Acetylcholine Receptors in Cell Migrationis

项目摘要 乙酰胆碱(ACh)是一种古老的信号分子，存在于许多生物中，包括细菌、原生动物、 植物和动物。通过烟碱和毒扁豆碱受体检测ACh存在于人体许多器官中 除了神经系统以外，这些都比它的神经功能研究要少得多。一种非突触 ACh信号在细胞正常发育过程中起着迁移作用。比如，平滑肌细胞， 上皮角质形成细胞和间充质干细胞对ACh的反应迁移，但详细的机制 目前还不清楚这种信号是如何在迁移中被利用的，特别是在体内。我们有 开发了一种独特的系统，用于研究Ach信号在体内细胞迁移中的作用。秀丽线虫 连接细胞(Linker cell，LC)是一种特殊的上皮细胞，负责引导发育中的男性性腺迁移。我们发现 通过单细胞转录，认为LC表达了许多神经元受体和离子通道，导致 认为LC使用神经元线索进行迁移的假设。我们将研究乙酰胆碱的作用 LC中的信号，因为我们观察到缺乏特定毒碱和烟碱的突变体的迁移缺陷 感受器。我们首先将重点放在LC中唯一表达的M受体，GAR-3，我们发现它能 激活单元格方向的更改。我们将同时研究多种尼古丁受体的功能。 在LC迁移和潜在的受体类型之间的合作。LC将其方向从 乙酰胆碱酯酶诱导的ACh信号过量反应的后位向前位 我们将使用一种分析来确定LC中GAR-3途径的下游成分。 由于已经在其他上下文中识别了分支GAR-3信号通路的下游目标(例如， 突触传递)，我们将测试这些基因的突变，以确定用于 调制液晶取向。为了研究ACh分布的作用，我们将重新编程神经元在 腹神经索(VNC)建立人工前/后ACh梯度以研究ACh如何分布 大小影响LC的迁移。我们将合作使用相关电子显微镜来检查GAR- 3高分辨率定位与LC及其周围组织超微结构的关系。我们将重复使用这些资源 研究六种烟碱受体亚基的潜在作用。

Project Summary Acetylcholine (ACh) is an ancient signaling molecule found in many organisms including bacteria, protozoa, plants, and animals. ACh detection through nicotinic and muscarinic receptors occurs in many human organs besides the nervous system but these much less studied than its neuronal function. One of the non-synaptic functions of ACh signaling is in cell migration during normal development. For example, smooth muscle cells, epithelial keratinocytes, and mesenchymal stem cells migrate in response to ACh, but a detailed mechanistic understanding of how this signaling is utilized in migration, particularly in vivo, is not available. We have developed a unique system with which to study the role of Ach signaling in cell migration in vivo. The C. elegans linker cell (LC) is a specialized epithelial cell that leads the migration of the developing male gonad. We found through single-cell transcriptomics that the LC expresses many neuronal receptors and ion channels, leading to the hypothesis that the LC uses neuronal cues for its migration. We will investigate the role of acetylcholine signaling in the LC because we observed migration defects in mutants lacking particular muscarinic and nicotinic receptors. We will initially focus on the only muscarinic receptor expressed in the LC, GAR-3, that we found to activate changes in cell orientation. We will in parallel investigate the function of the multiple nicotinic receptors in LC migration and potential cooperation between the receptor types. The LC reverses its orientation from the posterior to anterior in response to excess ACh signaling induced by treatment with the acetylcholinesterase inhibitor aldicarb, an assay we will use to identify the downstream components of the GAR-3 pathway in the LC. Since downstream targets of the branching gar-3 signaling pathway have been identified in other contexts (e.g., synaptic transmission), we will test mutants of those genes to identify the specific downstream pathway used to modulate LC orientation. To investigate the role of ACh distribution, we will reprogram the fate of neurons in the ventral nerve cord (VNC) to create artificial anterior/posterior ACh gradients to investigate how ACh distribution and amount affects LC migration. We will collaborate to use Correlation Electron Microscopy to examine GAR- 3 localization relative to the ultrastructure of LC and surrounding tissue at high resolution. We will reuse these assays to study potential effects of six nicotinic receptor subunits.