Control of transport by epithelia

上皮细胞对运输的控制

• 批准号: RGPIN-2017-05196
• 负责人: Marshall, William
• 金额: $ 2.04万
• 依托单位: St. Francis Xavier University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711242
• 关键词: Control; transport; epithelia

We study mechanisms that animals use to regulate ion and water transport across epithelia. The main model system we use is a hardy teleost fish that frequents estuaries of N. America and is robust to environmental extremes (salinity, temperature, low oxygen, pollution), the mummichog (Fundulus heteroclitus), an important physiological and genomic model species and important as a bioindicator species. The models of the gill osmoregulatory function we use are flat epithelia rich in salt transporting ionocytes, the opercular epithelium in adults and the yolk sac epithelium of embryos. The research focuses on revealing regulatory proteins (kinases and phosphatases) that modulate the transport proteins directly or via kinase cascades. Aim 1. We will discover the regulatory components of the transport complexes that control the anion channel Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and the cotransporters Na,K,2Cl cotransporter (NKCC1) and NaCl cotransporter (NCC) which include many kinases as well as a calcium-dependent inhibitory pathway. These pathways are activated by osmotic stimuli, neurotransmitters and hormones. The overarching hypothesis is that membrane transport proteins have multiple regulatory components in apparently redundant pathways that regulate ion transport, but in hardy animals such as Fundulus and less hardy tropical fish (Nile tilapia,Oreochromis nilotica), the extreme variation in the environment determines which pathways are best-suited for operation in extreme environments. Aim 2. We will overlay the above with cellular dynamics of this highly regulated transport system using simple salinity changes and immunocytochemistry of transporter proteins to generate cellular succession vs. remodeling of existing ionocytes and their various phenotypic variations in freshwater, seawater and hypersaline conditions. Aim 3. We will use the yolk sac membrane of Fundulus embryos (also salt hardy and rich in ionocytes) to test regulatory pathways using short interfering mRNA (siRNA) injection to block specific kinase/phosphatase genes (confirmed by quantitative PCR) in the yolk sac and see how transport regulation (hypotonic, hypertonic shock, adrenergic agonists) are selectively disrupted to identify the critical components of the regulatory pathways. Importance: Understanding of ion transport and its regulation also informs fisheries and aquaculture and advances comparative physiology. The research will reveal new relationships among regulatory kinases controlling ion transport proteins also associated with disease, particularly CFTR (responsible for cystic fibrosis), NKCC (implicated in pseudohypoaldosteronism) and NCC (involved in Gitelman's syndrome). The work will contribute 19 HQP (BSc and MSc) to Canadian research.

我们研究了动物用来调节离子和水在上皮细胞中运输的机制。 我们使用的主要模型系统是一个哈代硬骨鱼，经常在河口的N。美洲和是强大的极端环境（盐度，温度，低氧，污染），mummichog（底heteroclitus），一个重要的生理和基因组模式物种和重要的生物指示物种。我们所用的鳃膜调节功能的模型是富含盐转运细胞的扁平上皮、成体的鳃盖上皮和胚胎的卵黄囊上皮。 研究重点是揭示直接或通过激酶级联调节转运蛋白的调节蛋白（激酶和磷酸酶）。 目标1.我们将发现控制阴离子通道囊性纤维化跨膜电导调节器（CFTR）和共转运体Na，K，2Cl共转运体（NKCC 1）和NaCl共转运体（NCC）的转运复合物的调节组分，其中包括许多激酶以及钙依赖性抑制途径。这些通路由渗透刺激、神经递质和激素激活。最重要的假设是，膜转运蛋白在调节离子转运的明显冗余途径中具有多种调节组分，但在哈代动物如底栖动物和不太哈代的热带鱼（尼罗罗非鱼、尼罗罗非鱼）中，环境的极端变化决定了哪些途径最适合在极端环境中运行。 目标2.我们将使用简单的盐度变化和转运蛋白的免疫细胞化学将上述内容与这种高度调节的转运系统的细胞动力学叠加，以产生细胞演替与现有离子细胞的重塑及其在淡水，海水和高盐条件下的各种表型变化。 目标3.我们将使用卵黄囊膜底胚胎（也盐哈代和丰富的离子细胞），以测试监管途径，使用短干扰mRNA（siRNA）注射，以阻止特定的激酶/磷酸酶基因（通过定量PCR证实）在卵黄囊，看看如何运输调节（低渗，高渗休克，肾上腺素能激动剂）被选择性地破坏，以确定关键组成部分的监管途径。重要性：对离子运输及其调节的理解也为渔业和水产养殖业提供了信息，并促进了比较生理学的发展。这项研究将揭示控制离子转运蛋白的调节激酶之间的新关系，这些蛋白也与疾病有关，特别是CFTR（负责囊性纤维化），NKCC（涉及假性醛固酮减少症）和NCC（涉及Gitelman综合征）。 这项工作将为加拿大的研究贡献19名HQP（理学士和硕士）。