DSU Sub-Project 2

指导发育中的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。CXCR4是一种趋化因子受体，参与了趋化、神经元迁移和轴突引导的调节。神经母细胞瘤细胞起源于神经脊，在培养过程中能够分化为更成熟的交感神经元，胰岛素样生长因子-1(IGF-1)被证明可以促进这些细胞的迁移和突起生长。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，并且根据分化程度的不同，能够产生完全延伸的轴突。细胞骨架变化的中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受体都参与了神经元的生长，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分化的神经元形态，并直接参与肌动蛋白，而IGF-IR刺激导致非常不成熟的神经元形态，突起更短、更宽。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，CXCL12激活CXCR4促进了神经突起的延长，我们已经沿着这些投射发现了CXCR4。在这项工作中，我们将测试三个具体的假设： 1)细胞环境，包括细胞外基质的存在和细胞所接触的CXCL12配体的浓度，在决定激活的信号通路，从而决定细胞迁移的能力方面起着重要作用； 2)CXCL12激活CXCR4可促进神经母细胞瘤细胞突起长度的增加； 3)CXCR4通过与肌动蛋白结合蛋白DBN调节神经元突起的伸长； 我们将使用免疫细胞化学/共聚焦显微镜、轴突分析和培养的神经母细胞瘤和交感神经元的免疫沉淀来验证这些假设。CXCR4的翻译后修饰将用质谱学进行分析。

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.