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100万到200万个肾单位，它们被认为是最小的生理亚基。尽管肾单位的肾上皮细胞在功能和形态上非常不同，但它们都表现出明显的细胞极化并形成细胞-细胞接触。细胞的极性和细胞接触的形成都取决于质膜中Crumbs(或Crumbs蛋白质网络)的不对称分布。然而，到目前为止，关于这种不对称性是如何在分子水平上实现和维持的，人们知之甚少。在肾脏中表达Crumbs蛋白Crb2、Crb3A和Crb3B。在初步研究中，我们发现Crb3A和Crb3B在质膜中的侧向迁移率不同。同样，载有Crb3A或Crb3B的囊泡表现出不同的运动模式。与Crb3A囊泡相比，Crb3B阳性囊泡表现出更强的流动性和更少的固定化。我们认为，内部朝向质膜的迁移模式对于实现和维持肾脏中不对称组织的膜是重要的。另一方面，移动性或定向碎屑运动的扰动可能会扰乱肾细胞与细胞的接触，例如在狭缝隔膜。这一点尤其重要，因为最近有研究表明，人类CRB2基因的某些突变可以伴随着肾小球滤过屏障的破坏而来。Crb3A和Crb3B在所有肾上皮细胞中均有表达，但Crb2主要在足细胞中表达。因此，我们打算分析Crb2、Crb3A和Crb3B在足细胞和MDCK细胞膜中的迁移模式，这两种细胞膜代表了肾小管上皮细胞膜的细胞模型。通过这种方式，应阐明CRUMBS移动性在影响细胞极性、细胞接触、肌动蛋白动力学和肾细胞内信号传递过程中的作用。在我们的合作项目中，我们将首次将细胞生物学方法与定量光学显微镜技术(单分子/粒子跟踪和荧光光漂白测量)相结合，以揭示Crb2等关键分子在肾质膜上的运动。

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.