DSU Sub-Project 2

DSU 子项目 2

基本信息

批准号：
8461871
负责人：
Cynthia M. van Golen
金额：
$ 20.89万
依托单位：
DELAWARE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8461871
关键词：
AMD3100 Actin-Binding Protein Actins Axon Binding CXCL12 gene CXCR4 Receptors CXCR4 gene Cell Line Cells Cellular Morphology Centers of Research Excellence Chemicals Chemotaxis Confocal Microscopy Cues Data Defect Delaware Development Extracellular Matrix Human Immigration Immunoprecipitation In Vitro Insulin-Like Growth Factor I Insulin-Like-Growth Factor I Receptor Laboratories Length Ligands Mass Spectrum Analysis Mediating Mediator of activation protein Morphology Nervous system structure Neural Crest Neural Crest Cell Neurites Neuroblastoma Neurons Neurosciences Research Play Population Post-Translational Protein Processing Process Proteins Regulation Role Signal Pathway Synapses Testing Work axonal guidance base cell motility chemokine receptor immunocytochemistry in vivo inhibitor/antagonist migration neuroblastoma cell neutralizing antibody polymerization receptor receptor function research study

项目摘要

The external cues that guide the migration of developing neurons and outgrowth of neurons upon differentiation have been intensely studied for decades. CXCR4, a chemokine receptor, has been implicated in the regulation of chemotaxis, neuronal migration, and axonal guidance. Neuroblastoma cells, which are of neural crest origin, are capable of differentiating into more mature sympathetic neurons in culture, and insulin-like growth factor I (IGF-1), has been shown to promote both migration and neurite outgrowth in these cells. In addition, neuroblastoma cells often express high levels of CXCR4, are responsive to CXCLI 2 and, depending upon the level of differentiation, are capable of producing fully extended axons. The mediator of the cytoskeletal changes seen in during process extension is actin polymerization. We have shown that in the neuroblastoma cell line, SHSY-5Y, both CXCR4 and IGF I receptors are involved in neuronal outgrowth, however, treatment with ligands for these receptors results in different cellular morphologies. CXCR4 stimulation stimulated the cells to take on a more differentiated neuronal form and directly involved actin, while IGF-IR stimulation resulted in a very immature neuronal morphology with shorter, broader processes. Based on these results, our overall hypothesis is that CXCR4 promotes neuronal migration and neurite extension through direct regulation of actin dynamics. Our preliminary data suggest that activation of CXCR4 by CXCL12 in cultured neuroblastoma cells promotes the elongation of neurites, and we have found CXCR4 along these projections. In this work we will be testing three specific hypotheses: 1) Cellular context, including the extracellular matrix present and the concentration of CXCL12 ligand to which the cells are exposed play a large role in determining the signaling pathways that are activated, and thereby the ability of the cells to migrate; 2) CXCR4 activation by CXCL12 promotes an increase in neurite length in neuroblastoma cells; 3) CXCR4 regulates elongation of neuronal processes through interaction with actin using the actinbinding protein Dbn; We will use immunocytochemistry/confocal microscopy, neurite analysis, and immunoprecipitation with cultured neuroblastoma and sympathetic neurons to test these hypotheses. Posttranslational modification of CXCR4 will be analyzed using Mass Spectroscopy.

几十年来，人们对引导发育中的神经元迁移和分化时神经元生长的外部线索进行了深入研究。 CXCR4 是一种趋化因子受体，与趋化性、神经元迁移和轴突引导的调节有关。源自神经嵴的神经母细胞瘤细胞能够在培养物中分化为更成熟的交感神经元，而胰岛素样生长因子 I (IGF-1) 已被证明可以促进这些细胞的迁移和神经突生长。此外，神经母细胞瘤细胞通常表达高水平的CXCR4，对CXCLI 2有反应，并且根据分化水平，能够产生完全延伸的轴突。在过程延伸过程中看到的细胞骨架变化的介体是肌动蛋白聚合。我们已经证明，在神经母细胞瘤细胞系 SHSY-5Y 中，CXCR4 和 IGF I 受体均参与神经元生长，然而，用这些受体的配体处理会导致不同的细胞形态。 CXCR4刺激刺激细胞呈现更加分化的神经元形式并直接涉及肌动蛋白，而IGF-IR刺激导致非常不成熟的神经元形态，具有更短、更宽的过程。基于这些结果，我们的总体假设是 CXCR4 通过直接调节肌动蛋白动力学来促进神经元迁移和神经突延伸。我们的初步数据表明，在培养的神经母细胞瘤细胞中，CXCL12 激活 CXCR4 会促进神经突的伸长，并且我们在这些突起上发现了 CXCR4。在这项工作中，我们将测试三个具体假设： 1) 细胞环境，包括细胞外基质的存在和细胞所暴露的CXCL12配体的浓度，在决定被激活的信号通路以及细胞迁移的能力方面发挥着重要作用； 2) CXCL12激活CXCR4促进神经母细胞瘤细胞神经突长度增加； 3) CXCR4 通过肌动蛋白结合蛋白 Dbn 与肌动蛋白相互作用来调节神经元过程的伸长；我们将使用免疫细胞化学/共聚焦显微镜、神经突分析以及培养的神经母细胞瘤和交感神经元的免疫沉淀来检验这些假设。将使用质谱分析 CXCR4 的翻译后修饰。