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。人叶酸受体是 正常神经管发育所必需的，并且经常在癌症中过度表达， 有助于癌症的发展。在这两个过程中，有新的证据表明， 叶酸受体的作用不依赖于提供叶酸作为一碳代谢的维生素。的 小蛔虫C.线虫从其食物来源细菌中获得叶酸。我们已经发现 一种特殊的细菌叶酸盐（10-甲酰-THF）增加了C.秀丽线虫 干细胞，从而作为一个信号，将增殖速度与动物的可用性联系起来。 细菌饮食此外，我们发现10-甲酰-THF作为信号激活NSM 神经元NSM神经元的激活导致饥饿的动物停止前进 当他们遇到细菌时。这两个过程都需要C。线虫叶酸受体， FOLR-1，并且不依赖于提供叶酸在一碳代谢中的典型作用。 该提案的目标是确定FOLR-1介导的分子途径 信号从刺激叶酸到生殖细胞和NSM神经元。我们将使用荧光标记 为了确定FOLR-1在细胞中的定位以及在存在FOLR-1的情况下该定位是否改变， 刺激性叶酸遗传实验表明，FOLR-1在神经元和非神经元细胞中都发挥作用。 NSM神经元的活化。我们发现GON-2 钙通道是伴随NSM神经元激活的钙内流所必需的。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。 使用遗传学方法，我们将鉴定FOLR-1和GON-2各自发挥功能的组织， 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增殖，这表明钙水平的增加促进了 生殖细胞增殖我们将确定钙水平是否因生长条件而异， 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 将被用来确定下游效应，改变生殖细胞增殖反应， 改变了生殖细胞的钙水平无偏见的基因筛选将用于识别组件 和刺激-叶酸途径的调节剂。鉴定的基因将使用 遗传和生物化学方法，以确定它们如何促进途径。这 研究将揭示叶酸调节多种细胞的分子途径， 过程独立于其在新陈代谢中的作用。这将为理解 非经典叶酸途径，具有深入了解神经管缺陷和癌症的潜力。

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.