DSU Sub-Project 2

DSU 子项目 2

• 批准号: 9132807
• 负责人: Cynthia M. van Golen
• 金额: $ 16.18万
• 依托单位: DELAWARE STATE UNIVERSITY
• 依托单位国家: 美国
• 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/9132807
• 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

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.

引导发育中神经元迁移和分化后神经元生长的外部线索已经被深入研究了几十年。趋化因子受体CXCR 4参与趋化性、神经元迁移和轴突导向的调节。神经嵴起源的神经母细胞瘤细胞能够在培养中分化成更成熟的交感神经元，并且胰岛素样生长因子I（IGF-1）已显示促进这些细胞中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CXCR 4，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。在突起延伸过程中，细胞骨架变化的介体是肌动蛋白聚合。 我们已经表明，在神经母细胞瘤细胞系SHSY-5 Y中，CXCR 4和IGF I受体都参与神经元生长，然而，用这些受体的配体处理导致不同的细胞形态。CXCR 4刺激刺激细胞呈现更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、更宽的突起。基于这些结果，我们的总体假设是CXCR 4通过直接调节肌动蛋白动力学促进神经元迁移和轴突延伸。我们的初步数据表明，在培养的神经母细胞瘤细胞中，CXCL 12激活CXCR 4可促进神经突的伸长，我们已经发现CXCR 4沿着这些突起。在这项工作中，我们将测试三个具体的假设： 1)细胞环境，包括存在的细胞外基质和细胞所暴露的CXCL 12配体的浓度，在确定被激活的信号传导途径中起重要作用，从而确定细胞迁移的能力; 2)CXCL 12激活CXCR 4促进神经母细胞瘤细胞中神经突长度的增加; 3)CXCR 4通过肌动蛋白结合蛋白Dbn与肌动蛋白相互作用调节神经元突起的延伸; 我们将使用免疫细胞化学/共聚焦显微镜，神经突分析，免疫沉淀与培养的神经母细胞瘤和交感神经元来测试这些假设。将使用质谱分析CXCR 4的翻译后修饰。