molecules but are prominently trafficked in internal compartments including the endosome/lysosome system. It is hypothesized that this intracellular trafficking might be related to the anti-adhesive properties of Pcdhs. An amino acid segment of the cytoplasmic domain from one Pcdh family member that controls intracellular trafficking (termed the VCD motif) has been mapped. The function of this segment and similar segments in other Pcdh family members needs to be elaborated and how Pcdhs cause cell membranes to detach needs to be studied. In this project, VCD motifs from representatives of Pcdh subfamilies will be studied by site-directed mutagenesis and trafficking assays. The ability of these motifs to physically interact will also be characterized. Critical residues for VCD motif function will be identified and used to create trafficking defective Pcdh mutants that will be used to study the requirements for correct intracellular trafficking in the anti-adhesive process. The ability of wild-type and mutant Pcdhs to undergo trans-endocytosis from one interacting cell to the other, a proposed mechanism for anti-adhesion, will be characterized by correlative light and electron microscopy. Finally, how Pcdh mediated cell-cell interaction affects the levels of other cell-surface adhesion molecules by altering the rate of lysosomal degradation will be characterized and proteins up- or down-regulated by Pcdh interaction identified by quantitative proteomics. Completion of these experiments will lead to a better understanding of how Pcdhs affect interacting cells in neural development.