GENETICS OF GROWTH CONE BEHAVIOR IN C ELEGANS

线虫生长锥行为的遗传学

• 批准号: 6056669
• 负责人: MIKEL W DAVIS
• 金额: $ 3.03万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6056669
• 关键词: Caenorhabditis elegans; actins; gene mutation; genetically modified animals; growth cones; guanine nucleotide binding protein; guanosinetriphosphatases; molecular cloning; neurogenetics; neuronal guidance

The GTPase proteins Rho, Rac, and CDC42 have each been implicated in the formation of different cytoskeletal structures in migrating cells. Thus, these proteins act as a signaling intermediaries between extracellular signals such as growth factors or migration guidance cues and the cytoskeletal rearrangements and cell movements that these signals cause. They function not only in growth cones, but also in most migrating cells, including not only cells that migrate as part of their developmental program, but also metastatic cancer cells. The C. elegans VD motor neuron growth cone can be specifically labeled with green fluorescent protein and visualized with time- lapse confocal microscopy. The VD growth cone responds to specific substrates with reproducible rounded, lamellar, and extended spike morphologies. Aim 1A: I will construct transgenic worms expressing dominant negative and dominant active forms of the C. elegans Rho family GTPase genes in the VD neuron. Aim 1B: I will examine the VD growth cone behavior in these worms and in worms carrying mutations that affect the function of Rho GTPases. I expect to discover whether any or all of these GTPase genes affects specific aspects of the growth cone cytoskeleton. Aim 2A: I will carry out a screen using both chemical and transposon mediated mutagenesis to find mutations in genes affecting growth cone dynamics. I intend to concentrate my efforts on mutations that affect the growth cone in ways that resemble the defects caused by mutations in the small GTPase signaling pathways. Aim 2B: Mutations will be cloned by conventional positional cloning strategies or by using the technique of transposon display. For transposon mutagenesis, I intend to take advantage of transgenic control of Tc3 transposition, an emerging technology under development in Dr. Jorgensen's lab.

GTPase 蛋白 Rho、Rac 和 CDC42 均与迁移细胞中不同细胞骨架结构的形成有关。 因此，这些蛋白质充当细胞外信号（例如生长因子或迁移引导线索）与这些信号引起的细胞骨架重排和细胞运动之间的信号中介。 它们不仅在生长锥中发挥作用，而且在大多数迁移细胞中发挥作用，不仅包括作为其发育程序一部分而迁移的细胞，还包括转移性癌细胞。线虫 VD 运动神经元生长锥可以用绿色荧光蛋白特异性标记，并用延时共聚焦显微镜观察。 VD 生长锥以可重复的圆形、层状和延长的尖峰形态响应特定的基质。 目标 1A：我将构建在 VD 神经元中表达秀丽隐杆线虫 Rho 家族 GTPase 基因的显性失活和显性活性形式的转基因蠕虫。 目标 1B：我将检查这些蠕虫以及携带影响 Rho GTPases 功能的突变的蠕虫中的 VD 生长锥行为。我期望发现这些 GTP 酶基因中的任何一个或全部是否影响生长锥细胞骨架的特定方面。 目标 2A：我将使用化学和转座子介导的诱变进行筛选，以发现影响生长锥动态的基因突变。 我打算集中精力研究影响生长锥的突变，其方式类似于小 GTP 酶信号通路突变引起的缺陷。 目标 2B：通过传统的定位克隆策略或使用转座子展示技术来克隆突变。 对于转座子诱变，我打算利用 Tc3 转座的转基因控制，这是 Jorgensen 博士实验室正在开发的一项新兴技术。