Folate-directed signaling in C. elegans

项目摘要 叶酸受体是叶酸转运蛋白的三种主要类型之一。人类的叶酸受体是 对神经管的正常发育是必需的，在癌症中经常过度表达 会导致癌症的发展。在这两个过程中，都有新的证据表明 叶酸受体的作用独立于提供叶酸作为一碳代谢的维生素。这个 小的线虫从其食物细菌中获取叶酸。我们发现 一种特定的细菌叶酸(10-甲酰-四氢呋喃)可提高线虫胚芽的增殖速度 干细胞，从而作为一个信号，将增殖速度与动物的可获得性联系起来 细菌饮食。此外，我们还发现，10-甲酰-四氢呋喃作为激活NSM的信号 神经元。NSM神经元的激活导致饥饿的动物停止向前运动 当它们遇到细菌时。这两个过程都需要线虫的叶酸受体， FOLR-1，并且不依赖于提供叶酸，因为它们在一碳代谢中起着典型的作用。 这项提议的目标是确定FOLR-1介导的分子途径 从刺激性叶酸到生殖细胞和NSM神经元的信号。我们将使用荧光标签 确定FOLR-1在细胞中的定位位置以及在存在的情况下定位是否发生变化 刺激性叶酸。遗传学实验表明，FOLR-1在神经元和非神经元中都有功能 神经组织来调节NSM神经元的激活。我们发现Gon-2 伴随着NSM神经元激活的钙内流需要钙通道。类似 对于FOLR-1，神经元和非神经元组织中也需要Gon-2来激活NSM。 利用遗传学方法，我们将确定FOLR-1和Gon-2各自发挥作用的组织 允许NSM激活。我们发现Gon-2也是维持体内钙水平所必需的。 生殖系和正常生殖细胞的增殖，表明增加的钙水平促进 生殖细胞增殖。我们将确定钙水平是否会因生长条件和 刺激性叶酸，以及Gon-2和FOLR-1在改变钙水平中的作用。遗传方法 将用于识别改变生殖细胞增殖的下游效应物 改变了生殖系中的钙水平。将使用无偏见的基因筛查来识别成分 和叶酸刺激途径的调节者(S)。识别出的基因将被分析使用 用遗传和生化方法来确定它们对该途径的贡献(S)。这 研究将揭示叶酸调节多种细胞的分子途径(S) 独立于其在新陈代谢中的作用的过程。这将为理解 非典型的叶酸途径，有可能洞察神经管缺陷和癌症。

Project Summary The folate receptor is one of three major types of folate transporters. The human folate receptor is required for proper neural tube development, and is often overexpressed in cancers where it contributes to cancer progression. In both of these processes, there is emerging evidence that the foIate receptor acts independently of providing folates as vitamins for one-carbon metabolism. The small roundworm C. elegans obtains folates from its food source of bacteria. We have discovered that a specific bacterial folate (10-formyl-THF) increases the rate of proliferation of C. elegans germ stem cells, thereby acting as a signal to link the rate of proliferation to the availability of the animal’s bacterial diet. Additionally, we discovered that 10-formyl-THF acts as a signal to activate the NSM neurons. The activation of the NSM neurons causes starved animals to stop their forward motion when they encounter bacteria. Both of these processes require the C. elegans folate receptor, FOLR-1, and are independent of providing folates for their canonical role in one-carbon metabolism. The goal of this proposal is to identify the molecular pathways through which FOLR-1 mediates signals from the stimulatory folate to germ cells and the NSM neurons. We will use fluorescent tags to determine where FOLR-1 is localized in cells and whether the localization changes in the presence of stimulatory folates. Genetic experiments suggest that FOLR-1 functions in both neuronal and non- neuronal tissues to mediate the activation of the NSM neuron. We discovered that the GON-2 calcium channel is required for the calcium influx that accompanies NSM neuronal activation. Similar to FOLR-1, GON-2 is also required in both neuronal and non-neuronal tissues for NSM activation. Using genetic approaches, we will identify the tissues in which FOLR-1 and GON-2 each function to allow NSM activation. We discovered that GON-2 is also required both to maintain calcium levels in the germline and for normal germ cell proliferation, suggesting that increased calcium levels promote germ cell proliferation. We will determine if calcium levels differ in response to growth conditions and stimulatory folate, and the role of GON-2 and FOLR-1 in altering calcium levels. Genetic approaches will be used to identify the downstream effectors that alter germ cell proliferation in response to altered calcium levels in the germline. Unbiased genetic screens will be used to identify components and regulators of the stimulatory-folate pathway(s). The identified genes will be analyzed using genetic and biochemical approaches to determine how they contribute to the pathway(s). This research will uncover the molecular pathway(s) through which folates regulate diverse cellular processes independently of their role in metabolism. This will provide a paradigm for understanding non-canonical folate pathways, with the potential for insights into neural tube defects and cancer.