TUBULIN BOUND DEOXY GTP AND NGF TREATED NEURONS

描述：(改编自申请者摘要)微管蛋白AB- 杂二聚体与GTP或GDP在其可交换的(E-)和 不可交换(N-)位点，E位GTP水解酶是驱动因素 动力不稳定性与微管细胞骨架 细胞周期中的重排。大鼠PC12嗜铬细胞瘤细胞 MT细胞骨架实现形态变化，尤其是 神经生长因子对分支神经突起生长的影响 (NGF)。利用高效液相色谱法对组装的MTS的核苷酸含量进行分析， 调查人员最近发现dGTP替代了GTP在 在NGF存在下生长的PC12细胞的微管蛋白N位点。 经NGF处理后生物合成的微管蛋白中约有80%-85%含有 DGTP，基于一对一结合化学计量比。这项研究 该项目将检验以下总体假设：“NGF刺激 轴突生长、微管蛋白生物合成和dGTP掺入 微管蛋白；考虑GTP调节转导的中心作用 系统在细胞增殖中，这种迄今未被认识到的联系 细胞生长条件和微管蛋白N位核苷酸组成可能 提供调整细胞GTP水平和隔离的机制 研究计划有四个具体目标：1)确定 NGF处理的PC12细胞dGTP池变化的时程 对NGF的响应；2)确定新合成的周转率 微管蛋白亚型与N-位dGTP周转率的关系 PC12细胞培养前后线粒体dGTP含量的研究 用NGF突变体进行治疗，该突变体仅能与高- 亲和NGF受体；4)使用视频显微镜建立时间- 神经生长因子诱导细胞突起生长的过程及探讨 从细胞体和细胞体分离的MTS的组装/拆卸动力学 神经突起中富含dGTP的MTS。

DESCRIPTION: (adapted from Applicant's Abstract) Tubulin aB- heterodimers bind GTP or GDP at their exchangeable (E-) and nonexchangeable (N-) sites, and E-site GTP hydrolysis is the driving force for dynamic instability and microtubule (MT) cytoskeletal rearrangements in the cell cycle. Rat PC12 pheochromocytoma cells use the MT cytoskeleton to achieve morphologic changes, most particularly outgrowth of branched neurites, in response to nerve growth factor (NGF). Using HPLC analysis for nucleotide content of assembled MTs, the investigators recently discovered that dGTP substitutes for GTP in the tubulin N-site of MTs from PC12 cells grown in the presence of NGF. About 80- 85% of tubulin biosynthesized after NGF treatment contained dGTP, based on a one-to-one binding stoichiometry. This research project will test the following overall hypothesis: "NGF stimulates neurite outgrowth, tubulin biosynthesis, and incorporation of dGTP into tubulin; considering the central role of GTP-regulatory transduction systems in cell proliferation, this hitherto unrecognized link between cell growth conditions and tubulin N-site nucleotide composition may provide a mechanism for adjusting cell GTP levels and sequestering dGTP." The research plan has four specific aims: 1) to determine the time-courses for changes in dGTP pools in NGF-treated PC12 cells in response to NGF; 2) to determine turnover rates of newly synthesized tubulin isotypes relative to the turnover rate of N-site dGTP; 3) to investigate dGTP content of MTs isolated from PC12 cells before and after treatment with a mutant of NGF capable of binding only to the high- affinity NGF receptor; 4) to use video microscopy to establish time- courses of neurite outgrowth in cells treated with NGF and to explore the assembly/disassembly dynamics of MTs isolated from cell bodies and of the dGTP-rich MTs from neurites.