Cell adhesion mediated by LINKIN

项目摘要 细胞黏附是发育、受精、组织内稳态、保护和相互作用的关键方面 和其他生物体一起。许多黏附蛋白已经被深入研究了几十年，它是一个开放的 质疑我们是否对粘连有足够的了解，因为有大量相互作用的蛋白质和 与粘合有关的相互作用。Linkin是一种保守的跨膜蛋白，我们发现它与 在细胞黏附方面，表明通过研究Linkin及其相互作用的蛋白质，我们可以定义一个新的细胞 参与细胞黏附的途径。Linkin序列和基因拷贝数的显著保守性 整个真核生物的进化表明了它在发育中的关键作用。我们发现了林肯在细胞中的作用 线虫发育过程中的黏附，特别是迁移连接细胞与 输精管，这为其结构、功能和相互作用提供了一个强有力的分析方法。我们确认了 在人类细胞中相互作用的蛋白质，并为两个生物体中类似的分子相互作用提供了证据。 我们鉴定了RUVBL1和RUVBL2AAA-ATPase和α-微管蛋白是Linkin的细胞质相互作用因子， 提示Linkin在调节微管动力学中的作用，但需要进一步的研究才能联系起来 连接到其他相互作用者和信号通路。我们建议进一步描述Linkin在细胞中的作用 黏附，以便将这种新的黏附分子连接到其他已知(或未知)的细胞途径。一个 人类遗传、基因组和系统生物学方法的主要局限性是我们对其功能的理解 由许多基因组成。通过发现一条新的途径，我们将增加基因组、临床和 基因研究将是可解释的。我们将分析细胞外和细胞外分子的功能和相互作用 Linkin的胞内结构域。以进化守恒为指导，我们将突变保守残基 在线虫中，并在转基因蠕虫中测试它们的功能。我们将使用人类Linkin蛋白 免疫沉淀细胞系的同源突变和定量质谱学蛋白质组学 鉴定生理上相关的相互作用蛋白。我们将测试相互作用的蛋白质的功能 线虫和细胞系的蛋白质组学。我们还将使用线虫的遗传学来筛选优先顺序 使用连接细胞附着试验进行黏附实验的候选对象。候选人的优先顺序将是 在连接细胞中的表达，我们对它有一个很深的转录图谱，预测编码 跨膜或分泌蛋白、系统发育共生以及相互作用的网络预测 基于其他深入研究的生物体中的同源蛋白质。通过这种方式，我们将识别部分 在黏附和生理相关的相互作用蛋白中起关键作用的Linkin 其他可能介导黏附的细胞和同一细胞，以及在细胞质中实现黏附和 细胞骨架的相互作用。这组有效的功能相互作用的蛋白质将定义一条新的途径 细胞黏附并为各种人类正常发育和疾病状态的遗传学研究提供信息。

Project Summary Cell adhesion is crucial aspect of development, fertilization, tissue homeostasis, protection from and interaction with other organisms. Many adhesion proteins have been intensively studied for decades and it was an open question whether we have sufficient knowledge of adhesion, given the large number of interacting proteins and interactions involved in adhesion. LINKIN is a conserved, transmembrane protein that we found to be involved in cell adhesion, suggesting that by studying LINKIN and its interacting proteins we can define a new cellular pathway involved in cell adhesion. The striking conservation of LINKIN sequence and gene copy number across eukaryotic evolution suggests a critical role in development. We discovered LINKIN's role in cell adhesion in the context of C. elegans development, specifically the attachment of the migrating linker cell to the vas deferens, which provides a powerful assay for its structure, function and interactions. We identified interacting proteins in human cells and provided evidence for similar molecular interactions in both organisms. We identified the RUVBL1 and RUVBL2 AAA-ATPases, and α-tubulin as cytoplasmic interactors of LINKIN, suggesting a role for LINKIN in regulating microtubule dynamics, but further investigation is required to connect LINKIN to other interactors and signaling pathways. We propose to further characterize LINKIN's role in cell adhesion in order to connect this new adhesion molecule to other known (or unknown) cellular pathways. A major limitation of human genetic, genomic and systems biology approach is our understanding of the function of many genes. By discovering a new pathway, we will increase the likelihood that genomic, clinical and genetic studies will be interpretable. We will analyze the function and interactors of both the extracellular and intracellular domains of LINKIN. Using evolutionary conservation as a guide we will mutate conserved residues in C. elegans LINKIN and test their function in transgenic worms. We will use human LINKIN proteins with cognate mutations in cell lines for immunoprecipitation and quantitative mass spectrometry proteomics to identify physiologically relevant interacting proteins. We will test the function of interacting proteins identified by the proteomics in C. elegans and cell lines. We will also use C. elegans genetics to screen prioritized candidates for a role in adhesion using the linker cell attachment assay. Candidates will be prioritized by expression in the linker cell, for which we have a deep transcriptomic profile, predictions of encoding transmembrane or secreted proteins, phylogenetic co-occurrence as well as network predictions of interactions based on orthologous proteins in other intensively-studied organisms. In this way, we will identify the portions of LINKIN that are crucial to its function in adhesion and physiologically-relevant interacting proteins both on other cells and same cell that likely mediate adhesion, and in the cytoplasm that implement the adhesive and cytoskeletal interactions. This validated set of functionally interacting proteins will define a new pathway of cellular adhesion and inform a variety of human genetic studies in normal development and disease states.