Regulation und gating von neuen Aquaporinen aus dem Schleimpilz Dictyostelium disciodeum und deren Funktion für die Zellmotilität

我们从盘基网柄菌中鉴定了一种新的水通道蛋白AqpB。AqpB是水特异的，O-糖基化的，并表现出一种新的门控机制。它定位于液泡结构、质膜和膜突起中。到目前为止，它是Dictyostelia中唯一具有功能的水通道蛋白，并且在所有发育阶段都有表达。门控的关键结构域包括D-环中的七个氨基酸位置。到目前为止，基因敲除尝试已经产生了非特异性的基因组整合。除AqpB外，我们还在基因和蛋白质水平上鉴定了阿米巴中的第二个水通道蛋白AqpC。现在我们将进一步阐明AqpB的门控机制，并主要分析AqpB的生理功能，更初步地分析AqpC的生理功能。我们将产生新的AqpB突变，特别是在保守的酪氨酸-218周围的序列中，用于生物物理分析。我们将构建具有更长连接臂的新的敲除质粒，并在AqpB的编码区选择富含GC的序列，以提高基因组整合的特异性。如果基因敲除是致命的，我们将应用琥珀抑制基因敲除方法，这种方法可以将蛋白质水平降低高达90%。通过使用去调节的AqpB变异体的显性过表达，我们将测试门控在细胞生理学中的作用。我们将从形态、胞质分裂、发育、运动性、体积调节和巨噬细胞吞噬等方面分析敲除/敲除/过表达培养物的表型。我们希望获得有关水通道蛋白在细胞生理学中的作用的基础知识，并有可能转化为可运动的人类细胞，如免疫细胞和扩散中的肿瘤细胞。

We have characterized a novel aquaporin, AqpB, from Dictyostelium discoideum. AqpB is water-specific, O-glycosylated and exhibits and a novel gating mechanism. It is localized in vacuolar structures, the plasma membrane, and in membrane protrusions. So far, it represents the sole functional aquaporin in Dictyostelia und is expressed in all developmental stages. The critical domain for the gating comprises seven amino acid positions in the D-loop. Until now, knockout attempts have yielded unspecific genome integration. Besides AqpB, we have identified a second aquaporin, AqpC, in amoebae on the cDNA and protein level. Now we will further elucidate the gating mechanism of AqpB and analyze the physiological function mainly of AqpB and more initially of AqpC. We will generate new mutations of AqpB especially in the sequence stretch around the conserved tyrosine-218 for biophysical analysis. We will construct new knockout plasmids with longer linker arms and select GC-rich sequences within the coding region of AqpB to improve the specificity of genomic integration. If the knockout is lethal, we will apply the amber-suppression knockdown method, which reduces protein levels up to 90%. By using dominant overexpression of de-regulated AqpB-Variants, we will test for the role of gating with respect to cell physiology. We will analyze the phenotypes of the knockout/knockdown/overexpression cultures regarding morphology, cytokinesis, development, motility, volume regulation, and macropinocytosis. We expect to gain basic knowledge on the role of aquaporins in cell physiology with the potential for translation to motile human cells, such as immune cells and spreading tumor cells.