The impact of intracellular membrane-associated Crumbs mobility for cell polarity, cell contacts and signal transduction in renal epithelial cells.

哺乳动物的肾脏含有100万至200万个肾单位，它们被认为是最小的生理亚基。虽然肾单位的肾上皮在功能和形态上非常不同，但它们都显示出不同的细胞极化并形成细胞-细胞接触。细胞极性和细胞接触形成都依赖于质膜内Crumbs（或Crumbs蛋白网络）的不对称分布。然而，到目前为止，人们对这种Crumbs不对称性是如何在分子水平上实现和维持的知之甚少。在肾脏中，Crumbs蛋白Crb 2、Crb 3A和Crb 3B表达。在初步研究中，我们发现，Crb 3A和Crb 3B不同，在质膜内的横向流动性。此外，载有Crb 3A或Crb 3B的囊泡表现出不同的迁移率模式。Crb 3B-阳性囊泡一般出现更多的移动的和不太经常固定相比Crb 3A vesicle.We假设的流动性模式内分别朝向质膜是重要的，以实现和维持不对称的组织膜在肾脏。另一方面，移动性或定向碎屑移动的扰动可能破坏肾细胞-细胞接触，例如在狭缝隔膜中。这是特别重要的，因为最近已经表明，人CRB 2基因中的某些突变可以伴随着肾小球滤过屏障的破坏而出现沿着。Crb 3A和Crb 3B在所有肾上皮细胞中表达，但Crb 2优先在足细胞中表达。因此，我们打算分析的流动性模式的Crb 2，Crb 3A和Crb 3B在足细胞细胞膜，以及在MDCK细胞膜，这代表细胞模型的管状顶端上皮膜。通过这种方式，将阐明碎屑移动性在肾细胞中影响细胞极性、细胞接触、肌动蛋白动力学和信号传导过程的作用。在我们的合作项目中，我们将联合收割机首次将细胞生物学方法与定量光学显微镜技术（单分子/颗粒跟踪和荧光光漂白测量）相结合，以揭示关键分子如Crb 2在肾质膜上的运动。

The mammalian kidney contains 1 to 2 million nephrons, which are considered as the smallest physiological subunits. Although kidney epithelia of the nephron are functionally and morphologically very divergent, they all display a distinct cell polarization and form cell-cell contacts. Both cell polarity and cell contact formation are dependent on the asymmetric distribution of the Crumbs (or the Crumbs protein network) within the plasma membrane. However, so far only little is known about how this Crumbs asymmetry is realized and maintained on a molecular level. In the kidney, the Crumbs proteins Crb2, Crb3A and Crb3B are expressed. In preliminary studies we showed that Crb3A and Crb3B differ in their lateral mobility within the plasma membrane. Also vesicles loaded with either Crb3A or Crb3B exhibit different mobility patterns. Crb3B-positive vesicles appeared generally more mobile and were less often immobilized compared to Crb3A vesicles.We assume that the mobility pattern within respectively towards the plasma membrane is important to achieve and maintain asymmetrically organized membranes in the kidney. On the other hand perturbations of the mobility or directed Crumbs movement could disrupt renal cell-cell contacts, e.g. in the slit diaphragm. This is especially important since it has recently been shown that certain mutations in the human CRB2 gene can come along with a disruption of the glomerular filtration barrier. Crb3A and Crb3B are expressed in all renal epithelia, but Crb2 preferentially in podocytes. Thus, we intend to analyze the mobility patterns of Crb2, Crb3A and Crb3B in podocyte cell membranes as well as in MDCK cell membranes, which represent cellular models for tubular apical epithelial membranes. In this manner the role of Crumbs mobility for influencing cell polarity, cell cell contacts, actin dynamics and signaling processes in renal cells shall be elucidated. In our cooperation project we will combine for the first time cell biological approaches with quantitative light microscopy techniques (single molecule/particle tracking and fluorescence photobleaching measurements) to uncover the movement of key molecules like Crb2 on the renal plasma membrane.