Rab蛋白透過果蠅腎管調控身體水分含量

Abstract

水分的恆定是生物體維持生存不可或缺的要件之一。而腎臟排尿作用是個體排除體內水分的主要機制。對於未演化出完善腎臟的物種而言亦會發展出調控水分的器官，藉調控多數離子的通透來維持體液恆定，同時也將代謝廢物及有毒物質排出體外，免於個體受到危害。 黑腹果蠅 (Drosophila melanogaster）的馬氏管 (Malpighian tubules) 在功能上同等於脊椎動物的腎臟，調控離子的平衡及體液的滲透壓。馬氏管外觀細長，絕大部份片段由主細胞 (principal cells) 及星狀細胞 (stellate cells) 兩種細胞組成，主細胞上有許多運輸蛋白，可調控陽離子運輸；而星狀細胞缺少此主動運輸的機制。此兩類細胞亦含有大量的水通道蛋白，為可專一性通透水分子，調節滲透壓的重要渠道，然而相關研究對於馬氏管整體的運輸機轉並不完備，仍需更深一層的探討。 本篇的研究重點為馬氏管中Rab GTPase在水分運輸功能上所扮演的角色。Rab GTPase是一類控制細胞內膜狀囊胞運輸的蛋白，具有將不同蛋白質，例如水通道蛋白或離子通道蛋白經由內膜系統從內質網運輸至細胞膜上表現的功能，且在先前研究也已證實Rab GTPase具有組織專一性，會在不同的組織表現出不同Rab蛋白組合，行使特定功能。在這篇研究中，我們將範圍限縮至果蠅的馬氏管系統，探討兩種細胞上Rab蛋白的表現專一性，以及這些多樣的Rab蛋白是否與馬氏管的水分運輸有關。我們利用Gal4/UAS 系統，在受特定調控因子 (promoter) 的控制下活化下游UAS-CD8-GFP，得知Rab在馬氏管的表現形態，接著針對其中專一性表現在主細胞 (principal cells) 的Rab蛋白著手，抑制這些Rab蛋白的表現量，觀察果蠅的水分運輸能力變化，藉此分析這些Rab蛋白在調節水分運輸，體液滲透的生理功能及角色。 實驗結果顯示不同Rab蛋白在馬氏管的表現形態均有差異，且降低專一性表現在主細胞 (principal cells) 的Rab蛋白表現量，並無顯著影響該身體的水分含量。另外在特定抑制RabX4時，我們發現水分分泌能力會些微增加。同樣的現象在完全剔除其中4個Rab的情 結論，本篇論文利用基因轉殖果蠅，得知不同Rab蛋白在馬氏管不同部位的表現形態，且個別降低其中表現在主細胞的Rab蛋白表現量，並不會對馬氏管功能造成顯著影響。

Water homeostasis is an indispensable element for organism to maintain life. In human, water is taken from feeding and drinking, and then removed through sweating, excretion, and breath. Among with these, urination of kidney is the major mechanism to remove water from body fluid. For other species, which have no such complete organ like kidney, will develop other organs for maintaining water balance. These organs maintain fluid balance by regulating most ion channels, and then remove metabolic waste and toxin to prevent from disease. The malpighian tubule system of the fruit fly Drosophila melanogaster is the functional equivalent to the vertebrate kidney that regulates ionic balance and fluid osmolality. Malpighian tubules are long, thin tubes comprised of two major cell types of distinct functions: the principal cells and the stellate cells. While the principal cells regulate transport of electrolytes, the morphology and previous evidence suggest that the stellate cells are not transport-active cells. There is a large number of aquaporins transported water in these two types of cell to regulate fluid osmolality. Although many kinds of aquaporin in malpighain tubules have been known, the mechanism about intracellular trafficking and function has not been comprehensive characterized. In this study, we investigated the role of Rab GTPase mediating water transport via Drosophila malpighian tubules. Rab GTPase are master regulators of intracellular membrane trafficking. While these proteins interact with different proteins, such as aquaporins and ion channels, they facilitate the delivery of transport vesicles from endoplasmic reticulum to plasma membrane. Previous publications have demonstrated that Rab proteins are tissue-specific. We, therefore, investigated whether Rab proteins also express specifically in two types of cell in malpighian tubules and regulate water homeostasis. Using of the Gal4-UAS technique, the Gal4-lines controlled by the regulatory elements of each rab gene drive the expression of wild-type GFP-tagged CD8 in malpighian tubules, so that the expression pattern of Rab proteins could be observed. Then, we focus on the principle-cell-specific Rab proteins and try to figure out whether interfering the expression of these Rabs influences water homeostasis in malpighian tubules. For this purpose, we analyzed several physiological functions like water secretion, water content, and uric acid accumulation in malpighian tubules. Here, we show that different Rabs have different forms of manifestations in malpighian tubules, and then we focus on the interesting Rabs which is only present in principle cell, not stellate cell. However, knocking down these principle-cell-specific Rabs wouldn’t influence water content of whole fly. Furthermore, down-regulating these expressions wouldn’t affect water secretion but RabX4. Moreover, these physiological phenotypes appear the similar result when Rabs are endogenous knock-out. In summary, these results demonstrate that different Rabs have different expression patterns in malpighian tubules, and reducing principle-cell-specific Rabs would not influence the water regulation in malpighian tubules.