Collaborative Research: Mechanisms of Iron Transport in Insects

铁对动物生活的许多方面都是必不可少的，但铁也有潜在的毒性。由于铁是必需的，也是有害的，因此必须严格控制动物体内铁的数量、位置和形式。在人类和其他哺乳动物中，在正确的时间提供适量铁的生理过程被部分理解。然而，在昆虫和其他无脊椎动物中，这些过程尚未被发现。该研究项目的目标是了解铁是如何从一个细胞运输到另一个昆虫使用果蝇作为模型系统。该项目将为来自不同背景的本科生提供研究经验。从这项研究中获得的知识将为未来研究吸血昆虫（如蚊子）的铁运输提供一个框架，蚊子在摄取血餐时必须科普看似有毒的铁量。该项目的一个潜在成果是发现一种更古老的铁运输机制，不仅在昆虫中起作用，而且在其他无脊椎动物中也起作用。此外，从这项研究中获得的信息可能会深入了解人类铁转运的机制。最后，由于昆虫管理策略依赖于有关昆虫生物学的信息，因此该项目的结果可能有助于新的昆虫控制策略的开发。该研究的目的是了解昆虫铁转运的生理机制。四个模型，描述了铁运输到细胞的可能机制，将测试使用果蝇作为模式昆虫物种。目标1：第一个目的是检验受体介导的铁负载铁蛋白或铁转铁蛋白的内吞作用将铁从血淋巴转运到细胞中的假设。1A）铁蛋白和转铁蛋白将用荧光团标记，然后观察以确定是否发生细胞摄取。1B）共聚焦显微镜将用于评价铁蛋白或转铁蛋白是否存在于核内体中。1C）内吞途径的必需组分将被阻断，并且将测量对铁摄取的影响。目的2：第二个目的是检验血淋巴中铁离子的还原导致铁转运到细胞中的假设。血淋巴含有三价铁形式的铁，但动物体内的铁转运蛋白对亚铁具有特异性;因此，通过流入转运蛋白摄取铁可能需要还原步骤。将研究已知铁还原酶CG 8399和候选铁还原酶CG 1275的功能。2A）RNAi将用于确定CG 8399或CG 1275是否有助于细胞表面铁还原酶活性。2B）RNAi将用于确定CG 8399或CG 1275的活性是否影响细胞的铁含量。2C）评价膜不渗透亚铁螯合剂抑制铁摄取的能力。

Iron is essential for many aspects of an animal's life, but iron is also potentially toxic. Because iron is both necessary and harmful, the amount, location and form of iron within an animal must be tightly controlled. In humans and other mammals, the physiological processes that provide the right amount of iron at the right times are partially understood. However, in insects and other invertebrate animals, these processes have not yet been discovered. The goal of this research project is to understand how iron is transported from one cell to another in insects using the fruit fly as a model system. The project will provide research experiences for undergraduate students from diverse backgrounds. Knowledge gained from this research will provide a framework for future studies of iron transport in blood-feeding insects such as mosquitoes, which must cope with a seemingly toxic amount of iron when they ingest a blood meal. A potential outcome of this project is the discovery of a more evolutionarily ancient iron transport mechanism that functions not only in insects but also in other invertebrate animals. In addition, information learned from this research may provide insight into the less-understood mechanisms of iron transport in humans. Finally, because insect management strategies rely on information about insect biology, the results of this project may contribute to the development of a new insect control strategy. The goal of the proposed research is to understand the physiological mechanisms of iron transport in insects. Four models that describe possible mechanisms of iron transport into cells will be tested using Drosophila melanogaster as the model insect species. OBJECTIVE 1: The first objective is to test the hypothesis that receptor-mediated endocytosis of iron-loaded ferritin or ferric-transferrin transports iron from hemolymph into cells. 1A) Ferritin and transferrin will be labeled with a fluorophore and then observed to determine whether cellular uptake occurs. 1B) Confocal microscopy will be used to evaluate whether ferritin or transferrin are present in endosomes. 1C) Essential components of the endocytosis pathways will be blocked, and the effect on iron uptake will be measured. OBJECTIVE 2: The second objective is to test the hypothesis that reduction of ferric ions in hemolymph leads to iron transport into cells. Hemolymph contains iron in the ferric form, but iron transporters in animals are specific to ferrous iron; therefore, iron uptake via an influx transporter is likely to require a reduction step. The functions of a known ferric reductase, CG8399, and a candidate ferric reductase, CG1275, will be studied. 2A) RNAi will be used to determine whether CG8399 or CG1275 contribute to cell surface ferric reductase activity. 2B) RNAi will be used to determine whether the activity of CG8399 or CG1275 influences the iron content of cells. 2C) A membrane-impermeant ferrous chelator will be evaluated for its ability to inhibit iron uptake.